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Lake Dziani Dzaha (Mayotte Island, Indian Ocean) is a tropical thalassohaline lake which geochemical and biological conditions make it a unique aquatic ecosystem considered as a modern analogue of Precambrian environments. In the present study, we focused on the diversity of phytoplanktonic communities, which produce very high and stable biomass (mean2014–2015 = 652 ± 179 μg chlorophyll a L−1). As predicted by classical community ecology paradigms, and as observed in similar environments, a single species is expected to dominate the phytoplanktonic communities. To test this hypothesis, we sampled water column in the deepest part of the lake (18 m) during rainy and dry seasons for two consecutive years. Phytoplanktonic communities were characterized using a combination of metagenomic, microscopy-based and flow cytometry approaches, and we used statistical modeling to identify the environmental factors determining the abundance of dominant organisms. As hypothesized, the overall diversity of the phytoplanktonic communities was very low (15 OTUs), but we observed a co-dominance of two, and not only one, OTUs, viz., Arthrospira fusiformis (Cyanobacteria) and Picocystis salinarum (Chlorophyta). We observed a decrease in the abundance of these co-dominant taxa along the depth profile and identified the adverse environmental factors driving this decline. The functional traits measured on isolated strains of these two taxa (i.e., size, pigment composition, and concentration) are then compared and discussed to explain their capacity to cope with the extreme environmental conditions encountered in the aphotic, anoxic, and sulfidic layers of the water column of Lake Dziani Dzaha. Electronic supplementary material The online version of this article (10.1007/s00248-019-01332-8) contains supplementary material, which is available to authorized users.

International audience; Thalassohaline ecosystems are hypersaline environments originating from seawater in which sodium chloride is the most abundant salt and the pH is alkaline. Studies focusing on microbial diversity in thalassohaline lakes are still scarce compared with those on athalassohaline lakes such as soda lakes that have no marine origin. In this work, we investigated multiple facets of bacterial, archaeal and eukaryotic diversity in the thalassohaline Lake Dziani Dzaha using a metabarcoding approach. We showed that bacterial and archaeal diversity were mainly affected by contrasting physicochemical conditions retrieved at different depths. While photosynthetic microorganisms were dominant in surface layers, chemotrophic phyla (Firmicutes or Bacteroidetes) and archaeal methanogens dominated deeper layers. In contrast, eukaryotic diversity was constant regardless of depth and was affected by seasonality. A detailed focus on eukaryotic communities showed that this constant diversity profile was the consequence of the high predominance of Picocystis salinarum, while nondominant eukaryotic groups displayed seasonal diversity turnover. Altogether, our results provided an extensive description of the diversity of the three domains of life in an unexplored extreme environment and showed clear differences in the responses of prokaryotic and eukaryotic communities to environmental conditions.

Discovery after discovery, host-associated microbiota reveal a growing list of positive effects on host homeostasis by contributing to host nutrition, improving hosts' immune systems and protecting hosts against pathogens. In that context, a collection of oyster associated bacteria producing antibacterial compounds have been established to evaluate their role in non-host-derived immunity. Here, we described alterins, potent anti-Gram negative compounds produced by Pseudoalteromonas hCg-6 and hCg-42 isolated from different healthy oyster hemolymph. The strains hCg-6 and hCg-42 produce a set of at least seven antibacterial compounds, ranging from 926 to 982 Da structurally characterized as cyclolipopeptides (CLPs). Alterins share the same cationic heptapeptidic cycle connected via an amido bond to different hydrophobic hydrocarbon tails. Their MICs disclosed a potent antibacterial activity directed against Gram-negative bacteria including oyster and human pathogens that may confer a beneficial defense mechanism to the host but also represents an untapped source of new antibiotics. The alterins&rsquo, mechanisms of action have been deciphered: after binding to lipopolysaccharides (LPS), alterins provoke a membrane depolarization and permeabilization leading to bacterial lysis. As hCg-6 and hCg-42 produced a set of natural derivatives, the structure/activity relationship linked to the carbon tail is clarified. We showed that the hydrocarbon tail determines the LPS-binding properties of alterins and consequently their antibacterial activities. Its length and saturation seem to play a major role in this interaction.

International audience; Since 2008, juvenile Crassostrea gigas oysters have suffered from massive mortalities in European fanning areas. This disease of complex etiology is still incompletely understood. Triggered by an elevated seawater temperature, it has been associated to infections by a herpes virus named OsHV-1 as well as pathogenic vibrios of the Splendidus Glade. Ruling out the complexity of the disease, most of our current knowledge has been acquired in controlled experiments. Among the many unsolved questions, it is still ignored what role immunity plays in the capacity oysters have to survive an infectious episode. Here we show that juvenile oysters susceptible to the disease mount an inefficient immune response associated with microbial permissiveness and death. We found that, in contrast to resistant adult oysters having survived an earlier episode of mortality, susceptible juvenile oysters never exposed to infectious episodes died by more than 90% in a field experiment. Susceptible oysters were heavily colonized by OsHV-1 herpes virus as well as bacteria including vibrios potentially pathogenic for oysters, which proliferated in oyster flesh and body fluids during the mortality event. Nonetheless, susceptible oysters were found to sense microbes as indicated by an overexpression of immune receptors and immune signaling pathways. However, they did not express important immune effectors involved in antimicrobial immunity and apoptosis and showed repressed expression of genes involved in ROS and metal homeostasis. This contrasted with resistant oysters, which expressed those important effectors, controlled bacterial and viral colonization and showed 100% survival to the mortality event. Altogether, our results demonstrate that the immune response mounted by susceptible oysters lacks some important immune functions and fails in controlling microbial proliferation. This study opens the way to more holistic studies on the "mass mortality syndrome", which are now required to decipher the sequence of events leading to oyster mortalities and determine the relative weight of pathogens, oyster genetics and oyster-associated microbiota in the disease.

The toxicity of polycyclic aromatic hydrocarbons (PAHs) mixtures was evaluated on natural phytoplankton communities sampled from lagoons of Bizerte (South-western Mediterranean Sea) and Thau (North-western Mediterranean Sea). PAHs induced short-term dose and ecosystem-dependant decreases in photosynthetic potential. Chlorophyll a was negatively affected by increasing PAHs concentrations, together with dramatic changes in phytoplankton community composition. Size classes were strongly affected in the Bizerte compare to the Thau lagoon, with a decrease in nano- and microphytoplankton densities compare to picophytoplankton. In both locations, the diatom Entomoneis paludosa appeared favoured under PAH exposure as evidenced by increase in cell density, whereas autotrophic flagellates and dinophytes were strongly reduced. Smaller cells were more tolerant to exposure to highest PAHs concentrations, with persistent picophytoplankton carbon biomass at the end of the incubations. Apparent recovery of photosynthetic potential, accompanied with a regrowth of chlorophyll a under the lowest PAH doses, coincided with a significantly altered community composition in both lagoons. Furthermore, sensitivity to PAHs was not related to the phytoplankton cell size, and toxicity-induced modification of top-down control by grazers during the experiment cannot be excluded. (C) 2018 Elsevier Ltd. All rights reserved.

International audience; The grazing activity by specific marine organisms represents a growing threat to the survival of many scleractinian species. For example, the recent proliferation of the corallivorous gastropod Drupella now constitutes a critical case in all South-East Asian waters. If the damaging effects caused by this marine snail on coral polyps are relatively well known, the indirect incidence of predation on coral microbial associates is still obscure and might also potentially impair coral health. In this study, we compared the main ecological traits of coral-associated bacterial and viral communities living in the mucus layer of Acropora formosa and Acropora millepora, of healthy and predated individuals (i.e., colonized by Drupella rugosa), in the Bay of Van Phong (Vietnam). Our results show a substantial impact of the gastropod on a variety of microbiological markers. Colonized corals harbored much more abundant and active epibiotic bacteria whose community composition shifted toward more pathogenic taxa (belonging to the Vibrionales, Clostridiales, Campylobacterales, and Alteromonadales orders), together with their specific phages. Viral epibionts were also greatly influenced by Drupella corallivory with spectacular modifications in their concentrations, life strategies, genotype richness, and diversity. Novel and abundant circular Rep-encoding ssDNA viruses (CRESS-DNA viruses) were detected and characterized in grazed corals and we propose that their occurrence may serve as indicator of the coral health status. Finally, our results reveal that corallivory can cause severe dysbiosis by altering virus-bacteria interactions in the mucus layer, and ultimately favoring the development of local opportunistic infections.

International audience; The impact of fish and oysters on components of the pelagic microbial food web (MFW) was studied in a 10 d mesocosm experiment using Mediterranean coastal waters. Two mesocosms contained natural water only (‘Controls’), 2 contained natural water with Crassostrea gigas (‘Oyster’), and 2 contained natural water with Atherina spp. (‘Fish’). Abundances and biomasses of microorganisms (viruses, bacteria, phytoplankton, heterotrophic flagellates, and ciliates) were measured to estimate their contribution to the total microbial carbon biomass. Two MFW indices, the microbial autotroph:heterotroph C biomass ratio (A:H) structural index and the gross primary production:respiration ratio (GPP:R) functional index, were defined. In the Fish mesocosms, selective predation on zooplankton led to a trophic cascade with 51% higher phyto- plankton C biomass and consequently higher A:H and GPP:R than in the Controls. By the end of the experiment, the Oyster mesocosms had a bacterial C biomass 87% higher and phytoplankton C biomass 93% lower than the Controls, giving significantly lower A:H and GPP:R (1), whereas oyster activities made the MFW more hetero- trophic (both indices

International audience; Coral reef and atoll lagoons are among the most diversified marine ecosystems but also the most affected by the combined effects of climate change and human activities. The Iles Eparses (Scattered Islands) in the Western Indian Ocean have been little affected by human pressure and can be considered to be "pristine" ecosystems. Metazooplankton plays a major role in the functioning and productivity of aquatic ecosystems, and this study was undertaken: (i) to determine the spatial abundance, distribution and species composition of metazooplankton, (ii) to assess the effect of metazooplankton grazing on pico- and nanophytoplankton and (iii) to analyze the trophic positions of metazooplankton by using the stable isotope signatures of a wide variety of taxa and particulate organic matter from the Iles Eparses and Mayotte. Tromelin Island (which is not located in the Mozambique Channel) had the lowest metazooplankton abundance with no cyanobacteria Trichodesmium spp. or mollusks (pteropods) presence, and with δ(15)N signatures of organisms that were higher than for the islands in the Mozambique Channel. Trichodesmium spp. was found in the Mozambique Channel and the plankton food web was probably based preferentially on these cyanobacteria with lower δ(15)N signatures indicating direct or indirect trophic transfer of diazotrophic nitrogen to metazooplankton. Three of the islands were distinct: Europa had the highest proportion of copepods, with oithonids being dominant, which is typical of rich mangrove systems, while Juan de Nova and Mayotte seemed to be the sites most affected by human activity with a high abundance of appendicularians and distinct particulate organic matter ∂(13)C signatures. Grazing experiments showed that food could be a limiting factor for metazooplankton in the Iles Eparses. However, the effect of metazooplankton grazing on phytoplankton appeared to be very low (0.01-2.32% of the total phytoplankton per day).

Salmonella enterica serovar Typhimurium strains were isolated during a monitoring survey of the carpet shell clam (Ruditapes decussatus) collected from 23 stations along the Tunisian coast. The effect of starvation on Salmonella Typhimurium survival was investigated in vitro using microcosms composed of filtered (0.2 µm) marine water under dark and ambient temperature conditions (25C for 30 days). Eventual changes in physiological, biochemical properties, serotyping, biotyping and antimicrobial sensitivity for the environmentally adapted Salmonella Typhimurium were monitored. In response to stress conditions, the Salmonella Typhimurium strains progressively lost culturability in the absence of notable changes in the total cell count (during all the period of 30 days). Strains appeared to enter into a viable but noncultureable state as determined by epifluorescence method. We have also observed that the T90 value (time required for the reduction of 90% of cells) for the five different Salmonella strains ranged between 25 and 30 h; this indicated the survivability of Salmonella under stress conditions. This state was also characterized by biochemical and antimicrobial changes. PRACTICAL APPLICATION This research highlighted the acquisition of the viable but nonculturable state of bacteria. This form of adaptation of certain pathogens does not really reflect their presence or absence in the marine environment and thus in foods of marine origin. Hence the use of conventional methods of bacterial identification must be completed more advanced research tools before declaring a healthy product or environment.

International audience; Size fractionation was performed using water from the Great Reef of Toliara (Madagascar) taken from two different habitats (ocean and lagoon) during the dry and wet seasons, to study the growth and mortality rates of bacterioplankton. Experiments were conducted with 1 and 100% of heterotrophic nanoflagellate (HNF) concentrations and virus-free water was obtained by tangential filtration (10 kDa). During the dry season, in both environments, bacterial abundance and production were significantly lower than values recorded during the wet season. Bacterial growth rates without grazers were 0.88 day–1 in the lagoon and 0.58 day–1 in the ocean. However, growth rates were statistically higher without grazers and viruses (1.58 day–1 and 1.27 day–1). An estimate of virus-induced bacterial mortality revealed the important role played by viruses in the lagoon (0.70 day–1) and the ocean (0.69 day–1). During the wet season, bacterial growth rates without grazers were significantly higher in both environments than were values obtained in the dry season. However, the bacterial growth rates were paradoxally lower in the absence of viruses than with viruses in both environments. Our results suggest that changes in nutrient concentrations can play an important role in the balance between viral lysis and HNF grazing in the bacterial mortality. However, virus-mediated bacterial mortality is likely to act simultaneously with nanoflagellates pressure in their effects on bacterial communities.

Vibrio splendidus, strain LGP32, is an oyster pathogen associated with the summer mortalities affecting the production of Crassostrea gigas oysters worldwide. Vibrio splendidus LGP32 was shown to resist to up to 10 µM Cg-Def defensin and Cg-BPI bactericidal permeability increasing protein, two antimicrobial peptides/proteins (AMPs) involved in C. gigas immunity. The resistance to both oyster Cg-Def and Cg-BPI and standard AMPs (polymyxin B, protegrin, human BPI) was dependent on the ompU gene. Indeed, upon ompU inactivation, minimal bactericidal concentrations decreased by up to fourfold. AMP resistance was restored upon ectopic expression of ompU. The susceptibility of bacterial membranes to AMP-induced damages was independent of the ompU-mediated AMP resistance. Besides its role in AMP resistance, ompU proved to be essential for the adherence of V. splendidus LGP32 to fibronectin. Interestingly, in vivo, ompU was identified as a major determinant of V. splendidus pathogenicity in oyster experimental infections. Indeed, the V. splendidus-induced oyster mortalities dropped from 56% to 11% upon ompU mutation (Kaplan-Meier survival curves, P < 0.01). Moreover, in co-infection assays, the ompU mutant was out competed by the wild-type strain with competitive indexes in the range of 0.1-0.2. From this study, ompU is required for virulence of V. splendidus. Contributing to AMP resistance, conferring adhesive properties to V. splendidus, and being essential for in vivo fitness, the OmpU porin appears as an essential effector of the C. gigas/V. splendidus interaction.

Planktothrix agardhii dynamics, microcystin concentration and limnological variables were monitored every 2 weeks for 2 years (2004-2006) in a shallow hypereutrophic artificial lake (BNV, Viry-Chatillon, France). Time-series analysis identified two components in the P. agardhii biomass dynamics: (1) a significant decreasing trend in P. agardhii biomass (65% of the overall variance) and (2) a residual component without significant seasonal periodicity. A path-analysis model was built to determine the main factors controlling the P. agardhii dynamics over the period studied. The model explained 66% of P. agardhii biomass changes. The decreasing trend in P. agardhii biomass was significantly related to a decrease in the PO(4)(3-) concentration resulting from an improved treatment of the incoming watershed surface water. The residual component was related to zooplankton dynamics (cyclopoid abundances), supporting the hypothesis of a top-down control of P. agardhii, but only when the biomass was low. Forty-nine percent of the variability in the microcystin (MC) concentration (min:< 0.1 mu g equivalent MC-LR L(-1); max: 7.4 mu g equivalent MC-LR L(-1)) could be explained by changes in the P. agardhii biomass. The highest toxin content was observed when P. agardhii biomass was the lowest, which suggests changes in the proportion of microcystin-producing and -nonproducing subpopulations and/or the physiological status of cells.

We investigated the characteristics of the sulfur-oxidizing symbiont hosted in the gills of Codakia orbicularis, a bivalve living in shallow marine tropical environments. Special attention was paid to describing the heterogeneity of the population by using single-cell approaches including flow cytometry (FCM) and different microscopic techniques and by analyzing a cell size fractionation experiment. Up to seven different subpopulations were distinguished by FCM based on nucleic acid content and light side scattering of the cells. The cell size analysis of symbionts showed that the symbiotic population was very heterogeneous in size, i.e., ranging from 0.5 to 5 m in length, with variable amounts of intracellular sulfur. The side-scatter signal analyzed by FCM, which is often taken as a proxy of cell size, was greatly influenced by the sulfur content of the symbionts. FCM revealed an important heterogeneity in the relative nucleic acid content among the subclasses. The larger cells contained exceptionally high levels of nucleic acids, suggesting that these cells contained multiple copies of their genome, i.e., ranging from one copy for the smaller cells to more than four copies for the larger cells. The proportion of respiring symbionts (5-cyano-2,3-ditolyl-terazolium chloride positive) in the bacteriocytes of Codakia revealed that around 80% of the symbionts hosted by Codakia maintain respiratory activity throughout the year. These data allowed us to gain insight into the functioning of the symbionts within the host and to propose some hypotheses on how the growth of the symbionts is controlled by the host. Thioautotrophic symbioses in marine invertebrates have been studied for the last 25 years. Most of those studies were focused on hydrothermal vent environments. Nevertheless, similar types of symbiosis occur in sulfide-rich environments in the coastal zone with invertebrate hosts belonging to annelids, bivalves, gastropods, and protozoans (19, 42). These coastal organisms may represent attractive models to study thioautotrophic symbiosis, since these animals are much easier to sample and can often be kept alive in the laboratory with their symbionts for some extended period. This is an advantage for studies aiming to unravel the functioning of the symbionts within the host tissues and their interactions with the host.

The trophic relationships between metazooplankton and natural particles were studied in May 2002 in the Senegal River Estuary (16 degrees N, 16 degrees W) in low water conditions (dry season). Environmental factors, micro-organism and metazooplankton were analyzed through sampling at a fixed station. Gut fluorescence measurements of and field experiments on zooplankton metabolism were also performed. Chlorophyll a concentrations ranged from 6.5 to 10.2 mu g 1(-1). The phytoplankton was dominated by picoplanktonic cells (83-94% of total numbers). The particulate organic carbon (1.2-2.7 mg l(-1)) originated for a large amount from organic detritus (20-70%). The zooplankton biomass was dominated by Cirripedia larvae and calanoid copepods (Acartia clausi, Temora stylifera and Paracalanus spp.). These taxa showed diel vertical migrations and maximal gut fluorescence at night, independently of tidal effects. Metabolic budgets show that their daily ingestion rates on phytoplankton (27-55% of body carbon weight) were insufficient to balance their respiration needs (40-51% of body carbon) and suggest that a selective feeding upon micro-heterotrophs (Heterotrophic NanoFlagellates, HNF) and/or detritus would be necessary to complete their energetic needs. The daily grazing pressure of metazooplankton represented only 5% of the in situ chlorophyll a and 14% of the primary production, but this pressure was mainly orientated towards nanophytoplankton. The daily recycling of nutrients by the metazooplankton excretion was rather high (83 and 46% of the in situ NH4-N and PO4-P concentrations, respectively). Therefore, the impact of metazooplankton on phytoplankton through top-down (grazing) and bottom-up (nutrient recycling) processes seemed substantial in this tropical estuary. (c) 2005 Elsevier Ltd. All rights reserved.

on microbial plankton isolated from the Senegal River Estuary. Experiments performed in microcosms were based on size-class fractionation (3, 12, 20 and 60 m size classes) of the microbial community in order to obtain different assemblages, including bacteria, heterotrophic nanoflagellates (HNF), ciliates, pico- and nanophytoplankton. Removal of bacterial predators >3 m increased the net growth rate of heterotrophic bacteria from 0.437 day � 1 in the presence of all predators (fraction 12 m caused an increased net growth rate of flagellates from 0.102 day � 1 (fraction 12 m by this copepod, allowing an increase in HNF growth rates through indirect effect. The highest ingestion rate over 60 h of experiment was recorded for the copepod Temora (61.1 m 3 ngC � 1 h � 1 ) and the lowest value for barnacle larvae (25.6 m 3 ngC � 1 h � 1 ). An intermediate value (47.5m 3 ngC � 1 h � 1 )was recorded for thecopepodAcartia clausi.Alltheseingestion ratesweremainlychanneled in the size classes between

We conducted experiments to determine whether bacterial growth, with or without predators, is limited by inorganic (N and P) and organic (C) substrates in Selingue, a mesotrophic reservoir located in Mali, West Africa. Significant increases (relative to controls) in bacterial cell vol- umes and thymidine incorporation rates were observed after 24 h incubation only for samples amended with the combination +PC and +CNP. The data revealed a colimitation of bacterioplankton growth by organic carbon during the dry season. Flow cytometry discriminated 3 groups of bacteria (Bact I, Bact II, Bact III) differing in increases in nucleic acid content and cell size. The Bact I group, comprising cells with low nucleic acid content and of small size, was the dominant population in all experiments. In the absence of bacterial predators, only the Bact II group showed significant differ- ences between the control and the +PC and +CNP treatments, indicating that this bacterial group was the most sensitive to nutrient additions. The Bact II group, corresponding to cells with high DNA content, are active members of the bacterioplankton community. The Bact III group did not increase in any treatment but the proportions of these cells always increased in the presence of bacterial predators. These cells may be, at least partly, grazing-resistant bacteria.

Blanchet, Marine ... et. al.-- Special issue Microbial Ecology of the Continental and Coastal Environments.-- 16 pages, 8 figures, 2 tables, electronic supplementary material http://dx.doi.org/10.1007/s11356-014-3848-x, Spatial increases and temporal shifts in outbreaks of gelatinous plankton have been observed over the past several decades in many estuarine and coastal ecosystems. The effects of these blooms on marine ecosystem functioning and particularly on the dynamics of the heterotrophic bacteria are still unclear. The response of the bacterial community from a Mediterranean coastal lagoon to the addition of dissolved organic matter (DOM) from the jellyfish Aurelia aurita, corresponding to an enrichment of dissolved organic carbon (DOC) by 1.4, was assessed for 22 days in microcosms (8 l). The high bioavailability of this material led to (i) a rapid mineralization of the DOC and dissolved organic nitrogen from the jellyfish and (ii) the accumulation of high concentrations of ammonium and orthophosphate in the water column. DOM from jellyfish greatly stimulated heterotrophic prokaryotic production and respiration rates during the first 2 days; then, these activities showed a continuous decay until reaching those measured in the control microcosms (lagoon water only) at the end of the experiment. Bacterial growth efficiency remained below 20 %, indicating that most of the DOM was respired and a minor part was channeled to biomass production. Changes in bacterial diversity were assessed by tag pyrosequencing of partial bacterial 16S rRNA genes, DNA fingerprints, and a cultivation approach. While bacterial diversity in control microcosms showed little changes during the experiment, the addition of DOM from the jellyfish induced a rapid growth of Pseudoalteromonas and Vibrio species that were isolated. After 9 days, the bacterial community was dominated by Bacteroidetes, which appeared more adapted to metabolize high-molecular-weight DOM. At the end of the experiment, the bacterial community shifted toward a higher proportion of Alphaproteobacteria. Resilience of the bacterial community after the addition of DOM from the jellyfish was higher for metabolic functions than diversity, suggesting that jellyfish blooms can induce durable changes in the bacterial community structure in coastal lagoons. © 2014, Springer-Verlag Berlin Heidelberg, This study was supported by the CNRS-EC2CO program through the GELPROC project (2010–2012): “Interactions between gelatinous plankton and dynamics of prokaryotic communities.”

International audience; While considered as sustainable and low-cost agricultural amendments, the impacts of organic fertilizers on downstream aquatic microbial communities remain poorly documented. We investigated the quantity and quality of the dissolved organic matter leaching from agricultural soil amended with compost, vermicompost or biochar and assessed their effects on lake microbial communities, in terms of viral and bacterial abundances, community structure and metabolic potential. The addition of compost and vermicompost significantly increased the amount of dissolved organic carbon in the leachate compared to soil alone. Leachates from these additions, either with or without biochar were highly bioavailable to aquatic microbial communities, though reducing the metabolic potential of the community and harboring more specific communities. Although not affecting bacterial richness or taxonomic distributions, the specific addition of biochar affected the original lake bacterial communities resulting in a strongly different community. This could be partly explained by viral burst and converging bacterial abundances throughout the samples. These results underline the necessity to include off-site impacts of agricultural amendments when considering their cascading effect on downstream aquatic ecosystems. This article is protected by copyright. All rights reserved.

cited By 3; International audience; A microcosm experiment was setup to examine (1) the effect of phenanthrene contamination on meiofauna and bacteria communities and (2) the effects of different bioremediation strategies on phenanthrene degradation and on the community structure of free-living marine nematodes. Sediments from Bizerte lagoon were contaminated with (100 mg kg-1) phenanthrene and effects were examined after 20 days. Biostimulation (addition of nitrogen and phosphorus fertilizer or mineral salt medium) and bioaugmentation (inoculation of a hydrocarbonoclastic bacterium) were used as bioremediation treatments. Bacterial biomass was estimated using flow cytometry. Meiofauna was counted and identified at the higher taxon level using a stereomicroscope. Nematodes, comprising approximately two thirds of total meiofauna abundance, were identified to genus or species. Phenanthrene contamination had a severe impact on bacteria and meiofauna abundances with a strong decrease of nematodes with a complete disappearance of polychaetes and copepods. Bioremediation counter balanced the toxic effects of phenanthrene since meiofauna and bacteria abundances were significantly higher (p < 0.01) than those observed in phenanthrene contamination. Up to 98 % of phenanthrene removal was observed. In response to phenanthrene contamination, the nematode species had different behavior: Daptonema fallax was eliminated in contaminated microcosms, suggesting that it is an intolerant species to phenanthrene; Neochromadora peocilosoma, Spirinia parasitifera, and Odontophora n. sp., which significantly (p < 0.05) increased in contaminated microcosms, could be considered as "opportunistic" species to phenanthrene whereas Anticoma acuminata and Calomicrolaimus honestus increased in the treatment combining biostimulation and bioaugmentation. Phenanthrene had a significant effect on meiofaunal and bacterial abundances (p < 0.05), with a strong reduction of density and change in the nematode communities. Biostimulation using mineral salt medium strongly enhanced phenanthrene removal, leading to a decrease of its toxicity. This finding opens exciting axes for the future use of biostimulation to reduce toxic effects of PAHs for meiofauna and bacteria in lagoon sediment.

International audience; The effects of grazing pressure and inorganic nutrient availability on the direct carbon transfer from freshly produced phytoplankton exudates to heterotrophic bacteria biomass production were studied in Mediterranean coastal waters. The short-term incorporation of 13C (H13CO3) in phytoplankton and bacterial lipid biomarkers was measured as well as the total bacterial carbon production (BP), viral lysis and the microbial community structure under three experimental conditions: (1) High inorganic Nutrient and High Grazing (HN + HG), (2) High inorganic Nutrient and Low Grazing (HN + LG) and (3) under natural in situ conditions with Low inorganic Nutrient and High Grazing (LN + HG) during spring. Under phytoplankton bloom conditions (HN + LG), the bacterial use of freshly produced phytoplankton exudates as a source of carbon, estimated from 13C enrichment of bacterial lipids, contributed more than half of the total bacterial production. However, under conditions of high grazing pressure on phytoplankton with or without the addition of inorganic nutrients (HN + HG and LN + HG), the 13C enrichment of bacterial lipids was low compared with the high total bacterial production. BP therefore seems to depend mainly on freshly produced phytoplankton exudates during the early phase of phytoplankton bloom period. However, BP seems mainly relying on recycled carbon from viral lysis and predators under high grazing pressure.

The effects of starvation and salinity on the physiology of Salmonella typhimurium were investigated in a microcosm study. The physiological changes were monitored by using fluorochromes dyes such as DAPI (4',6-diamidino-2-phenylindole) for evaluation of the genomic content, CTC (5-cyano-2,3-ditolyl tetrazolium chloride) for respiratory activity and syto 9 and propidium iodide for cytoplasmic membrane damages. The metabolic activity of the cellular population was assessed with the method of Kogure (direct viable count), to enumerate the substrate-responsive cells. These different staining procedures were objectively analysed by an image analysis system. This paper describes the progressive alteration of Salmonella typhimurium physiology under salinity and starvation conditions.

The release of enteric pathogenic bacteria in aquatic environments poses the problem of the fate of these bacteria under the effects of environmental factors (solar radiation, salt concentration, temperature, nutrient level, pH, competition). Frequently, these bacterial cells, potentially pathogenic, enter into a non-culturable state on routine bacteriological plating media. However, the use of direct detection methods (DAPI stained cells) allows the visualization of these Viable but Non Culturable cells (VNC). But, beyond the characterization of the viability of the cells (electron transport activity, metabolic activity, membrane integrity, structure and/or quantity of DNA), what happens with the virulence of these cells? This problem was experimentally investigated according to the bacterial model Salmonella Typhimurium. The virulence of this strain, which is the agent of the murine typhoid, was evaluated on a mouse model. Experimentally, the effects of some environmental factors on the survival and on the maintenance of virulence of Salmonella Typhimurium were measured in microcosms exposed to UV radiation (four germicidal lamps 8 mW s(-1) cm(-2), wave length: 254 nm), salt concentration (Sea Salt Sigma, 37), nutrient starvation. The microcosms were simultaneously submitted to these three factors, with variable exposure times. For each of those times, the viability of the nonculturable cells (which became nonculturable because of the exposure to the three factors) was measured through different physiological states notable in the cells, after using different fluorescent dyes. The stained cells were observed by epifluorescence microscopy and analysed by image cytometry. So, the cellular populations are characterised by enumeration of respiring bacteria (CTC, [39]), metabolising bacteria (YEK, [22] modified), bacteria owning an undamaged cytoplasmic membrane (LD, Live/Dead BacLight Viability Kit. Molecular Probes Inc.); we also determined the quantity and/or structure of DNA of the cells (fluorescence level of DAPI stained cells). After exposure to the three factors for one hour (13.56 J cm(-2)), while the plate count cell density rapidly decreased from #10(7) cells mL(-1) to < 0.1 cell mL(-1), physiological states of these viable but non-culturable cells are similar to those of nonexposed cells. On the other hand, after exposure for three hours, only 10 % of the cells deposit a CTC formazan-crystal and 20 % are substrate responsive cells (enlarged cells in presence of Yeast Extract and Cephalexin: YEC). Half of the cellular population presents an undamaged cytoplasmic membrane and the level of fluorecence of DAPI stained cells is close to 85 %, which shows that the DNA of these cells is weakly damaged. After exposure to the three experimental factors for 24 hours (315 J cm(-2)), weak replies to the physiological tests used in this study to characterize the viability of the non-culturable cellular population are observed (CTC: 4 %; YEC: 2 %; LD: 11.8 %) while the fluorescence level of DAPI stained cells remains firm at 80 %. At the same time, the virulence expression of VNC cells of Salmonella Typhimurium, evaluated by intraperitoneal injection to the mouse (route which excludes uncontrolled parameters, unlike the per os route) does not seem to be correlated with the cellular viability such as it has been evaluated in this study. A 30 min exposure (6.73 J cm(-2)) to the three environmental factors, leading to the non-culturability of almost the entire exposed cell population (0.08 culturable cell mL(-1)) whereas the level of viability of those culturable cells is closed to the one of non-exposed cells. The injection of 1000 of those cells (, Le rejet de bactéries entériques pathogènes dans l'environnement aquatique pose le problème du devenir de ces microorganismes sous les effets des facteurs environnementaux (rayonnement solaire, salinité, température, oligotrophie, pH, compétition). L'évolution fréquente de ces cellules bactériennes potentiellement pathogènes vers un état non cultivable implique l'absence de détection de celles-ci par des méthodes traditionnelles. Cependant, l'utilisation des méthodes de détection directes permet de visualiser ces cellules non cultivables. Au stade suivant, il est nécessaire de caractériser le niveau de viabilité de ces cellules non cultivables. Mais au-delà de la caractérisation de l'état viable de la cellule (activité des chaînes respiratoires, activité métabolique, intégrilé membranaire, structure et/ou quantité d'ADN) se pose parallèlement le problème du maintien du pouvoir pathogène de ces bactéries. Cette problématique a été abordée expérimentalement sur le modèle bactérien Salmonella Typhimurium. Pour évaluer le pouvoir pathogène de cette souche, il est fait appel au modèle souris, Salmonella Typhimurium étant l'agent de la typhoïde murine. Pour évaluer les effets de certains facteurs environnementaux sur cette souche pathogène, il a été fait appel sur le plan expérimental en microcosmes au rayonnement UV (quatre lampes germicides 8 mW s−1 cm −2à 254 nm), à la salinité (SeaSalt Sigma, 37) et à l'oligotrophie. Ces trois facteurs sont appliqués simultanément avec des temps d'exposition variables. Pour chaque temps d'exposition, le niveau de viabilité des cellules qui ont perdu le pouvoir de cultiver sous les effets de ces facteurs est mesuré à travers différents états physiologiques, au moyen de marqueurs fluorochromiques. La réaction marqueur-cellule est observée par microscopic en épifluorescence et analysée par cytométrie en images. Ainsi, les populations cellulaires sont caractérisées sur le plan de la respiration (CTC, [39]), de la capacité à métaboliser un nutriment (YEC, [22] modifié), de l'intégrité membranaire (LD, Kit Live/Dead Baclight-Molecular Probes Inc) et de la structure et/ou quantité d'ADN [36]. Alors que la disparition du pouvoir de cultiver de toutes les cellules exposées (zéro cultivabilité) se réalise en 1 h (13,56 J cm−2), les états physiologiques que présentent ces cellules non cultivables sont proches de ceux présentés par les cellules d'une culture non exposée. Par contre, après 3 h d'exposition, 10 % seulement des cellules présentent une activité de leur chaîne respiratoire (CTC) et 20 % présentent une activité métabolique (extrait de levure en présence de céphalexine). La moitié des cellules présente une atteinte de la membrane cytoplasmique et le niveau de fluorescence de la population cellulaire est voisin de 85 % montrant par là que ces cellules ne présentent à ce stade qu'une faible atteinte de l'ADN. Après 24 h d'exposition (315 J cm−2) à ces trois facteurs stressants expérimentaux, les tests physiologiques appliqués dans cette étude sur les cellules non cultivables sont quasiment négatifs alors que le niveau de fluorescence de leur ADN marqué se maintient à 80 %. En parallèle, le suivi du pouvoir pathogène de ces cellules de Salmonella Typhimurium, évalué par injection intrapéritonéale à la souris (voie permettant de s'affranchir de paramètres non contrôlés comme la voie per os), ne paraît pas évoluer en fonction de la disparition de la viabilité cellulaire mesurée par les tests utilisés dans cette étude. Une exposition de 30 min (6,73 J cm−2) aux trois facteurs stressants expérimentaux fait disparaître le pouvoir de cultiver de la quasi-totalité des cellules exposées (0,08 cellules cultivables mL−1) alors que ces cellules présentent un état de viabilité proche de celui d'une souche non exposée. L'injection de 1000 de ces cellules ( 0,001 cellule cultivable dans 100 μL injecté) à la souris (un lot de dix souris) n'entraîne aucun état pathologique ni mortalité sur quatre semaines d'observations alors que l'injection du même nombre de cellules non exposées aux trois facteurs stressants entraîne la mort du lot de souris en une semaine. D'après ces quelques expérimentations préliminaires sur Salmonella Typhimurium, la perte du pouvoir de cultiver paraît s'accompagner de la perte de la virulence testée chez la souris par voie intrapéritonéale après exposition aux effets associés de l'irradiation UV, de la salinité 37 et de l'oligotrophie.

International audience; Mangroves are threatened ecosystems that provide numerous ecosystem services, especially through their wide biodiversity, and their bioremediation capacity is a challenging question in tropical areas. In a mangrove in Mayotte, we studied the potential role of microbial biofilm communities in removing nutrient loads from pre-treated wastewater. Microbial community samples were collected from tree roots, sediments, water, and from a colonization device, and their structure and dynamics were compared in two areas: one exposed to sewage and the other not. The samples from the colonization devices accurately reflected the natural communities in terms of diversity. Communities in the zone exposed to sewage were characterized by more green algae and diatoms, higher bacteria densities, as well as different compositions. In the area exposed to sewage, the higher cell densities associated with specific diversity patterns highlighted adapted communities that may play a significant role in the fate of nutrients.

International audience; Few studies on the consequences of future global warming and increased ultraviolet-B radiation (UVBR, 280-320nm) have been carried out at the food web scale and very few have considered the effect of the combination of these two climatic stressors. The single and combined effect of 3°C warming and 20% increased UVBR on planktonic carbon and nitrogen dynamics was investigated during a 10-day mesocosm experiment in coastal Mediterranean waters. Planktonic organisms from Thau Lagoon were sampled and incubated into eight in situ moored 2000L mesocosms and exposed to 4 different temperature and light conditions in duplicates; 1) natural light and temperature conditions; 2) +3°C above in situ water temperature; 3) +20% above incident UVBR, and 4) simultaneous +3°C and +20% above natural conditions. Compared to the natural in situ temperature and light conditions, increasing UVBR alone reduced bacterial carbon production and biomass by an average of 20%, with some reduction in ammonium net production. Warming alone significantly reduced bacterial carbon production by about 40% as a consequence of a trophic cascade effect on bacterial biomass. Warming also increased phytoplankton carbon production and associated nitrate uptake rates by an average of 90% and 200%, respectively. Thus, the results showed a direct positive effect of temperature increase on phytoplankton metabolism and greater nutrient availability owing to the reduction in potential bacterial competition. The combination of temperature and increased UVBR significantly increased the autotrophic production but without accumulation of freshly produced particulate organic matter. This suggests an accumulation of dissolved organic matter under these climatic conditions.

The aim of this study was to determine the extent and source of inaccuracies for total counts of microorganisms by filtration followed by epifluorescent microscopy. The extent of these inaccuracies were determined by comparing the microscopic method with viable plate counts while their possible sources were investigated using flow cytometry and fluorescent microbeads. One factor responsible for these accuracies was found to be the microscope conversion factor which did not take into account the total filter surface area on which the bacteria were impacted. Approximately a two-fold underestimation was observed when the filtration area rather than the effective area over which the cells are disturbed were compared. Thus it is recommended that the latter be accurately measured when calculating the conversion factor in order to reduce the inaccuracy of direct microscopic counts.

OmpU porins are increasingly recognized as key determinants of pathogenic host Vibrio interactions. Although mechanisms remain incompletely understood, various species, including the human pathogen Vibrio cholera , require OmpU for host colonization and virulence. We have shown previously that OmpU is essential for virulence in the oyster pathogen Vibrio splendidus LGP32. Here, we showed that V. splendidus LGP32 invades the oyster immune cells, the hemocytes, through subversion of host-cell actin cytoskeleton. In this process, OmpU serves as an adhesin/invasin required for β-integrin recognition and host cell invasion. Furthermore, the major protein of oyster plasma, the extracellular superoxide dismutase Cg -EcSOD, is used as an opsonin mediating the OmpU-promoted phagocytosis through its RGD sequence. Finally, the endocytosed bacteria were found to survive intracellularly, evading the host defense by preventing acidic vacuole formation and limiting reactive oxygen species production. We conclude that ( i ) V. splendidus is a facultative intracellular pathogen that manipulates host defense mechanisms to enter and survive in host immune cells, and ( ii ) that OmpU is a major determinant of host cell invasion in Vibrio species, used by V. splendidus LGP32 to attach and invade oyster hemocytes through opsonisation by the oyster plasma Cg -EcSOD.

International audience; This study investigated survival and virulence of Escherichia coli strains exposed to natural conditions in brackish water. Two E. coli strains (O126:B16 and O55:B5) were incubated in water microcosms in the Bizerte lagoon in Northern Tunisia and exposed for 12 days to natural sunlight in June (231 to 386 W/m(2), 26 +/- 1 degrees C, 30 g/L) and in April (227 to 330 W/m(2), 17 +/- 1 degrees C, 27 g/L) or maintained in darkness for 21 days (17 +/- 1 degrees C, 27 g/L). The results revealed that sunlight was the most significant inactivating factor (decrease of 3 Ulog within 48 hours for the two strains) compared to salinity and temperature (in darkness). Survival time of the strains was prolonged as they were maintained in darkness. Local strain (E. coli O55:B5) showed better survival capacity (T(90) = 52 hours) than E. coli O126:B16 (T(90) = 11 h). For both, modifications were noted only for some metabolic activities of carbohydrates hydrolysis. Cytotoxicity of the two strains, tested on Vero cell, was maintained during the period of survival. Water Environ. Res., 82, 2249 (2010).

The existence of Salmonella enterica serovar Typhimurium viable-but-nonculturable (VBNC) cells is a public health concern since they could constitute unrecognized sources of infection if they retain their pathogenicity. To date, many studies have addressed the ability of S . Typhimurium VBNC cells to remain infectious, but their conclusions are conflicting. An assumption could explain these conflicting results. It has been proposed that infectivity could be retained only temporarily after entry into the VBNC state and that most VBNC cells generated under intense stress could exceed the stage where they are still infectious. Using a Radioselectan density gradient centrifugation technique makes it possible to increase the VBNC-cell/culturable-cell ratio without increasing the exposure to stress and, consequently, to work with a larger proportion of newly VBNC cells. Here, we observed that (i) in the stationary phase, the S . Typhimurium population comprised three distinct subpopulations at 10, 24, or 48 h of culture; (ii) the VBNC cells were detected at 24 and 48 h; (iii) measurement of invasion gene ( hilA , invF , and orgA ) expression demonstrated that cells are highly heterogeneous within a culturable population; and (iv) invasion assays of HeLa cells showed that culturable cells from the different subpopulations do not display the same invasiveness. The results also suggest that newly formed VBNC cells are either weakly able or not able to successfully initiate epithelial cell invasion. Finally, we propose that at entry into the stationary phase, invasiveness may be one way for populations of S . Typhimurium to escape stochastic alteration leading to cell death.

The bivalve Codakia orbicularis , hosting sulfur-oxidizing gill endosymbionts, was starved (in artificial seawater filtered through a 0.22-μm-pore-size membrane) for a long-term experiment (4 months). The effects of starvation were observed using transmission electron microscopy, fluorescence in situ hybridization and catalyzed reporter deposition (CARD-FISH), and flow cytometry to monitor the anatomical and physiological modifications in the gill organization of the host and in the symbiotic population housed in bacteriocytes. The abundance of the symbiotic population decreased through starvation, with a loss of one-third of the bacterial population each month, as shown by CARD-FISH. At the same time, flow cytometry revealed significant changes in the physiology of symbiotic cells, with a decrease in cell size and modifications to the nucleic acid content, while most of the symbionts maintained a high respiratory activity (measured using the 5-cyano-2,3-ditolyl tetrazolium chloride method). Progressively, the number of symbiont subpopulations was reduced, and the subsequent multigenomic state, characteristic of this symbiont in freshly collected clams, turned into one and five equivalent genome copies for the two remaining subpopulations after 3 months. Concomitant structural modifications appeared in the gill organization. Lysosymes became visible in the bacteriocytes, while large symbionts disappeared, and bacteriocytes were gradually replaced by granule cells throughout the entire lateral zone. Those data suggested that host survival under these starvation conditions was linked to symbiont digestion as the main nutritional source.

This study focused on the effects of two pesticides, paraquat (herbicide) and deltamethrin (insecticide), which are two common molecules used intensively in Burkina Faso. Natural bacterial populations, phytoplankton cultures (one cyanobacterium, Cylindrospermopsis raciborskii, and one chlorophycea, Monoraphidium sp.), and two species of zooplankton (Diaphanosoma excisum and Moina micrura) were isolated from aquatic communities and were used as biological targets in the experimental protocols. Paraquat was moderately toxic to bacteria and phytoplankton, whereas deltamethrin was significantly toxic only to the zooplankton species. Paradoxically, the chlorophycea Monoraphidium sp. exhibited a significant increase of in vitro fluorescence after 48 h at the highest doses. Preliminary tests were also performed from natural water extract of the main drinking water supply of the country (Loumbila Reservoir) by using solid-phase extraction. Obviously, the natural extract proved to be toxic to the same biological targets. Despite the absence of any determination of pesticides in the natural extract, the question of contamination and toxicity of these waters affects concerns about the safety of water supply and the effect of human pressure on the dynamics of planktonic communities of freshwater reservoirs in arid regions of western Africa.

Temporal variability in bacterial communities was studied on a daily scale in the estuar- ine part of the largest river on the West African coast, the Senegal River. Duplicate mesocosms (3 m 3 volume) were placed in the upper part of the estuary at the end of the dry season (May 2002) and treated with low and high inorganic nutrient (N and P) enrichment. High nutrient additions were followed by a 7-fold increase in phytoplankton biomass and a 6-fold increase in bacterial abundance after 4 and 9 d, respectively. Heterotrophic nanoflagellates (HNF) showed their maximal abundance (1 × 10 6 ml -1 ) 2 d after the bacterial peak. The low bacteria to flagellate ratios recorded on Day 10 may suggest enhanced bacterivory from HNF. Simultaneous measurements of growth and grazing rates on bacteria during the bacterial growth phase were performed with the dilution method and seemed to indicate that the HNF community was capable of quickly controlling bacterial development. How- ever, estimates of the carbon demand of HNF during their growth phase (915 µg C l -1 d -1 ) appeared to be more elevated than the bacterial carbon production (63% of the HNF carbon demand). To cover HNF carbon requirements, an alternative/complementary prey might be the picophytoplanktonic cells, which were very numerous during the study (i.e. 85% of the total phytoplankton count). During the period of high grazing pressure, bacterial populations were characterized by higher specific activity (from tritiated thymidine incorporation) and culturability (from plate counts). The presence of very large bacteria, as detected by epifluorescence microscopy and flow cytometry measure- ments, may be an escape response from flagellate grazing. 16S rDNA sequences from bacterial isolates showed the presence of 2 types of taxonomic units (Vibrio natriegens- and Flexibacter maritimus-like bacteria), which can be considered by their forms and growth rates to be strains that have developed strategies to protect against grazing. Thus, as demonstrated in various temperate systems, predation by HNF in estuarine tropical ecosystems may also be of importance in shaping the structure and functions of the bacterial community.

Maintenance of pathogenicity of viable but nonculturable Salmonella typhimurium cells experimentally stressed with UV-C and seawater, was investigated relative to the viability level of the cellular population. Pathogenicity, tested in a mouse model, was lost concomitantly with culturability, whereas cell viability remained undamaged, as determined by respiratory activity and cytoplasmic membrane and genomic integrities.