Importance of Local and Regional Scales in Shaping Mycobacterial Abundance in Freshwater Lakes.

Biogeographical studies considering the entire bacterial community may underestimate mechanisms of bacterial assemblages at lower taxonomic levels. In this context, the study aimed to identify factors affecting the spatial and temporal dynamic of the Mycobacterium, a genus widespread in aquatic ecosystems. Nontuberculous mycobacteria (NTM) density variations were quantified in the water column of freshwater lakes at the regional scale (annual monitoring of 49 lakes in the Paris area) and at the local scale (2-year monthly monitoring in Créteil Lake) by real-time quantitative PCR targeting the atpE gene. At the regional scale, mycobacteria densities in water samples ranged from 6.7 × 103 to 1.9 × 108 genome units per liter. Density variations were primarily explained by water pH, labile iron, and dispersal processes through the connection of the lakes to a river. In Créteil Lake, no spatial variation of mycobacterial densities was noticed over the 2-year monthly survey, except after large rainfall events. Indeed, storm sewer effluents locally and temporarily increased NTM densities in the water column. The temporal dynamic of the NTM densities in Créteil Lake was associated with suspended solid concentrations. No clear seasonal variation was noticed despite a shift in NTM densities observed over the 2012-2013 winter. Temporal NTM densities fluctuations were well predicted by the neutral community model, suggesting a random balance between loss and gain of mycobacterial taxa within Créteil Lake. This study highlights the importance of considering multiple spatial scales for understanding the spatio-temporal dynamic of bacterial populations in natural environments.

Global Metabolomic Characterizations of Microcystis spp. Highlights Clonal Diversity in Natural Bloom-Forming Populations and Expands Metabolite Structural Diversity.

Cyanobacteria are photosynthetic prokaryotes capable of synthesizing a large variety of secondary metabolites that exhibit significant bioactivity or toxicity. Microcystis constitutes one of the most common cyanobacterial genera, forming the intensive blooms that nowadays arise in freshwater ecosystems worldwide. Species in this genus can produce numerous cyanotoxins (i.e., toxic cyanobacterial metabolites), which can be harmful to human health and aquatic organisms. To better understand variations in cyanotoxin production between clones of Microcystis species, we investigated the diversity of 24 strains isolated from the same blooms or from different populations in various geographical areas. Strains were compared by genotyping with 16S-ITS fragment sequencing and metabolite chemotyping using LC ESI-qTOF mass spectrometry. While genotyping can help to discriminate among different species, the global metabolome analysis revealed clearly discriminating molecular profiles among strains. These profiles could be clustered primarily according to their global metabolite content, then according to their genotype, and finally according to their sampling location. A global molecular network of all metabolites produced by Microcystis species highlights the production of a wide set of chemically diverse metabolites, including a few microcystins, many aeruginosins, microginins, cyanopeptolins, and anabaenopeptins, together with a large set of unknown molecules. These components, which constitute the molecular biodiversity of Microcystis species, still need to be investigated in terms of their structure and potential bioactivites (e.g., toxicity).

Drivers and ecological consequences of dominance in periurban phytoplankton communities using networks approaches.

Evaluating the causes and consequences of dominance by a limited number of taxa in phytoplankton communities is of huge importance in the current context of increasing anthropogenic pressures on natural ecosystems. This is of particular concern in densely populated urban areas where usages and impacts of human populations on water ecosystems are strongly interconnected. Microbial biodiversity is commonly used as a bioindicator of environmental quality and ecosystem functioning, but there are few studies at the regional scale that integrate the drivers of dominance in phytoplankton communities and their consequences on the structure and functioning of these communities. Here, we studied the causes and consequences of phytoplankton dominance in 50 environmentally contrasted waterbodies, sampled over four summer campaigns in the highly-populated Île-de-France region (IDF). Phytoplankton dominance was observed in 32-52% of the communities and most cases were attributed to Chlorophyta (35.5-40.6% of cases) and Cyanobacteria (30.3-36.5%). The best predictors of dominance were identified using multinomial logistic regression and included waterbody features (surface, depth and connection to the hydrological network) and water column characteristics (total N, TN:TP ratio, water temperature and stratification). The consequences of dominance were dependent on the identity of the dominant organisms and included modifications of biological attributes (richness, cohesion) and functioning (biomass, RUE) of phytoplankton communities. We constructed co-occurrence networks using high resolution phytoplankton biomass and demonstrated that networks under dominance by Chlorophyta and Cyanobacteria exhibited significantly different structure compared with networks without dominance. Furthermore, dominance by Cyanobacteria was associated with more profound network modifications (e.g. cohesion, size, density, efficiency and proportion of negative links), suggesting a stronger disruption of the structure and functioning of phytoplankton communities in the conditions in which this group dominates. Finally, we provide a synthesis on the relationships between environmental drivers, dominance status, community attributes and network structure.

Specificity of the metabolic signatures of fish from cyanobacteria rich lakes.

With the increasing impact of the global warming, occurrences of cyanobacterial blooms in aquatic ecosystems are becoming a main worldwide ecological concern. Due to their capacity to produce potential toxic metabolites, interactions between the cyanobacteria, their cyanotoxins and the surrounding freshwater organisms have been investigated during the last past years. Non-targeted metabolomic analyses have the powerful capacity to study simultaneously a high number of metabolites and thus to investigate in depth the molecular signatures between various organisms encountering different environmental scenario, and potentially facing cyanobacterial blooms. In this way, the liver metabolomes of two fish species (Perca fluviatilis and Lepomis gibbosus) colonizing various peri-urban lakes of the Île-de-France region displaying high biomass of cyanobacteria, or not, were investigated. The fish metabolome hydrophilic fraction was analyzed by 1H NMR analysis coupled with Batman peak treatment for the quantification and the annotation attempt of the metabolites. The results suggest that similar metabolome profiles occur in both fish species, for individuals collected from cyanobacterial blooming lakes compared to organism from non-cyanobacterial dominant environments. Overall, such environmental metabolomic pilot study provides new research perspectives in ecology and ecotoxicology fields, and may notably provide new information concerning the cyanobacteria/fish ecotoxicological interactions.

Collapse of a Planktothrix agardhii perennial bloom and microcystin dynamics in response to reduced phosphate concentrations in a temperate lake.

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-Châtillon, 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 PO4(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 microg equivalent MC-LR L(-1); max: 7.4 microg 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.

Design and application of a stratified sampling strategy to study the regional distribution of cyanobacteria (Ile-de-France, France).

This study describes the design and application of a stratified sampling strategy of waterbodies to assess and analyze the distribution of cyanobacteria at a regional scale (Ile-de-France, IDF). Ten groups of hydrographical zones were defined within the IDF on the basis of their anthropogenic and geomorphologic characteristics. Sampling effort (n=50) was then randomly allocated according to the number of waterbodies in each group. This sampling strategy was tested in August 2006, using a field probe to estimate total phytoplankton as well as cyanobacteria biomasses. The sampled waterbodies exhibited a wide range of phytoplankton (< 1-375 microg equiv.Chla L(-1)) and cyanobacteria biomasses (< 1-278 microg equiv.ChlaL(-1)). 72% of the waterbodies in the IDF were classified as eutrophic (42% hypereutrophic), and 24% of the sites studied were dominated by cyanobacteria. Waterbodies connected to hydrographical networks (n=26) showed significantly higher total (p<0.0001; 3.5 times greater) and cyanobacterial (p<0.001, 3.2 times greater) biomasses than the isolated ones (n=24). No significant overall relationship was found through contingency analysis between waterbody trophic status and global land use categories (urban, periurban, and rural) within their hydrographical zones. However, concerning the waterbodies linked to hydrographical networks, the percentage of land covered by forest appeared as a good indicator of phytoplankton and cyanobacterial biomasses. This observation may be a consequence of lower amounts of nutrients being discharged into waterbodies from highly forested hydrological zone than from urban and/or agricultural areas. Our results illustrate a successful means of selecting representative waterbodies to conduct a regional assessment of cyanobacteria distribution using accessible GIS analyses.

Author Correction: Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms: an integrative approach combining proteomic and metabolomic analyses.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.

Physiological effects caused by microcystin-producing and non-microcystin producing Microcystis aeruginosa on medaka fish: A proteomic and metabolomic study on liver.

Cyanobacterial blooms have become a common phenomenon in eutrophic freshwater ecosystems worldwide. Microcystis is an important bloom-forming and toxin-producing genus in continental aquatic ecosystems, which poses a potential risk to Human populations as well as on aquatic organisms. Microcystis is known to produce along with various bioactive peptides, the microcystins (MCs) that have attracted more attention notably due to their high hepatotoxicity. To better understand the effects of cyanobacterial blooms on fish, medaka fish (Oryzias latipes) were sub-chronically exposed to either non-MC-producing or MC-producing living strains and, for this latter, to its subsequent MC-extract of Microcystis aeruginosa. Toxicological effects on liver have been evaluated through the combined approach of histopathology and 'omics' (i.e. proteomics and metabolomics). All treatments induce sex-dependent effects at both cellular and molecular levels. Moreover, the modalities of exposure appear to induce differential responses as the direct exposure to the cyanobacterial strains induce more acute effects than the MC-extract treatment. Our histopathological observations indicate that both non-MC-producing and MC-producing strains induce cellular impairments. Both proteomic and metabolomic analyses exhibit various biological disruptions in the liver of females and males exposed to strain and extract treatments. These results support the hypothesis that M. aeruginosa is able to produce bioactive peptides, other than MCs, which can induce toxicological effects in fish liver. Moreover, they highlight the importance of considering cyanobacterial cells as a whole to assess the realistic environmental risk of cyanobacteria on fish.

Microcystin ecotypes in a perennial Planktothrix agardhii bloom.

The dynamics and microcystins (MC) concentrations of a perennial Planktothrix agardhii bloom were investigated in a eutrophic lake (Viry-Châtillon, France). A weak relationship was observed between P. agardhii population biomass and the MC concentrations in a 1-year survey. To further investigate the causes of MC concentration changes, we concurrently conducted experiments on 41 strains isolated from this lake. We first checked the clonal diversity of P. agardhii population (i) by molecular techniques, to assess the presence of MC synthetase gene (mcyB), (ii) by biochemical assay (PP2A inhibition assay), for MC production, and (iii) by mass spectrometry (MS), to identify the MC chemotypes. Our results illustrated the diversity of genotype and MC chemotypes within a P. agardhii natural population. Eleven chemotypes among the 16 possible ones were found by MS. Furthermore, we noticed major differences in the MC content of isolated strains (from 0.02 to 1.86 microg equiv. MC-LR mg DW(-1), n=25). Growth and MC production of one MC-producing strain and one non-MC-producing strain were also assessed at two temperatures (10 and 20 degrees C). We showed that growth capacities of these strains were similar at the two tested temperatures, and that the MC production rate was correlated to the growth rate for the MC-producing strain. On the basis of these results, several hypotheses are discussed to explain the weakness of relationships between natural P. agardhii biomass and MC concentration. One of the main reasons could lie in the proportion of MC-producing clones and non-MC-producing clones that may change during the sampling period. Also, the MC-producing clones may present different intracellular MC content due to (i) MC chemotypes diversity, (ii) changes in MC variants proportions within a strain, and (iii) changes in MC rate production depending on the physiological state of cells. Finally, we concluded that various biological organization levels have to be considered (population, cellular and molecular), through an integrative approach, in order to provide a better understanding of P. agardhii in situ MC production.

Global metabolome changes induced by cyanobacterial blooms in three representative fish species.

Cyanobacterial blooms induce important ecological constraints for aquatic organisms and strongly impact the functioning of aquatic ecosystems. In the past decades, the effects of the cyanobacterial secondary metabolites, so called cyanotoxins, have been extensively studied in fish. However, many of these studies have used targeted approaches on specific molecules, which are thought to react to the presence of these specific cyanobacterial compounds. Since a few years, untargeted metabolomic approaches provide a unique opportunity to evaluate the global response of hundreds of metabolites at a glance. In this way, our study provides the first utilization of metabolomic analyses in order to identify the response of fish exposed to bloom-forming cyanobacteria. Three relevant fish species of peri-urban lakes of the European temperate regions were exposed for 96h either to a microcystin (MC)-producing or to a non-MC-producing strain of Microcystis aeruginosa and metabolome changes were characterized in the liver of fish. The results suggest that a short-term exposure to those cyanobacterial biomasses induces metabolome changes without any response specificity linked to the fish species considered. Candidate metabolites are involved in energy metabolism and antioxidative response, which could potentially traduce a stress response of fish submitted to cyanobacteria. These results are in agreement with the already known information and could additionally bring new insights about the molecular interactions between cyanobacteria and fish.

Metabolic changes in Medaka fish induced by cyanobacterial exposures in mesocosms: an integrative approach combining proteomic and metabolomic analyses.

Cyanobacterial blooms pose serious threats to aquatic organisms and strongly impact the functioning of aquatic ecosystems. Due to their ability to produce a wide range of potentially bioactive secondary metabolites, so called cyanotoxins, cyanobacteria have been extensively studied in the past decades. Proteomic and metabolomic analyses provide a unique opportunity to evaluate the global response of hundreds of proteins and metabolites at a glance. In this study, we provide the first combined utilization of these methods targeted to identify the response of fish to bloom-forming cyanobacteria. Medaka fish (Oryzias latipes) were exposed for 96 hours either to a MC-producing or to a non-MC-producing strain of Microcystis aeruginosa and cellular, proteome and metabolome changes following exposure to cyanobacteria were characterized in the fish livers. The results suggest that a short-term exposure to cyanobacteria, producing or not MCs, induces sex-dependent molecular changes in medaka fish, without causing any cellular alterations. Globally, molecular entities involved in stress response, lipid metabolism and developmental processes exhibit the most contrasted changes following a cyanobacterial exposure. Moreover, it appears that proteomic and metabolomic analyses are useful tools to verify previous information and to additionally bring new horizons concerning molecular effects of cyanobacteria on fish.

Patterns and multi-scale drivers of phytoplankton species richness in temperate peri-urban lakes.

Local species richness (SR) is a key characteristic affecting ecosystem functioning. Yet, the mechanisms regulating phytoplankton diversity in freshwater ecosystems are not fully understood, especially in peri-urban environments where anthropogenic pressures strongly impact the quality of aquatic ecosystems. To address this issue, we sampled the phytoplankton communities of 50 lakes in the Paris area (France) characterized by a large gradient of physico-chemical and catchment-scale characteristics. We used large phytoplankton datasets to describe phytoplankton diversity patterns and applied a machine-learning algorithm to test the degree to which species richness patterns are potentially controlled by environmental factors. Selected environmental factors were studied at two scales: the lake-scale (e.g. nutrients concentrations, water temperature, lake depth) and the catchment-scale (e.g. catchment, landscape and climate variables). Then, we used a variance partitioning approach to evaluate the interaction between lake-scale and catchment-scale variables in explaining local species richness. Finally, we analysed the residuals of predictive models to identify potential vectors of improvement of phytoplankton species richness predictive models. Lake-scale and catchment-scale drivers provided similar predictive accuracy of local species richness (R(2)=0.458 and 0.424, respectively). Both models suggested that seasonal temperature variations and nutrient supply strongly modulate local species richness. Integrating lake- and catchment-scale predictors in a single predictive model did not provide increased predictive accuracy; therefore suggesting that the catchment-scale model probably explains observed species richness variations through the impact of catchment-scale variables on in-lake water quality characteristics. Models based on catchment characteristics, which include simple and easy to obtain variables, provide a meaningful way of predicting phytoplankton species richness in temperate lakes. This approach may prove useful and cost-effective for the management and conservation of aquatic ecosystems.

Neutral community model explains the bacterial community assembly in freshwater lakes.

Over the past decade, neutral theory has gained attention and recognition for its capacity to explain bacterial community structure (BCS) in addition to deterministic processes. However, no clear consensus has been drawn so far on their relative importance. In a metacommunity analysis, we explored at the regional and local scale the effects of these processes on the bacterial community assembly within the water column of 49 freshwater lakes. The BCS was assessed using terminal restriction fragment length polymorphism (T-RFLP) of the 16S rRNA genes. At the regional scales, results indicated that the neutral community model well predicted the spatial community structure (R(2) mean = 76%) compared with the deterministic factors - which explained only a small fraction of the BCS total variance (less than 14%). This suggests that the bacterial compartment was notably driven by stochastic processes, through loss and gain of taxa. At the local scale, the bacterial community appeared to be spatially structured by stochastic processes (R(2) mean = 65%) and temporally governed by the water temperature, a deterministic factor, even if some bacterial taxa were driven by neutral dynamics. Therefore, at both regional and local scales the neutral community model appeared to be relevant in explaining the bacterial assemblage structure.

Projecting the impact of regional land-use change and water management policies on lake water quality: an application to periurban lakes and reservoirs.

As the human population grows, the demand for living space and supplies of resources also increases, which may induce rapid change in land-use/land-cover (LULC) and associated pressures exerted on aquatic habitats. We propose a new approach to forecast the impact of regional land cover change and water management policies (i.e., targets in nutrient loads reduction) on lake and reservoir water eutrophication status using a model that requires minimal parameterisation compared with alternative methods. This approach was applied to a set of 48 periurban lakes located in the Ile de France region (IDF, France) to simulate catchment-scale management scenarios. Model outputs were subsequently compared to governmental agencies' 2030 forecasts. Our model indicated that the efforts made to reduce pressure in the catchment of seepage lakes might be expected to be proportional to the gain that might be obtained, whereas drainage lakes will display little improvement until a critical level of pressure reduction is reached. The model also indicated that remediation measures, as currently planned by governmental agencies, might only have a marginal impact on improving the eutrophication status of lakes and reservoirs within the IDF region. Despite the commitment to appropriately managing the water resources in many countries, prospective tools to evaluate the potential impacts of global change on freshwater ecosystems integrity at medium to large spatial scales are lacking. This study proposes a new approach to investigate the impact of region-scale human-driven changes on lake and reservoir ecological status and could be implemented elsewhere with limited parameterisation. Issues are discussed that relate to model uncertainty and to its relevance as a tool applied to decision-making.

Effects of a toxic cyanobacterial bloom (Planktothrix agardhii) on fish: insights from histopathological and quantitative proteomic assessments following the oral exposure of medaka fish (Oryzias latipes).

Cyanobacterial toxic blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to fish and other aquatic organisms. Microcystin-LR (the cyanotoxin most commonly detected in the freshwater environment) is a potent hepatotoxin, deregulating the kinase pathway by inhibiting phosphatases 1 and 2A. Although toxicological effects have been clearly linked to the in vitro exposure of fish to purified microcystins, cyanotoxins are produced by the cyanobacteria together with numerous other potentially toxic molecules, and their overall and specific implications for the health of fish have still not been clearly established and remain puzzlingly difficult to assess. The medaka fish (Oryzias latipes) was chosen as an in vitro model for studying the effects of a cyanobacterial bloom on liver protein contents using a gel free quantitative approach, iTRAQ, in addition to pathology examinations on histological preparations. Fish were gavaged with 5 µL cyanobacterial extracts (Planktothrix agardhii) from a natural bloom (La Grande Paroisse, France) containing 2.5 µg equiv. MC-LR. 2h after exposure, the fish were sacrificed and livers were collected for analysis. Histological observations indicate that hepatocytes present glycogen storage loss, and cellular damages, together with immunological localization of MCs. Using a proteomic approach, 304 proteins were identified in the fish livers, 147 of them with a high degree of identification confidence. Fifteen of these proteins were statistically significantly different from those of controls (gavaged with water only). Overall, these protein regulation discrepancies clearly indicate that oxidative stress and lipid regulation had occurred in the livers of the exposed medaka fish. In contrast to previous pure microcystin-LR gavage experiments, marked induction of vitellogenin 1 protein was observed for the first time with a cyanobacterial extract. This finding was confirmed by ELISA quantification of vitellogenin liver content, suggesting that the Planktothrix bloom extract had induced the occurrence of an endocrine-disrupting effect.