New sensitive tools to characterize meta-metabolome response to short- and long-term cobalt exposure in dynamic river biofilm communities.

Untargeted metabolomics is a non-a priori analysis of biomolecules that characterizes the metabolome variations induced by short- and long-term exposures to stressors. Even if the metabolite annotation remains lacunar due to database gaps, the global metabolomic fingerprint allows for trend analyses of dose-response curves for hundreds of cellular metabolites. Analysis of dose/time-response curve trends (biphasic or monotonic) of untargeted metabolomic features would thus allow the use of all the chemical signals obtained in order to determine stress levels (defense or damage) in organisms. To develop this approach in a context of time-dependent microbial community changes, mature river biofilms were exposed for 1 month to four cobalt (Co) concentrations (from background concentration to 1 × 10-6 M) in an open system of artificial streams. The meta-metabolomic response of biofilms was compared against a multitude of biological parameters (including bioaccumulation, biomass, chlorophyll a content, composition and structure of prokaryotic and eukaryotic communities) monitored at set exposure times (from 1 h to 28 d). Cobalt exposure induced extremely rapid responses of the meta-metabolome, with time range inducing defense responses (TRIDeR) of around 10 s, and time range inducing damage responses (TRIDaR) of several hours. Even in biofilms whose structure had been altered by Co bioaccumulation (reduced biomass, chlorophyll a contents and changes in the composition and diversity of prokaryotic and eukaryotic communities), concentration range inducing defense responses (CRIDeR) with similar initiation thresholds (1.41 ± 0.77 × 10-10 M Co2+ added in the exposure medium) were set up at the meta-metabolome level at every time point. In contrast, the concentration range inducing damage responses (CRIDaR) initiation thresholds increased by 10 times in long-term Co exposed biofilms. The present study demonstrates that defense and damage responses of biofilm meta-metabolome exposed to Co are rapidly and sustainably impacted, even within tolerant and resistant microbial communities.

Meta-metabolomic responses of river biofilms to cobalt exposure and use of dose-response model trends as an indicator of effects.

The response of the meta-metabolome is rarely used to characterize the effects of contaminants on a whole community. Here, the meta-metabolomic fingerprints of biofilms were examined after 1, 3 and 7 days of exposure to five concentrations of cobalt (from background concentration to 1 × 10-5 M) in aquatic microcosms. The untargeted metabolomic data were processed using the DRomics tool to build dose-response models and to calculate benchmark-doses. This approach made it possible to use 100% of the chemical signal instead of being limited to the very few annotated metabolites (7%). These benchmark-doses were further aggregated into an empirical cumulative density function. A trend analysis of the untargeted meta-metabolomic feature dose-response curves after 7 days of exposure suggested the presence of a concentration range inducing defense responses between 1.7 × 10-9 and 2.7 × 10-6 M, and of a concentration range inducing damage responses from 2.7 × 10-6 M and above. This distinction was in good agreement with changes in the other biological parameters studied (biomass and chlorophyll content). This study demonstrated that the molecular defense and damage responses can be related to contaminant concentrations and represents a promising approach for environmental risk assessment of metals.

How do biomarkers dance? Specific moves of defense and damage biomarkers for biological interpretation of dose-response model trends.

Omics studies are currently increasingly used in ecotoxicology to highlight the induction of known or novel biomarkers when organisms are exposed to contaminants. Although it is virtually impossible to identify all biomarkers from all organisms, biomarkers can be grouped as defense or damage biomarkers, exhibiting a limited number of response trends. Our working hypothesis is that defense and damage biomarkers follow different dose-response patterns. A meta-analysis of 156 articles and 2595 observations of dose-response curves of defense and damage biomarkers was carried out in order to characterize the response trends of these biological parameters in a large panel of living organisms (18 phyla) exposed to inorganic or organic contaminants (176 in total). Using multinomial logistic regression models, defense biomarkers were found to describe biphasic responses (bell- and U-shaped) to a greater extent (2.5 times) than damage biomarkers. In contrast, damage biomarkers varied mainly monotonically (decreasing or increasing), representing 85% of the observations. Neither the nature of the contaminant nor the type of organisms belonging to 4 kingdoms, influence these specific responses. This result suggests that cellular defense and damage mechanisms are not specific to stressors and are conserved throughout life. Trend analysis of dose-response models as a biological interpretation of biomarkers could thus be a valuable way to exploit large omics datasets.

Multi-omics analyses from a single sample: prior metabolite extraction does not alter the 16S rRNA-based characterization of prokaryotic community in a diversity of sample types.

Massive sequencing of the 16S rRNA gene has become a standard first step to describe and compare microbial communities from various samples. Parallel analysis of high numbers of samples makes it relevant to the statistical testing of the influence of natural or experimental factors and variables. However, these descriptions fail to document changes in community or ecosystem functioning. Nontargeted metabolomics are a suitable tool to bridge this gap, yet extraction protocols are different. In this study, prokaryotic community compositions are documented by 16S rRNA gene sequencing after direct DNA extraction or after metabolites extraction followed by DNA extraction. Results obtained using the V3-V4 region on nonaxenic cultures of cyanobacteria, lake water column, biofilm, and gut of wild and lab-reared fish indicate that prior extraction of metabolites does not influence the obtained image of prokaryotic communities. This validates sequential extraction of metabolites followed by DNA as a way to combine 16S rRNA sequencing with metabolome characterization from a single sample. This approach has the potential to complement community structure characterization with a proxy of their functioning, without the uncertainties associated with the use of separate samples.

Anatoxin-a: Overview on a harmful cyanobacterial neurotoxin from the environmental scale to the molecular target.

Anatoxin-a (ATX-a) is a neurotoxic alkaloid, produced by several freshwater planktonic and benthic cyanobacteria (CB). Such CB have posed human and animal health issues for several years, as this toxin is able to cause neurologic symptoms in humans following food poisoning and death in wild and domestic animals. Different episodes of animal intoxication have incriminated ATX-a worldwide, as confirmed by the presence of ATX-a-producing CB in the consumed water or biofilm, or the observation of neurotoxic symptoms, which match experimental toxicity in vivo. Regarding toxicity parameters, toxicokinetics knowledge is currently incomplete and needs to be improved. The toxin can passively cross biological membranes and act rapidly on nicotinic receptors, its main molecular target. In vivo and in vitro acute effects of ATX-a have been studied and make possible to draw its mode of action, highlighting its deleterious effects on the nervous systems and its effectors, namely muscles, heart and vessels, and the respiratory apparatus. However, very little is known about its putative chronic toxicity. This review updates available data on ATX-a, from the ecodynamic of the toxin to its physiological and molecular targets.

Toxicity, transfer and depuration of anatoxin-a (cyanobacterial neurotoxin) in medaka fish exposed by single-dose gavage.

The proliferations of cyanobacteria are increasingly prevalent in many rivers and water bodies due especially to eutrophication. This work aims to study in female medaka fish the toxicity, the transfer and the depuration of the anatoxin-a, a neurotoxin produced by benthic cyanobacterial biofilms. This work will provide answers regarding acute toxicity induced by single gavage by anatoxin-a and to the risks of exposure by ingestion of contaminated fish flesh, considering that data on these aspects remain particularly limited. The oral LD50 and NOAEL of a single dose of (±)-anatoxin-a were determined at 11.50 and 6.67 µg.g-1, respectively. Subsequently, the toxico-kinetics of the (±)-anatoxin-a was observed in the guts, the livers and the muscles of female medaka fish for 10 days. Anatoxin-a was quantified by high-resolution qTOF mass spectrometry coupled upstream to a UHPLC chromatographic chain. The toxin could not be detected in the liver after 12 h, and in the gut and muscle after 3 days. Overall, the medaka fish do not appear to accumulate (±)-anatoxin-a and to largely recover after 24 h following a single sub-acute oral liquid exposure at the NOAEL.