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.

Insights on the Organ-Dependent, Molecular Sexual Dimorphism in the Zebra Mussel, Dreissena polymorpha, Revealed by Ultra-High-Performance Liquid Chromatography-Tandem Mass Spectrometry Metabolomics.

The zebra mussel, Dreissena polymorpha, is extensively used as a sentinel species for biosurveys of environmental contaminants in freshwater ecosystems and for ecotoxicological studies. However, its metabolome remains poorly understood, particularly in light of the potential molecular sexual dimorphism between its different tissues. From an ecotoxicological point of view, inter-sex and inter-organ differences in the metabolome suggest variability in responsiveness, which can influence the analysis and interpretation of data, particularly in the case where males and females would be analyzed indifferently. This study aimed to assess the extent to which the molecular fingerprints of functionally diverse tissues like the digestive glands, gonads, gills, and mantle of D. polymorpha can reveal tissue-specific molecular sexual dimorphism. We employed a non-targeted metabolomic approach using liquid chromatography high-resolution mass spectrometry and revealed a significant sexual molecular dimorphism in the gonads, and to a lesser extent in the digestive glands, of D. polymorpha. Our results highlight the critical need to consider inter-sex differences in the metabolome of D. polymorpha to avoid confounding factors, particularly when investigating environmental effects on molecular regulation in the gonads, and to a lesser extent in the digestive glands.

Dataset on metabolome dimorphism in different organs of mature Palaemon serratus prawn.

The prawn Palaemon serratus exhibits a large distribution (occurring along the Northeastern Atlantic coast to the Mediterranean), and has thus been found suitable as model organism valuable for various ecotoxicological studies. However, little is still known about the potential input of its metabolome and particularly concerning a potential molecular sexual dimorphism observable in the different tissues of this organism. In an ecotoxicological point of view, inter-sex and inter-organ differences of the metabolomes may introduce analytical bias and impact the robustness of the analysis and its interpretation. To explore such possibilities, we obtained qualitative metabolomic data from the analysis of different organs of mature male and female Palaemon serratus. We used ultra-high-performance liquid chromatography-electrospray ionization-high resolution tandem mass spectrometry (UHPLC-ESI-HRMS on positive mode) to characterize the 75%-extracted metabolome of both gills, hepatopancreas, nervous gland, muscle and gonads. The data were dereplicated using specific metabolomic software (MetaboScape 4) and 2,782 features were extracted, 1,720 of them being also analysed on MS/MS mode, supporting molecular networking investigations with Metgem 1.3.6. These metabolites were thus putatively identified using GNPS (Global Natural Product Social) Molecular Networking databases for de-novo annotation followed by manual curation of 84 metabolites. This data provides essential information on the important sexual dimorphism occurring at the molecular level in the different organs and supports further research on physiology and ecotoxicology in common European prawn.

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.

Establishment of the Bacterial Microbiota in a Lab-Reared Model Teleost Fish, the Medaka Oryzias latipes.

Oryzias latipes is an important model organism for physiology, genetics, and developmental studies, and has also emerged as a relevant vertebrate model for aquatic ecotoxicology. Knowledge regarding its associated microbiota on the other hand is still scarce and limited to adults, despite the relevance of the associated microbiome to the host's biology. This study provides the first insights into the establishment of bacterial microbiota during early developmental stages of laboratory-reared medaka using a 16S-rRNA-sequencing-based approach. Major shifts in community compositions are observed, from a Proteobacteria-dominated community in larvae and juveniles to a more phylum-diverse community towards adulthood, with no obvious difference between female and male specimens. Major bacterial taxa found in adults, including genera Cetobacterium and ZOR0006, establish progressively and are rare during early stages. Dominance shifts are comparable to those documented in another major model teleost, the zebrafish. Results from this study provide a basis for future work investigating the influence of medaka-associated bacteria during host development.

Gut microbiota and holobiont metabolome composition of the medaka fish (Oryzias latipes) are affected by a short exposure to the cyanobacterium Microcystis aeruginosa.

Blooms of toxic cyanobacteria are a common stress encountered by aquatic fauna. Evidence indicates that long-lasting blooms affect fauna-associated microbiota. Because of their multiple roles, host-associated microbes are nowadays considered relevant to ecotoxicology, yet the respective timing of microbiota versus functional changes in holobionts response needs to be clarified. The response of gut microbiota and holobiont's metabolome to exposure to a dense culture of Microcystis aeruginosa was investigated as a microcosm-simulated bloom in the model fish species Oryzias latipes (medaka). Both gut microbiota and gut metabolome displayed significant composition changes after only 2 days of exposure. A dominant symbiont, member of the Firmicutes, plummeted whereas various genera of Proteobacteria and Actinobacteriota increased in relative abundance. Changes in microbiota composition occurred earlier and faster compared to metabolome composition. Liver and muscle metabolome were much less affected than guts, supporting that the gut and associated microbiota are in the front row upon exposure. This study highlights that even short cyanobacterial blooms, that are increasingly frequent, trigger changes in microbiota composition and holobiont metabolome. It emphasizes the relevance of multi-omics approaches to explore organism's response to an ecotoxicological stress.

Fish gut-associated bacterial communities in a tropical lagoon (Aghien lagoon, Ivory Coast).

Aghien lagoon (Ivory Coast) is a eutrophic freshwater lagoon that harbors high biomasses of phytoplankton. Despite Increasing interest in fish gut microbiomes diversity and functions, little data is currently available regarding wild species from tropical west African lakes. Here, gut-associated bacterial communities are investigated in four fish species that are consumed by locale populations, namely the Cichlidae Hemichromis fasciatus, Tilapia guineensis and Sarotherodon melanotheron, and the Claroteidae Chrysichthys nigrodigitatus. Species-related differences are identified, that can be attributed to host phylogeny and diet. Important variations throughout the year are observed in T. guineensis and C. nigrodigitatus. This result emphasized the importance of time-series sampling and comparison with environmental variables even in tropical regions, that are not often conducted in wild populations. Effects of environmental factors (anthropogenic or not) on the microbiota and potential outcomes for fish health and populations sustainability need to be further explored. Interestingly, fish appear as major reservoirs of bacterial diversity, suggesting that they could contribute to the overall stability and resilience of bacterial communities present in the Aghien lagoon.