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.

Disruption of fish gut microbiota composition and holobiont's metabolome during a simulated Microcystis aeruginosa (Cyanobacteria) bloom.

BACKGROUND: Cyanobacterial blooms are one of the most common stressors encountered by metazoans living in freshwater lentic systems such as lakes and ponds. Blooms reportedly impair fish health, notably through oxygen depletion and production of bioactive compounds including cyanotoxins. However, in the times of the "microbiome revolution", it is surprising that so little is still known regarding the influence of blooms on fish microbiota. In this study, an experimental approach is used to demonstrate that blooms affect fish microbiome composition and functions, as well as the metabolome of holobionts. To this end, the model teleost Oryzias latipes is exposed to simulated Microcystis aeruginosa blooms of various intensities in a microcosm setting, and the response of bacterial gut communities is evaluated in terms of composition and metabolome profiling. Metagenome-encoded functions are compared after 28 days between control individuals and those exposed to highest bloom level. RESULTS: The gut bacterial community of O. latipes exhibits marked responses to the presence of M. aeruginosa blooms in a dose-dependent manner. Notably, abundant gut-associated Firmicutes almost disappear, while potential opportunists increase. The holobiont's gut metabolome displays major changes, while functions encoded in the metagenome of bacterial partners are more marginally affected. Bacterial communities tend to return to original composition after the end of the bloom and remain sensitive in case of a second bloom, reflecting a highly reactive gut community. CONCLUSION: Gut-associated bacterial communities and holobiont functioning are affected by both short and long exposure to M. aeruginosa, and show evidence of post-bloom resilience. These findings point to the significance of bloom events to fish health and fitness, including survival and reproduction, through microbiome-related effects. In the context of increasingly frequent and intense blooms worldwide, potential outcomes relevant to conservation biology as well as aquaculture warrant further investigation. Video Abstract.

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.

Fish metabolome from sub-urban lakes of the Paris area (France) and potential influence of noxious metabolites produced by cyanobacteria.

The recent democratization of high-throughput molecular phenotyping allows the rapid expansion of promising untargeted multi-dimensional approaches (e.g. epigenomics, transcriptomics, proteomics, and/or metabolomics). Indeed, these emerging omics tools, processed for ecologically relevant species, may present innovative perspectives for environmental assessments, that could provide early warning of eco(toxico)logical impairments. In a previous pilot study (Sotton et al., Chemosphere 2019), we explore by 1H NMR the bio-indicative potential of metabolomics analyses on the liver of 2 sentinel fish species (Perca fluviatilis and Lepomis gibbosus) collected in 8 water bodies of the peri-urban Paris' area (France). In the present study, we further investigate on the same samples the high potential of high-throughput UHPLC-HRMS/MS analyses. We show that the LC-MS metabolome investigation allows a clear separation of individuals according to the species, but also according to their respective sampling lakes. Interestingly, similar variations of Perca and Lepomis metabolomes occur locally indicating that site-specific environmental constraints drive the metabolome variations which seem to be influenced by the production of noxious molecules by cyanobacterial blooms in certain lakes. Thus, the development of such reliable environmental metabolomics approaches appears to constitute an innovative bio-indicative tool for the assessment of ecological stress, such as toxigenic cyanobacterial blooms, and aim at being further follow up.

Draft Genome Sequence of the Toxic Freshwater Microcystis aeruginosa Strain PMC 728.11 (Cyanobacteria, Chroococcales).

Microcystis aeruginosa is one of the major species that cause toxic cyanobacterial blooms in freshwater systems worldwide. Here, we report the draft genome sequence of M. aeruginosa PMC 728.11, a microcystin-producing cyanobacterium isolated from the freshwater reservoir of Juanon in Valence, France. The genome sequence contains 276 contigs, consisting of 5,536,025 bp and 5,594 putative protein-coding genes, among which are several biosynthetic gene clusters encoding enzyme complexes involved in the production of various bioactive and toxic metabolites.

Microbiome-Aware Ecotoxicology of Organisms: Relevance, Pitfalls, and Challenges.

Over the last 15 years, the advent of high-throughput "omics" techniques has revealed the multiple roles and interactions occurring among hosts, their microbial partners and their environment. This microbiome revolution has radically changed our views of biology, evolution, and individuality. Sitting at the interface between a host and its environment, the microbiome is a relevant yet understudied compartment for ecotoxicology research. Various recent works confirm that the microbiome reacts to and interacts with contaminants, with consequences for hosts and ecosystems. In this paper, we thus advocate for the development of a "microbiome-aware ecotoxicology" of organisms. We emphasize its relevance and discuss important conceptual and technical pitfalls associated with study design and interpretation. We identify topics such as functionality, quantification, temporality, resilience, interactions, and prediction as major challenges and promising venues for microbiome research applied to ecotoxicology.

Low-diversity bacterial microbiota in Southern Ocean representatives of lanternfish genera Electrona, Protomyctophum and Gymnoscopelus (family Myctophidae).

Myctophids are among the most abundant mesopelagic teleost fishes worldwide. They are dominant in the Southern Ocean, an extreme environment where they are important both as consumers of zooplankton as well as food items for larger predators. Various studies have investigated myctophids diet, but no data is yet available regarding their associated microbiota, despite that the significance of bacterial communities to fish health and adaptation is increasingly acknowledged. In order to document microbiota in key fish groups from the Southern Ocean, the bacterial communities associated with the gut, fin, gills and light organs of members of six species within the three myctophid genera Electrona, Protomyctophum and Gymnoscopelus were characterized using a 16S rRNA-based metabarcoding approach. Gut communities display limited diversity of mostly fish-specific lineages likely involved in food processing. Fin and skin communities display diversity levels and compositions resembling more those found in surrounding seawater. Community compositions are similar between genera Electrona and Protomyctophum, that differ from those found in Gymnoscopelus and in water. Low abundances of potentially light-emitting bacteria in light organs support the hypothesis of host production of light. This first description of myctophid-associated microbiota, and among the first on fish from the Southern Ocean, emphasizes the need to extend microbiome research beyond economically-important species, and start addressing ecologically-relevant species.