Favored by global changes, freshwater cyanobacterial harmful blooms generate major ecological, economic, and public health challenges. Microcystis, one of the most widespread cyanobacterial genera, grows within a phycosphere where specialized interactions with its microbiome occur, that are suspected to influence bloom appearance and its potential toxicity. Using a combination of metagenomics, metabolomics, and metabolic modeling, we characterized the culture-associated phycospheres of 12 Microcystis strains isolated from a French pond. The distribution of metabolic reactions within Microcystis was consistent with their genospecies, whereas the metabolic landscape at the community level diverged from cyanobacterial phylogeny, indicating partial functional decoupling between cyanobacteria and their associated microbiomes. Bacteria associated with the simplified phycospheres substantially expanded the metabolic repertoire of the system, while maintaining functional redundancy within and across communities. On the other hand, endometabolomic profiles were largely driven by cyanobacterial metabolic outputs, whereas exometabolomic analysis did not reveal metabolites involved in exchange processes. Metabolic modeling, together with the identification of toxic specialized metabolites produced by specific biosynthetic gene clusters, further highlighted differences in metabolic potential among phycospheres. Together, these findings deepen the understanding of Microcystis' phycosphere functioning and demonstrate the value of multi-omics systems biology approaches, while suggesting that metabolic complementarity between species and across phycospheres could play a role in bloom-associated microbiome structure.

Recent advances in molecular phenotyping have driven the rapid growth of untargeted, multi-dimensional approaches such as epigenomics, transcriptomics, proteomics, and metabolomics. When applied to ecology, these high-throughput omics tools offer powerful new molecular trait descriptors for investigating biological and environmental processes. Using UHPLC-HRMS/MS, we analyzed metabolome variations in gut, liver, and muscle tissues of chubs and gudgeons collected in summer 2019 from French rivers affected by benthic cyanobacterial blooms. Tissue-specific metabolomic profiles were evident, with muscle metabolomes showing the most distinct species differentiation. The different tissue metabolomes of both fish species also varied by sampling location, indicating local environmental influences. Notably, fish from the Vienne site exhibited molecular signatures of metabolic stress, including elevated oxidized glutathione and bile acids, and decreased purines, amino acids, peptides, and lipids—potentially linked to anatoxin-a-producing cyanobacterial mats. These findings underscore the potential of environmental metabolomics as a sensitive tool for assessing ecological stress and support its integration into routine environmental bio-indicator programs.

Bivalves of the family Lucinidae, Loripes orbiculatus and Lucinoma borealis , are sympatric species inhabiting coastal seagrass beds in Roscoff Bay. These bivalves harbor chemoautotrophic symbionts within their gills that provide autotrophic nutrition to the host by oxidizing hydrogen sulfide (H₂S) present in the sediment. Although Lucinidae are typically considered fully autotrophic in these environments, seagrass beds are subject to fluctuations in sulfide availability due to tides, seasonal changes, and anthropogenic disturbances. This study investigates how Lucinidae cope with periods of low sulfide availability by exploring their nutritional strategies under sulfide starvation. Lucinidae species were incubated for 15 days in the presence of sediment bacteria or a mixture of two phytoplankton species labeled with ¹⁵N and ¹³C, with or without addition of sulfide, to trace assimilation pathways into the gill and visceral mass. Results show that both ¹⁵N and ¹³C were incorporated into tissues within seven days, indicating that lucinids are capable of assimilating both autotrophy- and heterotrophy-derived sources of nutrition. Composition of their associated bacterial communities was not affected. These findings provide evidence of mixotrophy in coastal Lucinidae, indicating that they can shift to filter-feeding under low sulfide availability, probably contributing to their ecological success. Nutritional plasticity of the Lucinidae may be key to their resilience in fluctuating coastal environments.

Limnospira platensis is the most widely cultivated cyanobacterium worldwide and is valued by the food industry for its high content of useful compounds like peptides, amino acids, long-chain polyunsaturated fatty acids, vitamins, carotenoids, phenolics, and other antioxidants. Its rapid cellular multiplication capacity makes it a highly promising source for sustainable biomass production and synthesis of commercially valuable compounds. Growing food industry interest has stimulated studies on optimizing culture parameters such as growth medium composition, growth temperature, and light to maximize biomass and the production of high-value biomolecules. Given the demonstrated genomic diversity within each species of cyanobacterium, we hypothesised that different L. platensis strains would exhibit varying genomic and phenotypic characteristics, resulting in strain-specific biomolecule production, growth and productivity rates. In the context of Access and Benefit-Sharing under the 1992 Convention on Biological Diversity, the selection of French L. platensis strains for local industrial applications requires a large-scale isolation process followed by a selection protocol. The novel aspects of this study include the isolation and characterization of a large number (n = 85) of French L. platensis strains and a multistage workflow procedure involving several steps to select the most promising strains for industrial culture in France. The L. platensis PMC 1246.20 strain (from Camargue, France) exhibited exceptional performance, achieving some of the highest recorded growth rates as well as the highest biomass, phycobiliproteins and carotenoids production when cultured under red light. These selection experiments highlight the importance of identifying the optimal strain-specific culture conditions when selecting L. platensis strains for scaling up production to industrial levels.

Microcystis is one of the most common bloom-forming cyanobacteria colonizing freshwater ecosystems worldwide. This genus remarkably produces numerous bio-active accessory metabolites, which are believed to be potentially involved in different ecological and/or physiological processes. However, their genuine contribution to the evolutionary success of Microcystis blooms remains undetermined. To better depict the potential relationship between the local genetic diversity of blooming Microcystis populations and their respective associated chemical diversity, we conducted a joint genomic and metabolomic analysis of 65 Microcystis strains collected from various lakes in France and surrounding Western European countries. Interestingly, both core and pan-gene phylogenetic analysis place 57 of these strains in 11 distinct genotypes with at least 2 genomes, being widely distributed along the entire Microcystis phylogeny and presenting specific signatures of accessory metabolite biosynthesis. The direct chemical analysis of metabolite diversity produced by these strains, cultured under laboratory conditions, reveals the production of stable metabolite cocktails, with minimal variations over replication, growth phases and culture conditions. Remarkably, these strains belonging to 11 different genotypes correspond to 13 distinct chemotypes according to an accurate one-chemotype-for-one-genotype rule. Furthermore, these genotypes also appear distinguishable regarding their respective ecotoxicological traits and might be considered as specific toxico-ecotypes. Overall, our investigations reveal that the production of accessory metabolites constitute well conserved chemical traits across the different Microcystis genotypes, suggesting these molecules may be involved in key adaptive and selective processes, that still remain under-explored. A combined genomic and metabolomic approach sheds light on the consistency between genotypes, chemotypes, and toxico-ecotypes of European Microcystis strains, suggesting that different clades may induce different ecotoxicological threats.

Effects of trophic status on lake microeukaryote community dynamics remain underexplored. Spanning oligotrophic to hypereutrophic conditions, peri-urban lakes located in the Greater Paris region (France) offer unique opportunities to compare these dynamics along a eutrophication gradient. Here, community composition was characterised using 18S rRNA gene metabarcoding, and analyzed in the context of environmental parameters and chlorophyll a-derived trophic status throughout an 18-month sampling period. Microeukaryote assemblages were dominated by Cryptophyceae, Spirotrichea, Chrysophyceae, and Chlorophyceae, with mixotrophic and heterotrophic taxa dominating across all lakes. Taxa richness peaked at intermediate trophic levels, consistent with the intermediate disturbance hypothesis. Beta-diversity and network analyses revealed increasing community modularity, reduced connectivity, and enhanced temporal variability with higher trophic status. In lakes reaching the hypereutrophic status, communities diverged progressively over time. Conversely, oligo- to mesotrophic lakes maintained more connected and stable assemblages. These findings demonstrate that eutrophication fosters more diverse and increasingly divergent microeukaryote communities, underscoring its role as a central driver of microbial community restructuring in urban freshwater systems.

Global change is expected to increase cyanobacterial dominance and cyanotoxin production. However, cyanobacterial responses to meteorological events remain uncertain due to limited integrative approaches. Eco-metabolomics offers a promising tool to investigate drivers of phytoplankton successions and metabolite production. We investigated the spatio-temporal dynamics of phytoplankton and their metabolomes in relation to local meteorological conditions in Lake Aydat (France) during summer 2021, sampling both near the Veyre inflow and at the lake center. High-resolution mass spectrometry, combined with phytoplankton biovolumes and physicochemical parameters, revealed two distinct metabolomic fingerprints positively correlated with phytoplankton composition. The first metabolome is composed of mainly lipids and positively correlated with the presence of diatoms throughout the summer until the end of the drought. In contrast, the second metabolome is correlated with the presence of Dolichospermum, that occurrs after a 22-day period without rain and when there is a low nitrate concentration ($NO_3^ - $ ∼ 0.4 mg.l-1). Interestingly, despite similar phytoplankton composition across sites, significant higher abundance of cyanopeptides are detected close to the river inflow during the second rain period, likely driven by higher nutrient inputs and warmer waters compared to the lake center. Overall, these findings demonstrate that fine-scale spatial monitoring can reveal distinct ecometabolomic niches, separated by only 200 m.

Cyanobacterial blooms represent one prevalent stressor in aquatic ecosystems worldwide, exposing aquatic animals to complex mixtures of live cells and dissolved bioactive compounds, including cyanotoxins. The gut microbiota, which plays fundamental roles in digestion, immunity, and metabolic regulation, has been recognized as a key interface between environmental stressors and host health. Exposure to cyanobacteria occurs primarily through ingestion, making the gut the main site of interaction with live cells, cyanobacterial metabolites, and associated bacteria. While dissolved bioactive compounds can also penetrate via gills and skin, their role is secondary in most animals. Understanding how cyanobacteria interact with the host gut microbiota is therefore essential to fully assess their impacts on animal health. This review summarizes current knowledge of gut microbiota responses to cyanobacterial stress in crustaceans, mollusks, and chordates, highlighting dynamic and context-dependent changes in microbial communities. We discuss the cascading effects on host physiology, immune function, and metabolic homeostasis, and explore how the microbiota may modulate host resilience, including potential roles in cyanotoxin degradation.

ABSTRACT Limnospira can colonize a wide variety of environments (e.g., freshwater, brackish, alkaline, or alkaline-saline water) and develop dominant and even permanent blooms that overshadow and limit the diversity of adjacent phototrophs, especially in alkaline and saline environments. Previous phylogenomic analysis of Limnospira allowed us to distinguish two major phylogenetic clades (I and II) but failed to clearly segregate strains according to their respective habitats in terms of salinity or biogeography. In the present work, we attempted to determine whether Limnospira displays metabolic signatures specific to its different habitats, particularly brackish and alkaline-saline ecosystems. The impact of accessory gene repertoires on respective chemical adaptations was also determined. In complement of our previous phylogenomic investigation of Limnospira (Roussel et al., 2023), we develop a specific analysis of the metabolomic diversity of 93 strains of Limnospira , grown under standardized lab culture conditions. Overall, this original work showed distinct chemical fingerprints that were correlated with the respective biogeographic origins of the strains. The molecules that most distinguished the different Limnospira geographic groups were sugars, lipids, peptides, photosynthetic pigments, and antioxidants. Interestingly, these molecular enrichments might represent consequent adaptations to conditions of salinity, light, and oxidative stress in their respective sampling environments. Although the genes specifically involved in the production of these components remain unknown, we hypothesized that within extreme environments, such as those colonized by Limnospira , a large set of flexible genes could support the production of peculiar metabolite sets providing remarkable adaptations to specific local environmental conditions. IMPORTANCE Limnospira are ubiquitous cyanobacteria with remarkable adaptive strategies allowing them to colonize and dominate a wide range of alkaline-saline environments worldwide. Phylogenomic analysis of Limnospira revealed two distinct major phylogenetic clades but failed to clearly segregate strains according to their habitats in terms of salinity or biogeography. We hypothesized that the genes found within this variable portion of the genome of these clades could be involved in the adaptation of Limnospira to local environmental conditions. In the present paper, we attempted to determine whether Limnospira displayed metabolic signatures specific to its different habitats. We also sought to understand the impact of the accessory gene repertoire on respective chemical adaptations.

Bivalves of the family Lucinidae thrive in sulfidic sediments thanks to their chemoautotrophic bacterial symbionts. However, how different Lucinidae species respond to sulfide deprivation and associated symbiont loss remains poorly understood. Here, we investigated the responses of Lucinoma borealis and Loripes orbiculatus, two species that co-occur in temperate seagrass beds, exposed to prolonged sulfide starvation. Using metabolomics, ultrastructural TEM analyses and 16S rRNA-based metabarcoding, we monitored and compared responses in gills and visceral mass over a 4-month period. Both host species as well as their symbionts survived sulfide-free conditions. Hosts tissues displayed limited impact on ultrastructure and metabolites. Despite decrease in numbers and activity level, symbionts remained present throughout the experiment and no evidence for bacteremia or infection was detected. Our results also revealed differences, in particular in host apoptosis response, suggesting species-specific stress strategies. Altogether, both holobionts can survive extended low-sulfide periods without critical damage and without completely losing their symbionts. These could be adaptations to the extended low-sulfide periods that are associated with low primary production and the cold season in seagrass beds. Adaptations could involve a switch in the symbionts' physiological state to preserve a dormant symbiotic population. These findings highlight the importance of stress tolerance mechanisms in coastal Lucinidae, and raise questions about the nature of host–symbiont dependency in these periods.

With more than 12 million inhabitants, the Greater Paris offers a “natural laboratory” to explore the effects of eutrophication on freshwater lake’s microbiomes within a relative restricted area ( 70 km radius). Here, a 4-months survey was carried out during summertime to monitor planktonic microbial communities of nine lakes located around Paris (Île-de-France, France) of comparable morphologies, yet distinct trophic statuses from mesotrophic to hypereutrophic. By thus minimizing the confounding factors, we investigated how trophic status could influence prokaryotic community structures (16S rRNA gene sequencing) and functions (shotgun metagenomics). These freshwater lakes harbored highly distinct and diverse prokaryotic communities, and their trophic status appears as the main driver explaining both differences in community structure and functional potential. Although their gene pool was quite stable and shared among lakes, taxonomical and functional changes were correlated. According to trophic status, differences in phosphorus metabolism-related genes were highlighted among the relevant functions involved in the biogeochemical cycles. Overall, hypereutrophic lakes microbiomes displayed the highest contrast and heterogeneity over time, suggesting a specific microbial regime shift compared to eutrophic and mesotrophic lakes.

Microcystis spp., prolific bloom-forming freshwater cyanobacteria, produce a wide range of bioactive secondary metabolites. While microcystins (MCs) are well-characterized hepatotoxins, the ecotoxicological roles of other Microcystis-derived compounds remain poorly understood. This study evaluates the toxic potential of four Microcystis strains - two MC-producing (PMC 728.11, 807.12) and two non-MC-producing (PMC 810.12, 826.12) - in Medaka fish (Oryzias latipes) at different life stages. Embryos and larvae exposed to extracts from non-MC-producing strains exhibited pronounced developmental toxicity and malformations, linked to the presence of microginins, aeruginosins, and microcyclamides. MC-rich strain PMC 728.11 induced significant post-hatch toxicity. In adult females exposed under microcosm conditions to environmentally relevant concentrations, strain-specific disruptions were observed in gut microbiota composition and tissue metabolomes. Notably, PMC 728.11 caused microbial dysbiosis, while PMC 826.12 impaired digestive functions, enhancing susceptibility to toxicant uptake. The remaining strains produced milder effects. Our results demonstrate that Microcystis toxicity extends beyond MCs, driven by complex and variable metabolite mixtures. This biochemical diversity poses challenges for bloom risk assessment and raises concerns over unrecognized environmental hazards. These findings advocate for broader monitoring of cyanobacterial metabolites and their mechanistic impacts in aquatic toxicology and environmental health.

Various studies suggest that global change is causing an increase in phytoplankton biomass, cyanobacteria prevalence and cyanotoxin production. However, there are conflicting reports regarding the response of cyanobacteria blooms to global warming and meteorological events, probably because of the lack of global approaches. Metabolomics approaches in natural system hold great promise in investigating the factors leading to variations in phytoplankton successions and subsequent cyanotoxin production. However, eco-metabolomics studies are still scares in literature and suffer to adequately unravel the biologically relevant variables under environmental changes. In this study, we investigate the temporal and spatial dynamics of phytoplankton community and the production of their primary and secondary untargeted metabolites in response to local meteorological events. Thus, we collected water samples in two points of the Aydat Lake (France): near the inflowing waters from Veyre River and at the middle of the lake during the 2021 summer. Untargeted intracellular metabolites were measured using ultra-high-performance liquid chromatography coupled with a high-resolution mass spectrometer, as well as phytoplankton biovolume and diversity and physicochemical lake’s parameters. Primarily, our results show the increase of the biovolume of diazotrophic cyanobacteria at the end of the drought and after rain events at both sites. During the drought, we observe a strong increase of intracellular lipid contents, probably in response to sudden nitrogen and phosphorus limitation. Differently, during the wet periods, we observe an increase of the phytoplankton glycerophospholipid content, especially at the middle of the lake, whereas significantly higher abundance of secondary metabolites was monitored at site near the wetland area. Since then, we report a strong correlation between the abundance of different cyanopeptides and the biovolume of Dolichospermum, which is present at both sites, we suggest acclimative responses to cope with the phytoplankton growing stimulation related with the increase of the nutritive ion influx following the rain events. The significant difference in the intra-cellular content in metabolites between the 2 sampling sites, separated by only 200m, while phytoplankton communities were similar suggests the existence of local metabolomic niches.

Cyanobacteria are ancient photosynthetic microorganisms with a long evolutionary history that have adapted to inhabit diverse environments, such as thermal waters and muds. To do so, they are known to produce a wide range of bioactive molecules likely involved in adaptative traits such as high light and/or high temperature resistance mechanisms, which makes them particularly interesting for inclusion in thermal treatments and various therapeutic applications. In this study, the impact of higher temperature and light intensity culture conditions on the metabolome of a promising cyanobacterial strain isolated from the muds of the Balaruc-les-Bains thermal station, Planktothricoides raciborskii PMC 877.14, was investigated through liquid chromatography and mass spectrometry analyses. Statistical analyses of the biological data and molecular network construction allowed for the exploration of potential metabolite induction under different culture conditions, in order to drive the production of specific metabolites aimed at coping with cellular stress. A global shift on intracellular metabolic composition was observed over time with increased biomass production. Higher light intensity was found to stimulate both growth and production of antioxidant and/or photoprotective molecules (such as ergothioneine, mycosporine-like amino acids or carotenoids) while an increase in temperature influenced the higher production of phycobilins (such as phycoerythrin and phycocyanin). These results highlight the importance of culture/environmental conditions in driving intracellular metabolite differentiation and open up new perspectives in selecting of the optimal parameters for the growth and production of bioactive molecules for therapeutic applications in the cyanobacterium P. raciborskii PMC 877.14.

Biofilm-forming cyanobacteria are abundant in mangrove ecosystems, colonizing various niches including sediment surface and periphyton where they can cover large areas, yet have received limited attention. Several filamentous isolates were recently isolated from Guadeloupe, illustrating the diversity and novelty present in these biofilms. In this study, nine strains belonging to three novel lineages found abundantly in Guadeloupe biofilms are characterized by genome sequencing, morphological and ultrastructural examination, metabolome fingerprinting and searched for secondary metabolites biosynthesis pathways. Assignation of two lineages to known genera is confirmed, namely Scytonema and Jaaginema. The third lineage corresponds to a new Coleofasciculales genus herein described as Karukerafilum gen. nov. The four strains belonging to this genus group into two subclades, one of which displays genes necessary for nitrogen fixation as well as the complete pathway for geosmin production. This study gives new insights into the diversity of mangrove biofilm-forming cyanobacteria, including genome-based description of a new genus and the first genome sequence available for the genus Jaaginema.

One of the most prevalent and notorious bloom-forming freshwater cyanobacterial genus is Microcystis, whom toxicological impairs yet remain incompletely investigated. Based on our previous studies, we hypothesize that some emerging Microcystis metabolites, in addition to microcystins (MCs), are of (eco)toxicological concerns and should be further investigated. To this end, we explore the ecotoxicological potential of different Microcystis genotypes producing various bio-active metabolite cocktails, particularly cyanopeptides of different structural families including MCs, cyanopeptolins, microginins, anabaenopetins, aeruginosins or microcyclamides, on embryo/larvae and adult Medaka fish model. Embryo and larvae exposures to the extracts of the four distinct Microcystis genotypes - comprising two MC-producing (PMC 728.11 and 807.12) and two non-MC-producing (PMC 810.12 and 826.12) strains - showed that PMC 810.12 and 826.12 respectively producing microginins, aeruginosins and microcyclamides exhibit early toxicity and teratogenicity, while PMC 728.11 presented rather larvae toxicity on hatched larvae, in agreement with its high MC content. In addition, we conducted a 4-days microcosm experiment with adult female Medaka exposed to environmental concentrations of these four Microcystis strain cultures to document the microbiome and metabolome responses. Fishes exposed to PMC 728.11 exhibited microbiota dysbiosis signature, while exposure to PMC 826.12 perturbated the fish digestion process inducing even more pronounced microbial and metabolic alterations. The two other strains provoked more moderate perturbations. These findings highlight toxic effects on fish exposed to both MC- as well as non-MC-producing cyanobacteria, suggesting complex interplay and effects of undocumented cyanobacterial bio-active and toxic compounds basides MCs during blooms of Microcystis. ### Competing Interest Statement The authors have declared no competing interest.

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. The combination of untargeted metabolomic features and benchmark-dose (BMD) calculations then makes it possible to determine concentration range inducing defense responses (CRIDeR) and concentration range inducing damage responses (CRIDaR). 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 (background concentration, 1 x 10 -7 , 5 x 10 -7 and 1 x 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 hour to 28 days). Cobalt exposure induced extremely rapid responses of the meta-metabolome, with time range inducing defense responses (TRIDeR) of around ten seconds, 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), CRIDeRs with similar initiation thresholds (1.41 ± 0.77 x 10 -10 M Co 2+ added in the exposure medium) were set up at the meta-metabolome level at every time point. In contrast, the 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. Graphical abstract Highlights Prokaryotic community structures were impacted after 1 h of exposure to Co Biofilm meta-metabolome was impacted after 36 s of exposure to Co Biofilm meta-metabolome response was faster than changes in biofilm communities Short- and long-term exposed biofilms have similar CRIDeR initiation thresholds Long-term exposed biofilms have higher CRIDaR initiation thresholds

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 x 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 doseresponse curves after 7 days of exposure suggested the presence of a concentration range inducing defense responses between 1.7 x 10 -9 and 2.7 x 10 -6 M, and of a concentration range inducing damage responses from 2.7 x 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.