The success of the bloom-forming cyanobacteria Planktothrix: Genotypes variability supports variable responses to light and temperature stress.

Cyanobacterial blooms can modify the dynamic of aquatic ecosystems and have harmful consequences for human activities. Moreover, cyanobacteria can produce a variety of cyanotoxins, including microcystins, but little is known about the role of environmental factors on the prevalence of microcystin producers in the cyanobacterial bloom dynamics. This study aimed to better understand the success of Planktothrix in various environments by unveiling the variety of strategies governing cell responses to sudden changes in light intensity and temperature. The cellular responses (photosynthesis, photoprotection, heat shock response and metabolites synthesis) of four Planktothrix strains to high-light or high-temperature were studied, focusing on how distinct ecotypes (red- or green-pigmented) and microcystin production capability affect cyanobacteria's ability to cope with such abiotic stimuli. Our results showed that high-light and high-temperature impact different cellular processes and that Planktothrix responses are heterogeneous, specific to each strain and thus, to genotype. The ability of cyanobacteria to cope with sudden increase in light intensity and temperature was not related to red- or green-pigmented ecotype or microcystin production capability. According to our results, microcystin producers do not cope better to high-light or high-temperature and microcystin content does not increase in response to such stresses.

Insights into the Diversity of Secondary Metabolites of Planktothrix Using a Biphasic Approach Combining Global Genomics and Metabolomics.

Cyanobacteria are an ancient lineage of slow-growing photosynthetic bacteria and a prolific source of natural products with diverse chemical structures and potent biological activities and toxicities. The chemical identification of these compounds remains a major bottleneck. Strategies that can prioritize the most prolific strains and novel compounds are of great interest. Here, we combine chemical analysis and genomics to investigate the chemodiversity of secondary metabolites based on their pattern of distribution within some cyanobacteria. Planktothrix being a cyanobacterial genus known to form blooms worldwide and to produce a broad spectrum of toxins and other bioactive compounds, we applied this combined approach on four closely related strains of Planktothrix. The chemical diversity of the metabolites produced by the four strains was evaluated using an untargeted metabolomics strategy with high-resolution LC-MS. Metabolite profiles were correlated with the potential of metabolite production identified by genomics for the different strains. Although, the Planktothrix strains present a global similarity in terms of a biosynthetic cluster gene for microcystin, aeruginosin, and prenylagaramide for example, we found remarkable strain-specific chemodiversity. Only few of the chemical features were common to the four studied strains. Additionally, the MS/MS data were analyzed using Global Natural Products Social Molecular Networking (GNPS) to identify molecular families of the same biosynthetic origin. In conclusion, we depict an efficient, integrative strategy for elucidating the chemical diversity of a given genus and link the data obtained from analytical chemistry to biosynthetic genes of cyanobacteria.

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).

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.

Subcellular localization of microcystin in the liver and the gonads of medaka fish acutely exposed to microcystin-LR.

Among the diverse toxic components produced by cyanobacteria, microcystins (MCs) are one of the most toxic and notorious cyanotoxin groups. Besides their potent hepatotoxicity, MCs have been revealed to induce potential reproductive toxicity in various animal studies. However, little is still known regarding the distribution of MCs in the reproductive organ, which could directly affect reproductive cells. In order to respond to this question, an acute study was conducted in adult medaka fish (model animal) gavaged with 10 µg.g-1 body weight of pure MC-LR. The histological and immunohistochemical examinations reveal an intense distribution of MC-LR within hepatocytes along with a severe liver lesion in the toxin-treated female and male fish. Besides being accumulated in the hepatocytes, MC-LR was also found in the connective tissue of the ovary and the testis, as well as in oocytes and degenerative spermatocyte-like structures but not spermatocytes. Both liver and gonad play important roles in the reproductive process of oviparous vertebrates. This observation constitutes the first observation of the presence of MC-LR in reproductive cells (female, oocytes) of a vertebrate model with in vivo study. Our results, which provide intracellular localization of MC-LR in the gonad, advance our understanding of the potential reproductive toxicity of MC-LR in fish.

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.

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.

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.

An integrated omic analysis of hepatic alteration in medaka fish chronically exposed to cyanotoxins with possible mechanisms of reproductive toxicity.

Cyanobacterial blooms threaten human health as well as the population of other living organisms in the aquatic environment, particularly due to the production of natural toxic components, the cyanotoxin. So far, the most studied cyanotoxins are microcystins (MCs). In this study, the hepatic alterations at histological, proteome and transcriptome levels were evaluated in female and male medaka fish chronically exposed to 1 and 5 µg L-1 microcystin-LR (MC-LR) and to the extract of MC-producing Microcystis aeruginosa PCC 7820 (5 µg L-1 of equivalent MC-LR) by balneation for 28 days, aiming at enhancing our understanding of the potential reproductive toxicity of cyanotoxins in aquatic vertebrate models. Indeed, both MC and Microcystis extract adversely affect reproductive parameters including fecundity and egg hatchability. The liver of toxin treated female fish present glycogen storage loss and cellular damages. The quantitative proteomics analysis revealed that the quantities of 225 hepatic proteins are dysregulated. In particular, a notable decrease in protein quantities of vitellogenin and choriogenin was observed, which could explain the decrease in reproductive output. Liver transcriptome analysis through Illumina RNA-seq reveals that over 100-400 genes are differentially expressed under 5 µg L-1 MC-LR and Microcystis extract treatments, respectively. Ingenuity pathway analysis of the omic data attests that various metabolic pathways, such as energy production, protein biosynthesis and lipid metabolism, are disturbed by both MC-LR and the Microcystis extract, which could provoke the observed reproductive impairment. The transcriptomics analysis also constitutes the first report of the impairment of circadian rhythm-related gene induced by MCs. This study contributes to a better understanding of the potential consequences of chronic exposure of fish to environmental concentrations of cyanotoxins, suggesting that Microcystis extract could impact a wider range of biological pathways, compared with pure MC-LR, and even 1 µg L-1 MC-LR potentially induces a health risk for aquatic organisms.

Deep sexual dimorphism in adult medaka fish liver highlighted by multi-omic approach.

Sexual dimorphism describes the features that discriminate between the two sexes at various biological levels. Especially, during the reproductive phase, the liver is one of the most sexually dimorphic organs, because of different metabolic demands between the two sexes. The liver is a key organ that plays fundamental roles in various physiological processes, including digestion, energetic metabolism, xenobiotic detoxification, biosynthesis of serum proteins, and also in endocrine or immune response. The sex-dimorphism of the liver is particularly obvious in oviparous animals, as the female liver is the main organ for the synthesis of oocyte constituents. In this work, we are interested in identifying molecular sexual dimorphism in the liver of adult medaka fish and their sex-variation in response to hepatotoxic exposures. By developing an integrative approach combining histology and different high-throughput omic investigations (metabolomics, proteomics and transcriptomics), we were able to globally depict the strong sexual dimorphism that concerns various cellular and molecular processes of hepatocytes comprising protein synthesis, amino acid, lipid and polysaccharide metabolism, along with steroidogenesis and detoxification. The results of this work imply noticeable repercussions on the biology of oviparous organisms environmentally exposed to chemical or toxin issues.

Gender-Specific Toxicological Effects of Chronic Exposure to Pure Microcystin-LR or Complex Microcystis aeruginosa Extracts on Adult Medaka Fish.

Cyanobacterial blooms often occur in freshwater lakes and constitute a potential health risk to human populations, as well as to other organisms. However, their overall and specific implications for the health of aquatic organisms that are chronically and environmentally exposed to cyanobacteria producing hepatotoxins, such as microcystins (MCs), together with other bioactive compounds have still not been clearly established and remain difficult to assess. The medaka fish was chosen as the experimental aquatic model for studying the cellular and molecular toxicological effects on the liver after chronic exposures (28 days) to environmentally relevant concentrations of pure MC-LR, complex extracts of MC producing or nonproducing cyanobacterial biomasses, and of a Microcystis aeruginosa natural bloom. Our results showed a higher susceptibility of females to the different treatments compared to males at both the cellular and the molecular levels. Although hepatocyte lysis increased with MC-containing treatments, lysis always appeared more severe in the liver of females compare to males, and the glycogen cellular reserves also appeared to decrease more in the liver of females compared to those in the males. Proteomic investigations reveal divergent responses between males and females exposed to all treatments, especially for proteins involved in metabolic and homeostasis processes. Our observations also highlighted the dysregulation of proteins involved in oogenesis in female livers. These results suggest that fish populations exposed to cyanobacteria blooms may potentially face several ecotoxicological issues.