Assessment of estrogenic potential from exudates of microcystin-producing and non-microcystin-producing Microcystis by metabolomics, machine learning and E-screen assay.

Cyanobacterial blooms, often dominated by Microcystis aeruginosa, are capable of producing estrogenic effects. It is important to identify specific estrogenic compounds produced by cyanobacteria, though this can prove challenging owing to the complexity of exudate mixtures. In this study, we used untargeted metabolomics to compare components of exudates from microcystin-producing and non-microcystin-producing M. aeruginosa strains that differed with respect to their ability to produce microcystins, and across two growth phases. We identified 416 chemicals and found that the two strains produced similar components, mainly organoheterocyclic compounds (20.2%), organic acids and derivatives (17.3%), phenylpropanoids and polyketides (12.7%), benzenoids (12.0%), lipids and lipid-like molecules (11.5%), and organic oxygen compounds (10.1%). We then predicted estrogenic compounds from this group using random forest machine learning. Six compounds (daidzin, biochanin A, phenylethylamine, rhein, o-Cresol, and arbutin) belonging to phenylpropanoids and polyketides (3), benzenoids (2), and organic oxygen compound (1) were tested and exhibited estrogenic potency based upon the E-screen assay. This study confirmed that both Microcystis strains produce exudates that contain compounds with estrogenic properties, a growing concern in cyanobacteria management.

Identification of neurotoxic compounds in cyanobacteria exudate mixtures.

Release of toxic cyanobacterial secondary metabolites threatens biosecurity, foodwebs and public health. Microcystis aeruginosa (Ma), the dominant species in global freshwater cyanobacterial blooms, produces exudates (MaE) that cause adverse outcomes including nerve damage. Previously, we identified > 300 chemicals in MaE. It is critical to investigate neurotoxicity mechanisms of active substances among this suite of Ma compounds. Here, we screened 103 neurotoxicity assays from the ToxCast database to reveal targets of action of MaE using machine learning. We then built a potential Adverse Outcome Pathway (AOP) to identify neurotoxicity mechanisms of MaE as well as key targets. Finally, we selected potential neurotoxins matched with those targets using molecular docking. We found 38 targets that were inhibited and eight targets that were activated, collectively mainly related to neurotransmission (i.e. cholinergic, dopaminergic and serotonergic neurotransmitter systems). The potential AOP of MaE neurotoxicity could be caused by blocking calcium voltage-gated channel (CACNA1A), because of antagonizing neurotransmitter receptors, or because of inhibiting solute carrier transporters. We identified nine neurotoxic MaE compounds with high affinity to those targets, including LysoPC(16:0), 2-acetyl-1-alkyl-sn-glycero-3-phosphocholine, egonol glucoside, polyoxyethylene (600) monoricinoleate, and phytosphingosine. Our study enhances understanding of neurotoxicity mechanisms and identifies neurotoxins in cyanobacterial bloom exudates, which may help identify priority compounds for cyanobacteria management.

Corrigendum: Comparative metabolomic analysis of exudates of high-microcystin-producing and low-microcystin-producing Microcystis aeruginosa strains.

[This corrects the article DOI: 10.3389/fmicb.2022.1075621.].

Abnormal neurobehavior in fish early life stages after exposure to cyanobacterial exudates.

Cyanobacterial harmful algal blooms (cHABs) pose a risk to exposed aquatic and terrestrial species. Numerous studies have addressed effects of single toxins while much less attention has been devoted to mixtures of cHAB metabolites that are continually released by living cyanobacteria. Neuro-impairment associated with cHABs has been reported in fish, though the mechanism remains unclear. Here we exposed embryos of Sinocyclocheilus grahami, an endangered fish, to Microcystis aeruginosa exudates (MaE) to evaluate neurotoxicity and the toxicity mechanism(s). We found that MaE affected embryonic development by increasing malformation and mortality rates and decreasing the fertilization rate. MaE also inhibited fish neurobehavior including touch response, social frequency, swimming distance, and aggravated light-stimulation response. Neurobehavior suppression resulted from a decrease in excitatory neurotransmitters acetylcholine and dopamine, even though receptors increased. MaE also affected gene and protein expression of neurotransmitters, synthetic and/or degrading enzymes, and receptors. Our findings shed light on specific mechanisms by which MaE induces neurotoxicity in early life stages in fish and contributes to improvement of the conservation strategy for this species.

Comparative metabolomic analysis of exudates of microcystin-producing and microcystin-free Microcystis aeruginosa strains.

Cyanobacterial harmful algal blooms (cHABs) dominated by Microcystis aeruginosa threaten the ecological integrity and beneficial uses of lakes globally. In addition to producing hepatotoxic microcystins (MC), M. aeruginosa exudates (MaE) contain various compounds with demonstrated toxicity to aquatic biota. Previously, we found that the ecotoxicity of MaE differed between MC-producing and MC-free strains at exponential (E-phase) and stationary (S-phase) growth phases. However, the components in these exudates and their specific harmful effects were unclear. In this study, we performed untargeted metabolomics based on liquid chromatography-mass spectrometry to reveal the constituents in MaE of a MC-producing and a MC-free strain at both E-phase and S-phase. A total of 409 metabolites were identified and quantified based on their relative abundance. These compounds included lipids, organoheterocyclic compounds, organic acid, benzenoids and organic oxygen compounds. Multivariate analysis revealed that strains and growth phases significantly influenced the metabolite profile. The MC-producing strain had greater total metabolites abundance than the MC-free strain at S-phase, whereas the MC-free strain released higher concentrations of benzenoids, lipids, organic oxygen, organic nitrogen and organoheterocyclic compounds than the MC-producing strain at E-phase. Total metabolites had higher abundance in S-phase than in E- phase in both strains. Analysis of differential metabolites (DMs) and pathways suggest that lipids metabolism and biosynthesis of secondary metabolites were more tightly coupled to growth phases than to strains. Abundance of some toxic lipids and benzenoids DMs were significantly higher in the MC-free strain than the MC-producing one. This study builds on the understanding of MaE chemicals and their biotoxicity, and adds to evidence that non-MC-producing strains of cyanobacteria may also pose a threat to ecosystem health.

Blooming cyanobacteria alter water flea reproduction via exudates of estrogen analogues.

Cyanobacteria blooms are increasing globally, with further increases predicted in association with climate change. Recently, some cyanobacteria species have been identified as a source of estrogenic effects in aquatic animals. To explore possible estrogenic effects of Microcystis aeruginosa (an often-dominant cyanobacteria species) on zooplankton, we examined effects of cyanobacteria exudates (MaE, 2 × 104 and 4 × 105 cells/ml) on reproduction in Daphnia magna. We analyzed physiological, biochemical and molecular characteristics of exposed Daphnia via both chronic and acute exposures. MaE at both low and high cell density enhanced egg number (15.4% and 23.3%, respectively) and reproduction (37.7% and 52.4%, respectively) in D. magna similar to 10 µg/L estradiol exposure. In addition, both MaE of low and high cell densities increased population growth rate (15.8% and 19.6%, respectively) and reproductive potential (60% and 83%, respectively) of D. magna. These exudates promoted D. magna reproduction by stimulating 17ß-hydroxysteroid-dehydrogenase (17ß-HSD) activity and production of ecdysone and juvenile hormone, and by enhancing vitellogenin biosynthesis via up-regulating expression of Vtg1 and Vtg2. However, increased expression (6.6 times higher than controls) of a detoxification gene (CYP360A8) indicated that MaE might also induce toxicity in D. magna. Reproductive interference of zooplankton by blooming cyanobacteria might negatively affect foodwebs because MaE-induced zooplankton population increase would enhance grazing and reduce abundance of edible algae, thereby adding to the list of known disruptive properties of cyanobacterial blooms.