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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 identified300 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.

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.

Monitoring of cyanobacterial bloom biomass in large lakes at high resolution is made possible by remote sensing. However, monitoring cyanobacterial toxins is only feasible with grab samples, which, with only sporadic sampling, results in uncertainties in the spatial distribution of toxins. To address this issue, we conducted two intensive “HABs Grabs” of microcystin (MC)-producing Microcystis blooms in the western basin of Lake Erie. These were one-day sampling events during August of 2018 and 2019 in which 100 and 172 grab samples were collected, respectively, within a six-hour window covering up to 2,270 km2 and analyzed using consistent methods to estimate the total mass of MC. The samples were analyzed for 57 parameters, including toxins, nutrients, chlorophyll, and genomics. There were an estimated 11,513 kg and 30,691 kg of MCs in the western basin during the 2018 and 2019 HABs Grabs, respectively. The bloom boundary poses substantial issues for spatial assessments because MC concentration varied by nearly two orders of magnitude over very short distances. The MC to chlorophyll ratio (MC:chl) varied by a factor up to 5.3 throughout the basin, which creates challenges for using MC:chl to predict MC concentrations. Many of the biomass metrics strongly correlated (r > 0.70) with each other except chlorophyll fluorescence and phycocyanin concentration. While MC and chlorophyll correlated well with total phosphorus and nitrogen concentrations, MC:chl correlated with dissolved inorganic nitrogen. More frequent MC data collection can overcome these issues, and models need to account for the MC:chl spatial heterogeneity when forecasting MCs.

Cyanobacterial harmful algal blooms (cyanoHABs) in freshwater lakes across the globe are often combined with other stressors. Pharmaceutical pollution, especially antibiotics in water bodies, poses a potential hazard in aquatic ecosystems. However, how antibiotics influence the risk of cyanoHABs remains unclear. Here, we investigated the effects of norfloxacin (NOR), one of the most widely used antibiotics globally, to a bloom-forming cyanobacterium (Microcystis aeruginosa) and a common green alga (Scenedesmus quadricauda), under both mono- and coculture conditions. Taxon-specific responses to NOR were evaluated in monoculture. In addition, the growth rate and change in ratio of cyanobacteria to green algae when cocultured with exposure to NOR were determined. In monocultures of Microcystis, exposure to low concentrations of NOR resulted in decreases in biomass, chlorophyll a and soluble protein content, while superoxide anion content and superoxide dismutase activity increased. However, NOR at high concentration only slightly affected Scenedesmus. During the co-culture trials of Microcystis and Scenedesmus, the 5 μg · L-1 NOR treatment increased the ratio of Microcystis to co-cultured Scenedesmus by 47.2%. Meanwhile, although Scenedesmus growth was enhanced by 4.2% under NOR treatment in monoculture, it was conversely inhibited by 63.4% and 38.2% when co-cultured with Microcystis with and without NOR, respectively. Our results indicate that antibiotic pollution has a potential risk to enhance the perniciousness of cyanoHABs by disturbing interspecific interaction between cyanobacteria and green algae. These results reinforce the need for scientists and managers to consider the influence of xenobiotics in shaping the outcome of interactions among multiple species in aquatic ecosystems.

Eichhornia crassipes is an invasive, floating macrophyte and may have biocontrol functions on harmful cyanobacteria blooms. Eichhornia produces N-phenyl-2-naphthylamine (PNA), a compound which can inhibit algae. However, the impact of PNA on allelopathic effects of the cyanobacterium Microcystis aeruginosa on other phytoplankton species is not clear. In this study, we tested whether PNA can influence the allelopathic effect of M. aeruginosa on the green alga Scenedesmus quadricauda. S. quadricauda was more sensitive to PNA than M. aeruginosa, with 72 h-EC50 values of 0.42 and 3.66 mg L−1, respectively. In addition, PNA significantly enhanced allelopathy by M. aeruginosa on S. quadricauda. Consequently, PNA had a greater impact on the green algae when Microcystis was present. In complex ecosystems with multiple species that release allelopathic substances, unintended interspecific interactions may occur, where allelopathy plays an important role under artificial or even natural contexts. It is hard to predict the competition outcomes among allelopathic organisms in these complex ecosystems, therefore a precautionary approach is required for biological control of algae.

The widespread use of silver nanoparticles (AgNPs) is becoming a growing concern because of the risk they pose to the ecological environment and human health. Unfortunately, little is known about how AgNPs' toxicity affects the mechanisms of metabolite shift in aquatic organisms. The aim of this work is thus to study systematically the cellular metabolism of the cyanobacteria Microcystis aeruginosa upon exposure to AgNPs by using metabolomics based on non-targeted liquid chromatography and mass spectrometry. The results reveal that a total of 97 biochemical metabolites and 16 specific metabolic pathways are significantly altered by exposure to AgNPs, many of which are associated with cellular stress response. Importantly, a metabolic analysis identified some novel metabolic pathways that are specifically regulated by AgNPs, including arginine and proline metabolism, indole alkaloid biosynthesis, and phospholipid metabolism (phosphatidylinositol-4,5-bisphosphate). Further examination revealed that exposure to AgNPs elevates superoxide dismutase activity and malondialdehyde generation, damages the thylakoids, and interrupts the endocytosis process in algae cells. Together, the manifestation of differential metabolic profiles combined with the perturbation of physiological indicators upon AgNP exposure is taken as evidence of damage to photosynthesis and membranes. These results provide holistic information about complex disturbances in metabolite profiles of M. aeruginosa upon exposure to AgNPs and provide new insights into the mechanisms of AgNPs' toxicity in the aquatic environment.

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 17β-hydroxysteroid-dehydrogenase (17β-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.

Allelopathic interactions among phytoplankton species are regarded as one of the important factors contributing to phytoplankton species competition and succession. The role and extent of allelopathic effects of blooming freshwater cyanobacteria on other phytoplankton species in eutrophied waters, however, are still unknown. We examined the allelopathic effect of Microcystis aeruginosa on two common green algae ( Scenedesmus quadricauda , Chlorella pyrenoidosa ) and a diatom ( Cyclotella meneghiniana ) by adding exudates from different growth phases and in co-culture tests. Exudates of M. aeruginosa from the exponential growth phase and the stationary phase significantly inhibited the growth of S. quadricauda , C. pyrenoidosa and C. meneghiniana , whereas those from the decline phase increased their growth. The presence of M. aeruginosa extremely inhibited the growth of all tested species in co-cultures within 24 h. Our results indicate that under the tested environmental conditions (25 °C, light 80 μmol quanta m −2 s −1 , manual shaking twice a day), allelopathic effects of M. aeruginosa on other phytoplankton species can significantly contribute to their competitive success.

Recovery of rare aquatic plant species in eutrophic lakes often fails, and mutual allelopathic effects between cyanobacteria and submerged macrophytes are one potential reason. In this study, we investigated allelopathic interactions between cyanobacteria and Ottelia acuminata, an endangered submerged macrophyte species in China. We tested effects of Microcystis aeruginosa on germination and seedling growth of two varieties of O. acuminata (O. acuminata var. crispa, OAC; and O. acuminata var. songmingensis, OAS) and vice versa effects of culture water of young seedlings and mature macrophytes on the growth of M. aeruginosa. We found that: (1) The culture water of mature O. acuminata (both OAC and OAS) allelopathically promoted M. aeruginosa growth, while seedlings had no effect. (2) M. aeruginosa exudates and extracts significantly promoted or inhibited seed germination of O. acuminata; (3) both exudates and extracts significantly decreased the seedling vitality of OAC and OAS. (4) M. aeruginosa exudates and extracts inhibited seedlings growth of O. acuminata, especially growth of roots and the second true leaf. (5) Allelopathic effects of M. aeruginosa exudates were stronger than those of extracts, and OAC showed stronger resistance to cyanobacteria than OAS. Our results indicate that cyanobacteria can allelopathically inhibit O. acuminata mainly by their exudates, while mature O. acuminata stimulate cyanobacteria growth. Such negative effects of cyanobacteria may contribute to the disappearance of O. acuminata and also slow down their recovery in eutrophic lakes.

Cyanobacterial blooms drive water-quality and aquatic-ecosystem deterioration in eutrophic lakes worldwide, mainly owing to their harmful, secondary metabolites. The response of fish exposed to these cyanobacterial chemicals, however, remains largely unknown. In this paper, we employed an endangered fish species (Sinocyclocheilus grahami) in Dianchi Lake, China to evaluate the risks of cell-free exudates (MaE) produced by a dominant cyanobacterium (Microcystis aeruginosa) on embryo development, as well as the molecular mechanisms responsible. MaE (3d cultured) caused a reduction of fertilization (35.4%) and hatching (15.5%) rates, and increased mortality rates (≤90.0%) and malformation rate (27.6%), typically accompanied by heart failure. Proteomics analysis revealed that two greatest changed proteins - protein S100A1 (over-expressed 26 times compared with control) and myosin light chain (under-expressed 25 fold) - are closely associated with heart function. Further study revealed that heart failure was due to calcium ion imbalance and malformed cardiac structure. We conclude that harmful secondary metabolites from cyanobacteria may adversely affect embryo development in this endangered fish, and possibly contribute to its disappearance and unsuccessful recovery in Dianchi Lake. Hazardous consequences of substances released by cyanobacteria should raise concerns for managers addressing recovery of this and other imperiled species in affected lakes.

Submerged macrophytes are known for their positive effects on water clarity, especially in shallow lake ecosystems. Eutrophication of these systems, however, has often resulted in a decline of submerged vegetation and a dominance of cyanobacteria, producing and releasing allelopathically active substances. Whether and how these allelochemicals affect submerged macrophytes is, however, largely unknown. We tested the effect of exudates and extracts of one of the most common cyanobacteria species, Microcystis aeruginosa, on seed germination and seedling growth and photosynthesis of the submerged macrophyte Potamogeton malaianus, a common species in Chinese shallow lakes. We found that: (1) low and high exudate concentrations significantly promoted or inhibited P. malaianus seed germination, respectively, while extracts had no effect; (2) low exudate and medium extract concentrations significantly increased, whereas high concentrations decreased the biomass of P. malaianus seedlings; (3) chlorophyll content and maximum quantum yield of PS II (Fv/Fm) of the seedlings were decreased by all levels of exudates; (4) of the various responses tested, seedling leaf Fv/Fm showed the greatest sensitivity to M. aeruginosa allelochemicals; (5) the allelopathic effects of M. aeruginosa exudates were stronger than those of extracts. Our results indicate that cyanobacteria exudates can allelopathically inhibit submerged macrophytes. The early growth of seedlings rather than seed germination seems to be a sensitive phase for the impact of cyanobacterial allelochemicals.

Harmful cyanobacterial blooms and their negative impacts on water ecosystems and human health have been reported all over the world, and Microcystis aeruginosa is one of the most dominant and ecologically damaging species in eutrophic lakes such as Dianchi Lake and Taihu Lake in China. Although physical or chemical control of the blooms is possible in short period, these methods could cause other problems. Thus, there is still a need for research on biological factors controlling cyanobacteria blooms. We screened part of the Yunnan University actinomycete strain library and found that the extract of Streptomyces hebeiensis YIM 001 T exhibited obvious inhibition on M. aeruginosa . The main component – nanaomycin A methyl ester (NAME) was isolated at a level of 20 mg L −1 by scale-up fermentation. Lytic mechanism experiments of NAME on M. aeruginosa demonstrated it could effectively inhibit the growth of M. aeruginosa (EC 50 at 72 h = 2.97 mg L −1 ), hinder cell division and enlarge cell size. NAME damaged M. aeruginosa cells as evidenced by decreases in algal activity, biomass, esterase activity and chlorophyll- a content. The SOD activity of the treated cells initially increased, and then decreased together with a gradual increase in both O 2 − and malondialdehyde (MDA) content, consistent with inhibitory effects of NAME due to lipid peroxidation damage of the algal cell membrane. The results suggest that NAME is effective for controlling undesired cyanobacteria and the Chl -fluorescence is a potential endpoint to assay the NAME eco-toxicity on cyanobacteria.

Plant-released allelopathic substances are increasingly suggested as potential natural measure to control cyanobacteria blooms in lakes. We tested whether the common cyanobacterium Microcystis aeruginosa , known to be sensitive to allelochemicals, remains suppressed when interacting with a less sensitive green alga. We investigated the effects of polyphenolic allelochemicals on single and mixed cultures of the common green alga Desmodesmus armatus and M. aeruginosa with three different inoculum compositions. Polyphenols were added as tannic acid or released by a macrophyte ( Myriophyllum verticillatum L.) in coexistence experiments with dialysis bags. Interaction with the green alga turned the inhibiting effect of allelochemicals on the cyanobacterium into an enhancement resulting in increased growth rates. The ratio between M. aeruginosa and the green alga consequently became significantly higher in treatments with allelochemicals as compared to controls after three days. When initial growth rates of M. aeruginosa and the green alga were similar, this resulted in an increasing abundance of the cyanobacterium under allelochemical presence. Interactions between phytoplankton species thus need to be taken into account when investigating the ecological relevance of macrophyte allelopathy and its potential to suppress cyanobacteria blooms.