Toxigenic Microcystis Research Articles

Seasonal dynamics of toxigenic Microcystis in a large, shallow Lake Peipsi (Estonia) using microcystin mcyE gene abundance.

Large and temperate Lake Peipsi is the fourth largest lake in Europe, where the massive cyanobacterial blooms are composed mostly of Microcystis spp., which have been common for several decades now. The seasonal dynamics of potentially toxic Microcystis were studied using microscopy and quantitative polymerase chain reaction (qPCR) by assessing the microcystin-encoding microcystin synthetase gene E (mcyE) abundances. Water samples were analyzed over the lake areas, varying in depth, trophic level, and cyanobacterial composition during the growing period of 2021. The Microcystis mcyE genes were detected through the growing period (May-October), forming peak abundances in September with decreasing temperatures (8.9-11.1°C). Total phosphorus (TP) and nitrate (NO3-) were the most relevant environmental variables influencing the Microcystis biomass as well as mcyE abundances. Comparison with previous years (2011, 2012) indicated that the abundance and seasonal dynamics of toxigenic Microcystis can be highly variable between the years and lake areas, varying also in dominant Microcystis species. Contrary to expectations, based on mcyE abundances, the increased risk of toxin-producing Microcystis can occur in Peipsi through the growing period, independently of the water temperature and biomasses of Microcystis.

Duplex Real-Time Fluorescent Quantitative PCR Assays for the Detection of Toxigenic Microcystis Genotypes Based on SNP/InDel Variation in mcy Gene Cluster.

In this study, the toxigenic characteristics of 14 strains of Microcystis were analyzed, and single nucleotide polymorphism (SNP) and insertion/deletion (InDel) loci in microcystinsynthetase (mcy)geneclusters were screened. Based on SNP and InDel loci associated with the toxigenic characteristics, primers andTaqMan or Cycling fluorescent probes were designed to develop duplex real-time fluorescent quantitative PCR (FQ-PCR) assays. After evaluating specificity and sensitivity, these assays were applied to detect the toxigenic Microcystis genotypes in a shrimp pond where Microcystis blooms occurred. The results showed a total of 2155 SNP loci and 66 InDel loci were obtained, of which 12 SNP loci and 5 InDel loci were associated with the toxigenic characteristics. Three duplex real-time FQ-PCR assays were developed, each of which could quantify two genotypes of toxigenic Microcystis. These FQ-PCR assays were highly specific, and two Cycling assays were more sensitive than TaqMan assay. In the shrimp pond, six genotypes of toxigenic Microcystis were detected using the developed FQ-PCR assays, indicating that above genotyping assays have the potential for quantitative analysis of the toxigenic Microcystis genotypes in natural water.

Phenotypic and Genotypic Methods for the Identification and Quantification of Cyanobacteria in Lake Water.

Early monitoring of Microcystis, a cyanobacterium that produces microcystin, is paramount in order to confirm the presence of Microcystis spp. Both phenotypic and genotypic methods have been used. The phenotypic methods provide the presence of the microcystis but do not confirm its species type and toxin produced. Additionally, phenotypic methods cannot differentiate toxigenic from non-toxigenic Microcystis. Therefore, the current protocol also describes genetic methods based on PCR to detect toxigenic Microcystis spp. based on microcystin synthetase E (mcy E) gene and 16-23S RNA genes for species-specific identification, which can effectively comprehend distinct lineages and discrimination of potential complexity of microcystin populations. The presence of these microcystin toxins in blood, in most cases, indicates contamination of drinking water by cyanobacteria. The methods presented herein are used to identify microcystin toxins in drinking water and blood.

Microbiome analysis reveals Microcystis blooms endogenously seeded from benthos within wastewater maturation ponds.

Toxigenic Microcystis blooms periodically disrupt the stabilization ponds of wastewater treatment plants (WWTPs). Dense proliferations of Microcystis cells within the surface waters (SWs) impede the water treatment process by reducing the treatment efficacy of the latent WWTP microbiome. Further, water quality is reduced when conventional treatment leads to Microcystis cell lysis and the release of intracellular microcystins into the water column. Recurrent seasonal Microcystis blooms cause significant financial burdens for the water industry and predicting their source is vital for bloom management strategies. We investigated the source of recurrent toxigenic Microcystis blooms at Australia's largest lagoon-based municipal WWTP in both sediment core (SC) and SW samples between 2018 and 2020. Bacterial community composition of the SC and SW samples according to 16S rRNA gene amplicon sequencing showed that Microcystis sp. was dominant within SW samples throughout the period and reached peak relative abundances (32%) during the summer. The same Microcystis Amplicon sequence variants were present within the SC and SW samples indicating a potential migratory population that transitions between the sediment water and SWs during bloom formation events. To investigate the potential of the sediment to act as a repository of viable Microcystis cells for recurrent bloom formation, a novel in-vitro bloom model was established featuring sediments and sterilized SW collected from the WWTP. Microcystin-producing Microcystis blooms were established through passive resuspension after 12 weeks of incubation. These results demonstrate the capacity of Microcystis to transition between the sediments and SWs in WWTPs, acting as a perennial inoculum for recurrent blooms.IMPORTANCECyanobacterial blooms are prevalent to wastewater treatment facilities owing to the stable, eutrophic conditions. Cyanobacterial proliferations can disrupt operational procedures through the blocking of filtration apparatus or altering the wastewater treatment plant (WWTP) microbiome, reducing treatment efficiency. Conventional wastewater treatment often results in the lysis of cyanobacterial cells and the release of intracellular toxins which pose a health risk to end users. This research identifies a potential seeding source of recurrent toxigenic cyanobacterial blooms within wastewater treatment facilities. Our results demonstrate the capacity of Microcystis to transition between the sediments and surface waters (SWs) of wastewater treatment ponds enabling water utilities to develop adequate monitoring and management strategies. Further, we developed a novel model to demonstrate benthic recruitment of toxigenic Microcystis under laboratory conditions facilitating future research into the genetic mechanisms behind bloom development.

Microcystis genotypes in a tropical freshwater lake: Discovery of novel MIB-producing Microcystis with potentially unique synthesis pathway

Harmful algal blooms (HABs) are a threat to freshwater systems over the world due to the production of hepatotoxins like microcystin (MC), and nuisance taste and odour (T&O) compounds like 2-methylisoborneol (MIB). While MCs are known to cause detrimental effects to both water quality and human health, MIB is only reported to cause aesthetical problems. In this study, we investigated a tropical, urban lake that was experiencing persistent MC and MIB events. Although it was dominated by Microcystis blooms, analysis revealed that the toxigenic Microcystis were not the only species driving the MC concentrations. Additionally, there was also a lack of causative species for the MIB events. Through isolation, we have identified three toxigenic Microcystis found to produce four different variants of MCs, and two novel non-toxigenic Microcystis that were capable of producing MIB. The ability to produce MIB had never been previously reported for this species. Compared to other major producers such as Planktothricoides sp. and Streptomyces sp., the MIB synthase genes of our Microcystis sp. strains were partial, illustrating the possibility of unique synthesis pathways. The Microcystis sp. strains were found to produce about 2.77–5.22 fg MIB cell−1, with a majority of the contents (70–80 %) existing in the extracellular phase. Correlation analysis of field study indicated that phosphorus limitation may have an indirect effect on non-toxigenic Microcystis abundance and proportion by influencing the toxigenic genotype, suggesting that current measures to control HABs may favour the proliferation of the non-toxigenic Microcystis. The potential for Microcystis sp. to produce MIB through unique synthesis pathway, coupled with the potential dominance of non-toxigenic genotypes in Microcystis blooms, signals the possibility that non-toxigenic Microcystis should be monitored as well.

Changes in microcystin concentration in Lake Taihu, 13 years (2007–2020) after the 2007 drinking water crisis

Since the 2007 water crisis occurred in Lake Taihu, substantial measures have been taken to restore the lake. This study evaluates the effectiveness of these restoration activities. We examined the physicochemical parameters and the distribution of microcystin and Microcystis in both the water column and sediment during the bloom period of May 2020 to October 2020. The mean value of extracellular and intracellular microcystin content was 0.12 μg L−1 and 16.26 μg L−1, respectively. The mean value of microcystin in sediment was 172.02 ng g−1 and peaked in August. The concentration in the water and sediment was significantly lower than the historical average concentration. The abundance of toxigenic Microcystis and total Microcystis in the water column ranged from 2.61 × 102 to 2.25 × 109 copies·L−1 and 8.28 × 105 to 2.76 × 109 copies·L−1, respectively. The proportion of toxic Microcystis in the sediment ranging from 31.2% to 19.12%. The highest and lowest region was Meiliang Bay and Grass-algae type zone, respectively. The copy number of the 16S rRNA gene was 1–4 orders of magnitude higher than that of mcyA gene in populations of Microcystis, indicating that non-toxic Microcystis was the dominant form in the majority of the lake. The abundance of toxic Microcystis in the water column was positively correlated with total phosphorus, PO43--P and pH, while the water temperature played distinct role to the distribution of toxic Microcystis in sediment. Our research indicated phosphorus remains a key factor influencing the toxic Microcystis and microcystins in the water column. pH played distinct roles in the distribution of microcystins in sediment and water column. The increasing water temperature is a threat. Explicit management actions and policies, which take into account nutrient concentrations, pH, and increasing temperatures, are necessary to understand and control the distribution of microcystin and Microcystis in Lake Taihu.

Year-Round Presence of Microcystins and Toxin-Producing Microcystis in the Water Column and Ice Cover of a Eutrophic Lake Located in the Continuous Permafrost Zone (Yakutia, Russia).

This study aimed to test the hypothesis of the year-round presence of toxigenic Microcystis and cyanotoxins in the water and ice of the shallow eutrophic Lake Ytyk-Kyuyol located in the continuous permafrost zone. Three independent approaches-mass-spectrometry, molecular methods and light microscopy-were applied in the study. The cyanobacterial biomass ranged from 1.0 × 10-4 to 4.8 mg L-1. Microcystis flos-aquae and M. aeruginosa were the dominant morphospecies in plankton throughout the observation. In environmental DNA, the presence of M. aeruginosa was supported and mcy gene regions responsible for microcystin biosynthesis were detected through a BLAST (Basic Local Alignment Search Tool) search and phylogenetic estimation based on newly obtained 16S rRNA, 16S-23S ITS rRNA, mcyA and mcyE nucleotide sequences. The intracellular microcystin concentration ranged from <0.1 to 803 ng L-1, and the microcystin quota in the Microcystis biomass was extremely low. For the first time, it was shown that Microcystis cells containing mcy genes and microcystins presented permanently in the water column, both during the ice-free period and under ice, as well as inside thick ice covers within 7 months of severe winter. We hypothesized that minor pelagic and ice populations of Microcystis could participate in increasing cell density in the spring. However, further studies are needed to confirm the viability of the overwintering Microcystis colonies in the water and inside the ice of Lake Ytyk-Kyuyol.

Phosphorus level impacts luteolin effect on Microcystis aeruginosa growth and microcystin-pollution risk − Novel perspective from correlation between exopolymers substances fractions and microcystin-production/release

Luteolin as a phytogenic algicide can inhibit the growth and microcystins (MCs) release of Microcystis, a dominant genus during cyanobacterial blooms, but how phosphorus (P) level impacts luteolin effect on its growth and MC-pollution risk is unclear. By employing Microcystis aeruginosa as test alga, this study addressed this concern and explored response mechanisms from novel insights of relationship between extracellular polysaccharide (ex-poly) and protein (ex-pro) contents and MC-production/release. At each P level (0.05–5 mg/L), rising luteolin dose more greatly inhibited Microcystis growth and MC-pollution risk, with growth inhibition ratio of around 10%–30%, 20%–50% and 40%–90% for 3, 6 and 12 mg/L luteolin, respectively, but almost increasingly enhanced cellular ability of MC-production/conservation and total and bound ex-poly/ex-pro production. Rising P level promoted Microcystis growth and intracellular/extracellular MCs content (IMC, EMC) in test system at each luteolin dose, thus higher P level weakened algicidal and MC-removal effects of luteolin, indicating that P-decrease was required for stronger application outcome of luteolin. Total and bound ex-poly/ex-pro amount were positively correlated with cellular MC-production/conservation ability, IMC and EMC, which constituted cooperative stress-defense of Microcystis at each P level. Besides, rising luteolin dose posed stronger algicidal effect by inactivating gene expression involving peroxidase synthesis (especially at P-limitation), photosynthesis and P acquisition, while rising P level alleviated algicidal and MC-pollution inhibition effects of luteolin by enhancing gene expression involving N acquisition and peroxidase synthesis. This study shed novel insights for P-dependent effect and mechanisms of luteolin on toxigenic Microcystis growth and MC-pollution control, which guided to mitigating toxigenic Microcystis-dominated cyanobacterial blooms in different P-level water areas.

Toxicity of the disinfectant benzalkonium chloride (C14) towards cyanobacterium Microcystis results from its impact on the photosynthetic apparatus and cell metabolism

Quaternary ammonium compounds (QACs) are commonly used in a variety of consumer and commercial products, typically as a component of disinfectants. During the COVID-19 pandemic, QACs became one of the primary agents utilized to inactivate the SARS-CoV-2 virus on surfaces. However, the ecotoxicological effects of QACs upon aquatic organisms have not been fully assessed. In this study, we examined the effects of a widely used QAC (benzalkonium chloride-C14, BAC-14) on two toxigenic Microcystis strains and one non-toxigenic freshwater Microcystis strain and carried out an analysis focused on primary, adaptive and compensatory stress responses at apical (growth and photosynthesis) and metabolic levels. This analysis revealed that the two toxic Microcystis strains were more tolerant than the non-toxic strain, with 96 hr-EC50 values of 0.70, 0.76, and 0.38 mg/L BAC-14 for toxigenic M. aeruginosa FACHB-905, toxigenic M. aeruginosa FACHB-469, and non-toxigenic M. wesenbergii FACHB-908, respectively. The photosynthetic activities of the Microcystis, assessed via Fv/Fm values, were significantly suppressed under 0.4 mg/L BAC-14. Furthermore, this analysis revealed that BAC-14 altered 14, 12, and 8 metabolic pathways in M. aeruginosa FACHB-905, M. aeruginosa FACHB-469, and M. wesenbergii FACHB-908, respectively. It is noteworthy that BAC-14 enhanced the level of extracellular microcystin production in the toxigenic Microcystis strains, although cell growth was not significantly affected. Collectively, these data show that BAC-14 disrupted the physiological and metabolic status of Microcystis cells and stimulated the production and release of microcystin, which could result in damage to aquatic systems.

Temporal and functional interrelationships between bacterioplankton communities and the development of a toxigenic Microcystis bloom in a lowland European reservoir

The cyanobacteria-associated microbiome is constantly reshaped by bloom development. However, the synergistic-antagonistic nature of the relationships between Microcystis and its microbiome still remains unclear. Therefore, temporal changes of bacterioplankton communities and their functional potential through different developing stages of a Microcystis toxigenic bloom were investigated, considering bacterioplankton assemblages as particle-attached (PAB) and free-living (FLB) bacteria. 16S rRNA sequencing revealed that PAB were represented by Proteobacteria and Cyanobacteria, while FLB by Proteobacteria and Actinobacteria. Network and ordination analyses indicated that PAB inter-relationships were more complex—numerous connections between taxa with stronger correlations, than FLB—rather influenced by physico-chemical parameters. PAB in pre-summer was diverse with Proteobacteria containing potential taxa involved in nitrogen-transforming processes. In mid-summer, PAB presented a mix-bloom dominated by Snowella, Aphanizomenon, and Microcystis, which were succeeded by toxigenic Microcystis in post-summer. Both periods were associated to potential taxa with parasitic/predatory lifestyles against cyanobacteria. In post-summer, Sutterellaceae were recognized as poor water quality indicators, and their strong association with Microcystis could have represented an increased threat for that period. Microcystis was a major factor significantly reducing PAB diversity and evenness, suggesting that it negatively influenced bacterioplankton assemblages, probably also altering the overall community functional potential.

Combined effects of increased water temperature and cyanobacterial compounds exert heterogeneous effects on survival and ecological processes in key freshwater species

Climate change is increasing water temperature and intensifying the incidence of cyanobacterial blooms worldwide. However, the combined effects of increased temperature and microcystin concentrations as co-stressors on survival and ecological processes in freshwater species are unclear. Here, using purified MC-LR and crude extract of toxigenic Microcystis aeruginosa, we tested the individual and combined effects of three water temperatures (15, 20, 25 °C) and a range of environmentally relevant concentrations of dissolved microcystin and crude extract (0.01–10 µg·L−1) on survival, growth inhibition, grazing and predation rates in three freshwater species: phytoplankton (Scenedesmus quadricauda), zooplankton (Daphnia pulex), and an invertebrate predator (Ischnura elegans). Purified MC-LR exerted a higher growth inhibitory effect on S. quadricauda compared to crude extract with the same concentration of MC-LR, while neither treatment affected its chlorophyll-a content or survival of D. pulex. Crude extract reduced grazing and survival of D. pulex and I. elegans, respectively. The combined effect of higher temperature and crude extract reduced I. elegans survival by 50%. Increased temperature reduced prey handing time in I. elegans by 49%, suggesting a higher predation rate. However, warming together with higher concentrations of crude extract jointly increased zooplankton grazing and reduced damselfly predation. Taken together, these results suggest crude extract, and not necessarily microcystin, can affect survival and productivity in freshwater species, although these effects may vary unevenly across trophic levels. Our findings highlight the importance of complex ecological mechanisms by which warming can exacerbate toxic effects of cyanobacterial bloom extracts on survival and functions among species in eutrophic freshwaters.

Characterization of Potential Threats from Cyanobacterial Toxins in Lake Victoria Embayments and during Water Treatment.

Africa’s water needs are often supported by eutrophic water bodies dominated by cyanobacteria posing health threats to riparian populations from cyanotoxins, and Lake Victoria is no exception. In two embayments of the lake (Murchison Bay and Napoleon Gulf), cyanobacterial surveys were conducted to characterize the dynamics of cyanotoxins in lake water and water treatment plants. Forty-six cyanobacterial taxa were recorded, and out of these, fourteen were considered potentially toxigenic (i.e., from the genera Dolichospermum, Microcystis, Oscillatoria, Pseudanabaena and Raphidiopsis). A higher concentration (ranging from 5 to 10 µg MC-LR equiv. L−1) of microcystins (MC) was detected in Murchison Bay compared to Napoleon Gulf, with a declining gradient from the inshore (max. 15 µg MC-LR equiv. L−1) to the open lake. In Murchison Bay, an increase in Microcystis sp. biovolume and MC was observed over the last two decades. Despite high cell densities of toxigenic Microcystis and high MC concentrations, the water treatment plant in Murchison Bay efficiently removed the cyanobacterial biomass, intracellular and dissolved MC to below the lifetime guideline value for exposure via drinking water (<1.0 µg MC-LR equiv. L−1). Thus, the potential health threats stem from the consumption of untreated water and recreational activities along the shores of the lake embayments. MC concentrations were predicted from Microcystis cell numbers regulated by environmental factors, such as solar radiation, wind speed in the N–S direction and turbidity. Thus, an early warning through microscopical counting of Microcystis cell numbers is proposed to better manage health risks from toxigenic cyanobacteria in Lake Victoria.

Nitrogen-dependent luteolin effect on Microcystis growth and microcystin-pollution risk — Novel mechanism insights unveiled by comparative proteomics and gene expression

Phytogenic allelochemical luteolin has potential to mitigate Microcystis-dominated cyanobacterial blooms (MCBs), but its algicidal effect against toxigenic Microcystis may be impacted by natural factors, especially nitrogen (N) level in waters. This study innovatively explored N-dependent effect of luteolin on Microcystis growth and its microcystins (MCs) production/release, and elucidated underlying mechanisms from proteomics and gene expression views. Generally, at each N level, rising luteolin dose progressively inhibited Microcystis growth by inhibiting proteins syntheses and genes expression involving light-capturing, photosynthetic electron transfer, Calvin cycle and phosphorus (P) acquisition, according to comparative proteomics and gene expression. At higher luteolin dose and lower N level, Microcystis cell tended to increase microcystins (MCs) production and conservation ability, with the highest increase degree observed at 12 mg/L luteolin and 0.5 mg/L N on day 10, reaching 1.96 and 2.68 folds of luteolin-free control, respectively, but decrease MC-release as extracellular MCs content (EMC), with inhibition ratio of 72.86%, 73.57%, 74.45% and 40.58%, 45.28%, 60.00% at rising N level under 12 mg/L luteolin stress on day 10 and 16, respectively. These enabled cellular defensive response of Microcystis to stronger stress and N limitation. Under luteolin stress, higher N level more strongly up-regulated numerous processes (e.g., oxidoreductase activity, ATP binding and transmembrane transport, oxidative phosphorylation, tricarboxylic acid cycle, fatty acid biosynthesis, glycolysis/gluconeogenesis, pyruvate, amino acids metabolism, metal ion-binding, P acquisition) as compensative protective responses to progressively down-regulated photosynthetic and ribosomal processes at higher N level, thus causing faster Microcystis growth than lower N level. This study provided novel insights for N-dependent effect and mechanisms of luteolin on MCBs mitigation and MCs risk control, and guided algicidal application of luteolin in different eutrophic-degree waters.

Joint effects and mechanisms of luteolin and kaempferol on toxigenic Microcystis growth—Comprehensive analysis on two isomers interaction in binary mixture

Allelochemicals are widely accepted as promising algaecide to mitigate Microcystis-dominated cyanobacterial blooms (MCBs). Allelopathic algicidal effect of single luteolin or kaempferol against Microcystis had been confirmed, but their joint effect against Microcystis was unclear. This study comprehensively explored time-dependent joint effect and mechanisms of luteolin and kaempferol on Microcystis growth during 14 day-test. The 50%-inhibitory threshold of their mixture (IC50 mix) was verified as 4.872 and 5.211 mg/L at equitoxic ratio, and 5.167 and 4.487 mg/L at equivalent ratio, respectively, on day 8 and 14. Using toxicity unit, isobologram and predictive models, results revealed that luteolin and kaempferol at equivalent ratio interacted additively at lower, median and higher dosages, while at equitoxic ratio interacted additively at lower dosage but synergistically at median and higher dosages in Microcystis on day 8 and 14, implying that their equitoxic mixture posed better algicidal effect against Microcystis. Various dosages of equitoxic mixture concurrently decreased aqueous and total microcystins (MCs) contents along test. Thus, luteolin and kaempferol could be jointly applied as high-efficacy and eco-safe algaecide with declined MCs pollution risks. As mixture dosage elevated, more strongly weakened cellular MCs retention and inhibited cellular photosynthetic pigments content during late stage, as well as decreased aqueous MCs content long test, jointly explained increasing growth inhibition ratio with rising mixture dosage. Yet, cell damage was gradually repaired due to early stimulated antioxidant defense at each mixture dosage, thus cell damage might not be a major reason for inhibited growth under mixture stress. This study provided novel insights and guidance to coupled application of luteolin and kamepferol for mitigating MCBs and decreasing MCs pollution risks.

Synergistic toxicity to the toxigenic Microcystis and enhanced microcystin release exposed to polycyclic aromatic hydrocarbon mixtures

With the continued influx and accumulation of polycyclic aromatic hydrocarbons (PAHs) in eutrophic waters, the effects of PAHs on cyanobacteria bloom need to be clarified. PAHs usually existed as mixtures in aquatic environments, but the combined toxicity of PAH mixtures to toxigenic cyanobacteria remained unknown. This study investigated the effects of phenanthrene (Phe) and benzo [a]pyrene (BaP), alone or in combination, on the growth and physiology of Microcystis aeruginosa. The results showed that a hormesis effect on growth at low doses of the single Phe (≤1 mg/L) or PAH mixtures (≤0.279 mg/L) was observed, whereas the single BaP induced significant growth inhibitions at all concentrations (≥0.2 mg/L). The median effective concentrations (96 h) for Phe, BaP and their mixtures were 4.29, 1.29 and 1.07 mg/L, respectively. Mixture toxicity models showed that Phe and BaP elicited a synergistic interaction on M. aeruginosa. The synergy may be ascribed to the excessive oxidative stress induced by PAH mixtures, which further led to membrane structure damages, photosynthesis inhibitions and decreased metabolic activity. Moreover, the microcystins (MCs) release significantly increased by 25.3% and 31.9% upon exposure to 0.558 and 1.116 mg/L of PAH mixtures. In all, this study suggested that the enhanced release of MCs by PAH mixtures might exacerbate potential risks to the aquatic environment.

Comparative growth and cellular responses of toxigenic Microcystis exposed to different types of microplastics at various doses

Microplastics (MPs) pollution frequently co-occur with Microcystis-dominated blooms in freshwaters, but MPs effects on toxigenic Microcystis growth and effect mechanisms remained poorly understood. This study used 0.5 μm-size polyethylene (PE) and polyvinyl chloride (PVC) to explore dose- and time-dependent effects of single and combined MPs (i.e., PE + PVC) on toxigenic Microcystis growth and cellular responses during 16 day-test. Results showed that Microcystis growth and cellular responses depended on exposure time, MPs dose and type. MPs elicited hormesis effect in early stage at low dose (5 mg/L), while increasingly inhibited growth with rising PVC or PE + PVC dose but declining PE dose (5, 10, 50 mg/L) in mid-late stage, with stress intensity of PE + PVC > PVC > PE. Further analyses revealed unobvious cell damage under MPs stress, largely because antioxidases were increasingly activated as MPs stress enhanced. Unicellular MCs release ability during mid stage almost coincided with total/bound amount and each fraction of ex-poly and ex-pro trends under MPs stress. Significant positive relationship existed between MCs release ability and ex-poly/ex-pro fractions and total amount of Microcystis cells along mid-late stage under MPs stress, validating that ex-poly/ex-pro production was regulated as a result of MCs release. Besides, unicellular MCs production ability was generally positively correlated with soluble, tightly-bound and total ex-poly and ex-pro at late stage. These suggested that cellular antioxidants, MCs production/release ability and ex-poly/ex-pro production of Microcystis could be coupled to exert integrated defense against MPs stress to protect surviving cells in Microcystis population. These findings are crucial for acquiring the fate of Microcystis-dominated blooms co-occurring with MPs pollution, and reasonably assessing and managing involved eco-risks.

Time- and dose-dependent allelopathic effects and mechanisms of kaempferol on toxigenic Microcystis growth

This study determined time-dependent IC50 and confirmed 3.5 mg/L as IC50 value for kaempferol inhibiting toxigenic Microcystis growth, based on which algicidal effects and mechanisms against toxigenic Microcystis exposed to various kaempferol doses (0.5–2 × IC50) were explored along 14 day-test. Results showed that growth inhibition ratio (GIR) almost elevated with increasing kaempferol dose, and at each dose GIR elevated firstly and fluctuated around 17.8%– > 40%, 53.6%–65.6% and 84.8%–89.3% at 1.75, 3.5 and 7 mg/L kaempferol during mid-late stage, respectively. With rising kaempferol dose, photosynthetic pigments contents (chlorophyll-a, phycobiliproteins), antioxidant response (superoxide dismutase and catalase (CAT) activities, glutathione (GSH) contents) and microcystins (MCs) production were almost increasingly stimulated as cellular protective responses during early-mid stage. However, these parameters (excluding CAT and GSH) were almost increasingly inhibited at late stage by prolonged stress and Microcystis cell was still more severely damaged as dose elevated along test, which could be reasons for increasing GIR with rising kamepferol dose. Persistent stimulation of CAT and GSH at each dose could alleviate cell damage until late stage, thus GIR no longer increased at late stage at each kaempferol dose. Moreover, fewer MCs release under kaempferol stress than control suggested kaempferol as eco-safe algaecide for migrating toxigenic Microcystis-dominated blooms (MCBs) and decreasing MCs risks. Compared with our previous data for luteolin inhibiting toxigenic Microcystis, this study supported formerly-proposed 'flavonoids structure - algicidal activity' relationship that the only OH-location difference between kaempferol and luteolin could affect algicidal activity and mechanisms against toxigenic Microcystis. Also, kaempferol and luteolin was revealed to exert additive effect on toxigenic Microcystis growth at equitoxic ratio. Our findings gave novel algicidal scenario of flavonoids and were greatly implicated in eco-friendly migrating toxigenic MCBs.

Growth and Cellular Responses of Toxigenic Microcystis to Chloramphenicol-Stress at Various Environmentally-Relevant Nitrogen Levels.

This study explored nitrogen (N)-dependent interaction between Microcystis and chloramphenicol (CAP) along 20day-test. Results showed that 5mg/L N largely alleviated inhibitory effects of CAP on Microcystis growth, while 50 and 0.5mg/L N exacerbated growth-inhibition by CAP especially in early (before day 8) and mid-late stage, respectively. At each N level, CAP-induced antioxidant defense and cell damage extents were negatively correlated to growth state in each stage, and CAP-biodegradation coincided with Microcystis growth and glutathione synthesis dynamics, implying that antioxidant defense, cell damage and CAP-removal closely linked to N-dependent Microcystis growth under CAP-stress. Microcystin (MC)-production and -release under CAP-stress were also N-dependent. Although Microcystis growth was greatly-inhibited by prolonged CAP-stress at 0.5mg/L N, delayed CAP-loss and high MC-release at 0.5mg/L N should be emphasized during Microcystis-dominated cyanobacterial blooms (MCBs) and CAP co-occurrence. This study had great implication in risk assessment for MCBs-CAP co-occurrence in different waters.

Utility of a PCR-based method for rapid and specific detection of toxigenic Microcystis spp. in farm ponds.

Microcystis is a widespread freshwater cyanobacterium that can produce microcystin, a potent hepatotoxin harmful to animals and humans. Therefore, it is crucial to monitor for the presence of toxigenic Microcystis spp. to provide early warning of potential microcystin contamination. Microscopy, which has been used traditionally to identify Microcystis spp., cannot differentiate toxigenic from non-toxigenic Microcystis. We developed a PCR-based method to detect toxigenic Microcystis spp. based on detection of the microcystin synthetase C (mcyC) gene and 16S rRNA gene. Specificity was validated against toxic and nontoxic M. aeruginosa strains, as well as 4 intergeneric freshwater cyanobacterial strains. Analytical sensitivity was as low as 747 fg/µL genomic DNA (or 3 cells/µL) for toxic M. aeruginosa. Furthermore, we tested 60 water samples from 4 farm ponds providing drinking water to swine facilities in the midwestern United States using this method. Although all water samples were positive for Microcystis spp. (i.e., 16S rRNA gene), toxigenic Microcystis spp. were detected in only 34 samples (57%). Seventeen water samples contained microcystin (0.1-9.1 μg/L) determined with liquid chromatography-mass spectrometry, of which 14 samples (82%) were positive for mcyC. A significant correlation was found between the presence of toxigenic Microcystis spp. and microcystin in water samples (p = 0.0004). Our PCR method can be a low-cost molecular tool for rapid and specific identification of toxigenic Microcystis spp. in farm ponds, improving detection of microcystin contamination, and ensuring water safety for farm animals.

Occurrence of Toxigenic Microcystis spp in Major Water Bodies of North East India

Toxigenic cyanobacterial blooms in the water bodies represent a major ecological problem around the world. Some species produces a diverse range of toxins that have hepatotoxic, neurotoxic, cytotoxic and dermatoxic activity and hence have deleterious effect on humans, animals and fishes leading to death as well. Cultural eutrophication of water bodies leads to increased incidence of these harmful cyanobacterial blooms worldwide. North-East India being a biodiversity hotspot harbor many species of cyanobacteria. Few reports suggested presence of few toxigenic cyanobacteria in the water bodies of Assam, but no systematic studies have been undertaken to evaluate their toxicity. This work is being conducted to gather information on major toxigenic cyanobacteria, with special emphasis to microcystin (a cyclic heptapeptides with high acute and chronic toxicities to humans and animals) producing strains. Water samples have been collected from few water bodies of North-East and enriched in specific media. The toxin Microcystin was detected using specific ELISA kit and positive results have been obtained. Further, 16s rDNA sequencing was employed for molecular identification of the strains.