The application of hydrogen peroxide (H 2 O 2 ) as a management tool to control Microcystis blooms has become increasingly popular due to its short lifetime and targeted action. H 2 O 2 increases intracellular reactive oxygen species resulting in oxidative stress and subsequently cell death. H 2 O 2 is naturally produced in freshwater bodies as a result of photocatalytic reactions between dissolved organic carbon and sunlight. Previously, some studies have suggested that this environmental source of H 2 O 2 selectively targets for toxigenic cyanobacteria strains in the genus Microcystis. Also, past studies only focused on the morphological and biochemical changes of H 2 O 2 -induced cell death in Microcystis with little information available on the effects of different H 2 O 2 concentrations on growth, esterase activity and membrane integrity. Therefore, this study investigated the effects of non-lethal (40-4000 nM) concentrations on percentage cell death; with a focus on sub-lethal (50 μM) and lethal (275 μM; 500 μM) doses of H 2 O 2 on growth, cells showing esterase activity and membrane integrity. The non-lethal dose experiment was part of a preliminary study. Results showed a general effect of dose and time dependent relationship in all three Microcystis strains post H 2 O 2 treatment. H 2 O 2 resulted in a significant increase in intracellular reactive oxygen species, decreased chlorophyll a content, decreased growth rate and esterase activity. Interestingly, at sub-lethal (50 µM H 2 O 2 treatment), percentage of dead cells in microcystin-producing strains were significantly higher (p<0.05) from non-microcystin producing strains at 72h. These findings further cement our understanding of the influence of H 2 O 2 on different strains of Microcystis and its impact on membrane integrity and metabolic physiology; important to future toxic bloom control programmes.
Freshwater cyanobacteria blooms represent a risk to ecological and human health through induction of anoxia and release of potent toxins; both conditions require water management to mitigate risks. Many cyanobacteria taxa may produce microcystins, a group of toxic cyclic heptapeptides. Understanding the relationships between the abiotic drivers of microcystins and their occurrence would assist in the implementation of targeted, cost-effective solutions to maintain safe drinking and recreational waters. Cyanobacteria and microcystins were measured by flow cytometry and liquid chromatography coupled to tandem mass spectrometry in two interconnected reservoirs varying in age and management regimes, in southern Britain over a 12-month period. Microcystins were detected in both reservoirs, with significantly higher concentrations in the southern lake (maximum concentration >7 µg L−1). Elevated microcystin concentrations were not positively correlated with numbers of cyanobacterial cells, but multiple linear regression analysis suggested temperature and dissolved oxygen explained a significant amount of the variability in microcystin across both reservoirs. The presence of a managed fishery in one lake was associated with decreased microcystin levels, suggestive of top down control on cyanobacterial populations. This study supports the need to develop inclusive, multifactor holistic water management strategies to control cyanobacterial risks in freshwater bodies.
Microbial subpopulations in field and laboratory studies have been shown to display high heterogeneity in morphological and physiological parameters. Determining the real time state of a microbial cell goes beyond live or dead categories, as microbes can exist in a dormant state, whereby cell division and metabolic activities are reduced. Given the need for detection and quantification of microbes, flow cytometry (FCM) with molecular probes provides a rapid and accurate method to help determine overall population viability. By using SYTOX Green and SYTOX Orange in the model cyanobacteria Microcystis aeruginosa to detect membrane integrity, we develop a transferable method for rapid indication of single cell mortality. The molecular probes used within this journal will be referred to as green or orange nucleic acid probes respectively (although there are other products with similar excitation and emission wavelengths that have a comparable modes of action, we specifically refer to the fore mentioned probes). Protocols using molecular probes vary between species, differing principally in concentration and incubation times. Following this protocol set out on M.aeruginosa the green nucleic acid probe was optimized at concentrations of 0.5 µM after 30 min of incubation and the orange nucleic acid probe at 1 µM after 10 min. In both probes concentrations less than the stated optimal led to an under reporting of cells with membrane damage. Conversely, 5 µM concentrations and higher in both probes exhibited a type of non-specific staining, whereby 'live' cells produced a target fluorescence, leading to an over representation of 'non-viable' cell numbers. The positive controls (heat-killed) provided testable dead biomass, although the appropriateness of control generation remains subject to debate. By demonstrating a logical sequence of steps for optimizing the green and orange nucleic acid probes we demonstrate how to create a protocol that can be used to analyse cyanobacterial physiological state effectively.
Despite free-living protozoa being a major factor in modifying aquatic autotrophic biomass, ciliate−cyanobacteria interactions and their functional ecological roles have been poorly described, especially with toxic cyanobacteria. Trophic relationships have been neglected and grazing experiments give contradictory evidence when toxic taxa such as Microcystis are involved. Here, 2 toxic Microcystis strains (containing microcystins), 1 non-toxic Microcystis strain and a non-toxic green alga, Chlorella vulgaris, were used to investigate predator−prey interactions with a phagotrophic ciliate, Blepharisma americanum. Flow cytometric analysis for microalgal measurements and a rapid ultra high performance liquid chromatography-tandem mass spectrometry protocol to quantify microcystins showed that non-toxic photosynthetic microbes were significantly grazed by B. americanum, which sustained ciliate populations. In contrast, despite constant ingestion of toxic Microcystis, rapid egestion of cells occurred. The lack of digestion resulted in no significant control of toxic cyanobacteria densities, a complete reduction in ciliate numbers, and no observable encystment or cannibalistic behaviour (gigantism). Individual B. americanum morphological responses (biovolume and cell width) showed a significant decrease over time when sustained on non-toxic Microcystis compared to grazed C. vulgaris populations, supporting previous studies that cyanobacteria may be a relatively poor source of nutrition. Results here provide insight into the ecological interactions of ciliates and cyanobacteria, and for the first time B. americanum is shown to have the capacity to suppress potentially bloom-forming cyanobacteria. However, grazing can be significantly altered by the presence of microcystins, which could have an impact on bloom dynamics and overall community structure.
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