Is Toxin-Producing Planktothrix sp. an Emerging Species in Lake Constance?

Recurring blooms of filamentous, red-pigmented and toxin-producing cyanobacteria Planktothrix rubescens have been reported in numerous deep and stratified prealpine lakes, with the exception of Lake Constance. In a 2019 and 2020 Lake Constance field campaign, we collected samples from a distinct red-pigmented biomass maximum below the chlorophyll-a maximum, which was determined using fluorescence probe measurements at depths between 18 and 20 m. Here, we report the characterization of these deep water red pigment maxima (DRM) as cyanobacterial blooms. Using 16S rRNA gene-amplicon sequencing, we found evidence that the blooms were, indeed, contributed by Planktothrix spp., although phycoerythrin-rich Synechococcus taxa constituted most of the biomass (>96% relative read abundance) of the cyanobacterial DRM community. Through UPLC-MS/MS, we also detected toxic microcystins (MCs) in the DRM in the individual sampling days at concentrations of ≤1.5 ng/L. Subsequently, we reevaluated the fluorescence probe measurements collected over the past decade and found that, in the summer, DRM have been present in Lake Constance, at least since 2009. Our study highlights the need for a continuous monitoring program also targeting the cyanobacterial DRM in Lake Constance, and for future studies on the competition of the different cyanobacterial taxa. Future studies will address the potential community composition changes in response to the climate change driven physiochemical and biological parameters of the lake.

Removal of Positively Buoyant Planktothrix rubescens in Lake Restoration.

The combination of a low-dose coagulant (polyaluminium chloride-'Floc') and a ballast able to bind phosphate (lanthanum modified bentonite, LMB-'Sink/Lock') have been used successfully to manage cyanobacterial blooms and eutrophication. In a recent 'Floc and Lock' intervention in Lake de Kuil (the Netherlands), cyanobacterial chlorophyll-a was reduced by 90% but, surprisingly, after one week elevated cyanobacterial concentrations were observed again that faded away during following weeks. Hence, to better understand why and how to avoid an increase in cyanobacterial concentration, experiments with collected cyanobacteria from Lakes De Kuil and Rauwbraken were performed. We showed that the Planktothrix rubescens from Lake de Kuil could initially be precipitated using a coagulant and ballast but, after one day, most of the filaments resurfaced again, even using a higher ballast dose. By contrast, the P. rubescens from Lake Rauwbraken remained precipitated after the Floc and Sink/Lock treatment. We highlight the need to test selected measures for each lake as the same technique with similar species (P. rubescens) yielded different results. Moreover, we show that damaging the cells first with hydrogen peroxide before adding the coagulant and ballast (a 'Kill, Floc and Lock/Sink' approach) could be promising to keep P. rubescens precipitated.

In Vitro Toxicological Screening of Stable and Senescing Cultures of Aphanizomenon, Planktothrix, and Raphidiopsis.

Toxicity of cyanobacteria is the subject of ongoing research, and a number of toxic metabolites have been described, their biosynthesis pathways have been elucidated, and the mechanism of their action has been established. However, several knowledge gaps still exist, e.g., some strains produce hitherto unknown toxic compounds, while the exact dynamics of exerted toxicity during cyanobacterial growth still requires further exploration. Therefore, the present study investigated the toxicity of extracts of nine freshwater strains of Aphanizomenon gracile, an Aphanizomenon sp. strain isolated from the Baltic Sea, a freshwater strain of Planktothrix agardhii, and two strains of Raphidiopsis raciborskii obtained from 25- and 70-day-old cultures. An in vitro experimental model based on Cyprinus carpio hepatocytes (oxidative stress markers, DNA fragmentation, and serine/threonine protein activity) and brain homogenate (cholinesterase activity) was employed. The studied extracts demonstrated toxicity to fish cells, and in general, all examined extracts altered at least one or more of considered parameters, indicating that they possess, to some degree, toxic potency. Although the time from which the extracts were obtained had a significant importance for the response of fish cells, we observed strong variability between the different strains and species. In some strains, extracts that originated from 25-day-old cultures triggered more harmful effects on fish cells compared to those obtained from 70-day-old cultures, whereas in other strains, we observed the opposite effect or a lack of a significant change. Our study revealed that there was no clear or common pattern regarding the degree of cyanobacterial bloom toxicity at a given stage of development. This means that young cyanobacterial blooms that are just forming can pose an equally toxic threat to aquatic vertebrates and ecosystem functioning as those that are stable or old with a tendency to collapse. This might be largely due to a high variability of strains in the bloom.

Microcystins in European Noble Crayfish Astacus astacus in Lake Steinsfjorden, a Planktothrix-Dominated Lake.

Lake Steinsfjorden, an important Norwegian location for noble crayfish (Astacus astacus), is often affected by cyanobacterial blooms caused by microcystin (MC)-producing Planktothrix spp. The impact of MCs on noble crayfish as a food source and crayfish health is largely unknown. We investigated the quantities and correlations of MCs in noble crayfish and lake water during and after a cyanobacterial bloom peaking in June-July 2015. Noble crayfish and water samples were collected monthly from June to October 2015 and in October 2016. The content of MCs was analysed by ELISA from tail muscle, intestine, stomach and hepatopancreas. PCR analysis for Planktothrix gene markers was performed on crayfish stomach content. Water samples were analysed for phytoplankton composition, biomass and MCs. PCR-positive stomach contents indicated Planktothrix to be part of the noble crayfish diet. Concentrations of MCs were highest in the hepatopancreas, stomach and intestine, peaking in August-September. Tail muscle contained low concentrations of MCs. Similar levels of MCs were found in crayfish from 2016. Except in September 2015, a normal portion of boiled noble crayfish tails was below the tolerable daily intake (TDI) for MCs for humans. Removing the intestine more than halved the content of MCs and seems a reasonable precautionary measure for noble crayfish consumers.

The OAAH Family: Anti-Influenza Virus Lectins.

High mannose (HM)-binding Oscillatoria agardhii agglutinin homologue (OAAH) lectin family is an important class of anti-viral proteins. The OAAH family lectins show potent anti-influenza virus activity with EC50 of nanomolar levels by binding to HM glycans of the envelope glycoprotein hemagglutinin (HA), thereby inhibiting the viral entry into host cells. No broadly effective neutralizing vaccines for influenza virus are available due to the frequent antigenic drift caused by rapid mutations. Alternatives for vaccines need to be developed to prepare for a possible risk of future emergence of a highly virulent virus. Possible use of antiviral lectins is a simple and useful strategy to prevent viral infection by interfering with the interaction between viral HA and the host sialic acid-containing receptor. High-density glycans of surface HA are primary targets for the lectins to inhibit viral entry. In general, the anti-influenza virus potency of lectins is evaluated by a series of inhibitory assays for infection, such as neutral red dye uptake assay to determine the extent of viral cytopathic effect, and immunofluorescence microscopy to detect the expression of viral proteins in infected cells. Direct interaction between lectins and HA could be evaluated by enzyme-linked immunosorbent assay or surface plasmon resonance analysis.

Investigation of In Vitro Endocrine Activities of Microcystis and Planktothrix Cyanobacterial Strains.

Cyanobacteria are cosmopolitan photosynthetic prokaryotes that can form dense accumulations in aquatic environments. They are able to produce many bioactive metabolites, some of which are potentially endocrine disrupting compounds, i.e., compounds that interfere with the hormonal systems of animals and humans. Endocrine disruptors represent potential risks to both environmental and human health, making them a global challenge. The aim of this study was to investigate the potential endocrine disrupting activities with emphasis on estrogenic effects of extracts from cultures of Microcystis or Planktothrix species. We also assessed the possible role of microcystins, some of the most studied cyanobacterial toxins, and thus included both microcystin-producing and non-producing strains. Extracts from 26 cyanobacterial cultures were initially screened in estrogen-, androgen-, and glucocorticoid-responsive reporter-gene assays (RGAs) in order to identify endocrine disruption at the level of nuclear receptor transcriptional activity. Extracts from selected strains were tested repeatedly in the estrogen-responsive RGAs, but the observed estrogen agonist and antagonist activity was minor and similar to that of the cyanobacteria growth medium control. We thus focused on another, non-receptor mediated mechanism of action, and studied the 17ß-estradiol (natural estrogen hormone) biotransformation in human liver microsomes in the presence or absence of microcystin-LR (MC-LR), or an extract from the MC-LR producing M. aeruginosa PCC7806 strain. Our results show a modulating effect on the estradiol biotransformation. Thus, while 2-hydroxylation was significantly decreased following co-incubation of 17ß-estradiol with MC-LR or M. aeruginosa PCC7806 extract, the relative concentration of estrone was increased.