Cyanobacterial Contribution to Travertine Deposition in the Hoyoux River System, Belgium.

Travertine deposition is a landscape-forming process, usually building a series of calcareous barriers differentiating the river flow into a series of cascades and ponds. The process of carbonate precipitation is a complex relationship between biogenic and abiotic causative agents, involving adapted microbial assemblages but also requiring high levels of carbonate saturation, spontaneous degassing of carbon dioxide and slightly alkaline pH. We have analysed calcareous crusts and water chemistry from four sampling sites along the Hoyoux River and its Triffoy tributary (Belgium) in winter, spring, summer and autumn 2014. Different surface textures of travertine deposits correlated with particular microenvironments and were influenced by the local water flow. In all microenvironments, we have identified the cyanobacterium Phormidium incrustatum (Nägeli) Gomont as the organism primarily responsible for carbonate precipitation and travertine fabric by combining morphological analysis with molecular sequencing (16S rRNA gene and ITS, the Internal Transcribed Spacer fragments), targeting both field populations and cultures to exclude opportunistic microorganisms responding favourably to culture conditions. Several closely related cyanobacterial strains were cultured; however, only one proved identical with the sequences obtained from the field population by direct PCR. This strain was the dominant primary producer in the calcareous deposits under study and in similar streams in Europe. The dominance of one organism that had a demonstrated association with carbonate precipitation presented a valuable opportunity to study its function in construction, preservation and fossilisation potential of ambient temperature travertine deposits. These relationships were examined using scanning electron microscopy and Raman microspectroscopy.

From genes to toxins: Profiling Prymnesium parvum during a riverine harmful algal bloom.

Blooms of Prymnesium parvum, a unicellular alga globally distributed in marine and brackish environments, frequently result in massive fish kills due to the production of toxins called prymnesins by this haptophyte. In August 2022, a harmful algal bloom (HAB) of this species occurred in the lower Oder River (Poland and Germany), which caused mass mortalities of fish and other organisms. This HAB was linked to low discharge of the Oder and mining activities that caused a significant increase in salinity. In this context, we report on the molecular detection and screening of this haptophyte and its toxins in environmental samples and clonal cultures derived thereof. Both conventional PCR and droplet digital PCR assays reliably detected P. parvum in environmental samples. eDNA metabarcoding using the V4 region of the 18S rRNA gene revealed a single Prymnesium sequence variant, but failed to identify it to species level. Four clonal cultures established from environmental samples were unambiguously identified as P. parvum by molecular phylogenetics (near full-length 18S rRNA gene) and light microscopy. Phylogenetic analysis (ITS1-5.8S-ITS2 marker region) placed the cultured phylotype within a clade containing other P. parvum strains known to produce B-type prymnesins. Toxin-screening of the cultures using liquid chromatography-electrospray ionization - time of flight mass spectrometry identified B-type prymnesins, which were also detected in extracts of filter residues from water samples of the Oder collected during the HAB. Overall, our investigation provides a detailed characterization of P. parvum, including their prymnesins, during this HAB in the Oder River, contributing valuable insights into this ecological disaster. In addition, the droplet digital PCR assay established here will be useful for future monitoring of low levels of P. parvum on the Oder River or any other salt-impacted and brackish water bodies.

Intracellular eukaryotic pathogens in brown macroalgae in the Eastern Mediterranean, including LSU rRNA data for the oomycete Eurychasma dicksonii .

For the Mediterranean Sea, and indeed most of the world's oceans, the biodiversity and biogeography of eukaryotic pathogens infecting marine macroalgae remains poorly known, yet their ecological impact is probably significant. Based on 2 sampling campaigns on the Greek island of Lesvos in 2009 and 1 in northern Greece in 2012, this study provides first records of 3 intracellular eukaryotic pathogens infecting filamentous brown algae at these locations: Eurychasma dicksonii, Anisolpidium sphacellarum, and A. ectocarpii. Field and microscopic observations of the 3 pathogens are complemented by the first E. dicksonii large subunit ribosomal RNA (LSU rRNA) gene sequence analyses of isolates from Lesvos and other parts of the world. The latter highlights the monophyly of E. dicksonii worldwide and confirms the basal position of this pathogen within the oomycete lineage (Peronosporomycotina). The results of this study strongly support the notion that the geographic distribution of the relatively few eukaryotic seaweed pathogens is probably much larger than previously thought and that many of the world's marine bioregions remain seriously undersampled and understudied in this respect.

Glacier melt-down changes habitat characteristics and unique microbial community composition and physiology in alpine lake sediments.

Glacial melt-down alters hydrological and physico-chemical conditions in downstream aquatic habitats. In this study, we tested if sediment-associated microbial communities respond to the decrease of glaciers and associated meltwater flows in high-alpine lakes. We analyzed 16 lakes in forefield catchments of three glaciers in the Eastern Swiss Alps on physico-chemical and biological parameters. We compared lakes fed by glacier meltwater with hydrologically disconnected lakes, as well as "mixed" lakes that received water from both other lake types. Glacier-fed lakes had a higher turbidity (94 NTU) and conductivity (47 µS/cm), but were up to 5.2°C colder than disconnected lakes (1.5 NTU, 26 µS/cm). Nutrient concentration was low in all lakes (TN < 0.05 mg/l, TP < 0.02 mg/l). Bacterial diversity in the sediments decreased significantly with altitude. Bacterial community composition correlated with turbidity, temperature, conductivity, nitrate, and lake age and was distinctly different between glacier-fed compared to disconnected and mixed water lakes, but not between catchments. Chemoheterotrophic processes were more abundant in glacier-fed compared to disconnected and mixed water lakes where photoautotrophic processes dominated. Our study suggests that the loss of glaciers will change sediment bacterial community composition and physiology that are unique for glacier-fed lakes in mountain and polar regions.

Detection of differential host susceptibility to the marine oomycete pathogen Eurychasma dicksonii by real-time PCR: not all algae are equal.

In the marine environment, a growing body of evidence points to parasites as key players in the control of population dynamics and overall ecosystem structure. However, their prevalence and impact on marine macroalgal communities remain virtually unknown. Indeed, infectious diseases of seaweeds are largely underdocumented, partly because of the expertise required to diagnose them with a microscope. Over the last few years, however, real-time quantitative PCR (qPCR) has emerged as a rapid and reliable alternative to visual symptom scoring for monitoring pathogens. Thus, we present here a qPCR assay suitable for the detection and quantification of the intracellular oomycete pathogen Eurychasma dicksonii in its ectocarpalean and laminarialean brown algal hosts. qPCR and microscopic observations made of laboratory-controlled cultures revealed that clonal brown algal strains exhibit different levels of resistance against Eurychasma, ranging from high susceptibility to complete absence of symptoms. This observation strongly argues for the existence of a genetic determinism for disease resistance in brown algae, which would have broad implications for the dynamics and genetic structure of natural populations. We also used qPCR for the rapid detection of Eurychasma in filamentous brown algae collected in Northern Europe and South America and found that the assay is specific, robust, and widely applicable to field samples. Hence, this study opens the perspective of combining large-scale disease monitoring in the field with laboratory-controlled experiments on the genome model seaweed Ectocarpus siliculosus to improve our understanding of brown algal diseases.

Microplastics Reduce Short-Term Effects of Environmental Contaminants. Part II: Polyethylene Particles Decrease the Effect of Polycyclic Aromatic Hydrocarbons on Microorganisms.

Microplastic particles in terrestrial and aquatic ecosystems are currently discussed as an emerging persistent organic pollutant and as acting as a vector for hydrophobic chemicals. Microplastic particles may ultimately deposit and accumulate in soil as well as marine and freshwater sediments where they can be harmful to organisms. In this study, we tested the sensitivity of natural freshwater sediment bacterial communities (by genetic fingerprint) to exposure to microplastics (polyethylene, 2 and 20 mg/g sediment) and microplastics loaded with polycyclic aromatic hydrocarbons (PAHs, phenanthrene and anthracene), using a laboratory-based approach. After two weeks of incubation, the bacterial community composition from an unpolluted river section was altered by high concentrations of microplastics, whereas the community downstream of a wastewater treatment plant remained unchanged. Low microplastic concentrations loaded with phenanthrene or anthracene induced a less pronounced response in the sediment communities compared to the same total amount of phenanthrene or anthracene alone. In addition, biodegradation of the PAHs was reduced. This study shows, that microplastic can affect bacterial community composition in unpolluted freshwater sediments. Moreover, the results indicate that microplastics can serve as a vehicle for hydrophobic pollutants but bioavailability of the latter is reduced by the sorption to microplastics.

Toxic Cyanobacteria in Svalbard: Chemical Diversity of Microcystins Detected Using a Liquid Chromatography Mass Spectrometry Precursor Ion Screening Method.

Cyanobacteria synthesize a large variety of secondary metabolites including toxins. Microcystins (MCs) with hepato- and neurotoxic potential are well studied in bloom-forming planktonic species of temperate and tropical regions. Cyanobacterial biofilms thriving in the polar regions have recently emerged as a rich source for cyanobacterial secondary metabolites including previously undescribed congeners of microcystin. However, detection and detailed identification of these compounds is difficult due to unusual sample matrices and structural congeners produced. We here report a time-efficient liquid chromatography-mass spectrometry (LC-MS) precursor ion screening method that facilitates microcystin detection and identification. We applied this method to detect six different MC congeners in 8 out of 26 microbial mat samples of the Svalbard Archipelago in the Arctic. The congeners, of which [Asp³, ADMAdda5, Dhb7] MC-LR was most abundant, were similar to those reported in other polar habitats. Microcystins were also determined using an Adda-specific enzyme-linked immunosorbent assay (Adda-ELISA). Nostoc sp. was identified as a putative toxin producer using molecular methods that targeted 16S rRNA genes and genes involved in microcystin production. The mcy genes detected showed highest similarities to other Arctic or Antarctic sequences. The LC-MS precursor ion screening method could be useful for microcystin detection in unusual matrices such as benthic biofilms or lichen.

Raman Characterization of the UV-Protective Pigment Gloeocapsin and Its Role in the Survival of Cyanobacteria.

Extracellular UV-screening pigments gloeocapsin and scytonemin present in the exopolysaccharide (EPS) envelopes of extremophilic cyanobacteria of freshwater and marine environments were studied by Raman spectroscopy and compared to their intracellular photosynthetic pigments. This Raman spectral analysis of the extracellular pigment gloeocapsin showed that it shared Raman spectral signatures with parietin, a radiation-protective pigment known in lichens. The UV-light spectra also show similarities. Gloeocapsin occurs in some cyanobacterial species, mostly with exclusion of scytonemin, indicating that these pigments have evolved in cyanobacteria as separate protective strategies. Both gloeocapsin and scytonemin are widely and species-specifically distributed in different cyanobacterial genera and families. The widespread occurrence of these pigments may suggest an early origin, while their detection by Raman spectroscopy makes them potential biosignatures for cyanobacteria in the fossil record and demonstrates the usefulness of nondestructive Raman spectroscopy analyses for the search for complex organics, including possible photosynthetic pigments, if preservable in early Earth and extraterrestrial samples.

Potent toxins in Arctic environments--presence of saxitoxins and an unusual microcystin variant in Arctic freshwater ecosystems.

Cyanobacteria are the predominant phototrophs in freshwater ecosystems of the polar regions where they commonly form extensive benthic mats. Despite their major biological role in these ecosystems, little attention has been paid to their physiology and biochemistry. An important feature of cyanobacteria from the temperate and tropical regions is the production of a large variety of toxic secondary metabolites. In Antarctica, and more recently in the Arctic, the cyanobacterial toxins microcystin and nodularin (Antarctic only) have been detected in freshwater microbial mats. To date other cyanobacterial toxins have not been reported from these locations. Five Arctic cyanobacterial communities were screened for saxitoxin, another common cyanobacterial toxin, and microcystins using immunological, spectroscopic and molecular methods. Saxitoxin was detected for the first time in cyanobacteria from the Arctic. In addition, an unusual microcystin variant was identified using liquid chromatography-mass spectrometry. Gene expression analyses confirmed the analytical findings, whereby parts of the sxt and mcy operon involved in saxitoxin and microcystin synthesis, were detected and sequenced in one and five of the Arctic cyanobacterial samples, respectively. The detection of these compounds in the cryosphere improves the understanding of the biogeography and distribution of toxic cyanobacteria globally. The sequences of sxt and mcy genes provided from this habitat for the first time may help to clarify the evolutionary origin of toxin production in cyanobacteria.

Microcystin congener- and concentration-dependent induction of murine neuron apoptosis and neurite degeneration.

Cyanobacterial microcystins (MCs) represent a toxin group with > 100 variants, requiring active uptake into cells via organic anion-transporting polypeptides, in order to irreversibly inhibit serine/threonine-specific protein phosphatases. MCs are a human health hazard with repeated occurrences of severe poisonings. In the well-known human MC intoxication in Caruaru, Brazil (1996), patients developed signs of acute neurotoxicity, e.g., deafness, tinnitus, and intermittent blindness, as well as subsequent hepatotoxicity. The latter data, in conjunction with some animal studies, suggest that MCs are potent neurotoxins. However, there is little data to date demonstrating MC neuron-specific toxicity. MC exposure-induced cytotoxicity, caspase activity, chromatin condensation, and microtubule-associated Tau protein hyperphosphorylation (epitopes serine199/202 and serine396) were determined. Neurite degeneration was analyzed with confocal microscopy and neurite length determined using image analysis. MC-induced apoptosis was significantly increased by MC-LF and MC-LW, however, only at high concentrations (≥ 3µM), whereas significant neurite degeneration was already observed at 0.5µM MC-LF. Moreover, sustained hyperphosphorylation of Tau was observed with all MC congeners. The concentration- and congener-dependent mechanisms observed suggest that low concentrations of MC-LF and MC-LW can induce subtle neurodegenerative effects, reminiscent of Alzheimer's disease type human tauopathies, and thus should be taken more seriously with regard to potential human health effects than the apical cytotoxicity (apoptosis or necrosis) demonstrated at high MC concentrations.

Investigation of microcystin congener-dependent uptake into primary murine neurons.

BACKGROUND: Contamination of natural waters by toxic cyanobacteria is a growing problem worldwide, resulting in serious water pollution and human health hazards. Microcystins (MCs) represent a group of > 80 cyclic heptapeptides, mediating cytotoxicity via specific protein phosphatase (PP) inhibition at equimolar concentrations (comparable toxicodynamics). Because of the structure and size of MCs, active uptake into cells occurs via organic anion-transporting polypeptides (OATP/Oatp), as confirmed for liver-specific human OATP1B1 and OATP1B3, mouse Oatp1b2 (mOatp1b2), skate Oatp1d1, and the more widely distributed OATP1A2 expressed, for example, at the blood-brain barrier. Tissue-specific and cell-type-specific expression of OATP/Oatp transporters and specific transport of MC congeners (toxicokinetics) therefore appear prerequisite for the reported toxic effects in humans and other species upon MC exposure. Beyond hepatotoxicity induced by the MC-LR congener, the effects of other MC congeners, especially neuronal uptake and toxicity, are unknown. OBJECTIVES: In this study we examined the expression of mOatps and the uptake of congeners MC-LR, MC-LW, and MC-LF in primary murine neurons. METHODS: Intracellular MC accumulation was indicated indirectly via uptake inhibition experiments and directly confirmed by Western blot analysis and a PP inhibition assay. Neuronal mOatp expression was verified at the mRNA and protein level. RESULTS: MCs can cross neuronal cell membranes, with a subsequent decrease of PP activity. Of 15 mOatps, 12 were expressed at the mRNA level, but we found detectable protein levels for only two: mOatp1a5 (Slco1a5) and the known MC-LR transporter mOatp1b2 (Slco1b2). CONCLUSIONS: These data suggest mOatp-mediated uptake of MC congeners into neurons, thus corroborating earlier assumptions of the neurotoxic potential of MCs.