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Radiosumins are a structurally diverse family of low molecular weight natural products that are produced by cyanobacteria and exhibit potent serine protease inhibition. Members of this family are dipeptides characterized by the presence of two similar non-proteinogenic amino acids. Here we used a comparative bioinformatic analysis to identify radiosumin biosynthetic gene clusters from the genomes of 13 filamentous cyanobacteria. We used direct pathway cloning to capture and express the entire 16.8 kb radiosumin biosynthetic gene cluster from Dolichospermum planctonicum UHCC 0167 in Escherichia coli. Bioinformatic analysis demonstrates that radiosumins represent a new group of chorismate-derived non-aromatic secondary metabolites. High-resolution liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and chemical degradation analysis revealed that cyanobacteria produce a cocktail of novel radiosumins. We report the chemical structure of radiosumin D, an N-methyl dipeptide, containing a special Aayp (2-amino-3-(4-amino-2-cyclohexen-1-ylidene) propionic acid) with R configuration that differs from radiosumin A-C, an N-Me derivative of Aayp (Amyp) and two acetyl groups. Radiosumin C inhibits all three human trypsin isoforms at micromolar concentrations with preference for trypsin-1 and -3 (IC50 values from 1.7 mu M to >7.2 mu M). These results provide a biosynthetic logic to explore the genetic and chemical diversity of the radiosumin family and suggest that these natural products may be a source of drug leads for selective human serine proteases inhibitors.

Chemotaxonomic biomarkers are needed to monitor and evaluate the nutritional quality of phytoplankton communities. The biomolecules produced by different phytoplankton species do not always follow genetic phylogeny. Therefore, we analyzed fatty acids, sterols, and carotenoids from 57 freshwater phytoplankton strains to evaluate the usability of these biomolecules as chemotaxonomic biomarkers. We found 29 fatty acids, 34 sterols, and 26 carotenoids in our samples. The strains were grouped into cryptomonads, cyanobacteria, diatoms, dinoflagellates, golden algae, green algae, and raphidophytes, and the phytoplankton group explained 61%, 54%, and 89% of the variability of fatty acids, sterols, and carotenoids, respectively. Fatty acid and carotenoid profiles distinguished most phytoplankton groups, but not flawlessly. For example, fatty acids could not distinguish golden algae and cryptomonads, whereas carotenoids did not separate diatoms and golden algae. The sterol composition was heterogeneous but seemed to be useful for distinguishing different genera within a phytoplankton group. The chemotaxonomy biomarkers yielded optimal genetic phylogeny when the fatty acids, sterols, and carotenoids were used together in multivariate statistical analysis. Our results suggest that the accuracy of phytoplankton composition modeling could be enhanced by combining these three biomolecule groups.

Monodominant (one species dominates) or polidominant (multiple species dominate) cyanobacterial blooms are pronounced in productive freshwater ecosystems and pose a potential threat to the biota due to the synthesis of toxins. Seasonal changes in cyanobacteria species and cyanometabolites composition were studied in two shallow temperate eutrophic lakes. Data on cyanobacteria biomass and diversity of dominant species in the lakes were combined with chemical and molecular analyses of fifteen potentially toxin-producing cyanobacteria species (248 isolates from the lakes). Anatoxin-a, saxitoxin, microcystins and other non-ribosomal peptides formed the diverse profiles in monodominant (Planktothrix agardhii) and polidominant (Aphanizomenon gracile, Limnothrix spp. and Planktolyngbya limnetica) lakes. However, the harmfulness of the blooms depended on the ability of the dominant species to synthesize cyanometabolites. It was confirmed that P. agardhii produced a greater amount and diverse range of MCs and other NRPs. In the polidominant lake, isolates of the co-dominant A. gracile, L. planctonica and P. limnetica synthesized no or only small amounts of cyanometabolites. In general, the profile of cyanometabolites was greater in cyanobacteria isolates than in environmental samples, indicating a high potential for toxic cyanobacteria bloom.

To build a forecasting tool for the state of eutrophication in the Archipelago Sea, we fitted a Generalized Additive Mixed Model (GAMM) to marine environmental monitoring data, which were collected over the years 2011–2019 by an automated profiling buoy at the Seili ODAS-station. The resulting “Seili-index” can be used to predict the chlorophyll-α (chl-a) concentration in the seawater a number of days ahead by using the temperature forecast as a covariate. An array of test predictions with two separate models on the 2019 data set showed that the index is adept at predicting the amount of chl-a especially in the upper water layer. The visualization with 10 days of chl-a level predictions is presented online at https://saaristomeri.utu.fi/seili-index/. We also applied GAMMs to predict abrupt blooms of cyanobacteria on the basis of temperature and wind conditions and found the model to be feasible for short-term predictions. The use of automated monitoring data and the presented GAMM model in assessing the effects of natural resource management and pollution risks is discussed.

Cyanobacteria of the order Nostocales, including Baltic Sea bloom-forming taxa Nodularia spumigena, Aphanizomenon flosaquae, and Dolichospermum spp., produce resting stages, known as akinetes, under unfavorable conditions. These akinetes can persist in the sediment and germinate if favorable conditions return, simultaneously representing past blooms and possibly contributing to future bloom formation. The present study characterized cyanobacterial akinete survival, germination, and potential cyanotoxin production in brackish water sediment archives from coastal and open Gulf of Finland in order to understand recent bloom expansion, akinete persistence, and cyanobacteria life cycles in the northern Baltic Sea. Results showed that cyanobacterial akinetes can persist in and germinate from Northern Baltic Sea sediment up to >40 and >400 years old, at coastal and open-sea locations, respectively. Akinete abundance and viability decreased with age and depth of vertical sediment layers. The detection of potential microcystin and nodularin production from akinetes was minimal and restricted to the surface sediment layers. Phylogenetic analysis of culturable cyanobacteria from the coastal sediment core indicated that most strains likely belonged to the benthic genus Anabaena. Potentially planktonic species of Dolichospermum could only be revived from the near-surface layers of the sediment, corresponding to an estimated age of 1–3 years. Results of germination experiments supported the notion that akinetes do not play an equally significant role in the life cycles of all bloom-forming cyanobacteria in the Baltic Sea. Overall, there was minimal congruence between akinete abundance, cyanotoxin concentration, and the presence of cyanotoxin biosynthetic genes in either sediment core. Further research is recommended to accurately detect and quantify akinetes and cyanotoxin genes from brackish water sediment samples in order to further describe species-specific benthic archives of cyanobacteria.

Cyanobacteria are an important part of phytoplankton communities, however, they are also known for forming massive blooms with potentially deleterious effects on recreational use, human and animal health, and ecosystem functioning. Emerging high-frequency imaging flow cytometry applications, such as Imaging FlowCytobot (IFCB), are crucial in furthering our understanding of the factors driving bloom dynamics, since these applications provide community composition information at frequencies impossible to attain using conventional monitoring methods. However, the proof of applicability of automated imaging applications for studying dynamics of filamentous cyanobacteria is still scarce. In this study we present the first results of IFCB applied to a Baltic Sea cyanobacterial bloom community using a continuous flow-through setup. Our main aim was to demonstrate the pros and cons of the IFCB in identifying filamentous cyanobacterial taxa and in estimating their biomass. Selected environmental parameters (water temperature, wind speed and salinity) were included, in order to demonstrate the dynamics of the system the cyanobacteria occur in and the possibilities for analyzing high-frequency phytoplankton observations against changes in the environment. In order to compare the IFCB results with conventional monitoring methods, filamentous cyanobacteria were enumerated from water samples using light microscopical analysis. Two common bloom forming filamentous cyanobacteria in the Baltic Sea, Aphanizomenon flosaquae and Dolichospermum spp. dominated the bloom, followed by an increase in Oscillatoriales abundance. The IFCB results compared well with the results of the light microscopical analysis, especially in the case of Dolichospermum. Aphanizomenon biomass varied slightly between the methods and the Oscillatoriales results deviated the most. Bloom formation was initiated as water temperature increased to over 15°C and terminated as the wind speed increased, dispersing the bloom. Community shifts were closely related to movements of the water mass. We demonstrate how using a high-frequency imaging flow cytometry application can help understand the development of cyanobacteria summer blooms.

Highlights • Fish mortalities due to harmful algae cause substantial economic and social costs for the fish farming industry in the northeastern Atlantic, North Sea and adjacent European waters • Toxin syndromes associated with Diarrhetic Shellfish Toxins and Paralytic Shellfish Toxins and their regulation have the most profound effect on the bivalve aquaculture industry in the northeastern Atlantic region • Cyanobacteria and cyanotoxins are mainly problems in brackish water areas, particularly in the Baltic Sea • Emerging threats to the shellfish and finfish industries include the known presence of the phycotoxins azaspiracids and goniodomins • The IOC-ICES-PICESHAEDAT contains time-series baseline information on harmful algal events in Europe Harmful algal blooms (HAB) are recurrent phenomena in northern Europe along the coasts of the Baltic Sea, Kattegat-Skagerrak, eastern North Sea, Norwegian Sea and the Barents Sea. These HABs have caused occasional massive losses for the aquaculture industry and have chronically affected socioeconomic interests in several ways. This status review gives an overview of historical HAB events and summarises reports to the Harmful Algae Event Database from 1986 to the end of year 2019 and observations made in long term monitoring programmes of potentially harmful phytoplankton and of phycotoxins in bivalve shellfish. Major HAB taxa causing fish mortalities in the region include blooms of the prymnesiophyte Chrysochromulina leadbeateri in northern Norway in 1991 and 2019, resulting in huge economic losses for fish farmers. A bloom of the prymesiophyte Prymnesium polylepis (syn. Chrysochromulina polylepis) in the Kattegat-Skagerrak in 1988 was ecosystem disruptive. Blooms of the prymnesiophyte Phaeocystis spp. have caused accumulations of foam on beaches in the southwestern North Sea and Wadden Sea coasts and shellfish mortality has been linked to their occurrence. Mortality of shellfish linked to HAB events has been observed in estuarine waters associated with influx of water from the southern North Sea. The first bloom of the dictyochophyte genus Pseudochattonella was observed in 1998, and since then such blooms have been observed in high cell densities in spring causing fish mortalities some years. Dinoflagellates, primarily Dinophysis spp., intermittently yield concentrations of Diarrhetic Shellfish Toxins (DST) in blue mussels, Mytilus edulis, above regulatory limits along the coasts of Norway, Denmark and the Swedish west coast. On average, DST levels in shellfish have decreased along the Swedish and Norwegian Skagerrak coasts since approximately 2006, coinciding with a decrease in the cell abundance of D. acuta. Among dinoflagellates, Alexandrium species are the major source of Paralytic Shellfish Toxins (PST) in the region. PST concentrations above regulatory levels were rare in the Skagerrak-Kattegat during the three decadal review period, but frequent and often abundant findings of Alexandrium resting cysts in surface sediments indicate a high potential risk for blooms. PST levels often above regulatory limits along the west coast of Norway are associated with A. catenella (ribotype Group 1) as the main toxin producer. Other Alexandrium species, such as A. ostenfeldii and A. minutum, are capable of producing PST among some populations but are usually not associated with PSP events in the region. The cell abundance of A. pseudogonyaulax, a producer of the ichthyotoxin goniodomin (GD), has increased in the Skagerrak-Kattegat since 2010, and may constitute an emerging threat. The dinoflagellate Azadinium spp. have been unequivocally linked to the presence of azaspiracid toxins (AZT) responsible for Azaspiracid Shellfish Poisoning (AZP) in northern Europe. These toxins were detected in bivalve shellfish at concentrations above regulatory limits for the first time in Norway in blue mussels in 2005 and in Sweden in blue mussels and oysters (Ostrea edulis and Crassostrea gigas) in 2018. Certain members of the diatom genus Pseudo-nitzschia produce the neurotoxin domoic acid and analogs known as Amnesic Shellfish Toxins (AST). Blooms of Pseudo-nitzschia were common in the North Sea and the Skagerrak-Kattegat, but levels of AST in bivalve shellfish were rarely above regulatory limits during the review period. Summer cyanobacteria blooms in the Baltic Sea are a concern mainly for tourism by causing massive fouling of bathing water and beaches. Some of the cyanobacteria produce toxins, e.g. Nodularia spumigena, producer of nodularin, which may be a human health problem and cause occasional dog mortalities. Coastal and shelf sea regions in northern Europe provide a key supply of seafood, socioeconomic well-being and ecosystem services. Increasing anthropogenic influence and climate change create environmental stressors causing shifts in the biogeography and intensity of HABs. Continued monitoring of HAB and phycotoxins and the operation of historical databases such as HAEDAT provide not only an ongoing status report but also provide a way to interpret causes and mechanisms of HABs.

Food quality is one of the key factors influencing zooplankton population dynamics. Eutrophication drives phytoplankton communities toward the dominance of cyanobacteria, which means a decrease in the availability of sterols and long-chain polyunsaturated fatty acids (EPA and DHA). The effects of different restoration measures on the nutritional quality of the phytoplankton community and subsequent impacts on zooplankton biomass have rarely been considered. We analyzed the nutritional quality of phytoplankton in the eutrophic Lake Vesijärvi in southern Finland over a 37-year period, and studied the impacts of two restoration measures, biomanipulation and hypolimnetic aeration, on the abundance of high-quality phytoplankton. We found that biomanipulation had a positive impact on the abundance of taxa synthesizing sterols, EPA, and DHA and, concurrently, on the biomass of the keystone species Daphnia. In contrast, hypolimnetic aeration did not result in such a beneficial outcome, manifested as a decrease in the abundance of Daphnia and frequent phytoplankton blooms dominated by cyanobacteria suggesting reduction in the nutritional quality of food for Daphnia. Our analysis shows that the determination of the nutritional value of algae and the contribution of essential fatty acids and sterols is an effective method to evaluate the success of various restoration measures.

The composition of phytoplankton community is the basis for environmental monitoring and assessment of the ecological status of aquatic ecosystems. Community composition studies of phytoplankton have been based on time-consuming and expertise-demanding light microscopy analyses. Molecular methods have the potential to replace microscopy, but the high copy number variation of ribosomal genes and the lack of universal primers for simultaneous amplification of prokaryotic and eukaryotic genes complicate data interpretation. In this study, we used our previously developed directional primer-independent high-throughput sequencing (HTS) approach to analyze 16S and 18S rRNA community structures. Comparison of 83 boreal lake samples showed that the relative abundances of eukaryotic phytoplankton at class level and prokaryotic cyanobacteria at order level were consistent between HTS and microscopy results. At the genus level, the results had low correspondence, mainly due to lack of sequences in the reference library. HTS was superior to identify genera that are extensively represented in the reference databases but lack specific morphological characteristics. Targeted metatranscriptomics proved to be a feasible method to complement the microscopy analysis. The metatranscriptomics can also be applied without linking the sequences to taxonomy. However, direct indexing of the sequences to their environmental indicator values needs collections of more comprehensive sample sets, as long as the coverage of molecular barcodes of eukaryotic species remains insufficient. peerReviewed

Summary Phytoplankton and bacteria interactions have a significant role in aquatic ecosystem functioning. Associations can range from mutualistic to parasitic, shaping biogeochemical cycles and having a direct influence on phytoplankton growth. How variations in phenotype and sampling location, affect the phytoplankton microbiome is largely unknown. A high‐resolution characterization of the bacterial community in cultures of the dinoflagellate Alexandrium was performed on strains isolated from different geographical locations and at varying anthropogenic impact levels. Microbiomes of Baltic Sea Alexandrium ostenfeldii isolates were dominated by Betaproteobacteria and were consistent over phenotypic and genotypic Alexandrium strain variation, resulting in identification of an A. ostenfeldii core microbiome. Comparisons with in situ bacterial communities showed that taxa found in this A. ostenfeldii core were specifically associated to dinoflagellate dynamics in the Baltic Sea. Microbiomes of Alexandrium tamarense and minutum, isolated from the Mediterranean Sea, differed from those of A. ostenfeldii in bacterial diversity and composition but displayed high consistency, and a core set of bacterial taxa was identified. This indicates that Alexandrium isolates with diverse phenotypes host predictable, species‐specific, core microbiomes reflecting the abiotic conditions from which they were isolated. These findings enable in‐depth studies of potential interactions occurring between Alexandrium and specific bacterial taxa.

Microcystins are a family of chemically diverse hepatotoxins produced by distantly related cyanobacteria and are potent inhibitors of eukaryotic protein phosphatases 1 and 2A. Here we provide evidence for the biosynthesis of rare variants of microcystin that contain a selection of homo-amino acids by the benthic cyanobacterium Phormidium sp. LP904c. This strain produces at least 16 microcystin chemical variants many of which contain homophenylalanine or homotyrosine. We retrieved the complete 54.2 kb microcystin (mcy) gene cluster from a draft genome assembly. Analysis of the substrate specificity of McyB1 and McyC adenylation domain binding pockets revealed divergent substrate specificity sequences, which could explain the activation of homo-amino acids which were present in 31% of the microcystins detected and included variants such as MC-LHty, MC-HphHty, MC-LHph and MC-HphHph. The mcy gene cluster did not encode enzymes for the synthesis of homo-amino acids but may instead activate homo-amino acids produced during the synthesis of anabaenopeptins. We observed the loss of microcystin during cultivation of a closely related strain, Phormidium sp. DVL1003c. This study increases the knowledge of benthic cyanobacterial strains that produce microcystin variants and broadens the structural diversity of known microcystins.

Blooms of cyanobacteria are recurrent phenomena in coastal estuaries. Their maximum abundance coincides with the productive period of zooplankton and pelagic fish. Experimental studies indicate that diazotrophic, i.e. dinitrogen (N2)-fixing cyanobacterial (taxonomic order Nostocales) blooms affect zooplankton, as well as other phytoplankton. We used multidecadal monitoring data from one archipelago station (1992–2013) and ten open sea stations (1979–2013) in the Baltic Sea to explore the potential bottom-up connections between diazotrophic and non-diazotrophic cyanobacteria and phyto- and zooplankton in natural plankton communities. Random forest regression, combined with linear regression analysis showed that the biomass of cyanobacteria (both diazotrophic and non-diazotrophic) was barely connected to any of the phytoplankton and zooplankton variables examined. Instead, physico-chemical variables (salinity, temperature, total phosphorus), as well as spatial and temporal variability seemed to have more significant connections to both phytoplankton and zooplankton variables. Zooplankton variables were also connected to the biomass of phytoplankton groups other than cyanobacteria (such as chrysophytes, cryptophytes and prymnesiophytes), and phytoplankton variables had connections with the biomass of different zooplankton groups, especially copepods. Overall, negative relationships between cyanobacteria and other plankton taxa were scarcer than expected based on previous experimental studies.

Almost every summer, dense blooms of filamentous cyanobacteria are formed in the Baltic Sea. These blooms may cause problems for tourism and ecosystem services, where surface accumulations and beach fouling are commonly occurring. Future changes in environmental drivers, including climate change and other anthropogenic disturbances, may further enhance these problems. By compiling monitoring data from countries adjacent to the Baltic Sea, we present spatial and temporal genus-specific distribution of diazotrophic filamentous cyanobacteria (Nostocales) during four decades (1979–2017). While the summer surface salinity decreased with a half up to one unit, the surface temperature in July-August increased with 2–3 °C in most sub-basins of the Baltic Sea, during the time period. The biovolumes of the toxic Nodularia spumigena did not change in any of the sub-basins during the period. On the other hand, the biovolume of the non-toxic Aphanizomenon sp. and the potentially toxic Dolichospermum spp. increased in the northern parts of the Baltic Sea, along with the decreased salinity and elevated temperatures, but Aphanizomenon sp. decreased in the southern parts despite decreased salinity and increased temperatures. These contradictory changes in biovolume of Aphanizomenon sp. between the northern and southern parts of the Baltic Sea may be due to basin-specific effects of the changed environmental conditions, or can be related to local adaptation by sub-populations of the genera. Overall, this comprehensive dataset presents insights to genus-specific bloom dynamics by potentially harmful diazotrophic filamentous cyanobacteria in the Baltic Sea. Highlights • Biovolumes of bloom-forming cyanobacteria during four decades in the Baltic Sea. • Aphanizomenon sp. has increased with decreased salinity in the Bothnian Sea. • Dolichospermum spp. has increased with temperature in Bothnian Sea. • The total biovolume of Nostocales has decreased in the Southern Baltic Proper. • The biovolume of the toxic Nodularia spumigena has not changed since the 1980s.

Hassallidins are cyclic glycolipopeptides produced by cyanobacteria and other prokaryotes. The hassallidin structure consists of a peptide ring of eight amino acids where a fatty acid chain, additional amino acids, and sugar moieties are attached. Hassallidins show antifungal activity against several opportunistic human pathogenic fungi, but does not harbor antibacterial effects. However, they have not been studied on mammalian cells, and the mechanism of action is unknown. We purified hassallidin D from cultured cyanobacterium Anabaena sp. UHCC 0258 and characterized its effect on mammalian and fungal cells. Ultrastructural analysis showed that hassallidin D disrupts cell membranes, causing a lytic/necrotic cell death with rapid presence of disintegrated outer membrane, accompanied by internalization of small molecules such as propidium iodide into the cells. Furthermore, artificial liposomal membrane assay showed that hassallidin D selectively targets sterol-containing membranes. Finally, in silico membrane modeling allowed us to study the interaction between hassallidin D and membranes in detail, and confirm the role of cholesterol for hassallidin-insertion into the membrane. This study demonstrates the mechanism of action of the natural compound hassallidin, and gives further insight into how bioactive lipopeptide metabolites selectively target eukaryotic cell membranes. publishedVersion

The pederin family includes a number of bioactive compounds isolated from symbiotic organisms of diverse evolutionary origin. Pederin is linked to beetle-induced dermatitis in humans, and pederin family members possess potent antitumor activity caused by selective inhibition of the eukaryotic ribosome. Their biosynthesis is accomplished by a polyketide/nonribosomal peptide synthetase machinery employing an unusual trans-acyltransferase mechanism. Here, we report a novel pederin type compound, cusperin, from the free-living cyanobacterium Cuspidothrix issatschenkoi (earlier Aphanizomenon). The chemical structure of cusperin is similar to that of nosperin recently isolated from the lichen cyanobiont Nostoc sharing the tehrahydropyran moiety and major part of the linear backbone. However, the cusperin molecule is extended by a glycine residue and lacks one hydroxyl substituent. Pederins were previously thought to be exclusive to symbiotic relationships. However, C. issatschenkoi is a nonsymbiotic planktonic organism and a frequent component of toxic water blooms. Cusperin is devoid of the cytotoxic activity reported for other pederin family members. Hence, our findings raise questions about the role of pederin analogues in cyanobacteria and broaden the knowledge of ecological distribution of this group of polyketides.

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect effects of temperature were the main drivers of the spatial distribution in the toxins produced by the cyanobacterial community, the toxin concentrations and toxin quota. Generalized linear models showed that a Toxin Diversity Index (TDI) increased with latitude, while it decreased with water stability. Increases in TDI were explained through a significant increase in toxin variants such as MC-YR, anatoxin and cylindrospermopsin, accompanied by a decreasing presence of MC-LR. While global warming continues, the direct and indirect effects of increased lake temperatures will drive changes in the distribution of cyanobacterial toxins in Europe, potentially promoting selection of a few highly toxic species or strains. © 2018 by the authors. Licensee MDPI, Basel, Switzerland., The authors acknowledge COST Action ES 1105 “CYANOCOST—Cyanobacterial blooms and toxins in water resources: Occurrence impacts and management” and COST Action ES 1201 “NETLAKE—Networking Lake Observatories in Europe” for contributing to this study through networking and knowledge sharing with European experts in the field. Evanthia Mantzouki was supported by a grant from the Swiss State Secretariat for Education, Research and Innovation (SERI) to Bas Ibelings and by supplementary funding from the University of Geneva. We thank Clare Ahnlund, Ena Suarez and Irene Gallego for helping out with the Swiss survey. We thank Wendy Beekman and Els J. Faassen for the nutrient and toxin analysis.

Under ongoing climate change and increasing anthropogenic activity, which continuously challenge ecosystem resilience, an in-depth understanding of ecological processes is urgently needed. Lakes, as providers of numerous ecosystem services, face multiple stressors that threaten their functioning. Harmful cyanobacterial blooms are a persistent problem resulting from nutrient pollution and climate-change induced stressors, like poor transparency, increased water temperature and enhanced stratification. Consistency in data collection and analysis methods is necessary to achieve fully comparable datasets and for statistical validity, avoiding issues linked to disparate data sources. The European Multi Lake Survey (EMLS) in summer 2015 was an initiative among scientists from 27 countries to collect and analyse lake physical, chemical and biological variables in a fully standardized manner. This database includes in-situ lake variables along with nutrient, pigment and cyanotoxin data of 369 lakes in Europe, which were centrally analysed in dedicated laboratories. Publishing the EMLS methods and dataset might inspire similar initiatives to study across large geographic areas that will contribute to better understanding lake responses in a changing environment., The authors acknowledge COST Action ES 1105 “CYANOCOST – Cyanobacterial blooms and toxins in water resources: Occurrence impacts and management” and COST Action ES 1201 “NETLAKE – Networking Lake Observatories in Europe” for contributing to this study through networking and knowledge sharing with European experts in the field. Evanthia Mantzouki was supported by a grant from the Swiss State Secretariat for Education, Research and Innovation (SERI) to Bas Ibelings and by supplementary funding from University of Geneva. We thank Wendy Beekman for the nutrient analysis and the University of Wageningen for covering the costs of this analysis from the personal funding of dr. Miquel Lürling. We thank Pieter Slot for assisting with the pigment analysis and the University of Amsterdam for covering the costs of the analysis through funding from the group of Prof. Jef Huisman and dr. Petra Visser (IBED). We would like to thank the Environmental Data Initiative for covering the cost of archiving the EMLS dataset. We would like to thank the Leibniz Institute of Freshwater Ecology and Inland Fisheries, Dept. of Experimental Limnology and the Aquatic Microbial Ecology Group for logistic and technical support of J. Fonvielle and H.-P. Grossart; and the Leibniz Association for financial support. The collection of data for Lough Erne and Lough Neagh were funded by the Department of Agriculture, Environment and Rural Affairs, Northern Ireland.

Nostoc is a cyanobacterial genus, common in soils and a prolific producer of natural products. This research project aimed to explore and characterize Brazilian cyanobacteria for new bioactive compounds. Here we report the production of hepatotoxins and new protease inhibitors from benthic Nostoc sp. CENA543 isolated from a small, shallow, saline-alkaline lake in the Nhecolandia, Pantanal wetland area in Brazil. Nostoc sp. CENA543 produces exceptionally high amounts of nodularin-R. This is the first free-living Nostoc that produces nodularin at comparable levels as the toxic, bloom-forming, Nodularia spumigena. We also characterized pseudospumigins A-F, which are a novel family of linear tetrapeptides. Pseudospumigins are structurally related to linear tetrapeptide spumigins and aeruginosins both present in N. spumigena but differ in respect to their diagnostic amino acid, which is Ile/Leu/Val in pseudospumigins, Pro/mPro in spumigins, and Choi in aeruginosins. The pseudospumigin gene cluster is more similar to the spumigin biosynthetic gene cluster than the aeruginosin gene cluster. Pseudospumigin A inhibited trypsin (IC50 4.5 mu M after 1 h) in a similar manner as spumigin E from N. spumigena but was almost two orders of magnitude less potent. This study identifies another location and environment where the hepatotoxic nodularin has the potential to cause the death of eukaryotic organisms.

The growth and reproduction of animals is affected by their access to resources. In aquatic ecosystems, the availability of essential biomolecules for filter-feeding zooplankton depends greatly on phytoplankton. Here, we analyzed the biochemical composition, i.e., the fatty acid, sterol and amino acid profiles and concentrations as well as protein, carbon, nitrogen, and phosphorus content of 17 phytoplankton monocultures representing the seven most abundant phytoplankton classes in boreal and sub-arctic lakes. To examine how the differences in the biochemical composition between phytoplankton classes affect their nutritional quality for consumers, we assessed the performance of Daphnia, on these diets. Furthermore, we defined the most important biomolecules regulating the somatic growth and reproduction of Daphnia, expecting that higher concentrations of certain biomolecules are needed for reproduction than for growth. Finally, we combined these results with phytoplankton field data from over 900 boreal and sub-arctic lakes in order to estimate whether the somatic growth of Daphnia is sterol-limited when the natural phytoplankton communities are cyanobacteria-dominated. Our analysis shows that Daphnia grows best with phytoplankton rich in sterols, ω-3 fatty acids, protein, and amino acids. Their reproduction follows food sterol and ω-3 concentration as well as C:P-ratio being two times higher in Daphnia feeding on cryptophytes than any other diet. Interestingly, we found that a high dietary ω-6 fatty acid concentration decreases both somatic growth and reproduction of Daphnia. When combined with phytoplankton community composition field data, our results indicate that zooplankton is constantly limited by sterols in lakes dominated by cyanobacteria (≥40% of total phytoplankton biomass), and that the absence of cryptophytes can severely hinder zooplankton production in nature. peerReviewed

For 50 years persistent cyanobacterial blooms have been observed in Lake Ludo&scaron, (Serbia), a wetland area of international significance listed as a Ramsar site. Cyanobacteria and cyanotoxins can affect many organisms, including valuable flora and fauna, such as rare and endangered bird species living or visiting the lake. The aim was to carry out monitoring, estimate the current status of the lake, and discuss potential resolutions. Results obtained showed: (a) the poor chemical state of the lake, (b) the presence of potentially toxic (genera Dolichospermum, Microcystis, Planktothrix, Chroococcus, Oscillatoria, Woronichinia and dominant species Limnothrix redekei and Pseudanabaena limnetica) and invasive cyanobacterial species Raphidiopsis raciborskii, (c) the detection of microcystin (MC) and saxitoxin (STX) coding genes in biomass samples, (d) the detection of several microcystin variants (MC-LR, MC-dmLR, MC-RR, MC-dmRR, MC-LF) in water samples, (e) histopathological alterations in fish liver, kidney and gills. The potential health risk to all organisms in the ecosystem and the ecosystem itself is thus still real and present. Although there is still no resolution in sight, urgent remediation measures are needed to alleviate the incessant cyanobacterial problem in Lake Ludo&scaron, to break this ecosystem out of the perpetual state of limbo in which it has been trapped for quite some time.

We modified the stable isotope mixing model MixSIR to infer primary producer contributions to consumer diets based on their fatty acid composition. To parameterize the algorithm, we generated a 'consumer-resource library' of FA signatures of Daphnia fed different algal diets, using 34 feeding trials representing diverse phytoplankton lineages. This library corresponds to the resource or producer file in classic Bayesian mixing models such as MixSIR or SIAR. Because this library is based on the FA profiles of zooplankton consuming known diets, and not the FA profiles of algae directly, trophic modification of consumer lipids is directly accounted for. To test the model, we simulated hypothetical Daphnia comprised of 80% diatoms, 10% green algae, and 10% cryptophytes and compared the FA signatures of these known pseudo-mixtures to outputs generated by the mixing model. The algorithm inferred these simulated consumers were comprised of 82% (63-92%) [median (2.5th to 97.5th percentile credible interval)] diatoms, 11% (4-22%) green algae, and 6% (0-25%) cryptophytes. We used the same model with published phytoplankton stable isotope (SI) data for delta C-13 and delta N-15 to examine how a SI based approach resolved a similar scenario. With SI, the algorithm inferred that the simulated consumer assimilated 52% (4-91%) diatoms, 23% (1-78%) green algae, and 18% (1-73%) cyanobacteria. The accuracy and precision of SI based estimates was extremely sensitive to both resource and consumer uncertainty, as well as the trophic fractionation assumption. These results indicate that when using only two tracers with substantial uncertainty for the putative resources, as is often the case in this class of analyses, the underdetermined constraint in consumer-resource SI analyses may be intractable. The FA based approach alleviated the underdetermined constraint because many more FA biomarkers were utilized (n < 20), different primary producers (e.g., diatoms, green algae, and cryptophytes) have very characteristic FA compositions, and the FA profiles of many aquatic primary consumers are strongly influenced by their diets.

Salinity is one of the main factors that explain the distribution of species in the Baltic Sea. Increased precipitation and consequent increase in freshwater inflow is predicted to decrease salinity in some areas of the Baltic Sea. Clearly such changes may have profound effects on the organisms living there. Here we investigate the response of the commonly occurring cyanobacterium Dolichospermum spp. to three salinities, 0, 3 and 6. For the three strains tested we recorded growth, intracellular toxicity (microcystin) and allelopathic properties. We show that Dolichospermum can grow in all the three salinities tested with highest growth rates in the lowest salinity. All strains showed allelopathic potential and it differed significantly between strains and salinities, but was highest in the intermediate salinity and lowest in freshwater. Intracellular toxin concentration was highest in salinity 6. In addition, based on monitoring data from the northern Baltic Proper and the Gulf of Finland, we show that salinity has decreased, while Dolichospermum spp. biomass has increased between 1979 and 2013. Thus, based on our experimental findings it is evident that salinity plays a large role in Dolichospermum growth, allelopathic properties and toxicity. In combination with our long-term data analyses, we conclude that decreasing salinity is likely to result in a more favourable environment for Dolichospermum spp. in some areas of the Baltic Sea.

The smaller the phytoplankton, the greater effort is required to distinguish individual cells by optics-based methods. Flow cytometry is widely applied in marine picophytoplankton research, but in freshwater research its role has remained minor. We compared epifluorescence microscopy and flow cytometry in assessing the composition, abundance and cell sizes of autofluorescent picophytoplankton in epilimnia of 46 Finnish lakes. Phycocyaninrich picocyanobacteria were the most dominant. The two methods yielded comparable total picophytoplankton abundances, but the determination of cell sizes, and thus total biomasses, were on average an order of magnitude higher in the microscopy results. However, flow cytometry yielded higher cell sizes when applied on small-celled cultured algae. Our study demonstrated that both epifluorescence microscopy and flow cytometry are useful methods in assessing abundances of phycocyanin-rich and phycoerythrin-rich picocyanobacteria and eukaryotic picophytoplankton in lakes. However, accurate determination of cell size and biomass remain challenges for microscopy and especially for flow cytometry. peerReviewed