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The increasing frequency of cyanobacteria blooms in waterbodies caused by ecosystem eutrophication could endanger human health. This risk can be mitigated by effective monitoring incorporating molecular methods. To date, most molecular studies on toxigenic cyanobacteria have been limited to microcystins (MCs), disregarding other cyanotoxins, to freshwater planktic habitats while ignoring benthic habitats, and to limited geographic areas (usually one or a few specific waterbodies). In this study, we used PCR-based methods including PCR product sequencing and chemical-analytical methods (LC-MS/MS) to screen many plankton (n = 123) and biofilm samples (n = 113) originating from 29 Alpine lakes and 18 rivers for their cyanotoxin production potential. Both mcyE (indicating MC synthesis) and anaC (indicating anatoxin (ATX) synthesis) gene fragments were able to qualitatively predict MC or ATX occurrence. The abundance of mcyE gene fragments was significantly related to MC concentrations in plankton samples (R 2 = 0.61). mcyE gene fragments indicative of MC synthesis were most abundant in planktic samples (65 %) and were assigned to the genera Planktothrix and Microcystis. However, mcyE rarely occurred in biofilms of lakes and rivers, i.e., 4 % and 5 %, respectively, and were assigned to Microcystis, Planktothrix, and Nostoc. In contrast, anaC gene fragments occurred frequently in planktic samples (14 % assigned to Tychonema, Phormidium (Microcoleus), and Oscillatoria), but also in biofilms of lakes (49 %) and rivers (18 %) and were assigned to the genera Phormidium, Oscillatoria, and Nostocales. The cyrJ gene fragment indicating cylindrospermopsin synthesis occurred only once in plankton (assigned to Dolichospermum), while saxitoxin synthesis potential was not detected. For plankton samples, monomictic and less eutrophic conditions were positively related to mcyE/MC occurrence frequency, while oligomictic conditions were related to anaC/ATX frequency. The anaC/ATX frequency in biofilm was related to the lake habitats generally showing higher biodiversity as revealed from metabarcoding in a parallel study., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

The taxonomic identification of organisms based on the amplification of specific genetic markers (metabarcoding) implicitly requires adequate discriminatory information and taxonomic coverage of environmental DNA sequences in taxonomic databases. These requirements were quantitatively examined by comparing the determination of cyanobacteria and microalgae obtained by metabarcoding and light microscopy. We used planktic and biofilm samples collected in 37 lakes and 22 rivers across the Alpine region. We focused on two of the most used and best represented genetic markers in the reference databases, namely the 16S rRNA and 18S rRNA genes. A sequence gap analysis using blastn showed that, in the identity range of 99-100%, approximately 30% (plankton) and 60% (biofilm) of the sequences did not find any close counterpart in the reference databases (NCBI GenBank). Similarly, a taxonomic gap analysis showed that approximately 50% of the cyanobacterial and eukaryotic microalgal species identified by light microscopy were not represented in the reference databases. In both cases, the magnitude of the gaps differed between the major taxonomic groups. Even considering the species determined under the microscope and represented in the reference databases, 22% and 26% were still not included in the results obtained by the blastn at percentage levels of identity ≥95% and ≥97%, respectively. The main causes were the absence of matching sequences due to amplification and/or sequencing failure and potential misidentification in the microscopy step. Our results quantitatively demonstrated that in metabarcoding the main obstacles in the classification of 16S rRNA and 18S rRNA sequences and interpretation of high-throughput sequencing biomonitoring data were due to the existence of important gaps in the taxonomic completeness of the reference databases and the short length of reads. The study focused on the Alpine region, but the extent of the gaps could be much greater in other less investigated geographic areas., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Many studies have focused on the interactive effects of temperature increases due to global warming and nutrient enrichment on phytoplankton communities. Recently, non-temperature effects of climate change (e.g., decreases in wind speed and increases in solar radiation) on large lakes have received increasing attention. To evaluate the relative contributions of both temperature and non-temperature effects on phytoplankton communities in a large eutrophic subtropical shallow lake, we analyzed long-term monitoring data from Lake Taihu, China from 1997 to 2016. Results showed that Lake Taihu's spring phytoplankton biovolume and composition changed dramatically over this time frame, with a change in dominant species. Stepwise multiple linear regression models indicated that spring phytoplankton biovolume was strongly influenced by total phosphorus (TP), light condition, wind speed and total nitrogen (TN) (radj2 = 0.8, p < 0.01). Partial redundancy analysis (pRDA) showed that light condition accounted for the greatest variation of phytoplankton community composition, followed by TP and wind speed, as well as the interactions between TP and wind speed. Our study points to the additional importance of non-temperature effects of climate change on phytoplankton community dynamics in Lake Taihu., Competing Interests: The authors have declared that no competing interests exist.

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., Competing Interests: The authors declare no conflict of interest.

Lake water level fluctuations (WLF) are an important factor driving the selection and seasonal dynamics of phytoplankton and potentially toxigenic cyanobacteria. Nevertheless, the relative importance of environmental drivers connected to WLF may be completely different, depending on the typology and use of waterbodies, latitude and climatic regimes. In this study, we investigated the impact of WLF in a large subtropical reservoir in south-eastern China (Hongfeng Reservoir, Guizhou Province). The study was based on monthly samplings carried out in 2014 in six stations. The strong increase in the water level observed in early summer caused a radical shift in the phytoplankton community. While in the pre-flooding period phytoplankton was composed of large diatoms, chrysophytes and Oscillatoriales (mostly Limnothrix sp.), the post-flooding period showed an increase in smaller and more competitive chlorophytes, smaller diatoms and cryptophytes better adapted to a fast colonisation of new and nutrient-rich environments. The environmental drivers that drove the change were dilution, flushing and interference with the seasonal water stratification processes. We concluded that, because WLF represents a complex variable integrating different physical effects in one explanatory descriptor, its value as a predictor of phytoplankton and cyanobacteria dynamics in lake ecosystems is difficult to generalise and needs to be investigated on a case-by-case basis. For this reason, considering the year-to-year hydrological variability that potentially characterise reservoirs, definite indications for management should be outlined considering more than 1-year study.

In the last decades, the cyanobacterium Dolichospermum lemmermannii showed an increasing spread to Southern Europe, raising serious concerns due to its ability to produce cyanotoxins. The widening of its geographic distribution and the observation of strains showing high optimum temperature underline its ecological heterogeneity, suggesting the existence of different ecotypes. To investigate its biogeography, new isolates from different European water bodies, together with strains maintained by the Norwegian Institute for Water Research Culture Collection of Algae, were genetically characterised for the 16S rRNA gene and compared with strains obtained from public repositories. Geographic distance highly influenced the differentiation of genotypes, further suggesting the concurrent role of geographic isolation, physical barriers and environmental factors in promoting the establishment of phylogenetic lineages adapted to specific habitats. Differences among populations were also examined by morphological analysis and evaluating the toxic potential of single strains, which revealed the exclusive ability of North European strains to produce microcystins, whereas the populations in Southern Europe tested negative for a wide range of cyanotoxins. The high dispersion ability and the existence of toxic genotypes indicate the possible spread of harmful blooms in other temperate regions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This work allowed assessing a widespread occurrence of Tychonema bourrellyi in the largest lakes south of the Alps (Garda, Iseo, Como and Maggiore). The taxonomy of the species was confirmed adopting a polyphasic approach, which included microscopic examinations, molecular (16S rRNA and rbcLX sequences) and (Lake Garda) ecological characterisations. Over 70% of the 36 isolates of Tychonema sampled from the four lakes tested positive for the presence of genes implicated in the biosynthesis of anatoxins (anaF and/or anaC) and for the production of anatoxin-a (ATX) and homoanatoxin-a (HTX). A detailed analysis carried out in Lake Garda showed strong ongoing changes in the cyanobacterial community, with populations of Tychonema developing with higher biovolumes compared to the microcystins (MCs) producer Planktothrix rubescens Moreover, the time × depth distribution of Tychonema was paralleled by a comparable distribution of ATX and HTX. The increasing importance of Tychonema in Lake Garda was also suggested by the opposite trends of ATX and MCs observed since 2009. These results suggest that radical changes are occurring in the largest lakes south of the Alps. Their verification and implications will require to be assessed by extending a complete experimental work to the other large perialpine lakes., (© FEMS 2016. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

In order to identify the cyanobacterial species responsible of anatoxin-a (ATX) production in Lake Garda (Northern Italy), an intensive isolation and culturing of filamentous cyanobacteria were established since 2014 from environmental samples. In this work, we report a detailed account of the strategy adopted, which led to the discovery of a new unexpected producer of ATX, Tychonema bourrellyi. So far, this species is the first documented example of cultured Oscillatoriales able to produce ATX isolated from pelagic freshwater ecosystems. The isolated filaments were identified adopting a polyphasic approach, which included microscopic species identification, genetic characterisation and phylogenetic analyses based on 16S rRNA genes. The taxonomic identification was further confirmed by the high (>99%) rbcLX sequence similarities of the T. bourrellyi strains of Lake Garda with those deposited in DNA sequence databases. More than half of the isolates were shown to produce a significant amount of ATX, with cell quota ranging between 0.1 and 2.6 μg mm(-3), and 0.01 and 0.35 pg cell(-1). The toxic isolates were tested positive for anaC of the anatoxin-a synthetase (ana) gene cluster. These findings were confirmed with the discovery of one ATX producing T. bourrellyi strain isolated in Norway. This strain and a further non-ATX producing Norwegian Tychonema bornetii strain tested positive for the presence of the anaF gene of the ana gene cluster. Conversely, none of the Italian and Norwegian Tychonema strains were positive for microcystins (MCs), which was also confirmed by the absence of mcyE PCR products in all the samples analysed. This work suggests that the only reliable strategy to identify cyanotoxins producers should be based on the isolation of strains and their identification with a polyphasic approach associated to a concurrent metabolomic profiling., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, we investigated the relationships between microcystin (MCs) concentrations and the biovolumes of Planktothrix rubescens (BPr) in 2 natural lakes (Pusiano and Garda) and 2 artificially dammed reservoirs (Occhito and Ledro) in Italy. In all the considered water bodies, P. rubescens was the dominant cyanobacterium. All the lakes were characterized by significant relationships between MCs and BPr, with limited variability in the MC quota (the content of MCs per unit of biovolume) within each water body compared with the variability between sites. The results were consistent with the development of specific MC-genotypes, with moderate seasonal and spatial changes in the proportion between toxic and non-toxic strains. The MC cell quota obtained in our work (ECQ, Environmental Cell Quota) were in the same range of values computed on the basis of analyses made on environmental samples dominated by P. rubescens or Planktothrix agardhii, and on isolates of the same two species (<1 to over 10 μg mm(-3)). Besides the usual ordinary least square regressions, models have been evaluated by using quantile regression, a method that allows estimating the conditional median or other quantiles of the response variable. We showed that the use of quantile regressions has different advantages, which included the computation of MC quota based on the whole range of available data, the robustness against outliers, and the ability to estimate models also in cases where there is no or only weak relationships. The highest ECQ values estimated from 95% quantile regressions in specific water bodies might be used to estimate the worst-case MC concentrations from algal abundances. Nevertheless, it was stressed that a realistic assessment of toxicity and potential adverse health effects necessarily should take into account the toxicity potential of the more abundant MC-congeners produced by specific cyanobacteria populations., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

In this study, we investigated the kinetic aspects of the microcystin (MC) transfer from Planktothrix rubescens to Daphnia magna by carrying out exposure experiments in small simple mesocosms. We hypothesized that higher fractions of toxic cyanobacteria in the diet of grazers would shift the balance towards a greater than linear, i.e. non-linear accumulation of MC in D. magna. This hypothesis was tested by exposing D. magna to varying initial densities of MC-producing P. rubescens. The evolving models of MC accumulation differed largely as a result of the duration of exposure and initial MC concentrations used. Within the first 24h of exposure, MC accumulation in D. magna was linear, irrespective of the initial densities of toxic P. rubescens and thus MC concentrations. After 48 h of exposure, MC accumulation in D. magna showed an exponential pattern, possibly due to a delayed digestion of P. rubescens and/or decreased MC detoxification capabilities when compared with higher ambient concentrations of MC. After 72 h toxin concentrations in Daphnia drop in all experiments as a consequence of the reduced cyanobacterial cells in the medium and the detoxification of MC within Daphnia. The results obtained suggest that in lakes with higher MC content and longer cyanobacterial bloom period MC accumulation in D. magna should be more pronounced than in mesotrophic lakes with lower MC content. The latter interpretation, however, should be verified investigating accumulation of MC both in larger mesocosms and in situ, in lakes of different trophic status., (Copyright © 2014 Elsevier B.V. All rights reserved.)