Andreas Ballot

Anatoxin-a producing Tychonema (Cyanobacteria) in European waterbodies

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 2.6xa0μgxa0mm(-3), and 0.01 and 0.35xa0pgxa0cell(-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.

A review of the phylogeny, ecology and toxin production of bloom-forming Aphanizomenon spp. and related species within the Nostocales (cyanobacteria)

The traditional genus Aphanizomenon comprises a group of filamentous nitrogen-fixing cyanobacteria of which several memebers are able to develop blooms and to produce toxic metabolites (cyanotoxins), including hepatotoxins (microcystins), neurotoxins (anatoxins and saxitoxins) and cytotoxins (cylindrospermopsin). This genus, representing geographically widespread and extensively studied cyanobacteria, is in fact heterogeneous and composed of at least five phylogenetically distant groups (Aphanizomenon, Anabaena/Aphanizomenon like cluster A, Cuspidothrix, Sphaerospermopsis and Chrysosporum) whose taxonomy is still under revision. This review provides a thorough insight into the phylogeny, ecology, biogeography and toxicogenomics (cyr, sxt, and ana genes) of the five best documented Aphanizomenon species with special relevance for water risk assessment: Aphanizomenon flos-aquae, Aphanizomenon gracile, Cuspidothrix issatschenkoi, Sphaerospermopsis aphanizomenoides and Chrysosporum ovalisporum. Aph. flos-aquae, Aph. gracile and C. issatschenkoi have been reported from temperate areas only whereas S. aphanizomenoides shows the widest distribution from the tropics to temperate areas. Ch. ovalisporum is found in tropical, subtropical and Mediterranean areas. While all five species show moderate growth rates (0.1-0.4day-1) within a wide range of temperatures (15-30°C), Aph. gracile and A. flos-aquae can grow from around (or below) 10°C, whereas Ch. ovalisporum and S. aphanizomenoides are much better competitors at high temperatures over 30°C or even close to 35°C. A. gracile has been confirmed as the producer of saxitoxins and cylindrospermopsin, C. issatschenkoi of anatoxins and saxitoxins and Ch. ovalisporum of cylindrospermopsin. The suspected cylindrospermopsin or anatoxin-a production of A. flos-aquae or microcystin production of S. aphanizomenoides is still uncertain. This review includes a critical discussion on the the reliability of toxicity reports and on the invasive potential of Aphanizomenon species in a climate change scenario, together with derived knowledge gaps and research needs. As a whole, this work is intended to represent a key reference for scientists and water managers involved in the major challenges of identifying, preventing and mitigating toxic Aphanizomenon blooms.

Thiol Derivatization for LC-MS Identification of Microcystins in Complex Matrices

Microcystins are a group of cyclic heptapeptides originating from cyanobacteria. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analyzed by LC-MS. Derivatization with appropriate thiols (e.g., mercaptoethanol) of the olefin in the α,β-unsaturated amide present in most microcystins was shown to simplify analysis of LC-MS chromatograms of sample extracts, making it much easier to identify peaks corresponding to candidate microcystins. Furthermore, interpretation of MS(2) spectra was facilitated by addition of the mass associated with the thiol to the α,β-unsaturated amide of microcystins. Cyanotoxins containing Mdha or Dha reacted readily with thiols, whereas Mser, Ser, Mdhb, and thiol-derivatives of Mdha or Dha did not react under the conditions used. This approach therefore provides a convenient LC-MS method to obtain evidence for the presence of Mdha or Dha and can likely be used to differentiate between the isobaric amino acids Mdha and Dhb in candidate cyanotoxin peaks. When O-(2-mercaptoethyl)-O-methyl-hexa(ethylene glycol) (MEMHEG) (M(w)t. 356) was used as the thiol, the resulting derivatives eluted in an LC-MS mass window that was largely free of interferences. This approach simplifies detection of candidate microcystin analogues even in the presence of complex mixtures of coeluting components. The method was used for qualitative analysis of a Microcystis aeruginosa culture from Lake Naivasha, Kenya, and the results were verified using precursor-ion scanning and high-resolution mass spectrometry.

Diversity of cyanobacteria and cyanotoxins in Hartbeespoort Dam, South Africa

The South African Hartbeespoort Dam is known for the occurrence of heavy Microcystis blooms. Although a few other cyanobacterial genera have been described, no detailed study on those cyanobacteria and their potential toxin production has been conducted. The diversity of cyanobacterial species and toxins is most probably underestimated. To ascertain the cyanobacterial composition and presence of cyanobacterial toxins in Hartbeespoort Dam, water samples werecollectedinApril2011.Inapolyphasicapproach,27isolatedcyanobacterialstrainswereclassifiedmorphologically and phylogenetically and tested for microcystins (MCs), cylindrospermopsin (CYN), saxitoxins (STXs) and anatoxin-a (ATX) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and screened for toxin-encoding gene fragments. The isolated strains were identified as Sphaerospermopsis reniformis, Sphaerospermopsis aphanizomenoides, Cylindrospermopsis curvispora, Raphidiopsis curvata, Raphidiopsis mediterrranea and Microcystis aeruginosa. Only one of the Microcystis strains (AB2011/53) produced microcystins (35 variants). Forty-one microcystin variants were detected in the environmental sample from Hartbeespoort Dam, suggesting the existence of other microcystin producing strains in Hartbeespoort Dam. All investigated strains tested negative for CYN, STXs and ATX and their encoding genes. ThemcyEgeneofthemicrocystingeneclusterwasfoundinthemicrocystin-producingMicrocystisstrainAB2011/53and in eight non-microcystin-producing Microcystis strains, indicating that mcyE is not a good surrogate for microcystin production in environmental samples. Additional keywords: Cylindrospermopsis, Hartbeespoort Dam, microcystin, Microcystis, Raphidiopsis, Sphaerospermopsis.

Multihapten Approach Leading to a Sensitive ELISA with Broad Cross-Reactivity to Microcystins and Nodularin

Microcystins (MCs) are a group of biotoxins (>150) produced by cyanobacteria, with a worldwide distribution. MCs are hepatotoxic, and acute exposure causes severe liver damage in humans and animals. Rapid and cheap methods of analysis are therefore required to protect people and livestock, especially in developing countries. To include as many MCs as possible in a single analysis, we developed a new competitive ELISA. Ovine polyclonal antibodies were raised using an immunogen made by conjugating a mixture of microcystins to cationised bovine serum albumin, and the plate-coating antigen was prepared by conjugating [Asp3]MC-RY to ovalbumin. This strategy was used also to minimize specificity for particular microcystin congeners. Cross-reactivity studies indicate that the ELISA has broad specificity to microcystins and also detects nodularin, providing a sensitive and rapid analytical method for screening large numbers of samples. The limit of quantitation for microcystins in drinking water is 0.04 μg/L, well below the WHOs maximum recommendation of 1 μg/L. The ELISA can be used for quantifying total microcystins in various matrices, including drinking water, cyanobacterial cultures, extracts, and algal blooms, and may be useful in detecting metabolites and conjugates of MCs.

Identification of microcystins in a Lake Victoria cyanobacterial bloom using LC–MS with thiol derivatization

Microcystins are cyclic heptapeptides from cyanobacteria which are responsible for poisonings of livestock and humans. Cyanobacteria also produce a range of peptides and other compounds that can result in complex chromatograms when samples are analysed by LC-MS. Thiol derivatization of the α,β-unsaturated amide present in most microcystins was recently shown to simplify analysis of LC-MS chromatograms of a Microcystis culture, making it easier to identify peaks corresponding to microcystins in complex mixtures. This method was applied to analysis of extracts taken from a natural cyanobacteria bloom in Mwanza Gulf, Lake Victoria, Tanzania, in 2010, revealing the presence of numerous putative microcystin analogues in the sample. Results were verified using LC-MS², LC-MS/MS with precursor-ion scanning, and LC-HRMS, leading to identification of 8 major and 17 minor microcystins in the sample, including analogues of microcystin-RY, -RL and -RA. Microcystin-YR (2), -RR (3), and -RY (9) were isolated from bloom material from Lake Victoria, and the structure of 9 was confirmed by NMR spectroscopic analysis and NMR spectral comparison with 2 and 3. Confirmation of the structure of MC-RY (9) facilitated detailed analysis of its MS² spectrum, thereby supporting the structures of related analogues tentatively established on the basis of MS analyses.

The occurrence and spread of Gonyostomum semen (Ehr.) Diesing (Raphidophyceae) in Norwegian lakes

The invasive and nuisance microalga Gonyostomum semen has recently expanded its geographical distribution and increased its biomass in Sweden and Finland. Temperatures, total organic carbon (TOC), water colour and phosphorous are main factors suggested as drivers for its success. Already in the 1980s there were indications of the same patterns also in Norway, and in this study we further examine these observations based on the hypotheses that (1) G. semen has continued its increase in abundance in Norway since then, (2) after settling in a lake, G. semen may increase its biomass, provided a simultaneous change in climatic factors. We use existing data from Norwegian monitoring databases for the study and additional water samples to investigate distribution. G. semen was detected by microscopy and quantitative PCR, while trends over time in G. semen biomass and possible explanatory variables were analysed by simple regression. We show that G. semen has increased its distribution in Norway since the 1980s, geographically and to new lakes. In eight of the nine lakes studied, the proportion of G. semen biomass in lake phytoplankton has increased with time. These changes seem to be explained only to some degree by temperature, TOC, water colour and phosphorous.

DNA barcoding the genus Chara: molecular evidence recovers fewer taxa than the classical morphological approach

Charophytes (Charales) are benthic algae with a complex morphology. They are vulnerable to ecosystem changes, such as eutrophication, and are red‐listed in many countries. Accurate identification of Chara species is critical for understanding their diversity and for documenting changes in species distribution. Species delineation is, however, complicated, because of high phenotypic plasticity. We used barcodes of the ITS2, matK and rbcL regions to test if the distribution of barcode haplotypes among individuals is consistent with species boundaries as they are currently understood. The study included freshly collected and herbarium material of 91 specimens from 10 European countries, Canada and Argentina. Results showed that herbarium specimens are useful as a source of material for genetic analyses for aquatic plants like Chara. rbcL and matK had highest sequence recoverability, but rbcL had a somewhat lower discriminatory power than ITS2 and matK. The tree resulting from the concatenated data matrix grouped the samples into six main groups contrary to a traditional morphological approach that consisted of 14 different taxa. A large unresolved group consisted of C. intermedia, C. hispida, C. horrida, C. baltica, C. polyacantha, C. rudis, C. aculeolata, and C. corfuensis. A second unresolved group consisted of C. virgata and C. strigosa. The taxa within each of the unresolved groups shared identical barcode sequences on the 977 positions of the concatenated data matrix. The morphological differences of taxa within both unresolved groups include the number and length of spine cells, stipulodes, and bract cells. We suggest that these morphological traits have less taxonomic relevance than hitherto assumed.

Biogeography of bloom-forming microcystin producing and non-toxigenic populations of Dolichospermum lemmermannii (Cyanobacteria)

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.

Conjugation of Microcystins with Thiols Is Reversible: Base-Catalyzed Deconjugation for Chemical Analysis

Microcystins are potent cyclic heptapeptide toxins found in many freshwater cyanobacteria. Most microcystins contain an α,β-unsaturated amide that can react with thiol-containing amino acids, peptides, and proteins in vivo and in vitro. While soluble conjugates formed from small peptides can be extracted and analyzed directly by LC-MS, microcystins conjugated to proteins are analyzed after oxidative cleavage of their Adda side chains, but information on which microcystin analogues were present is lost. Observations during the development of thiol-derivatization-based LC-MS methods for microcystin analysis indicated that the reaction of thiols with microcystins was reversible. The kinetics of deconjugation was investigated with mercaptoethanol as a model thiol to identify suitable reaction conditions. A range of microcystins conjugated to mercaptoethanol, methanethiol, cysteine, and glutathione were then successfully deconjugated, demonstrating the feasibility of releasing conjugated forms of microcystins for chemical analysis. Reagents for removing the released thiols or for trapping the released microcystins increased the reaction rate. Optimization of methodologies based on this reaction should increase the methods utility for measuring free and conjugated microcystins. The results also indicate that thiol-conjugated microcystins slowly release free microcystins, even at neutral pH, with consequences for assessment of toxin exposure, metabolism, and trophic transfer. A range of other common natural and environmental toxins, such as deoxynivalenol and acrylamide, also contain α,β-unsaturated carbonyl groups and can be expected to behave in a similar manner.