title: Anatoxin-a producing Tychonema (Cyanobacteria) in European waterbodies.
abstract: 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.
authors.affiliation: IASMA Research and Innovation Centre, Istituto Agrario di S. Michele all'Adige - Fondazione E. Mach, Via E. Mach 1, 38010 S. Michele all'Adige (Trento), Italy; Human and Environmental Toxicology Group, Department of Biology, University of Konstanz, P.O. Box X-622, D-78457 Konstanz, Germany.
authors.forename: S
authors.initials: S
authors.lastname: Shams
authors.affiliation: IASMA Research and Innovation Centre, Istituto Agrario di S. Michele all'Adige - Fondazione E. Mach, Via E. Mach 1, 38010 S. Michele all'Adige (Trento), Italy; Department of Biology, University of Florence, Via La Pira 4, 50121 Florence, Italy.
authors.forename: C
authors.initials: C
authors.lastname: Capelli
authors.affiliation: IASMA Research and Innovation Centre, Istituto Agrario di S. Michele all'Adige - Fondazione E. Mach, Via E. Mach 1, 38010 S. Michele all'Adige (Trento), Italy.
authors.forename: L
authors.initials: L
authors.lastname: Cerasino
authors.affiliation: Norwegian Institute for Water Research (NIVA), Gaustadalléen 21, NO-0349 Oslo, Norway.
authors.forename: A
authors.initials: A
authors.lastname: Ballot
authors.affiliation: Human and Environmental Toxicology Group, Department of Biology, University of Konstanz, P.O. Box X-622, D-78457 Konstanz, Germany.
authors.forename: D R
authors.initials: DR
authors.lastname: Dietrich
authors.affiliation: Department of Food and Environmental Sciences, Division of Microbiology and Biotechnology, P.O.Box 56, Biocenter 1 Viikki (Viikinkaari 9), FIN-00014, Helsinki University, Finland.
authors.forename: K
authors.initials: K
authors.lastname: Sivonen
authors.affiliation: IASMA Research and Innovation Centre, Istituto Agrario di S. Michele all'Adige - Fondazione E. Mach, Via E. Mach 1, 38010 S. Michele all'Adige (Trento), Italy. Electronic address: nico.salmaso@fmach.it.
authors.forename: N
authors.initials: N
authors.lastname: Salmaso
mesh_terms: D002853:Chromatography, Liquid; D000458:Cyanobacteria / Q000302:isolation & purification / Q000378:metabolism*; D000087522:Cyanobacteria Toxins; D004777:Environment; D007558:Italy; D060106:Lakes / Q000382:microbiology*; D013058:Mass Spectrometry; D009664:Norway; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D013499:Surface Properties; D014326:Tropanes / Q000378:metabolism*
chemical_list: D000087522:Cyanobacteria Toxins; D014326:Tropanes; C509783:anatoxin a
keywords: Anatoxin-a; Cell quota; European waterbodies; Phylogenetic analysis; Polyphasic approach; Tychonema