title: Molecular tools to detect anatoxin-a genes in aquatic ecosystems: Toward a new nested PCR-based method.
abstract: Over the last few decades, cyanobacterial mass occurrence has become a recurrent feature of aquatic ecosystems. This has led to ecosystem exposure and health hazards associated with cyanotoxin production. The neurotoxin anatoxin-a and its homologs can be synthesized by benthic cyanobacterial species in lotic systems, but also by planktonic lacustrine species such as Dolichospermum (also known as Anabaena). However, only a few studies have focused on anatoxin-a occurrence and its biosynthesis genes in freshwater lakes. The initial aim of this study was to evaluate the molecular tools available in the literature to detect anatoxin-a biosynthesis genes in lacustrine environments. Having tested different sets of PCR primers, we found that that some sets of primers, such as anxC, were too specific and did not amplify anatoxin-a biosynthesis genes in all producing strains. On the other hand, some sets of primers, such as atxoa, seemed not to be specific enough, amplifying numerous non-specific bands in environmental samples, especially those from sediments. Furthermore, anaC and anaF amplification exhibited different band intensities during electrophoresis, suggesting a high variation in number of gene copies between samples. As a result, we proposed a new nested PCR-based method which considerably improved the amplification of the anaC gene in our environmental samples, eliminating non-specific bands and weak detections. Using this tool, our study also highlighted that anatoxin-a genes are widely distributed throughout freshwater lakes. This suggests the need for further ecological investigations into anatoxin-a in these ecosystems.
authors.affiliation: Université Clermont Auvergne, Université Blaise Pascal, LMGE, BP 10448, Clermont-Ferrand F-63000, France; CNRS, UMR 6023, LMGE, Campus Universitaire des Cézeaux, France; Athos-Environnement, 112 Avenue du Brézet, Clermont-Ferrand F-63100, France. Electronic address: benjaminlegrand02000@yahoo.fr.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Legrand
authors.affiliation: Université Clermont Auvergne, Université Blaise Pascal, LMGE, BP 10448, Clermont-Ferrand F-63000, France; CNRS, UMR 6023, LMGE, Campus Universitaire des Cézeaux, France.
authors.forename: Jerome
authors.initials: J
authors.lastname: Lesobre
authors.affiliation: Université Clermont Auvergne, Université Blaise Pascal, LMGE, BP 10448, Clermont-Ferrand F-63000, France; CNRS, UMR 6023, LMGE, Campus Universitaire des Cézeaux, France.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Colombet
authors.affiliation: Université Clermont Auvergne, Université Blaise Pascal, LMGE, BP 10448, Clermont-Ferrand F-63000, France; CNRS, UMR 6023, LMGE, Campus Universitaire des Cézeaux, France.
authors.forename: Delphine
authors.initials: D
authors.lastname: Latour
authors.affiliation: Université Clermont Auvergne, Université Blaise Pascal, LMGE, BP 10448, Clermont-Ferrand F-63000, France; CNRS, UMR 6023, LMGE, Campus Universitaire des Cézeaux, France.
authors.forename: Marion
authors.initials: M
authors.lastname: Sabart
mesh_terms: D001427:Bacterial Toxins / Q000235:genetics*; D000458:Cyanobacteria / Q000235:genetics; D000087522:Cyanobacteria Toxins; D004784:Environmental Monitoring / Q000379:methods*; D005618:Fresh Water / Q000737:chemistry* / Q000382:microbiology; D016133:Polymerase Chain Reaction; D014326:Tropanes
chemical_list: D001427:Bacterial Toxins; D000087522:Cyanobacteria Toxins; D014326:Tropanes; C509783:anatoxin a
keywords: Anatoxin-a; Cyanobacteria; Freshwater lakes; Nested PCR; anaC and anaF genes