Occurrence of cylindrospermopsin, anatoxin-a and their homologs in the southern Czech Republic - Taxonomical, analytical, and molecular approaches.

Water bloom-forming cyanobacteria have a severe impact on freshwater quality. Although some cyanobacterial toxins such as microcystins have been studied extensively, other toxins like anatoxin-a (ATX) and their structural analogs - as well as cyanobacterial taxa producing these toxins remain to be explored in detail. The present study investigated levels of ATX, CYN and their homologs along with the occurrence of anaC and cyrJ genes in water blooms in 16 sites in the Czech Republic that were pre-selected concerning the presence of potential toxin producers. Besides, we also studied toxins and genes in a series of strains available in our laboratories. ATX and its congener HATX were detected in 5 natural biomass samples from the Czech Republic (maximum concentration 2.8 micrograms per gram d.w.). Interestingly, the anaC gene coding for ATX production was not detected in any of these toxin-positive biomass samples. The concentrations of ATX congeners in cyanobacterial laboratory strains were about 10-times higher than those of the original ATX, which calls for further research addressing levels and hazards of ATX analogs. Regarding the CYN and 7-deoxyCYN (other CYN congeners were not analyzed in this study) - these toxins were identified in a single small pond in the Czech Republic at concentrations 4.3 and 2.7 micrograms per gram of biomass d.w., respectively (corresponded to dissolved concentrations higher than 1 microgram per liter). The CYN-positive sample was dominated by CYN-producing taxa Raphidiopsis (basionym Cylindrospermopsis) and Cuspidothrix. We also confirmed the presence of a specific cyrJ gene in this natural bloom sample. To our knowledge, this is the first study pointing to Raphidiopsis (Cylindrospermopsis) and Cuspidothrix as producers of CYN in Europe. This observation calls for further research because of their increasing occurrence in (Central) Europe along with the global change. The present study demonstrates the importance of using combined (taxonomical, analytical, and molecular) approaches in the assessment of hazardous cyanobacteria and their toxins in freshwaters.

Development and Application of a Quantitative PCR Assay to Assess Genotype Dynamics and Anatoxin Content in Microcoleus autumnalis-Dominated Mats.

Microcoleus is a filamentous cyanobacteria genus with a global distribution. Some species form thick, cohesive mats over large areas of the benthos in rivers and lakes. In New Zealand Microcoleus autumnalis is an anatoxin producer and benthic proliferations are occurring in an increasing number of rivers nationwide. Anatoxin content in M. autumnalis-dominated mats varies spatially and temporally, making understanding and managing proliferations difficult. In this study a M. autumnalis-specific TaqMan probe quantitative PCR (qPCR) assay targeting the anaC gene was developed. The assay was assessed against 26 non-M. autumnalis species. The assay had a detection range over seven orders of magnitude, with a limit of detection of 5.14 × 10-8 ng µL-1. The anaC assay and a cyanobacterial specific 16S rRNA qPCR were then used to determine toxic genotype proportions in 122 environmental samples collected from 19 sites on 10 rivers in New Zealand. Anatoxin contents of the samples were determined using LC-MS/MS and anatoxin quota per toxic cell calculated. The percentage of toxic cells ranged from 0 to 30.3%, with significant (p < 0.05) differences among rivers. The anatoxin content in mats had a significant relationship with the percentage of toxic cells (R² = 0.38, p < 0.001), indicating that changes in anatoxin content in M. autumnalis-dominated mats are primarily related to the dominance of toxic strains. When applied to more extensive samples sets the assay will enable new insights into how biotic and abiotic parameters influence genotype composition, and if applied to RNA assist in understanding anatoxin production.

Benthic Archives Reveal Recurrence and Dominance of Toxigenic Cyanobacteria in a Eutrophic Lake over the Last 220 Years.

Akinetes are resistant cells which have the ability to persist in sediment for several decades. We have investigated the temporal distribution of akinetes of two species, Dolichospermum macrosporum and Dolichospermum flos-aquae, in a sediment core sampled in Lake Aydat (France), which covers 220 years. The upper part, from 1907 to 2016, the number of akinetes fluctuated but stayed at high concentrations, especially for D. macrosporum in surface sediment (with the maximal value close to 6.105 akinetes g DW-1 of sediment), suggesting a recurrence of blooms of this species which was probably closely related to anthropic eutrophication since the 1960s. Before 1907, the abundance of akinetes of both species was very low, suggesting only a modest presence of these cyanobacteria. In addition, the percentage of intact akinetes was different for each species, suggesting different ecological processes in the water column. This percentage also decreased with depth, revealing a reduction in germination potential over time. In addition, biosynthetic genes of anatoxin-a (anaC) and microcystin (mcyA) were detected. First results show a high occurrence of mcyA all down the core. In contrast, anaC gene was mostly detected in the surface sediment (since the 1980s), revealing a potentially more recent occurrence of this cyanotoxin in Lake Aydat which may be associated with the recurrence of blooms of D. macrosporum and thus with anthropic activities.