Marion Sabart

Spatiotemporal Variations in Microcystin Concentrations and in the Proportions of Microcystin-Producing Cells in Several Microcystis aeruginosa Populations

ABSTRACT With the aim of explaining the variations in microcystin (MC) concentrations during cyanobacterial blooms, we studied several Microcystis aeruginosa populations blooming in different freshwater ecosystems located in the same geographical area. As assessed by real-time PCR, it appeared that the potentially MC-producing cells (mcyB+) were predominant (70 to 100%) in all of these M. aeruginosa populations, with the exception of one population in which non-MC-producing cells always dominated. Apart from the population in the Grangent Reservoir, we found that the proportions of potentially MC-producing and non-MC-producing cells varied little over time, which was consistent with the fact that according to a previous study of the same populations, the intergenic transcribed spacer (ITS) genotype composition did not change (38). In the Grangent Reservoir, the MC-RR variant was the dominant microcystin variant throughout the bloom season, despite changes in the ITS composition and in the proportions of mcyB+ cells. Finally, the variations in total MC concentrations (0.3 to 15 μg liter−1) and in the MC cellular quotas (0.01 to 3.4 pg cell−1) were high both between and within sites, and no correlation was found between the MC concentrations and the proportion of mcyB+ cells. All of these findings demonstrate that very different results can be found for the proportions of potentially MC-producing and non-MC-producing cells and MC concentrations, even in M. aeruginosa populations living in more or less connected ecosystems, demonstrating the importance of the effect of very local environmental conditions on these parameters and also the difficulty of predicting the potential toxicity of Microcystis blooms.

Spatiotemporal changes in the genetic diversity in French bloom-forming populations of the toxic cyanobacterium, Microcystis aeruginosa

Microcystis aeruginosa is a toxic cyanobacterium, which is able to bloom in a wide range of freshwater ecosystems. By sequencing the Internal Transcribed Spacer (ITS) of the ribosomal operon, we compared the genetic composition of several French bloom-forming M. aeruginosa populations from two reservoirs located on the Loire River, at two sampling points located between these reservoirs, and finally in two ponds closely linked to this river. No significant difference was found in the genetic diversity of the six Microcystis populations but we evidenced a strong genetic differentiation between most of these populations. Indeed, the Microcystis population in the Grangent reservoir was genetically differentiated from the other three populations sampled further downstream, implying that no massive transfer of population occurs from this reservoir to downstream segments. We also found genetic differentiation between the populations from the two ponds, and between these populations and those from the Loire River. On the other hand, the same dominant genotype was found in the populations sampled both in the river and in the Villerest reservoir, suggesting the selection of a distinct genotype adapted to river conditions and also an accumulation of this genotype in the downstream reservoir. Finally, by comparing our ITS sequences with those available in the GenBank, no biogeographical differentiation could be detected at a global scale, suggesting that most of the Microcystis genotypes seem to be ubiquitous.

Co-occurrence of microcystin and anatoxin-a in the freshwater lake Aydat (France): analytical and molecular approaches during a three-year survey.

Cyanobacterial mass occurrence is becoming a growing concern worldwide. They notably pose a threat to water users when cyanotoxins are produced. The aim of this study was to evaluate the occurrence and the dynamics of two cyanotoxins: microcystin (MC) and anatoxin-a (ANTX-a), and of two of the genes responsible for their production (respectively mcyA and anaC) during three consecutive bloom periods (2011, 2012 and 2013) in Lake Aydat (Auvergne, France). MC was detected at all sampling dates, but its concentration showed strong inter- and intra-annual variations. MC content did not correlate with cyanobacterial abundance, nor with any genera taken individually, but it significantly correlated with mcyA gene abundance (R2=0.51; p=0.042). MC content and mcyA gene abundance were maximal when cyanobacterial abundance was low, either at the onset of the bloom or during a trough of biomass. The LC-MS/MS analysis showed the presence of ANTX-a in the 2011 samples. To our knowledge, this is the first report of the presence of this neurotoxin in a French lake. The presence of ANTX-a corresponded to the only year for which Anabaena did not dominate the cyanobacterial community alone, and several cyanobacterial genera were present, including notably Aphanizomenon. anaC gene detection by PCR was not coherent with ANTX-a presence, both gene and toxin were never found for a same sample. This implies that molecular tools to study genes responsible for the production of anatoxin-a are still imperfect and the development of new primers is needed. This study also highlights the need for better monitoring practices that would not necessarily focus only on the peak of cyanobacterial abundance and that would take cyanotoxins other than MC into account.

The importance of small colonies in sustaining Microcystis population exposed to mixing conditions: an exploration through colony size, genotypic composition and toxic potential

Microcystis is a toxic colony-forming cyanobacterium, which can bloom in a wide range of freshwater ecosystems. Despite the ecological advantage of the colonial form, few studies have paid attention to the size of Microcystis colonies in the field. With the aim of evaluating the impact of a fluctuating physical environment on the colony size, the genotypic composition and the toxic potential of a Microcystis population, we investigated five different colony size classes of a Microcystis bloom in the Grangent reservoir (France). By sequencing the internal transcribed spacer of the ribosomal operon, we evidenced changes in the genetic structure among size classes in response to environmental change. While similar genotypes were seen in every size class in stable conditions, new dominant genotypes appeared in the smallest colonies (< 160 μm) concomitantly with mixing conditions, strongly suggesting the importance of these colonies in response to disturbances. Moreover, these small colonies played a major role in microcystin production during this bloom, since very high microcystin contents (> 1 pg.cell.(-1)) were found in their cells. These findings indicate that the colony size distribution of a Microcystis population in response to disturbance could be an adaptive strategy that may explain its ecological success in freshwater ecosystems.

Characterization of akinetes from cyanobacterial strains and lake sediment: A study of their resistance and toxic potential

Nostocalean cyanobacteria are known to proliferate abundantly in eutrophic aquatic ecosystems, and to produce several cyanotoxins, including anatoxin-a. In this study, we investigated both the resistance and toxic potential of the akinetes (resistant cells), using cyanobacterial cultures and akinetes extracted from the sediment of Lake Aydat (France) sampled in the winter and spring. Intact and lysed akinetes were differentiated using a double control based on the autofluorescence of akinetes and SYTOX-green staining. The percentage of resistant akinetes found in several different abiotic stress conditions was highly variable, depending on the species and also on the sampling season. Thus, the resistance of akinetes and their ability to germinate seems to follow a species-specific process, and akinetes can undergo physiologic changes during the sedimentary phase of the Nostocale life cycle. This study also revealed the first evidence of anatoxin-a genes in akinetes, with anaC and anaF genes detected in akinetes from all cyanobacterial producer cultures. The low number of anaC genes, almost exclusively detected using nested PCR, in the sediment at Lake Aydat suggests a limited but existent past population of toxic Nostocales in this lake. Given the key role of akinetes in the annual cycle and subsequent summer proliferation, it can be interesting to integrate the surveillance of akinetes in the management of lakes exposed to recurrent cyanobacterial blooms.

Temporal evolution and vertical stratification of Microcystis toxic potential during a first bloom event

Abstract In order to study the setup of a Microcystis bloom and the evolution of its toxic potential, we studied the temporal and vertical variations in Microcystis aeruginosa abundance, microcystins (MC) concentrations (intracellular and extracellular), and the relative proportion of potentially microcystin-producing cells (MC-producing cells) in relation to physicochemical parameters in the recently setup Moroccan reservoir “Yaacoub Al Mansour.” The Microcystis bloom appeared relatively late in the season and was associated with a low proportion of MC-producing cells in the water surface layer, probably related to non-limiting nutrient concentrations. Interestingly, the setup of the bloom leads to a vertical gradient, showing a decrease in Microcystis cell abundance inversely coupled with an increase in the proportion of MC-producing cells. Thus, this can be the result of the growth where non-MC-producing cells remain in the lighted water layer easier than MC-producing ones. Nevertheless, parameters other than light intensity may influence the toxic potential of bloom as no vertical pattern was observed concerning microcystins cellular quotas. The high microcystins concentrations measured in the deep water layer have also proved the importance of considering the deep part of aquatic ecosystem in the management of health risks associated with cyanobacterial proliferations.

Molecular tools to detect anatoxin-a genes in aquatic ecosystems: Toward a new nested PCR-based method

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.

Genetic diversity along the life cycle of the cyanobacterium Microcystis: highlight on the complexity of benthic and planktonic interactions

Microcystis is a toxic freshwater cyanobacterium with an annual life cycle characterized by the alternation of a planktonic proliferation stage in summer and a benthic resting stage in winter. Given the importance of both stages for the development and the survival of the population, we investigated the genotypic composition of the planktonic and benthic Microcystis subpopulations from the Grangent reservoir (France) during two distinct proliferation periods. Our results showed a succession of different dominant genotypes in the sediment as well as in the water all along the study periods with some common genotypes to both compartments. Analysis of molecular variance and UniFrac analysis confirmed the similarity between some benthic and planktonic samples, thus evidencing exchanges of genotypes between water and sediment. Thanks to these data, recruitment and sedimentation were proven not to be restricted to spring and autumn, contrary to what was previously thought. Finally, genetic diversity was significantly higher in the sediment than in the water (P < 0.01; Students t-test). Taken together, our results shed light on the hidden contribution of the benthic compartment in maintaining the genetic diversity of Microcystis populations throughout their annual cycle, which could explain their ecological success in aquatic ecosystems.

Horizontal distribution of the cell abundance and toxicity of Microcystis in a hypereutrophic Moroccan reservoir

The first results of the horizontal distribution of the cell abundance and toxicity of Microcystis in the hypereutrophic Moroccan reservoir Lalla Takerkoust are reported. An unexpected spatio-temporal heterogeneity has been shown between Microcystis abundance and microcystins concentrations. The principal determining factors were analyzed in order to identify the most likely sites for the proliferation and/or accumulation of Microcystis in this reservoir. The horizontal heterogeneity seems to be mainly influenced by the wind direction and inflows. The results can serve as reference data for monitoring cyanobacterial water blooms and associated cyanotoxins in the lake.

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.