Delphine Latour

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.

SEDIMENTARY IMPRINT OF MICROCYSTIS AERUGINOSA (CYANOBACTERIA) BLOOMS IN GRANGENT RESERVOIR (LOIRE, FRANCE)1

Analysis of a sediment core taken from the Grangent reservoir in 2004 showed the presence of high concentrations of Microcystis aeruginosa Kütz. colonies at the sediment surface (250 colonies · mL sediment−1) and also at depths of 25–35 cm (2300 colonies·mL sediment−1) and 70 cm (600 colonies · mL sediment−1). Measurements of radioactive isotopes (7Be, 137Cs, and 241Am) along with photographic analysis of the core were used to date the deep layers: the layer located at −30 cm dates from summer 2003, and that located at −70 cm from 1990 to 1991. The physiological and morphological conditions of those benthic colonies were compared with those of planktonic colonies using several techniques (environmental scanning electron microscopy [ESEM], TEM, DNA markers, cellular esterases, and toxins). The ESEM observations showed that, as these colonies age, peripheral cells disappear, with no cells remaining in the mucilage of the deepest colonies (70 cm), an indication of the survival thresholds of these organisms. In the benthic phase, the physiological conditions (enzyme activity, cell division, and intracellular toxins) and ultrastructure (particularly the gas vesicles) of the cells surviving in the heart of the colony are comparable to those of the planktonic form, with all the potential needed for growth. Maintaining cellular integrity requires a process that can provide sufficient energy and is expressed in the reduced, but still existing, enzymatic activity that we measured, which is equivalent to a quiescent state.

Fungal Parasitism: Life Cycle, Dynamics and Impact on Cyanobacterial Blooms

Many species of phytoplankton are susceptible to parasitism by fungi from the phylum Chytridiomycota (i.e. chytrids). However, few studies have reported the effects of fungal parasites on filamentous cyanobacterial blooms. To investigate the missing components of bloom ecosystems, we examined an entire field bloom of the cyanobacterium Anabaena macrospora for evidence of chytrid infection in a productive freshwater lake, using a high resolution sampling strategy. A. macrospora was infected by two species of the genus Rhizosiphon which have similar life cycles but differed in their infective regimes depending on the cellular niches offered by their host. R. crassum infected both vegetative cells and akinetes while R. akinetum infected only akinetes. A tentative reconstruction of the developmental stages suggested that the life cycle of R. crassum was completed in about 3 days. The infection affected 6% of total cells (and 4% of akinètes), spread over a maximum of 17% of the filaments of cyanobacteria, in which 60% of the cells could be parasitized. Furthermore, chytrids may reduce the length of filaments of Anabaena macrospora significantly by “mechanistic fragmentation” following infection. All these results suggest that chytrid parasitism is one of the driving factors involved in the decline of a cyanobacteria blooms, by direct mortality of parasitized cells and indirectly by the mechanistic fragmentation, which could weaken the resistance of A. macrospora to grazing.

Microbial players involved in the decline of filamentous and colonial cyanobacterial blooms with a focus on fungal parasitism

In the forthcoming decades, it is widely believed that the dominance of colonial and filamentous bloom-forming cyanobacteria (e.g. Microcystis, Planktothrix, Anabaena and Cylindrospermopsis) will increase in freshwater systems as a combined result of anthropogenic nutrient input into freshwater bodies and climate change. While the physicochemical parameters controlling bloom dynamics are well known, the role of biotic factors remains comparatively poorly studied. Morphology and toxicity often - but not always - limit the availability of cyanobacteria to filter feeding zooplankton (e.g. cladocerans). Filamentous and colonial cyanobacteria are widely regarded as trophic dead-ends mostly inedible for zooplankton, but substantial evidence shows that some grazers (e.g. copepods) can bypass this size constraint by breaking down filaments, making the bloom biomass available to other zooplankton species. A wide range of algicidal bacteria (mostly from the Alcaligenes, Flavobacterium/Cytophaga group and Pseudomonas) and viruses (Podoviridae, Siphoviridae and Myoviridae) may also contribute to bloom control, via their lytic activity underpinned by a diverse array of mechanisms. Fungal parasitism by the Chytridiomycota remains the least studied. While each of these biotic factors has traditionally been studied in isolation, emerging research consistently point to complex interwoven interactions between biotic and environmental factors.

INVOLVEMENT OF MICROCYSTINS AND COLONY SIZE IN THE BENTHIC RECRUITMENT OF THE CYANOBACTERIUM MICROCYSTIS (CYANOPHYCEAE)1

The benthic recruitment of Microcystis was simulated in vitro in order to characterize the colonies of Microcystis recruited and to study the impact of intracellular and extracellular microcystins (MCs), and the influence of colony size on the recruitment process. We observed recruitment dynamics consisting of a lag phase followed by a peak and then a return to low recruitment rates, mainly controlled by passive resuspension throughout the experiment, and by physiological processes during the recruitment peak. Ninety‐seven percent of the Microcystis colonies recruited were <160 μm in maximum length, and their cells contained much greater amounts of MCs (0.26 ± 0.14 pg eq microcystin leucine‐arginine variant [MC‐LR] · cell−1) than those in benthic colonies (0.021 ± 0.004 pg eq MC‐LR · cell−1). The MC content of recruited Microcystis varied significantly over time and was not related to changes in the proportion of potentially toxic genotypes, determined using real‐time PCR. On the other hand, the changes in MC content in the potentially toxic Microcystis recruited were closely and negatively correlated with recruitment dynamics; the lowest MC contents corresponded to high recruitment rates, and the highest MC contents corresponded to low recruitment rates. Thus, depending on temperature and light conditions, these variations are thought to result from the selection of various subpopulations from among the smallest and the most toxic of the initial benthic population. Adding purified MC‐LR to experimental treatments led to a decreased recruitment of Microcystis and more specifically of mcyB genotypes.

A Double Staining Method Using SYTOX Green and Calcofluor White for Studying Fungal Parasites of Phytoplankton

ABSTRACT We propose a double staining method based on the combination of two fluorochromes, calcofluor white (CFW; specific chitinous fluorochrome) and SYTOX green (nucleic acid stain), coupled to epifluorescence microscopy for counting, identifying, and investigating the fecundity of parasitic fungi of phytoplankton and the putative relationships established between hosts and their chytrid parasites. The method was applied to freshwater samples collected over two successive years during the terminal period of autumnal cyanobacterial blooms in a eutrophic lake. The study focused on the uncultured host-parasite couple Anabaena macrospora (cyanobacterium) and Rhizosiphon akinetum (Chytridiomycota). Our results showed that up to 36.6% of cyanobacterial akinetes could be parasitized by fungi. Simultaneously, we directly investigated the zoosporic content inside the sporangia and found that both the host size and intensity of infection conditioned the final size and hence fecundity of the chytrids. We found that relationships linking host size, final parasite size, and chytrid fecundity were conserved from year to year and seemed to be host-chytrid couple specific. We concluded that our double staining method was a valid procedure for improving our knowledge of uncultured freshwater phytoplankton-chytrid couples and so of the quantitative ecology of chytrids in freshwater ecosystems.

Short- and long-term dynamics of the toxic potential and genotypic structure in benthic populations of Microcystis.

Microcystis colonies are known to overwinter on the surface of the sediment of freshwater ecosystems. However, little is known about the genotypic and toxicological dynamics of Microcystis populations during this benthic life stage. In this study, we report a two-year-long survey of benthic populations of Microcystis, which had spent from a few days to more than six years in the sediment. In order to avoid any interaction with the planktonic proliferations, we chose two deeply buried benthic populations, which could be easily dated. Quantitative PCR on mcyB gene and protein phosphatase inhibition assays were performed to measure their toxic potential, and their genotypic structure was assessed by Capillary Electrophoresis-Single Strand Conformation Polymorphism (CE-SSCP), based on 16S-23S Intergenic Transcribed Spacer (ITS). The microcystin content of the cells seemed to change sharply during the first few months of benthic survival, whereas this content was low and decreased steadily after several years of benthic life. No genetic selection was observed in either the proportion of potentially toxic clones or the ITS sequences for any of the populations considered. From these results, the benthic life stage of Microcystis appears to preserve the structure and the composition of the population over a far larger time scale than classical overwintering period. Finally, some genotypes were common in both of the benthic populations, even though they originated from planktonic blooms that had developed five years apart, suggesting a major overlap of planktonic proliferations in successive years.

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.