Analyses of high-throughput environmental sequencing data have become the ‘gold-standard’ to address fundamental questions of microbial diversity, ecology and biogeography. Findings that emerged from sequencing are, e.g. the discovery of the extensive ‘rare microbial biosphere’ and its potential function as a seed-bank. Even though applied since several years, results from high-throughput environmental sequencing have hardly been validated. We assessed how well pyrosequenced amplicons [the hypervariable eukaryotic V4 region of the small subunit ribosomal RNA (SSU rRNA) gene] reflected morphotype ciliate plankton. Moreover, we assessed if amplicon sequencing had the potential to detect the annual ciliate plankton stock. In both cases, we identified significant quantitative and qualitative differences. Our study makes evident that taxon abundance distributions inferred from amplicon data are highly biased and do not mirror actual morphotype abundances at all. Potential reasons included cell losses after fixation, cryptic morphotypes, resting stages, insufficient sequence data availability of morphologically described species and the unsatisfying resolution of the V4 SSU rRNA fragment for accurate taxonomic assignments. The latter two underline the necessity of barcoding initiatives for eukaryotic microbes to better and fully exploit environmental amplicon data sets, which then will also allow studying the potential of seed-bank taxa as a buffer for environmental changes.
It is generally agreed that the hepatotoxic microcystins (MCs) are the most abundant toxins produced by cyanobacteria in freshwater. In various freshwater lakes in East Africa MC-producing Microcystis has been reported to dominate the phytoplankton, however the regulation of MC production is poorly understood. From May 2007 to April 2008 the Microcystis abundance, the absolute and relative abundance of the mcyB genotype indicative of MC production and the MC concentrations were recorded monthly in five freshwater lakes in Uganda: (1) in a crater lake (Lake Saka), (2) in three shallow lakes (Lake Mburo, George, Edward), (3) in Lake Victoria (Murchison Bay, Napoleon Gulf). During the whole study period Microcystis was abundant or dominated the phytoplankton. In all samples mcyB-containing cells of Microcystis were found and on average comprised 20+/-2% (SE) of the total population. The proportion of the mcyB genotype differed significantly between the sampling sites, and while the highest mcyB proportions were recorded in Lake Saka (37+/-3%), the lowest proportion was recorded in Lake George (1.4+/-0.2%). Consequently Microcystis from Lake George had the lowest MC cell quotas (0.03-1.24 fg MC cell(-1)) and resulted in the lowest MC concentrations (0-0.5 microg L(-1)) while Microcystis from Lake Saka consistently showed maximum MC cell quotas (14-144 fg cell(-1)) and the highest MC concentrations (0.5-10.2 microg L(-1)). Over the whole study period the average MC content per Microcystis cell depended linearly on the proportion of the mcyB genotype of Microcystis. It is concluded that Microcystis populations differ consistently and independently of the season in mcyB genotype proportion between lakes resulting in population-specific differences in the average MC content per cell.
The filamentous cyanobacterium Planktothrix rubescens frequently occurs in deep and stratified lakes in the temperate region of the northern hemisphere and is a known producer of the hepatotoxic secondary metabolite microcystin. These cyclic heptapepids are synthesized nonribosomally via large enzyme complexes encoded by the microcystin (mcy) synthetase gene cluster. The occurrence of cyanobacterial strains lacking microcystin but containing the mcy gene cluster has been reported repeatedly; it was shown that this inactivation is due to mutations such as gene deletion events and the insertion of transposable elements. In the present study, twelve lakes in Austria, Germany, and Switzerland were sampled from July 2005 to October 2007, and the proportion of inactive mcy genotypes was quantified in relation to the total population of the redpigmented filamentous cyanobacterium Planktothrix by means of quantitative PCR. In total, four different mutations were quantified, namely two insertions affecting mcyD, one insertion affecting mcyA, and a deletion within mcyH and mcyA. The mutations occurred over a wide range of the population density (40 -570,000 filaments L −1 ) and their abundance was found to be positively correlated with population density. However, on average, all nontoxic mutants were found in a low proportion only (min 0%, mean 6.5% ± 1.1 (SE), max 52% of the total population). The genotype containing the mcyHA deletion had a significantly higher proportion (min 0%, mean 3.7% ± 1, max 52%) when compared with all the genotypes containing insertions within the mcy gene cluster (min 0%, mean 2.8% ± 0.7, max 24%). The results demonstrate that the occurrence of inactive mcy genotypes is linearly related to the population density and selective sweeps of nontoxic mutants did not occur during the transition from prebloom to bloom conditions.
Quantitative real-time PCR methods are increasingly being applied for the enumeration of toxic cyanobacteria in the environment. However, to justify the use of real-time PCR quantification as a monitoring tool, significant correlations between genotype abundance and actual toxin concentrations are required. In the present study, we aimed to explain the concentrations of three structural variants of the hepatotoxin microcystin ( During the last decade, genetic methods have significantly increased our understanding of the distribution of genes that are involved in the production of toxins within cyanobacteria that occur in fresh and brackish water (45). Although genetic methods can indicate only the potential risk of toxin synthesis and do not provide information about the actual toxin concentrations, quantitative real-time PCR has been increasingly applied for monitoring the toxin-producing genotypes of cyanobacteria in water (26,33,44). The development of real-time PCR methods was driven primarily by its potential (i) as an early-warning tool as well as to monitor toxin-producing cyanobacteria and (ii) to identify those factors that lead to a dominance/repression of toxin-producing genotypes versus nontoxic genotypes. For the first aim, it is essential that the abundance of toxin-producing cyanobacteria can be related to the concentration of the respective toxic substance in water. A few studies showed that the concentration of certain toxic genotypes was linearly related to the respective toxin concentrations, e.g., for the most common group of hepatotoxins, the microcystins (MCs) (7,12,14), and for the related nodularin (19). Both microcystins and nodularins are known to be potent inhibitors of eukaryotic protein phosphatases 1 and 2A, resulting in a health hazard to humans and the environment (9). In contrast, no correlation was found (37, 50), or even the opposite was reported, by other studies, i.e., that the measurement of microcystin-producing genotypes is not a satisfactory method for use in monitoring programs in order to predict the toxic risk associated with cyanobacterial proliferation (3). For microcystins, these contrasting results may be due to several reasons: (i) several genera producing microcystins frequently coexist in water bodies, and therefore, not all microcystin producers may have been identified; (ii) the semilogarithmic calibration curves limit the accuracy in estimations of genotype numbers and proportions (for example, the only laboratory comparison carried out so far revealed that among the three laboratories tested, the proportions of toxic genotypes were overestimated or underestimated by 0 to 72% and 0 to 50%, respectively [42]); and (iii) inactive mutants that contain the respective genes, however, which have been inactivated in toxin production through the insertion of transposable elements, may co-occur and decrease toxin production in a given population (6). Nevertheless, the real-time PCR technique is the only quantitative technique available for estimating the proportion of...
Parasitic chytrid fungi (phylum Chytridiomycota) are known to infect specific phytoplankton, including the filamentous cyanobacterium Planktothrix. Subspecies, or chemotypes of Planktothrix can be identified by the presence of characteristic oligopeptides. Some of these oligopeptides can be associated with important health concerns due to their potential for toxin production. However, the relationship between chytrid parasite and Planktothrix host is not clearly understood and more research is needed. To test the parasite - host relationship over time, we used a sediment core extracted from a Norwegian lake known to contain both multiple Planktothrix chemotype hosts and their parasitic chytrid. Sediment DNA of chytrids and Planktothrix was amplified and a 35-year coexistence was found. It is important to understand how these two antagonistic species can coexistence in a lake. Reconstruction of the time series showed that between 1979–1990 at least 2 strains of Planktothrix were present and parasitic pressure exerted by chytrids was low. After this period one chemotype became dominant and yet showed continued low susceptibility to chytrid parasitism. Either environmental conditions or intrinsic characteristics of Planktothrix could have been responsible for this continued dominance. One possible explanation could be found in the shift of Planktothrix to the metalimnion, an environment that typically consists of low light and decreased temperatures. Planktothrix are capable of growth under these conditions while the chytrid parasites are constrained. Another potential explanation could be due to the differences between cellular oligopeptide variations found between Planktothrix chemotypes. These oligopeptides can function as defense systems against chytrids. Our findings suggest that chytrid driven diversity was not maintained over time, but that the combination of environmental constraints and multiple oligopeptide production to combat chytrids could have allowed one Planktothrix chemotype to have dominance despite chytrid presence.
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