Claudia Wiedner

Effects of Light on the Microcystin Content of Microcystis Strain PCC 7806

ABSTRACT Many cyanobacteria produce microcystins, hepatotoxic cyclic heptapeptides that can affect animals and humans. The effects of photosynthetically active radiation (PAR) on microcystin production by Microcystis strain PCC 7806 were studied in continuous cultures. Microcystis strain PCC 7806 was grown under PAR intensities between 10 and 403 μmol of photons m−2 s−1 on a light-dark rhythm of 12 h -12 h. The microcystin concentration per cell, per unit biovolume and protein, was estimated under steady-state and transient-state conditions and on a diurnal timescale. The cellular microcystin content varied between 34.5 and 81.4 fg cell−1 and was significantly positively correlated with growth rate under PAR-limited growth but not under PAR-saturated growth. Microcystin production and PAR showed a significant positive correlation under PAR-limited growth and a significant negative correlation under PAR-saturated growth. The microcystin concentration, as a ratio with respect to biovolume and protein, correlated neither with growth rate nor with PAR. Adaptation of microcystin production to a higher irradiance during transient states lasted for 5 days. During the period of illumination at a PAR of 10 and 40 μmol of photons m−2 s−1, the intracellular microcystin content increased to values 10 to 20% higher than those at the end of the dark period. Extracellular (dissolved) microcystin concentrations were 20 times higher at 40 μmol of photons m−2 s−1 than at 10 μmol of photons m−2 s−1 and did not change significantly during the light-dark cycles at both irradiances. In summary, our results showed a positive effect of PAR on microcystin production and content of Microcystis strain PCC 7806 up to the point where the maximum growth rate is reached, while at higher irradiances the microcystin production is inhibited.

Paralytic shellfish poisoning toxin-producing cyanobacterium Aphanizomenon gracile in Northeast Germany.

ABSTRACT Neurotoxic paralytic shellfish poisoning (PSP) toxins, anatoxin-a (ATX), and hepatotoxic cylindrospermopsin (CYN) have been detected in several lakes in northeast Germany during the last 2 decades. They are produced worldwide by members of the nostocalean genera Anabaena, Cylindrospermopsis, and Aphanizomenon. Although no additional sources of PSP toxins and ATX have been identified in German water bodies to date, the observed CYN concentrations cannot be produced solely by Aphanizomenon flos-aquae, the only known CYN producer in Germany. Therefore, we attempted to identify PSP toxin, ATX, and CYN producers by isolating and characterizing 92 Anabaena, Aphanizomenon, and Anabaenopsis strains from five lakes in northeast Germany. In a polyphasic approach, all strains were morphologically and phylogenetically classified and then tested for PSP toxins, ATX, and CYN by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) and screened for the presence of PSP toxin- and CYN-encoding gene fragments. As demonstrated by ELISA and LC-MS, 14 Aphanizomenon gracile strains from Lakes Melang and Scharmützel produced four PSP toxin variants (gonyautoxin 5 [GTX5], decarbamoylsaxitoxin [dcSTX], saxitoxin [STX], and neosaxitoxin [NEO]). GTX5 was the most prevalent PSP toxin variant among the seven strains from Lake Scharmützel, and NEO was the most prevalent among the seven strains from Lake Melang. The sxtA gene, which is part of the saxitoxin gene cluster, was found in the 14 PSP toxin-producing A. gracile strains and in 11 non-PSP toxin-producing Aphanizomenon issatschenkoi, A. flos-aquae, Anabaena planktonica, and Anabaenopsis elenkinii strains. ATX and CYN were not detected in any of the isolated strains. This study is the first confirming the role of A. gracile as a PSP toxin producer in German water bodies.

Microcystins (hepatotoxic heptapeptides) in german fresh water bodies

In 1995 and 1996 a total of 55 German water bodies with different trophic states were investigated for the presence of potentially microcystin‐producing cyanobacteria. The seston biomass of over 500 samples was analyzed by HPLC to assess microcystin content. The highest microcystin content per dry weight was found when Planktothrix rubescens was dominant, followed by Planktothrix agardhii and Microcystis spp. The microcystin to chlorophyll‐a ratio mostly varied between 0.1 and 0.5, with maxima of 1–2. In over 70% of the samples from pelagic water, particulate microcystin concentrations were below 10 μg L−1. By contrast, spatial and temporal concentrations varied by 4 orders of magnitude (2–25,000 μg L−1) at bathing sites in 1997 during a mass development of Microcystis spp. The seasonal peak of development of Microcystis spp. occurred from June to September and of Planktothrix agardhii from September to November. Planktothrix rubescens, however, was almost perennial. The seasonal levels of these cyanobacteria were clearly reflected by microcystin concentrations in the water bodies. ©1999 John Wiley & Sons, Inc. Environ Toxicol 14: 13–22, 1999

Distribution of three alien cyanobacterial species (Nostocales) in northeast Germany: Cylindrospermopsis raciborskii, Anabaena bergii and Aphanizomenon aphanizomenoides

A. Stüken, J. Rücker, T. Endrulat, K. Preussel, M. Hemm, B. Nixdorf, U. Karsten and C. Wiedner. 2006. Distribution of three alien cyanobacterial species (Nostocales) in northeast Germany: Cylindrospermopsis raciborskii, Anabaena bergii and Aphanizomenon aphanizomenoides. Phycologia 45: 696–703. DOI: 10.2216/05-58.1 Cylindrospermopsis raciborskii is considered a cyanobacterium of tropical origin and an alien species to temperate waters. However, it has been detected as far north as northern Germany. While previous studies have shown that all isolated German C. raciborskii strains are hepatotoxic, little is known about the spatial occurrence and relative frequency of this species in temperate Germany. The aim of this study was to investigate the spatial distribution and relative frequency of C. raciborskii close to its northernmost distribution limit, to characterise the habitat in which it is most likely to occur in this climatic zone and to search for any other neocyanobacterial species that might be present in German waters but has so far been overlooked. One hundred forty-two water bodies in northeast Germany were sampled from June until September 2004. All cyanobacteria species were analysed qualitatively and semiquantitatively. Besides C. raciborskii, two additional neocyano-bacterial species were detected: Anabaena bergii and Aphanizomenon aphanizomenoides. For both taxa, these findings represent their northernmost occurrence and their first report from German waters. Cylindrospermopsis raciborskii was present in 27%, Anabaena bergii in 9% and Aphanizomenon aphanizomenoides in 7% of the samples. The occurrence of each species was analysed in relation to maximum lake depth, Secchi depth, lake volume and lake surface area. All three species were present in a wide range of habitats, but C. raciborskii and Anabaena bergii occurred significantly more often in shallow, turbid waters than in deep, transparent water bodies. None of the parameters investigated were significantly correlated with the occurrence of Aphanizomenon aphanizomenoides. In conclusion, alien thermophilic cyanobacterial species are much more widely distributed in temperate Germany than previously known. The results are discussed with respect to the possible mechanisms that enable these organisms to expand northwards.

Factors controlling the dominance of Planktothrix agardhii and Limnothrix redekei in eutrophic shallow lakes

Lakes in the Scharmutzelsee-region (East-Brandenburg, Germany) were found to be dominated either by Limnothrix redekei or Planktothrix agardhii. These dominance regimes were stable over the whole investigation period from 1993 to 1995. Six lakes (maximum depth between 2 and 12 m) were compared in regard to physical and chemical conditions to find reasons for the superiority of the first or the second species. All investigated lakes are polymictic but show, nevertheless differences in their mixing-behaviour. In lakes dominated by Limnothrix redekeistratification events occur more often than in the lakes dominated by Planktothrix agardhii. The latter are more windexposed due to their greater size and/or their surroundings. In combination with polymixis light and nutrient supply are discussed as the key factors for the species composition. A lower content of soluble reactive phosphorus and lower light supply are considered as possible advantages for Limnothrix redekeiover Planktothrix agardhiiin three lakes. A lower total nitrogen/total phosphorus ratio may be a reason for the dominance of Planktothrix agardhiiin the other three investigated lakes.

First report of anatoxin-a-producing cyanobacterium Aphanizomenon issatschenkoi in northeastern Germany.

The neurotoxin anatoxin-a (ATX), has been detected in several northeast German lakes during the last two decades, but no ATX producers have been identified in German water bodies so far. In 2007 and 2008, we analyzed phytoplankton composition and ATX concentration in Lake Stolpsee (NE Germany) in order to identify ATX producers. Sixty-one Aphanizomenon spp. strains were isolated, morphologically and phylogenetically characterized, and tested for ATX production potential by liquid chromatography-tandem mass spectrometry (LC-MS/MS). New primers were specifically designed to identify a fragment of a polyketide synthase gene putatively involved in ATX synthesis and tested on all 61 Aphanizomenon spp. strains from L. Stolpsee and 92 non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from German lakes Langersee, Melangsee and Scharmützelsee. As demonstrated by LC-MS/MS, ATX concentrations in L. Stolpsee were undetectable in 2007 and ranged from 0.01 to 0.12 microg l(-1) in 2008. Fifty-nine of the 61 strains isolated were classified as Aphanizomenon gracile and two as Aphanizomenon issatschenkoi. One A. issatschenkoi strain was found to produce ATX at concentrations of 2354+/-273 microg g(-1) fresh weight, whereas the other A. issatschenkoi strain and A. gracile strains tested negative. The polyketide synthase gene putatively involved in ATX biosynthesis was found in the ATX-producing A. issatschenkoi strain from L. Stolpsee but not in the non-ATX-producing Aphanizomenon spp., Anabaena spp. and Anabaenopsis spp. strains from lakes Stolpsee, Langersee, Melangsee, and Scharmützelsee. This study is the first confirming A. issatschenkoi as an ATX producer in German water bodies.

Diversity of Microcystis aeruginosa isolates (Chroococcales, Cyanobacteria) from East-African water bodies

With exception of South Africa, very little is known about the presence and abundance of toxic cyanobacteria and cyanobacterial blooms on the African continent. The close proximity between society and nature, and the use of the sparse water resources as drinking water in large parts of Africa, lead to the recognition that more knowledge on toxic cyanobacterial blooms is of major importance. The bloom forming cyanobacterium Microcystis aeruginosa is known to produce cyclic heptatoxins (microcystins) which can be toxic to humans. In this study the morphological, genetic, and chemical characters of 24 strains of M. aeruginosa from several water bodies in Kenya and Uganda, some of them used as drinking water sources, were examined. The M. aeruginosa strains possessed different levels of diversity depending on characterisation method. Four morphotypes were identified based on the traditional morphological approach, 10 genotypes by DNA sequence comparison of the PC-IGS and ITS1 rDNA regions, and 10 chemotypes based on MALDI-TOF-MS oligopeptide analysis. Only 4 of the 24 isolated strains from East Africa were found to produce microcystins, while oligopeptides belonging to the aeruginosin and cyanopeptolin class were detected in most strains.

Seasonal dynamics of cylindrospermopsin and cyanobacteria in two German lakes

Cylindrospermopsin (CYN) is a potent hepatotoxin produced by different cyanobacteria of the order Nostocales. Questions of major concern are: which species produce CYN, which are the seasonal patterns of CYN dynamics and how are they regulated? Therefore, we studied for the first time the seasonal dynamics of particulate and dissolved CYN concentrations, cyanobacterial abundance and environmental factors in two German lakes over 2 years. Total CYN reached maximum concentrations of 0.34 and 1.80 microg L(-1) in Melangsee and Langer See, respectively. In both lakes, maxima of the dissolved CYN fraction occurred later in the season than those of the particulate fraction, and it reached higher concentrations. This indicates that CYN is poorly decomposed and accumulates in the water. The cyanobacterial community in both lakes included several potentially CYN-producing species that did not correlate with CYN concentrations. Significant correlations between the particulate CYN concentrations and species biovolume were only found for Aphanizomenon gracile (r(s)=0.803) in Langer See indicating that this species is a CYN producer. Different correlations of CYN with abiotic factors in the two lakes indicate the presence of further undetected CYN producers as well as different regulation mechanisms of their dynamics and the variability of CYN.

Phylogeography of cylindrospermopsin and paralytic shellfish toxin-producing Nostocales cyanobacteria from Mediterranean Europe (Spain)

ABSTRACT Planktonic Nostocales cyanobacteria represent a challenge for microbiological research because of the wide range of cyanotoxins that they synthesize and their invasive behavior, which is presumably enhanced by global warming. To gain insight into the phylogeography of potentially toxic Nostocales from Mediterranean Europe, 31 strains of Anabaena (Anabaena crassa, A. lemmermannii, A. mendotae, and A. planctonica), Aphanizomenon (Aphanizomenon gracile, A. ovalisporum), and Cylindrospermopsis raciborskii were isolated from 14 freshwater bodies in Spain and polyphasically analyzed for their phylogeography, cyanotoxin production, and the presence of cyanotoxin biosynthesis genes. The potent cytotoxin cylindrospermopsin (CYN) was produced by all 6 Aphanizomenon ovalisporum strains at high levels (5.7 to 9.1 μg CYN mg−1 [dry weight]) with low variation between strains (1.5 to 3.9-fold) and a marked extracellular release (19 to 41% dissolved CYN) during exponential growth. Paralytic shellfish poisoning (PSP) neurotoxins (saxitoxin, neosaxitoxin, and decarbamoylsaxitoxin) were detected in 2 Aphanizomenon gracile strains, both containing the sxtA gene. This gene was also amplified in non-PSP toxin-producing Aphanizomenon gracile and Aphanizomenon ovalisporum. Phylogenetic analyses supported the species identification and confirmed the high similarity of Spanish Anabaena and Aphanizomenon strains with other European strains. In contrast, Cylindrospermopsis raciborskii from Spain grouped together with American strains and was clearly separate from the rest of the European strains, raising questions about the current assumptions of the phylogeography and spreading routes of C. raciborskii. The present study confirms that the nostocalean genus Aphanizomenon is a major source of CYN and PSP toxins in Europe and demonstrates the presence of the sxtA gene in CYN-producing Aphanizomenon ovalisporum.

Success of chrysophytes, cryptophytes and dinoflagellates over blue-greens (cyanobacteria) during an extreme winter (1995/96) in eutrophic shallow lakes

Phytoplankton succession in three shallow eutrophic and hypertrophic lakes located in the Scharmutzelseeregion (East Brandenburg, Germany) is described for the extreme winter of 1995/96 before and during ice-cover. The lakes have been investigated since 1993 and pass along the tophic gradient from eutrophic, Petersdorfer See and Melangsee to hypertrophic, Langer See. All lakes are dominated by cyanobacteria from early spring to late autumn. Due to low water temperature and low light intensity before ice-cover a decline of cyanobaterial biovolume initiated which led to the total disappearance of cyanobacteria in Melangsee for the whole ice-covered period, whereas their portion in Langer See with at least 6% (3 mm31−1) and in Petersdorfer See with 25% (5 mm31−1) of total biovolume was still considerable. The cyanobacterial breakdown was linked to a switch in dominance to flagellates which built up together with diatoms a subsequent peak of biovolume during ice-cover.