Thomas Rohrlack

Effects of Cell-Bound Microcystins on Survival and Feeding of Daphnia spp.

ABSTRACT The influence of cell-bound microcystins on the survival time and feeding rates of six Daphnia clones belonging to five common species was studied. To do this, the effects of the microcystin-producing Microcystis strain PCC7806 and its mutant, which has been genetically engineered to knock out microcystin synthesis, were compared. Additionally, the relationship between microcystin ingestion rate by the Daphnia clones andDaphnia survival time was analyzed. Microcystins ingested with Microcystis cells were poisonous to allDaphnia clones tested. The median survival time of the animals was closely correlated to their microcystin ingestion rate. It was therefore suggested that differences in survival amongDaphnia clones were due to variations in microcystin intake rather than due to differences in susceptibility to the toxins. The correlation between median survival time and microcystin ingestion rate could be described by a reciprocal power function. Feeding experiments showed that, independent of the occurrence of microcystins, cells of wild-type PCC7806 and its mutant are able to inhibit the feeding activity of Daphnia. Both variants of PCC7806 were thus ingested at low rates. In summary, our findings strongly suggest that (i) sensitivity to the toxic effect of cell-bound microcystins is typical for Daphnia spp., (ii) Daphnia spp. and clones may have a comparable sensitivity to microcystins ingested with food particles, (iii) Daphnia spp. may be unable to distinguish between microcystin-producing and -lacking cells, and (iv) the strength of the toxic effect can be predicted from the microcystin ingestion rate of the animals.

Cyanobacteria and Cyanotoxins: The Influence of Nitrogen versus Phosphorus

The importance of nitrogen (N) versus phosphorus (P) in explaining total cyanobacterial biovolume, the biovolume of specific cyanobacterial taxa, and the incidence of cyanotoxins was determined for 102 north German lakes, using methods to separate the effects of joint variation in N and P concentration from those of differential variation in N versus P. While the positive relationship between total cyanobacteria biovolume and P concentration disappeared at high P concentrations, cyanobacteria biovolume increased continually with N concentration, indicating potential N limitation in highly P enriched lakes. The biovolumes of all cyanobacterial taxa were higher in lakes with above average joint NP concentrations, although the relative biovolumes of some Nostocales were higher in less enriched lakes. Taxa were found to have diverse responses to differential N versus P concentration, and the differences between taxa were not consistent with the hypothesis that potentially N2-fixing Nostocales taxa would be favoured in low N relative to P conditions. In particular Aphanizomenon gracile and the subtropical invasive species Cylindrospermopsis raciborskii often reached their highest biovolumes in lakes with high nitrogen relative to phosphorus concentration. Concentrations of all cyanotoxin groups increased with increasing TP and TN, congruent with the biovolumes of their likely producers. Microcystin concentration was strongly correlated with the biovolume of Planktothrix agardhii but concentrations of anatoxin, cylindrospermopsin and paralytic shellfish poison were not strongly related to any individual taxa. Cyanobacteria should not be treated as a single group when considering the potential effects of changes in nutrient loading on phytoplankton community structure and neither should the N2-fixing Nostocales. This is of particular importance when considering the occurrence of cyanotoxins, as the two most abundant potentially toxin producing Nostocales in our study were found in lakes with high N relative to P enrichment.

Isolation, Characterization, and Quantitative Analysis of Microviridin J, a New Microcystis Metabolite Toxic to Daphnia

This paper describes the purification and characterization of microviridin J, a newly discovered metabolite of Microcystis that causes a lethal molting disruption in Daphnia spp., upon ingestion of living cyanobacterial cells. Microviridin J consists of an acetylated chain of 13 amino acids arranged in three rings and two side chains. Unlike other known isoforms of microviridin, microviridin J contains arginine that imparts a unique solution conformation characterized by proximal hydrophobic interactions between Arg and other regions of the molecule. This eventually results in the formation and stabilization of an additional ring system. Microviridin J potently inhibits porcine trypsin, bovine chymotrypsin, and daphnid trypsin-like proteases. The activity against trypsin is most likely due to Arg and its distinctive conformational interactions. Overall, the data presented for microviridin J emphasize once again the ability of cyanobacteria to produce numerous and potent environmental toxins.

Cyanobacterial Protease Inhibitor Microviridin J Causes a Lethal Molting Disruption in Daphnia pulicaria

ABSTRACT Laboratory experiments identified microviridin J as the source of a fatal molting disruption in Daphnia species organisms feeding on Microcystis cells. The molting disruption was presumably linked to the inhibitory effect of microviridin J on daphnid proteases, suggesting that hundreds of further cyanobacterial protease inhibitors must be considered potentially toxic to zooplankton.

Possible Implications of Chytrid Parasitism for Population Subdivision in Freshwater Cyanobacteria of the Genus Planktothrix

ABSTRACT Populations of the cyanobacterium Planktothrix comprise multiple coexisting oligopeptide chemotypes that can behave differently in nature. We tested whether this population subdivision can, in principle, be driven by parasitic chytrid fungi, which are almost neglected agents of Planktothrix mortality. Two chytrid strains, Chy-Lys2009 and Chy-Kol2008, were isolated from Planktothrix-dominated lakes in Norway. The two strains shared 98.2% and 86.2% of their 28S and internal transcribe spacer rRNA gene sequences, respectively. A phylogenetic analysis placed them in the order Rhizophydiales family Angulomycetaceae. Chy-Lys2009 and Chy-Kol2008 could completely lyse Planktothrix cultures within days, while they failed to infect other filamentous cyanobacteria. The effect on Planktothrix was chemotype dependent, and both chytrid strains showed distinct chemotype preferences. These findings identify chytrid fungi infecting Planktothrix as highly potent and specialized parasites which may exert strong selective pressure on their hosts. According to established hypotheses on host-parasite coevolution, parasitism with the above properties may result in subdivision of Planktothrix populations into coexisting chemotypes and periodic shifts in the relative Planktothrix chemotype composition. These predictions are in agreement with field observations. Moreover, a genetic analysis verified the co-occurrence of Chy-Lys2009 and Chy-Kol2008 or related chytrid strains along with distinct Planktothrix chemotypes in at least one water body. Our findings are consistent with a scenario where chytrid parasitism is one driving force of Planktothrix population subdivision, which in turn leads to polymorphism in parasitic chytrid fungi. Future studies should test the validity of this scenario under field conditions.

Optofluidic lab-on-a-chip for rapid algae population screening

The rapid identification of algae species is not only of practical importance when monitoring unwanted adverse effects such as eutrophication, but also when assessing the water quality of watersheds. Here, we demonstrate a lab-on-a-chip that functions as a compact robust tool for the fast screening, real-time monitoring, and initial classification of algae. The water-algae sample, flowing in a microfluidic channel, is side-illuminated by an integrated subsurface waveguide. The waveguide is curved to improve the device sensitivity. The changes in the transmitted optical signal are monitored using a quadrant-cell photo-detector. The signal-wavelets from the different quadrants are used to qualitatively distinguish different families of algae. The channel and waveguide are fabricated out of a monolithic fused-silica substrate using a femtosecond laser-writing process combined with chemical etching. This proof-of-concept device paves the way for more elaborate femtosecond laser-based optofluidic micro-instruments incorporating waveguide networks designed for the real-time field analysis of cells and microorganisms.

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.

Laboratory studies of dissolved radiolabelled microcystin-LR in lake water

The fate of dissolved microcystin-LR was studied in laboratory experiments using surface water taken from a eutrophic lake. Based on initial range finding, a concentration of 50 microg l(-1) dissolved 14C-microcystin-LR was selected for subsequent time-course experiments. The first was performed in May before the cyanobacterial bloom season and low increases in the radioactivity of particulate fractions occurred with an approx. halving of the cyano-toxin during 4 days. The radioactivity of the dissolved fraction remained stable and there was no significant formation of radiolabelled inorganic carbon. A second time-course experiment was performed in September during the cyanobacterial bloom season. At the end of the four-day incubation period, the microcystin-LR concentration had decreased to an undetectable level and 24% of the added radiolabelled substance was found in different particulate fractions. The study demonstrated that biodegradation of dissolved microcystin-LR occurred in water collected at a lake surface with carbon dioxide as a major end-product.

Optical classification of algae species with a glass lab-on-a-chip

The identification of submillimetre phytoplankton is important for monitoring environmental and climate changes, as well as evaluating water for health reasons. Current standard methods for phytoplankton species identification require sample collection and ex situ analysis, an expensive procedure which prevents the rapid identification of phytoplankton outbreaks. To address this, we use a glass-based microchip with a microchannel and waveguide included on a monolithic substrate, and demonstrate its use for identifying phytoplankton species. The microchannel and the specimens inside it are illuminated by laser light from the curved waveguide as algae-laden water is passed through the channel. The intensity distribution of the light collected from the biochip is monitored with an external photodetector. Here, we demonstrate that the characteristics of the photodiode signal from this simple and robust system can provide significant and useful information as to the contents of the channel. Specifically, we show first that the signals are correlated to the size of algae cells. Using a pattern-matching neural network, we demonstrate the successful classification of five algae species with an average 78% positive identification rate. Furthermore, as a proof-of-concept for field-operation, we show that the chip can be used to distinguish between detritus in field-collected water and the toxin-producing cyanobacterium Cyanothece.

Fate of intracellular microcystins in the cyanobacterium Microcystis aeruginosa (Chroococcales, Cyanophyceae)

T. Rohrlack and P. Hyenstrand. 2007. Fate of intracellular microcystins in the cyanobacterium Microcystis aeruginosa (Chroococcales, Cyanophyceae). Phycologia 46: 277–283. DOI: 10.2216/06-14.1 The aim of the present study was to evaluate the importance of microcystin loss processes in cells of Microcystis aeruginosa, either as export from the cells or intracellular breakdown, and to draw conclusions about the function of microcystins to their producers. By supplying radioactive inorganic carbon to M. aeruginosa cultures and suppressing a considerable production of radioactive microcystin after a certain period of time, an intracellular pool of labelled microcystin was built up. The fate of this pool was studied by following its radioactivity in different time-course experiment. No evidence for losses from the intracellular microcystin pool could be found. All radioactive carbon that had been incorporated into microcystin molecules remained in the intracellular microcystin pool irrespective of whether the cyanobacterial cells were subjected to low light or high light. This suggests that under the conditions tested here, microcystins are not subjects of significant loss processes such as export from the cells or intracellular breakdown. The present study therefore failed to produce evidence for an involvement of microcystins in quorum sensing, defence against epiphytic organisms, or other functions requiring an export of the compounds into the surrounding medium. The lack of an intracellular breakdown suggests that microcystins are not involved in metabolic cellular processes.