Abstract:

Cyanobacteria (blue-green algae) occur in freshwater all over the world and frequently dominate the phytoplankton community. Beside ecophysiological adaptations such as buoyancy due to the synthesis of gas vesicles or the efficient uptake of bicarbonate ions at high pH it is generally believed that the production of toxins and other bioactive substances contributes to their ecological success. The toxins produced by cyanobacteria include cyclic peptides, most prominently the hepatotoxic microcystins and alkaloids, such as the hepatotoxic cylindrospermopsin or the neurotoxic saxitoxins, anatoxin-a or anatoxin-a(S). While it is known that cyanobacteria in general play an important role in the saline and freshwater lakes in East Africa, the knowledge about their ability to produce toxins in freshwater in this region is very scarce. This lack of knowledge is of particular relevance, as a significant part of the population in Uganda daily uses the water as drinking water and consumes phytoplanktivorous fish harvested from the freshwater lakes. In order to estimate a potential health risk due to the toxin production by cyanobacteria in those countries, not only a sound estimate of the phytoplankton biovolume in the lakes is needed, but also the understanding, when toxins are produced and which are the factors that favour toxin production in those systems. It was the aim of the study, to estimate the production of the most widely distributed toxin microcystin seasonally in several large freshwater lakes in Uganda as well as to identify not only the producers of microcystin but also the environmental factors favoring microcystin production. During one year five lakes in Uganda were sampled monthly (Lake Victoria: Murchison Bay, Napoleon Gulf; three shallow lakes within the catchment area of the Ruwenzori: Lake George, Lake Edward, Lake Mburo; one Crater Lake: Lake Saka) and limnologically characterized using physical and chemical parameters. In parallel the composition of phytoplankton was determined qualitatively and quantitatively by counting from sedimentation chambers using the inverted microscope technique. The concentration of microcystin was analysed by means of high-performance liquid chromatography (HPLC- DAD). In addition, clonal strains of the genus Microcystis were isolated from field samples and characterized both genetically and morphologically according to their ability to produce microcystin. PCR based diagnostic approaches were used to identify microcystin-producing genotypes both in field samples and in the laboratory. In the first publication (chapter 2, Okello et al. 2009, Environmental Toxicology, 16 July 2009) it could be shown, that Microcystis is the only genus producing microcystin in Ugandan freshwaters. All PCR based tests revealed gene regions that only could be assigned to Microcystis and never to co-occurring other taxa such as Anabaena or Planktolyngbia. Further it could be shown that eutrophication of freshwater water bodies -either natural or human-induced - always result in dominance of Microcystis and therefore ultimately in the production of microcystin. Two structural microcystin variants have been determined that seem to be unique and dominant and have not been described previously. In the second publication (chapter 3, Okello und Kurmayer, Water Research, submitted) the production of microcystin was analysed in the five freshwater lakes in Uganda during one year (April 2007-April 2008). In all samples microcystins were detected. Corresponding to publication Nol it could be shown that the cell number of Microcystis as determined in the microscope correlated significantly with the concentration of microcystin. Other potential microcystin-producing genera such as Anabaena or Planktothrix also showed partly significant correlations with the microcystin concentration in water. However, since no evidence for the presence of microcystin genes that could be assigned to either Anabaena or Planktothrix was obtained during publication Nol and the correlation coefficients were always much lower when compared with those observed for Microcystis it is concluded that the significant correlations observed for Anabaena or Planktothrix are due to their co-occurrence with Microcystis. Surprisingly the lakes showed a relatively narrow range in microcystin concentrations during the season, while differed significantly (on average up to 28-fold) in microcystin production. Samples from Lake Saka had the maximum MC concentration (10 pg L'1) in July 2007. The minimum concentration (0.02 pg L'1) was recorded in Lake George (Kalender) in the months of May 07, June 07, January 08 and April 08. The difference in microcystin concentrations between lakes only partly could be explained by the variation in Microcystis cell numbers. For example, while on average the maximum Microcystis cell concentration was observed in L. George (1.2 ± 0.3 (1 standard error) Mio cells mF1), on average the microcystin concentrations were at the minimum (0.2 ± 0.1 pg L’1). In contrast, in L. Mburo on average a rather low cell concentration was recorded (0.17 ± 0.02 Mio cells ml'1) while the microcystin concentration on average was significantly higher (1.6 ± 0.2 pg L1). In order to test the hypothesis that populations of Microcystis differ in the proportion of genotypes containing the genes involved in microcystin synthesis (wcy) resulting in the significant variation. in MC cellular contents between the study lakes, the absolute and relative abundance of nicy genotypes was determined. The average proportion of nicy genotypes in L. George (1,14±0.13 (SE)% was 17-fold lower when compared with the average nicy genotype proportion in L. Mburo (19.6±4.8%). It is concluded that the variation in average MC content of Microcystis that is observed between lakes could be explained by the divergence in nicy genotype proportion between populations. In the third publication (chapter 4, Okello and Kurmayer, in preparation) the morphological characteristics of the genus Microcystis in field samples were compared with those observed under culture conditions in the laboratory. While in field samples, the morphotypes M. aeruginosa, M. flos-aquae and M. wesenbergii occurred most frequently, only M. aeruginosa, M. flos-aquae could be found under culture conditions. However, altogether the morphological characters that were observed under field conditions were found reproducible. In total only 13 of 96 strains (14%) contained the zncyE/B gene indicative of microcystin production. In addition, twelve isolates were sequenced for the intergenic spacer region of the phycocyanin region (PC-IGS) and assigned to the existing classification system of Microcystis by phylogenetic methods. To construct a phylogenetic tree 128 additional entries of Microcystis from the Genbank database (602 bp) were compared with the sequences obtained during this study. In general, Microcystis strains showed a non-random distribution among the phylogenetic lineages, for example while the strains of this study occurred in two clades (cluster A, D) only but were absent from another clade (cluster F) containing the largest number of strains. It is concluded that the identification of Microcystis according to morphological criteria is reliable, and even in the presence of morphologically similar genera such as Gomphospaeria, Synechocystis, Chroococcus can be used to estimate Microcystis cell numbers under the microscope. Altogether from publications No 1-3 it is concluded, that microscopical counting of Microcystis is a reliable tool, in order to provide a relatively quick and simple estimate of microcystin concentrations by multiplying the average cell numbers by the microcystin cell quotas as determined in this study.