The effects of environmental factors on biomass and microcystin production by the freshwater cyanobacterial genera Microcystis and Anabaena

Several cyanobacterial genera produce the hepatotoxins, microcystins. Microcystins are produced only in cells that have microcystin synthetase gene (mcy) clusters, which encode enzyme complexes involved in microcystin biosynthesis. Microcystin-producing and nonmicrocystin-producing genotypes of single cyanobacterial genus may occur simultaneously in situ. Previously, the effects of environmental factors on the growth and microcystin production of cyanobacteria have mainly been studied by means of isolated cyanobacteria cultures in the laboratory. Studies in the field have been difficult, owing to the lack of methods to identify and quantify the different genotypes. In this study, genus-specific microcystin synthetase E (mcyE) gene primers were designed and a method to identify and quantify the mcyE copy numbers was developed and used in situ. Microcystis and Anabaena mcyE genes were observed in two Finnish lakes. Microcystis appeared to be the most abundant microcystin producer in Lake Tuusulanjärvi and in one basin of Lake Hiidenvesi. Because the most potent microcystin-producing genus of a lake can be identified, it will be possible in the future to design genus-targeted strategies for lake restoration. Effects of P and N concentrations on the biomass of microcystin-producing and nonmicrocystin-producing Microcystis strains and an Anabaena strain were studied in cultures. P and N concentrations and their combined effect increased cyanobacterial biomass of all Microcystis strains. The biomass of microcystin-producing Microcystis was higher than that of nonmicrocystin-producing strains at high nutrient concentrations. The P concentration increased Anabaena biomass, but the effect of N concentration was statistically insignificant for growth yield, probably due to the ability of the genus to fix molecular N2. P and N concentrations and combined nutrients caused an increase in cellular microcystin concentrations of the Microcystis strain cultivated in chemostat cultures. Cyanobacteria are able to hydrolyse nutrients from organic matter through extracellular enzyme activities. Leucine aminopeptidase (LAP) activity was observed in an axenic N2-fixing Anabaena strain grown in batch cultures. The P concentration caused a statistically significant increase in LAP activity, whereas the effect of N concentration was insignificant. The highest LAP activities were observed in the most eutrophic basins of Lake Hiidenvesi. LAP activity probably originated mostly from attached heterotrophic bacteria and less from cyanobacteria.

Evolution and detection of cyanobacterial hepatotoxin synthetase genes

Mass occurrences (blooms) of cyanobacteria are common in aquatic environments worldwide. These blooms are often toxic, due to the presence of hepatotoxins or neurotoxins. The most common cyanobacterial toxins are hepatotoxins: microcystins and nodularins. In freshwaters, the main producers of microcystins are Microcystis, Anabaena, and Planktothrix. Nodularins are produced by strains of Nodularia spumigena in brackish waters. Toxic and nontoxic strains of cyanobacteria co-occur and cannot be differentiated by conventional microscopy. Molecular biological methods based on microcystin and nodularin synthetase genes enable detection of potentially hepatotoxic cyanobacteria. In the present study, molecular detection methods for hepatotoxin-producing cyanobacteria were developed, based on microcystin synthetase gene E (mcyE) and the orthologous nodularin synthetase gene F (ndaF) sequences. General primers were designed to amplify the mcyE/ndaF gene region from microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia strains. The sequences were used for phylogenetic analyses to study how cyanobacterial mcy genes have evolved. The results showed that mcy genes and microcystin are very old and were already present in the ancestor of many modern cyanobacterial genera. The results also suggested that the sporadic distribution of biosynthetic genes in modern cyanobacteria is caused by repeated gene losses in the more derived lineages of cyanobacteria and not by horizontal gene transfer. Phylogenetic analysis also proposed that nda genes evolved from mcy genes. The frequency and composition of the microcystin producers in 70 lakes in Finland were studied by conventional polymerase chain reaction (PCR). Potential microcystin producers were detected in 84% of the lakes, using general mcyE primers, and in 91% of the lakes with the three genus-specific mcyE primers. Potential microcystin-producing Microcystis were detected in 70%, Planktothrix in 63%, and Anabaena in 37% of the lakes. The presence and co-occurrence of potential microcystin producers were more frequent in eutrophic lakes, where the total phosphorus concentration was high. The PCR results could also be associated with various environmental factors by correlation and regression analyses. In these analyses, the total nitrogen concentration and pH were both associated with the presence of multiple microcystin-producing genera and partly explained the probability of occurrence of mcyE genes. In general, the results showed that higher nutrient concentrations increased the occurrence of potential microcystin producers and the risk for toxic bloom formation. Genus-specific probe pairs for microcystin-producing Anabaena, Microcystis, Planktothrix, and Nostoc, and nodularin-producing Nodularia were designed to be used in a DNA-chip assay. The DNA-chip can be used to simultaneously detect all these potential microcystin/nodularin producers in environmental water samples. The probe pairs detected the mcyE/ndaF genes specifically and sensitively when tested with cyanobacterial strains. In addition, potential microcystin/nodularin producers were identified in lake and Baltic Sea samples by the DNA-chip almost as sensitively as by quantitative real-time PCR (qPCR), which was used to validate the DNA-chip results. Further improvement of the DNA-chip assay was achieved by optimization of the PCR, the first step in the assay. Analysis of the mcy and nda gene clusters from various hepatotoxin-producing cyanobacteria was rewarding; it revealed that the genes were ancient. In addition, new methods detecting all the main producers of hepatotoxins could be developed. Interestingly, potential microcystin-producing cyanobacterial strains of Microcystis, Planktothrix, and Anabaena, co-occurred especially in eutrophic and hypertrophic lakes. Protecting waters from eutrophication and restoration of lakes may thus decrease the prevalence of toxic cyanobacteria and the frequency of toxic blooms.

Biodiversity and Phylogeny of Planktic Cyanobacteria in Temperate Freshwater Lakes

Currently, the classification used for cyanobacteria is based mainly on morphology. In many cases the classification is known to be incongruent with the phylogeny of cyanobacteria. The evaluation of this classification is complicated by the fact that numerous strains are only described morphologically and have not been isolated. Moreover, the phenotype of many cyanobacterial strains alters during prolonged laboratory cultivation. In this thesis, cyanobacterial strains were isolated from lakes (mainly Lake Tuusulanjärvi) and both morphology and phylogeny of the isolates were investigated. The cyanobacterial community composition in Lake Tuusulanjärvi was followed for two years in order to relate the success of cyanobacterial phenotypes and genotypes to environmental conditions. In addition, molecular biological methods were compared with traditional microscopic enumeration and their ability and usefulness in describing the cyanobacterial diversity was evaluated. The Anabaena, Aphanizomenon, and Trichormus strains were genetically heterogeneous and polyphyletic. The phylogenetic relationships of the heterocytous cyanobacteria were not congruent with their classification. In contrast to heterocytous cyanobacteria, the phylogenetic relationships of the Snowella and Woronichinia strains, which had not been studied before this thesis, reflected the morphology of strains and followed their current classification. The Snowella strains formed a monophyletic cluster, which was most closely related to the Woronichinia strain. In addition, a new cluster of thin, filamentous cyanobacterial strains identified as Limnothrix redekei was revealed. This cluster was not closely related to any other known cyanobacteria. The cyanobacterial community composition in Lake Tuusulanjärvi was studied with molecular methods [denaturant gradient gel electrophoresis (DGGE) and cloning of the 16S rRNA gene], through enumerations of cyanobacteria under microscope, and by strain isolations. Microcystis, Anabaena/Aphanizomenon, and Synechococcus were the major groups in the cyanobacterial community in Lake Tuusulanjärvi during the two-year monitoring period. These groups showed seasonal succession, and their success was related to different environmental conditions. The major groups of the cyanobacterial community were detected by all used methods. However, cloning gave higher estimates than microscopy for the proportions of heterocytous cyanobacteria and Synechococcus. The differences were probably caused by the high 16S rRNA gene copy numbers in heterotrophic cyanobacteria and by problems in the identification and detection of unicellular cyanobacteria.

Harmful algae in the planktonic food web of the Baltic Sea

This study deals with algal species occurring commonly in the Baltic Sea: haptophyte Prymnesium parvum, dinoflagellates Dinophysis acuminata, D. norvegica and D. rotundata, and cyanobacterium Nodularia spumigena. The hypotheses are connected to the toxicity of the species, to the factors determining toxicity, to the consequences of toxicity and to the transfer of toxins in the aquatic food web. Since the Baltic Sea is severely eutrophicated, the fast-growing haptophytes have potential in causing toxic blooms. In our studies, the toxicity (as haemolytic activity) of the haptophyte P. parvum was highest under phosphorus-limited conditions, but the cells were toxic also under nitrogen limitation and under nutrient-balanced growth conditions. The cellular nutrient ratios were tightly related to the toxicity. The stoichiometric flexibility for cellular phosphorus quota was higher than for nitrogen, and nitrogen limitation led to decreased biomass. Negative allelopathic effects on another algae (Rhodomonas salina) could be observed already at low P. parvum cell densities, whereas immediate lysis of R. salina cells occurred at P. parvum cell densities corresponding to natural blooms. Release of dissolved organic carbon from the R. salina cells was measured within 30 minutes, and an increase in bacterial number and biomass was measured within 23 h. Because of the allelopathic effect, formation of a P. parvum bloom may accelerate after a critical cell density is reached and the competing species are eliminated. A P. parvum bloom indirectly stimulates bacterial growth, and alters the functioning of the planktonic food web by increasing the carbon transfer through the microbial loop. Our results were the first reports on DSP toxins in Dinophysis cells in the Gulf of Finland and on PTX-2 in the Baltic Sea. Cellular toxin contents in Dinophysis spp. ranged from 0.2 to 149 pg DTX-1 cell-1 and from 1.6 to 19.9 pg PTX-2 cell-1 in the Gulf of Finland. D. norvegica was found mainly around the thermocline (max. 200 cells L-1), whereas D. acuminata was found in the whole mixed layer (max. 7 280 cells L-1). Toxins in the sediment trap corresponded to 1 % of DTX-1 and 0.01 % PTX-2 of the DSP pool in the suspended matter. This indicates that the majority of the DSP toxins does not enter the benthic community, but is either decomposed in the water column, or transferred to higher trophic levels in the planktonic food chain. We found that nodularin, produced by Nodularia spumigena, was transferred to the copepod Eurytemora affinis through three pathways: by grazing on filaments of small Nodularia, directly from the dissolved pool, and through the microbial food web by copepods grazing on ciliates, dinoflagellates and heterotrophic nanoflagellates. The estimated proportion of the microbial food web in nodularin transfer was 22-45 % and 71-76 % in our two experiments, respectively. This highlights the potential role of the microbial food web in the transfer of toxins in the planktonic food web.

The effects of abiotic and biotic factors on somatic embryogenesis and seedlings of Pinus sylvestris (L.)

Somatic embryogenesis (SE) is an asexual form of plant propagation that occurs in nature and mimics many of the events of sexual reproduction. Pinus sylvestris (L.) is an important source of timber in Northern Eurasia but it is recalcitrant to somatic embryogenesis. Several factors important for the success of the P. sylvestris embryogenic cultures have not been thoroughly investigated. In this study, we examined the effects of parental genotypes on the SE in P. sylvestris, the involvement of the gaseous plant growth regulator, ethylene in SE, and also biotic effects on somatic embryos as well as on seedlings. We tested parental effects on immature embryo initiation for different media, storage periods, and on the maturation process. Maternal effects were found to be crucial for SE in the absence of paternal effects. No maternal-paternal interaction was observed at any stage of somatic embryo production. Additionally the role of ethylene at different developmental stages of SE was investigated. Two ACC synthase genes, PsACS1 and PsACS2, were isolated and characterized. PsACS1 was expressed during the proliferation stage in all tested genotypes, whereas PsACS2 was only expressed in somatic embryos of each genotype. Ethylene production in embryos at stage 3 was significantly higher than the other stages. In a parallel study, the response of somatic embryos to fungal elicitors was investigated. Three fungi, a mutualistic ectomycorrhizal (ECM) fungus (Suillus bovinus), a weak Scots pine pathogen (Heterobasidion parviporum) and a strong pathogen (H. annosum) were used. The gene expression patterns for embryos exposed to the H. parviporum elicitor were found to be similar to that documented for S. bovinus among the tested genes. By contrast somatic embryos exposed to the H. annosum elicitor had a different pattern of regulation which was marked by a delayed response, and in some cases death of the embryos. Furthermore, interaction without direct contact between P. sylvestris seedlings and microbes (mutualistic and pathogenic fungus, cyanobacterium) were investigated. Several novel genes expressed in seedlings treated with ECM fungus were isolated which suggested that physical contact is not necessary for elicitation of host responses. The results suggest that somatic embryos and seedlings of P. sylvestris are genetically well equipped to respond to fungal elicitor/exudates and could serve as a suitable model for reproducible molecular studies in conifer tree patho- and symbiotic systems.