Cembella, Allan
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Cembella, Allan
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Cembella, Allan
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Cembella, Allan D.
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Item-typ:Veröffentlichung, Determination and characterization of genes involved in toxic mechanisms of the prymnesiophyte Prymnesium parvum(2011-03-31); ; ; This thesis represents a study of the ecophysiology and toxicity of the prymnesiophyte Prymnesium parvum. The first aim was to investigate changes in the relative toxicity of P. parvum following a series of physiological shock treatments, meant to simulate environmental conditions under which harmful blooms of this species have been observed. As blooms of this haptophyte often occur in dynamic coastal brackish water systems, Prymnesium parvum is noted for its physiological flexibility, which may contribute to providing a competitive advantage over other coexisting species. Due to the unconfirmed nature of the compounds involved in toxigenic processes, two bioassays were employed to characterize changes in lytic capacity (extracellular vs. intracellular). These bioassays are considered physiologically relevant, as observed icthyotoxicity occurs through lysis of the gill cell membranes, rendering the fish unable to perform gas-exchange processes and obtain oxygen. Additionally, the gene expression of three polyketide synthase genes (PKS) were analyzed via quantitative PCR (qPCR), based on current chemical characterizations of toxic compounds produced by P. parvum. Low salinity and high irradiance were observed to alter the lytic effects of P. parvum on the sensitive cryptophyte Rhodomonas salina and erythrocytes. Furthermore, these two shock treatments were found to increase the transcript copy number in selected PKS genes, suggesting a possible correlation between toxicity and the PKS biosynthetic pathway. Allelochemical mediation has been suggested to affect competition and predatory relationships associated with formation of P. parvum blooms. As interactions between species are an integral part of understanding plankton ecology, interspecific interactions between P. parvum and three coexisting species were accordingly investigated. Combining bioassays with a functional genomic approach allowed differential characterization of cell-cell contact vs. waterborne cues depending on the organism with which incubated. A unique response on both the levels of toxicity, gene expression profile as well as PKS transcript copy number to the potential predator Oxhyrris marina suggest a fundamentally different type of interaction between the two species. Additionally, a dose-response time series experiment showed that changes in gene expression and toxicity did not occur immediately in P. parvum, rather after 60-90 minutes. Such a response by P. parvum may in fact signify a co-evolutionarily adaptive defense. Finally, examination of the effects of phosphorous limitation and low salinity stress on the gene expression profile and lytic capacity showed that the combination of these two stressors induces secretion or extracellular transport of toxic substances to a much higher degree than either stressor individually. Whether this observation is due to changes in membrane integrity due to homeostatic processes needs further research. The pattern of gene expression, however, revealed regulation of among others genes associated with active cellular transport processes, suggesting that maintenance of intracellular-extracellular homeostasis may play a role in the observed toxicity. In summary, these studies integrate the concepts of ecophysiology and functional genomics, providing a useful platform for further research regarding environmental factors associated with the toxicity of P. parvum. As functional genomic methods become more accessible, such approaches illustrate their potential application within the field of harmful algal research.Dissertation453 244 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Characterization of grazer-induced responses in the marine dinoflagellate Alexandrium tamarense(2013-04-19); ; ; Harmful algal blooms (HABs) have increased worldwide over the last several decades. The characterization of processes that promote the ecological success of toxic algae species that form such blooms has therefore gained immense importance. The HAB-dinoflagellate Alexandrium tamarense causes outbreaks of Paralytic Shellfish Poisoning (PSP) due to the synthesis of the highly neurotoxic alkaloid saxitoxin and several of its analogues (Paralytic Shellfish Toxins, PSTs). While the ecological function of PST production is still unknown and their allelochemical nature is mainly supposed due to the negative effects they have on some copepod species, there are other unknown secondary metabolites produced by A. tamarense which are definitely known to possess allelochemical properties. These compounds are distinct from PSTs and have a lytic effect on co-occurring competitors and protistan grazers. The investigations in this thesis focus on the analysis of mechanisms that promote the success in grazer interactions related to secondary metabolite synthesis in A. tamarense. The implementation of functional genomic tools enabled a detailed characterization of processes that are induced due to the presence of grazers in A. tamarense. Such induced processes that either decrease grazing pressure and/or provide an advantage over co-occurring species in the presence of grazers can strongly promote a species success.Dissertation282 192 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Evolutionary ecology of Alexandrium(Dinophyceae)with special emphasis on genotypic and phenotypic variation in the toxigenic species A. tamarense(2009-12-03); ; ; In this thesis, broad phenotypic variation in the genus Alexandrium with respect to allelochemical properties targeting a wide range of other planktonic protists was observed and indications of co-evolutionary processes shaping the respective allelopathic phenotype were found. At the intra- and inter-population level in the species A. tamarense large genotypic variation was observed. A conceptual population genetic model on how this variation is preserved in the long run - despite evidence of clonal selection and frequency shifts of clonal lineages during vegetative planktonic growth - is presented. Phenotypic characters such as allelochemical properties and content and relative composition of paralytic shellfish poisoning toxins (PSP) may be such characters that are under directional selection during planktonic population growth and harmful algal bloom (HAB) development. However, isolates from the populations studied varied widely with respect to these quantitative genetic characters.Dissertation340 161 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Characterization and lytic activity of allelochemicals produced by the toxigenic marine dinoflagellate Alexandrium tamarense(2010-12-13); ; ; In this thesis, allelochemistry of Alexandrium tamarense, a well-known producer of saxitoxin and its analogues was investigated in an attempt to interpret the chemical characteristics and mode of action of the allelochemicals produced. As the response of target cells was lysis, the allelochemicals were thereafter designated as lytic compounds. One A. tamarense clonal strain, Alex2, with potent lytic activity, was chosen as the producer of the lytic compounds. Another A. tamarense clonal strain from the same geographical population, but without measurable lytic activity was introduced as a negative control. First, a cryptophyte Rhodomonas salina bioassay was used to guide the isolation and eventual structural characterization of the unknown lytic substances. Highest lytic activity per cell of A. tamarense cultures was achieved in the stationary growth phase, however, the allelopathic potency per cell remained constant throughout the entire exponential phase. The supernatant of stationery cell cultures prepared via centrifugation was chosen as the major source of lytic compounds for research through out the whole thesis. Further isolation and purification of the supernatant often resulted in major loss or even disappearance of lytic activity, prompting the physico/chemical characterization of the lytic compounds. Although temporal stability of the lytic compounds was high, in that the lytic activity was stable over wide ranges of temperatures and pH and was refractory to bacterial degradation (at least of the bacteria consortium present in the cultures), substantial losses were evident during purification. The lytic compounds possessed high adsorption to various materials including polycarbonate and other plastics, glass, etc. Additionally, the lytic activity was reduced when stored at low concentration, in deionized water, or in vessels with large surface area/volume ratios. Similar to the bioactivity of allelochemicals produced among other harmful algal bloom species, A. tamarense produced a suite of metabolites acting as lytic compounds. At least two groups of lytic compounds with various polarities were separated and quantified via reversed phase solid phase extraction (SPE). Emphasis was put on the less polar fraction because of its higher lytic activity. Further purification of this SPE fraction was performed via both C8 high performance liquid chromatography (HPLC) and hydrophilic interaction ion-chromatography (HILIC). Through matrix assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF MS) masses of the spectra between the lytic and non-lytic strain in SPE, C8 HPLC, and HILIC fractions were compared. Two small unique masses, 1061.6 Da and 1291.6 Da were found in lytic but not in non-lytic SPE fractions, however, they were later excluded to be related to lytic compounds by both HPLC and HILIC. Trypsic digestion and trypsic digestion-coupled size exclusion chromatography (SEC) suggested that the lytic compounds are large non-proteinaceous compounds less than 22.3 kDa. Although the large masses range from 7 to 15 kDa found only in HILIC lytic fraction haven t been proved to be lytic, the mass range deduced from SEC suggest that they are probably related to the lytic compounds. Total sugar content analysis suggested the percentage of sugar in dry mass equivalent in the lytic fraction was rather low, indicating the major composition of the lytic compounds may not be related to sugar. Alexandrium tamarense causes lysis of a broad spectrum of target protistan cells, and since the process is rapid, direct membrane disruption of target cell was suspected to be the mode of action. The lytic compounds increased permeability of the cell membrane for calcium ion from buffer external to the outer cell membrane, and such increase was not inhibited even during blockade of calcium ion channels with cadmium. This indicates that the lytic compounds lyse membrane directly, and the molecular targets are not ion channels. Membrane sterols were then suspected as the molecular target of lytic compounds. Adding sterols to a lysis bioassay with the R. salina for evaluation of competitive binding indicated that the lytic compounds possessed apparent affinity for free sterols. For three tested sterols, the lytic compounds showed highest affinity towards cholesterol followed by ergosterol and brassicasterol. However, analysis of sterol composition of isolates of A. tamarense and of five target protistan species showed that both lytic and non-lytic A. tamarense strains contain cholesterol and dinosterol as major sterols, whereas none of the other tested species contain dinosterol. Cholesterol comprised a higher percentage of total sterols in plasma membrane fractions of A. tamarense than in corresponding whole cell fractions. It is therefore concluded that the molecular targets of the lytic compounds are not, or are not exclusively, sterol components of the membranes.Dissertation288 305 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Functional genomic insights into cellular processes related to harmful bloom formation in ichthyotoxic prymnesiophytes(2011-11-24); ; ; Not much information is available about the genetic background of growth and toxicity- related processes in toxic Haptophyta species. The aim of my thesis was to contribute to better understanding of these issues using functional and comparative genomic approaches with the ichthyotoxic prymnesiophytes Chrysochromulina polylepis and Prymnesium parvum. In particular, I explored different gene-expression profiling methods in order to monitor the transcriptomic responses in these species to different environmental conditions. Through the sequencing of a cDNA library, a transcriptomic database (Expressed Sequence Tag library) was established for both prymnesiophyte species. Approximately 2900 and 6300 contigs were found in the Chrysochromulina polylepis and Prymnesium parvum datasets, respectively. The sequences were annotated and compared to similar data sets available from other Haptophyta species (Pavlova lutherii, Isochrysis galbana and Emiliania huxleyi). This analysis revealed a `core set` of approx. 1500 genes which were found in all Haptophyta species investigated in this study. Moreover, 67 and 362 genes were present only in C. polylepis and P. parvum, respectively. The physiological background and cellular regulation of synthesis and liberation of Chrysochromulina and Prymnesium toxin(s) is still poorly understood, but the involvement of PKS genes in the biosynthesis of certain compounds is likely. The presence of the conserved ketosynthase (KS) domains - an obligatory part of PKS genes were shown in both species, represented by fourteen and four copies in C. polylepis and P. parvum, respectively. In order to indirectly test the hypothesis invoking a role of PKS genes in toxin biosynthesis, the correlation between toxicity and PKS gene expression was monitored in both species. The observed positive correlation strengthens the hypothesis on the involvement of PKS genes in toxin production C. polylepis as well as in P. parvum. A gene expression microarray was generated based on the EST data originating from P. parvum, and this tool was used to monitor gene-expression changes during growth in nutrient replete and phosphorus (P)- or nitrogen (N)-deprived P. parvum cells. In accord with previously published data, elevated intracellular toxicity was observed in P-deprived cells, whereas it did not change in N-depleted or nutrient replete cells. As a response to P limitation, the upregulation of different genes related to transport and acquisition of phosphate could be observed. On the other hand, N limitation did not lead to such a clear effect on the gene expression level, since most genes likely involved in the uptake, storage and transport of N sources were not upregulated. Utilizing the tools of ecophysiology and functional genomics we identified gene-expression patterns indicative of physiological (nutrient, toxicity) and growth status of C. polylepis and P. parvum. With reference to this data set, knowledge about cellular processes in toxic Prymnesiophyceae species was expanded considerably, and pointed the way forward for incorporation of functional genomic approaches to determining regulatory factors involved in prymnesiophyte bloom dynamics through gene expression studies.Dissertation290 143 - Some of the metrics are blocked by yourconsent settings
Item-typ:Veröffentlichung, Physiogenomics of Cylindrospermopsis raciborskii and Raphidiopsis brookii (Cyanobacteria) with Emphasis on Evolution, Nitrogen Control and Toxin Biosynthesis(2010-06-18); ; ; The freshwater toxic cyanobacteria Cylindrospermopsis raciborskii CS-505 and Raphidiopsis brookii D9 belong to the order Nostocales of filamentous, heterocystous (nitrogen fixation) morphotypes. However, Raphidiopsis spp. do not develop heterocyst nor fix N2, contradicting the morphological classification. This thesis presents an integrated study of evolutionary relationships linked to nitrogen (N) metabolism and toxin production in filamentous cyanobacteria. The genome sequencing and comparison of CS-505 and D9 revealed the smallest genomes of filamentous cyanobacteria and an extent of similarity corresponding to conspecific isolates, therefore recommending a taxonomical reclassification of the genera Raphidiopsis. The presence of heterocyst-specific genes and their expression under alternative N-sources in D9, together with differences in genomic plasticity, allowed us to propose divergent evolutionary pathways for both species. Late transcriptional responses were observed in C. raciborskii CS-505 under N-deprivation, contrasting with those of other heterocystous cyanobacteria. The pattern of biosynthetic gene regulation suggested that additional mechanisms of N-control and post-transcriptional mechanisms are involved in toxin production. The role of repetitive elements and transposases in gene regulation may be critical but this aspect requires further investigation.Dissertation314 144