Development of molecular tools to study Daphnia - parasite dynamics

Evolutionary dynamics of hosts and their parasites are complex processes. In order to study these processes on genotype level, reliable molecular tools have to be developed. The goal of this thesis was to develop such tools for freshwater crustaceans - Daphnia longispina species complex and its parasites. On one hand, from the host side - an interspecific hybridization plays an important role. For tracing community dynamics and reticulate evolution in such a hybrid species complex, long-term comparative studies of natural populations are necessary. In order to conduct such a study, it is essential to access historical samples. These samples are usually suffering from low DNA quality due to the preservation chemical such as formaldehyde or denaturated ethanol, therefore traditional genotyping through length-based markers (such as microsatellites or allozymes) proved to be insufficient. For circumventing these issues, SNP- based markers were developed. Based on transcriptome data of one species belonging to the complex, it was possible to amplify and sequence several unlinked loci, which were then scanned for species-specific SNPs. Altogether 11 loci distinguishing all three species of the complex and their hybrids were developed and incorporated into PCR-RFLP assay. By comparing the taxon assignment from microsatellite and SNP data, there was found nearly perfect concordance. Finally, the genotyping method was successfully tested on samples dating back to the year 1960. On the other hand, parasite genetic studies are much more limited, in terms of availability of molecular markers. Only handful of parasites allows their cultivation under laboratory conditions inside the hosts. Even then, their disproportional amount of DNA compared to hosts and additional presence of other organisms present in media is forcing to rely on the traditional markers such as internal transcribed spacer (ITS). The traditional method of obtaining sequences to access the diversity (Sanger sequencing preceded by cloning – due to high intragenomic variation of this region) is becoming inefficient due to its high costs in terms of funds and time. Therefore, there was developed a molecular pipeline able to produce and process larger amount of sequence data with more accurate processing, specifically using a next-generation-sequencing platform (454). Afterwards a new bioinformatic pipeline QRS (quantification of representative sequences) was developed, inferring the representative sequences from the next generation sequencing (NGS) data sets (based on neighbor joining or statistical parsimony) and calculating their frequencies. Verification of the method was done by comparing the dataset with the previous study of population structure of Daphnia parasite Caullerya mesnili based on cloning and Sanger sequencing. Pipelines were then used for accessing genetic diversity of the two parasite microsporidian species (Berwaldia and MIC1) commonly infecting Daphnia longispina complex in Central Europe. Specifically, the patterns of geographic population structure, intraspecific genetic variation, and the recombination events were examined, which are necessary for better characterization of the biology of these parasites. The limited geographical variation that was observed in Berwaldia and the different lake origin of recombinant and parental sequences supports usage of a mobile secondary host hypothesis during the life cycle of this species. Similarly MIC1 seems to have a secondary host, however the secondary hosts of both parasites likely differ and the Berwaldia`s one is assumed to have a higher mobility than the one transmitting MIC1.Die evolutionäre Dynamik von Wirtsarten und ihren Parasiten beinhaltet komplexe Prozesse. Zur Untersuchung dieser Prozesse auf der Ebene des Genotyps ist die Entwicklung zuverlässiger molekularer Werkzeuge notwendig. Das Ziel der vorliegenden Arbeit war die Entwicklung solcher Werkzeuge für den Daphnia longispina-Artenkomplex (Süßwasser-Crustaceen) und seine Parasiten. Einerseits spielt auf Seiten des Wirts interspezifische Hybridisierung eine große Rolle. Um die Dynamik der Artengemeinschaft sowie die verästelte Evolution innerhalb eines solchen Hybridartenkomplexes nachzuvollziehen, sind vergleichende Langzeitstudien natürlicher Populationen notwendig. Zur Durchführung einer solchen Studie ist die Einbeziehung historischer Proben essentiell. Diese Proben haben üblicherweise den Nachteil einer niedrigen DNA-Qualität durch Konservierungsmittel wie Formaldehyd oder vergällter Ethanol. Daher erwiesen sich traditionelle Methoden zur Genotypisierung mittels längenbasierter Marker wie Mikrosatelliten oder Allozyme als unzureichend. Aus diesem Grund wurden SNP-basierte Marker entwickelt. Basierend auf Transkriptomdaten einer Art aus dem Daphnia longispina-Artenkomplex konnten mehrere ungekoppelte Loci amplifiziert, sequenziert und nach artspezifischen SNPs durchsucht werden. Insgesamt wurden 11 SNP-Marker entwickelt, die alle drei Arten und ihre Hybriden unterscheiden und mit Hilfe eines PCR-RFLP-Tests untersucht wurden. Beim Vergleich der Taxonzuordnung von Mikrosatelliten- und SNP-Daten wurde annähernd perfekte Übereinstimmung gefunden. Schlussendlich konnte die Genotypisierungsmethode erfolgreich angewendet werden auf Proben, die aus dem Jahr 1960 stammen. Andererseits sind Studien zur Parasitengenetik viel beschränkter in Hinblick auf die Verfügbarkeit molekularer Marker. Nur wenige Parasiten können unter Laborbedingungen in den Wirtsorganismen kultiviert werden. Außerdem lässt die unverhältnismäßig kleine DNA-Menge der Parasiten im Vergleich zum Wirt und die Anwesenheit anderer Organismen im Kulturmedium wenig Alternativen zur Verwendung traditioneller Marker wie ITS (interne transkribierte Spacer). Die traditionelle Methode, Sequenzdaten zur Beurteilung der Diversität zu erhalten (Sanger-Sequenzierung gefolgt von Klonierung wegen der hohen intragenomischen Variation dieser Region) ist mittlerweile ineffizient durch den hohen finanziellen und zeitlichen Aufwand. Daher wurde eine molekulare Pipeline entwickelt, um größere Datenmengen zu produzieren und fehlerfreier zu verarbeiten unter Verwendung einer Next Generation Sequencing-Plattform (454). Anschließend wurde eine neuartige bioinformatische Pipeline ‚QRS‘ (Quantification of Representative Sequences, Quantifizierung Repräsentativer Sequenzen) entwickelt, die mithilfe von Neighbor Joining- und Parsimony-Ansätzen repräsentative Sequenzen aus Next Generation Sequencing (NGS)- Datensätzen ermittelt und deren Frequenzen berechnet. Die Methode wurde verifiziert durch den Vergleich des Datensatzes mit einer vorangegangenen Studie zur Populationsstruktur des Daphnia-Parasiten Caullerya mesnili, die auf Klonierung und Sanger-Sequenzierung basierte. Die Pipelines wurden anschließend zur Untersuchung der genetischen Diversität zweier parasitärer Mikrosporidienarten (Berwaldia und MIC1) verwendet, die häufig den Daphnia longispina-Komplex in Zentraleuropa infizieren. Besonders die Muster geografischer Populationsstruktur, intraspezifische genetische Variation und Rekombinationsereignisse wurden untersucht, da sie eine bessere Charakterisierung der Parasitenbiologie ermöglichen. Die bei Berwaldia beobachtete begrenzte geografische Variation und die Herkunft der rekombinanten und parentalen Sequenzen aus unterschiedlichen Seen unterstützt die Hypothese eines sekundären mobilen Wirts im Lebenszyklus dieser Art. Ebenso scheint MIC1 einen sekundären Wirt zu haben. Allerdings unterscheiden sich die sekundären Wirte der beiden Parasiten wahrscheinlich, wobei derjenige von Berwaldia vermutlich eine höhere Mobilität aufweist als der Wirt, der MIC1 überträgt

New possibilities arise for studies of hybridization: SNP-based markers for the multi-species Daphnia longispina complex derived from transcriptome data

In order to trace community dynamics and reticulate evolution in hybrid species complexes, long-term comparative studies of natural populations are necessary. Such studies require the development of tools for fine-scale genetic analyses. In the present study, we developed species-diagnostic SNP-based markers for hybridizing freshwater crustaceans: the multispecies Daphnia longispina complex. Specifically, we took advantage of transcriptome data from a key species of this hybrid complex, the annotated genome of a related Daphnia species and well-defined reference genotypes from three parental species. Altogether eleven nuclear loci with several species-specific SNP sites were identified in sequence alignments of these reference genotypes from three parental species and their interspecific hybrids. A PCR-RFLP assay was developed for cost-efficient large population screening by SNP-based genotyping. Taxon assignment by RFLP patterns was nearly perfectly concordant with microsatellite genotyping across several screened populations from Europe. Finally, we were able to amplify two short regions of these loci in formaldehyde-preserved samples dating back to the year 1960. The species-specific SNP-based markers developed here provide valuable tools to study hybridization over time, including the long-term impact of various environmental factors on hybridization and biodiversity changes. SNP-based genotyping will finally allow eco-evolutionary dynamics to be revealed at different time scale

Do microplastic particles affect Daphnia magna at the morphological, life history and molecular level?

Microplastic particles are ubiquitous not only in marine but also in freshwater ecosystems. However, the impacts of microplastics, consisting of a large variety of synthetic polymers, on freshwater organisms remains poorly understood. We examined the effects of two polymer mixtures on the morphology, life history and on the molecular level of the waterflea Daphnia magna (three different clones). Microplastic particles of similar to 40 mu m were supplied at a low concentration (1% of the food particles) leading to an average of similar to 30 particles in the digestive tract which reflects a high microplastic contamination but still resembles a natural situation. Neither increased mortality nor changes on the morphological (body length, width and tail spine length) or reproductive parameters were observed for adult Daphnia. The analyses of juvenile Daphnia revealed a variety of small and rather subtle responses of morphological traits (body length, width and tail spine length). For adult Daphnia, alterations in expression of genes related to stress responses (i.e. HSP60, HSP70 & GST) as well as of other genes involved in body function and body composition (i.e. SERCA) were observed already 48h after exposure. We anticipate that the adverse effects of microplastic might be influenced by many additional factors like size, shape, type and even age of the particles and that the rather weak effects, as detected in a laboratory, may lead to reduced fitness in a natural multi-stressor environment

Daphnia parasite dynamics across multiple Caullerya epidemics indicate selection against common parasite genotypes

Studies of parasite population dynamics in natural systems are crucial for our understanding of host–parasite coevolutionary processes. Some field studies have reported that host genotype frequencies in natural populations change over time according to parasite-driven negative frequency-dependent selection. However, the temporal patterns of parasite genotypes have rarely been investigated. Moreover, parasite-driven negative frequency-dependent selection is contingent on the existence of genetic specificity between hosts and parasites. In the present study, the population dynamics and host-genotype specificity of the ichthyosporean Caullerya mesnili, a common endoparasite of Daphnia water fleas, were analysed based on the observed sequence variation in the first internal transcribed spacer (ITS1) of the ribosomal DNA. The Daphnia population of lake Greifensee (Switzerland) was sampled and subjected to parasite screening and host genotyping during C. mesnili epidemics of four consecutive years. The ITS1 of wild-caught C. mesnili-infected Daphnia was sequenced using the 454 pyrosequencing platform. The relative frequencies of C. mesnili ITS1 sequences differed significantly among years: the most abundant C. mesnili ITS1 sequence decreased and rare sequences increased over the course of the study, a pattern consistent with negative frequency-dependent selection. However, only a weak signal of host-genotype specificity between C. mesnili and Daphnia genotypes was detected. Use of cutting edge genomic techniques will allow further investigation of the underlying micro-evolutionary relationships within the Daphnia–C. mesnili system

Evaluation of Efficiency and Risk of Project of Revitalisation of Production Facilities

This diploma thesis is focused on evaluation of efficiency and risks of project revitalization of production facilities. The core of the thesis is: a characterization of investment projects and their cycles, general approaches to economic evaluation of private projects, a description of general approaches to risk analysis of private projects. The last part of the thesis is created as a case study which is focused on an evaluation of efficiency and coordinating risks of a private investment project

Financial and Economic Feasibility of Public project

Předmětem této bakalářské práce je finanční a ekonomická proveditelnost veřejného projektu. V teoretické části bakalářské práce je specifikována problematika veřejného sektoru. Dále je zde popsáno ekonomické hodnocení veřejného projektu. Základní charakteristika veřejného projektu. Dalším z bodů teoretické části bakalářské práce je uvedení přístupů pro posouzení finanční proveditelnosti veřejného projektu, kde jsou také popsány jednotlivé možnosti financování projektu. Součástí práce je uvedení nákladových metod hodnocení veřejného projektu. V poslední části je posouzení ekonomické a finanční proveditelnosti na praktickém příkladu.The topic of the bachelor’s thesis is financial and economic feasibility of public project. The theoretical part specifies issues of public sector, economic evaluation and basic characteristics of public project. This part provides assessment approaches of financial feasibility of public project and gives examples of individual ways of financing. The thesis also includes cost assessment methods of public project. The last part is practical evaluation of economic and financial feasibility applied on specific example.

Evaluation of Efficiency and Risk of Project of Revitalisation of Production Facilities

Diplomová práce je zaměřena na posouzení efektivnosti a rizik projektu revitalizace výrobních prostor. Předmětem zpracování diplomové práce je: charakteristika investičních projektů a jejich životní cyklus, obecné přístupy k ekonomickému hodnocení soukromých projektů, uvedení obecných přístupů k rizikové analýze soukromých projektů. V poslední části práce je zpracována případová studie zaměřující se na posouzení efektivnosti a řízení rizik soukromého investičního projektu.This diploma thesis is focused on evaluation of efficiency and risks of project revitalization of production facilities. The core of the thesis is: a characterization of investment projects and their cycles, general approaches to economic evaluation of private projects, a description of general approaches to risk analysis of private projects. The last part of the thesis is created as a case study which is focused on an evaluation of efficiency and coordinating risks of a private investment project.