Biology and ecology of trematodes parasitizing aquatic snails in the Ruhr reservoir system in Germany

Parasites are integral and important elements of ecosystems that occur in virtually every habitat on this planet (Poulin 1999, Hudson et al. 2006). However, except for the obviously important role of some species as disease agents, parasites have long been neglected and considered unimportant in the context of most ecological studies. Only in recent years, studies have begun to investigate the ecological importance of parasites, e.g. their structuring forces in trophic transmissions and food webs (Lafferty et al. 2008) or their contribution to an ecosystem’s biomass (Kuris et al. 2008, Thieltges et al. 2008). It has been argued, that it is even impossible to fully understand ecosystems without considering the parasites therein (Lafferty et al. 2006a). Furthermore, based on their often complex life cycles and strong interaction in ecosystems, parasites may serve as useful bioindicators to assess environmental conditions and changes (Lafferty 1997, Vidal-Martínez et al. 2010, Nachev & Sures, 2015). Environmental changes, such as climate change and global warming are expected to have major impacts on parasites and on entire ecosystems, often with yet unpredictable consequences (Marcogliese 2001, 2008). It is, therefore, essential that we understand host-parasite systems at the ecosystem level. Although there are some exceptionally well-studied aquatic ecosystems that focus on the roles of parasites in these habitats and allow predictions of the impact and possible effects of environmental changes (e.g. Kuris et al. 2008, Preston et al. 2013, Thieltges et al. 2013, Lagrue et al. 2015), our knowledge of host-parasite interaction in man-made waterbodies, such as impounded lakes and reservoirs, is still very limited and large-scale studies focussing on the ecological role of parasites in such systems are lacking. This is especially critical, since such waterbodies fulfil vital roles in densely populated areas, where they serve as drinking water storages, recreational areas and constitute important biodiversity hot spots. The Ruhr area in Germany is one of the largest and most densely populated urban areas in Europe and the eponymous extensive reservoir system of the Ruhr and its tributaries plays a vital role for the whole region. Our knowledge of the ecological role of parasites in these aquatic habitats is still fairly limited, however. In order to bridge this gap, this thesis aims at assessing the biology and ecology of trematodes parasitizing aquatic snails in the Ruhr reservoir system in Germany. Trematodes in snails have been identified as a particularly promising group of parasites that lend themselves to investigating and assessing environmental conditions and ecosystem changes in aquatic systems (Huspeni & Lafferty 2004), due to their complex multiple-host life cycles that enable us to study the complex roles of parasites in ecosystems at many levels and in great detail. This thesis, therefore, aims at addressing the importance of trematodes in the Ruhr reservoir systems by identifying larval trematode infections in snails in the reservoirs of the Ruhr river system. The individual studies of this work provide (i) detailed integrative approaches to taxonomically questionable trematode groups to assess the full diversity in the ecosystem, (ii) an analysis of the productivity and emergence of trematode cercariae to assess the parasites’ functional role in the ecosystem, (iii) an evaluation of the risk factors of swimmer’s itch via a detailed case study of one of the Ruhr lakes, and finally (iv) an assessment of the overall diversity, distribution and community structure of larval trematodes and the identification of potential trematode transmission pathways between their host groups. Altogether, this holistic approach, encompassing trematode taxonomy, their functional part in ecosystems, the role of medically relevant species and their contribution to an ecosystem’s diversity will provide a detailed and comprehensive insight into the complex role of trematodes in an important reservoir system in Europe. In order to assess the diversity and distribution patterns of trematodes in the Ruhr area, snails were collected at several sampling sites in five reservoirs of the Ruhr river catchment area in Germany, Baldeneysee, Hengsteysee, Hennetalsperre, Sorpetalsperre and Versetalsperre, during the summer months of the years 2012 and 2013. Sampling and analyses were mostly focused on gastropod snails belonging to two families, Lymaeidae and Planorbidae, since they proved to harbour the most diverse trematode fauna in Europe (Faltýnková & Haas 2006, Soldánová et al. 2011, Brown et al. 2011). A total of 3,171 lymnaeid snails belonging to four species, Radix auricularia, Stagnicola palustris, Radix peregra and Lymnaea stagnalis, and 2,176 planorbid snails belonging to two species, Gyraulus albus and Segmentina nitida, were collected and screened for trematode infections. Trematode stages were identified according to morphological features and, where necessary, with the aid of molecular methods. Of the 5,347 sampled snails, 1,049 showed patent or prepatent infections with trematodes, resulting in a total prevalence of 19.6%. Detailed integrative approaches of molecular and morphological methods revealed the presence of cryptic species within the trematode genera Echinostoma, Petasiger and Diplostomum. The analysis of Echinostoma spp. cercariae allowed the delineation of two cryptic species of the ‘revolutum’ group from larval stages found in R. auricularia, R. peregra and S. palustris. Likewise, analyses of infections from G. albus revealed the distinctive status of three species of Petasiger from this host that occurred in sympatry at one locality, highlighting the high diversity within this trematode genus, even at small spatial scales. Most distinctively, seven different species of Diplostomum were found in the lymnaeid snail populations from the Ruhr reservoirs, three named species, D. spathaceum, D. pseudospathaceum and D. parviventosum, and four species-level lineages, ‘Diplostomum sp. Clade Q’ and ‘D. mergi’ Lineages 2-4. Furthermore, the detailed approach provided evidence that the cercariae of ‘D. mergi’ Lineage 1 of Georgieva et al. (2013a) are actually D. parviventosum. Remarkably, the snail species L. stagnalis and S. palustris harboured only one species, D. pseudospathaceum, while R. auricularia populations revealed a highly diverse picture with six different lineages of Diplostomum, and it remains to be investigated why the diversity of Diplostomum in these hosts presents such contrary situation. Altogether, these integrative approaches of molecular and morphological data advance our knowledge of the taxonomic situation of these trematode taxa and reveal the remarkably high cryptic diversity of these parasites in the Ruhr reservoir system. However, the knowledge of the sheer diversity of trematodes in snails does not provide information on their functional role within an ecosystem. In order to assess the contribution of trematode cercariae to the biomass in European freshwaters, cercarial emergence of the bird schistosome Trichobilharzia szidati from naturally infected L. stagnalis was studied. The study revealed an average daily emergence rate of 2,621 cercariae per snail, with emissions peaks of up to 29,560 cercariae. Calculated for an individual snail’s lifetime this summed up to a cumulative cercarial biomass of 4.8 g, a mass equivalent of or even exceeding the snail‘s own body weight, illustrating the ecological importance a single trematode species that contributes a considerable amount of cercarial mass to an aquatic ecosystem. Since T. szidati is only one out of 37 species that produce large amounts of cercariae, the overall cercarial biomass emitted into the Ruhr reservoirs is comparable to the impressive numbers recently calculated for marine (Thieltges et al. 2008), estuary (Kuris et al. 2008) and North American freshwater ecosystems (Preston et al. 2013). The results of this study demonstrate how trematodes, despite their small individual size, significantly contribute to the biotic productivity in the Ruhr freshwater system. Since bird schistosomes are the causative agents of swimmer’s itch, a re-emerging disease in Europe (Soldánová et al. 2013), the disease risk factors in the Ruhr area based on the occurrence, distribution and biology of these parasites was assessed for one reservoir (Baldeneysee). Two bird schistosome species, Trichobilharzia franki and T. szidati, could be detected at several sampling sites in Baldeneysee, where abundant lymnaeid snail populations were present. Although these species showed only low prevalence, human infections are well possible, due to the high numbers of cercariae that can be released from individual infected snail hosts in short periods of time, as shown in the laboratory emission studies. With a total of 37 species, the trematode species richness in the Ruhr reservoir system was considerably higher than the trematode species richness in snail intermediate hosts described from most other well-studied ecosystems (e.g. Faltýnková 2005, Faltynkova & Haas 2006, Thieltges et al. 2006, Żbikowska 2007, Kuris et al. 2008, Lagrue & Poulin 2015). Altogether, Radix auricularia harboured by far the most prevalent, species rich and diverse trematode fauna of all studied hosts, supporting the assumption that R. auricularia plays the most important role in the life cycle of trematodes in large reservoirs and lakes, comparable to the dominant role of L. stagnalis described for small pond systems. Similarly, G. albus harboured more diverse and prevalent trematode communities compared to S. nitida, which contrasts with the situation observed for these two species in small ponds (Faltýnková et al. 2008a). The analysis of the data from the Ruhr, therefore, suggests a characteristic host-parasite dynamics in large reservoir systems. The majority of trematode species in the Ruhr requires fish-eating or anatid birds as final hosts and almost all trematode species have life cycles involving trophic transmission of the parasites to their respective final host, which provides information on trophic interactions and energy flow in the ecosystem. Since trematodes with trophic transmission strategies often directly or indirectly alter their host’s behaviour in order to facilitate transmission success to the next host (see e.g. Lafferty & Morris 1996), they actively shape the structure of food webs through which they are transmitted. Therefore, the rich and abundant trematode fauna in the Ruhr freshwaters plays a highly complex role in the food web connectivity in the Ruhr reservoirs. Overall, the selected aspects of trematodes in the Ruhr river system studied in the context of this thesis provide a broad and comprehensive overview of these parasites in a freshwater ecosystem that is typical for freshwater reservoirs in Europe for the first time. Trematodes are deeply embedded in and active elements of the ecological processes that shape and structure ecological communities, energy flow and the biodiversity of complex ecosystems and the rich trematode fauna in the Ruhr contributes to key aspects that make this ecosystem more diverse, productive and stable, and thus healthy

Global analysis of seasonal changes in trematode infection levels reveals weak and variable link to temperature

Seasonal changes in environmental conditions drive phenology, i.e., the annual timing of biological events ranging from the individual to the ecosystem. Phenological patterns and successional abundance cycles have been particularly well studied in temperate freshwater systems, showing strong and predictable synchrony with seasonal changes. However, seasonal successional changes in the abundance of parasites or their infection levels in aquatic hosts have not yet been shown to follow universal patterns. Here, using a compilation of several hundred estimates of spring-to-summer changes in infection by trematodes in their intermediate and defnitive hosts, spanning multiple species and habitats, we test for general patterns of seasonal (temperature) driven changes in infection levels. The data include almost as many decreases in infection levels from spring to summer as there are increases, across diferent host types. Our results reveal that the magnitude of the springto-summer change in temperature had a weak positive efect on the concurrent change in prevalence of infection in frst intermediate hosts, but no efect on the change in prevalence or abundance of infection in second intermediate or defnitive hosts. This was true across habitat types and host taxa, indicating no universal efect of seasonal temperature increase on trematode infections. This surprising variation across systems suggests a predominance of idiosyncratic and species-specifc responses in trematode infection levels, at odds with any clear phenological or successional pattern. We discuss possible reasons for the minimal and variable efect of seasonal temperature regimes, and emphasise the challenges this poses for predicting ecosystem responses to future climate change

Larval trematode communities in Radix auricularia and Lymnaea stagnalis in a reservoir system of the Ruhr River

<p>Abstract</p> <p>Background</p> <p>Analysis of the data available from traditional faunistic approaches to mollusc-trematode systems covering large spatial and/or temporal scales in Europe convinced us that a parasite community approach in well-defined aquatic ecosystems is essential for the substantial advancement of our understanding of the parasite response to anthropogenic pressures in urbanised areas which are typical on a European scale. Here we describe communities of larval trematodes in two lymnaeid species, <it>Radix auricularia </it>and <it>Lymnaea stagnalis </it>in four man-made interconnected reservoirs of the Ruhr River (Germany) focusing on among- and within-reservoir variations in parasite prevalence and component community composition and structure.</p> <p>Results</p> <p>The mature reservoir system on the Ruhr River provides an excellent environment for the development of species-rich and abundant trematode communities in <it>Radix auricularia </it>(12 species) and <it>Lymnaea stagnalis </it>(6 species). The lake-adapted <it>R. auricularia </it>dominated numerically over <it>L. stagnalis </it>and played a major role in the trematode transmission in the reservoir system. Both host-parasite systems were dominated by bird parasites (13 out of 15 species) characteristic for eutrophic water bodies. In addition to snail size, two environmental variables, the oxygen content and pH of the water, were identified as important determinants of the probability of infection. Between-reservoir comparisons indicated an advanced eutrophication at Baldeneysee and Hengsteysee and the small-scale within-reservoir variations of component communities provided evidence that larval trematodes may have reflected spatial bird aggregations (infection 'hot spots'). Two life history groupings of dominant species, the 'cyprinid' and 'anatid' parasites, that depict two aspects of progressive eutrophication in this mature reservoir system, were identified.</p> <p>Conclusions</p> <p>We conclude that trematode communities in the lake-adapted <it>R. auricularia </it>are better suited for monitoring the effect of environmental change on host-parasite associations in the reservoir system on the Ruhr River and other similar systems due to the important role of this host in trematode transmission in lakes. Whereas variations in trematode community diversity and abundance may indicate the degree of eutrophication on a larger scale (among reservoirs), the infection rates of the two life history groups of dominant species, the 'cyprinid' and 'anatid' assemblages, may be particularly useful in depicting environmental variability, eutrophication effects and infection 'hot spots' on smaller spatial scales.</p

New cryptic species of the ‘revolutum’ group of Echinostoma (Digenea: Echinostomatidae) revealed by molecular and morphological data

BACKGROUND: The digenean species of Echinostoma (Echinostomatidae) with 37 collar spines that comprise the so-called ‘revolutum’ species complex, qualify as cryptic due to the interspecific homogeneity of characters used to differentiate species. Only five species were considered valid in the most recent revision of the group but recent molecular studies have demonstrated a higher diversity within the group. In a study of the digeneans parasitising molluscs in central and northern Europe we found that Radix auricularia, R. peregra and Stagnicola palustris were infected with larval stages of two cryptic species of the ‘revolutum’ complex, one resembling E. revolutum and one undescribed species, Echinostoma sp. IG. This paper provides morphological and molecular evidence for their delimitation. METHODS: Totals of 2,030 R. auricularia, 357 R. peregra and 577 S. palustris were collected in seven reservoirs of the River Ruhr catchment area in Germany and a total of 573 R. peregra was collected in five lakes in Iceland. Cercariae were examined and identified live and fixed in molecular grade ethanol for DNA isolation and in hot/cold 4% formaldehyde solution for obtaining measurements from fixed materials. Partial fragments of the mitochondrial gene nicotinamide adenine dinucleotide dehydrogenase subunit 1 (nad1) were amplified for 14 isolates. RESULTS: Detailed examination of cercarial morphology allowed us to differentiate the cercariae of the two Echinostoma spp. of the ‘revolutum’ species complex. A total of 14 partial nad1 sequences was generated and aligned with selected published sequences for eight species of the ‘revolutum’ species complex. Both NJ and BI analyses resulted in consensus trees with similar topologies in which the isolates from Europe formed strongly supported reciprocally monophyletic lineages. The analyses also provided evidence that North American isolates identified as E. revolutum represent another cryptic species of the ‘revolutum’ species complex. CONCLUSION: Our findings highlight the need for further analyses of patterns of interspecific variation based on molecular and morphological evidence to enhance the re-evaluation of the species and advance our understanding of the relationships within the ‘revolutum’ group of Echinostoma

A Proteomic Survey of Host and Virus Reveals Differential Dynamics

We studied the dynamics of the proteome of influenza virus A/PR/8/34 (H1N1) infected Madin-Darby canine kidney cells up to 12 hours post infection by mass spectrometry based quantitative proteomics using the approach of stable isotope labeling by amino acids in cell culture (SILAC). We identified 1311 cell proteins and, apart from the proton channel M2, all major virus proteins. Based on their abundance two groups of virus proteins could be distinguished being in line with the function of the proteins in genesis and formation of new virions. Further, the data indicate a correlation between the amount of proteins synthesized and their previously determined copy number inside the viral particle. We employed bioinformatic approaches such as functional clustering, gene ontology, and pathway (KEGG) enrichment tests to uncover co- regulated cellular protein sets, assigned the individual subsets to their biological function, and determined their interrelation within the progression of viral infection. For the first time we are able to describe dynamic changes of the cellular and, of note, the viral proteome in a time dependent manner simultaneously. Through cluster analysis, time dependent patterns of protein abundances revealed highly dynamic up- and/or down-regulation processes. Taken together our study provides strong evidence that virus infection has a major impact on the cell status at the protein level

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7.

Insertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs)

Isolation of Human Colon Stem Cells Using Surface Expression of PTK7

SummaryInsertion of reporter cassettes into the Lgr5 locus has enabled the characterization of mouse intestinal stem cells (ISCs). However, low cell surface abundance of LGR5 protein and lack of high-affinity anti-LGR5 antibodies represent a roadblock to efficiently isolate human colonic stem cells (hCoSCs). We set out to identify stem cell markers that would allow for purification of hCoSCs. In an unbiased approach, membrane-enriched protein fractions derived from in vitro human colonic organoids were analyzed by quantitative mass spectrometry. Protein tyrosine pseudokinase PTK7 specified a cell population within human colonic organoids characterized by highest self-renewal and re-seeding capacity. Antibodies recognizing the extracellular domain of PTK7 allowed us to isolate and expand hCoSCs directly from patient-derived mucosa samples. Human PTK7+ cells display features of canonical Lgr5+ ISCs and include a fraction of cells that undergo differentiation toward enteroendocrine lineage that resemble crypt label retaining cells (LRCs)

Gene expression profiling in whole blood of patients with coronary artery disease

Owing to the dynamic nature of the transcriptome, gene expression profiling is a promising tool for discovery of disease-related genes and biological pathways. In the present study, we examined gene expression in whole blood of 12 patients with CAD (coronary artery disease) and 12 healthy control subjects. Furthermore, ten patients with CAD underwent whole-blood gene expression analysis before and after the completion of a cardiac rehabilitation programme following surgical coronary revascularization. mRNA and miRNA (microRNA) were isolated for expression profiling. Gene expression analysis identified 365 differentially expressed genes in patients with CAD compared with healthy controls (175 up- and 190 down-regulated in CAD), and 645 in CAD rehabilitation patients (196 up- and 449 down-regulated post-rehabilitation). Biological pathway analysis identified a number of canonical pathways, including oxidative phosphorylation and mitochondrial function, as being significantly and consistently modulated across the groups. Analysis of miRNA expression revealed a number of differentially expressed miRNAs, including hsa-miR-140-3p (control compared with CAD, P=0.017), hsa-miR-182 (control compared with CAD, P=0.093), hsa-miR-92a and hsa-miR-92b (post- compared with pre-exercise, P<0.01). Global analysis of predicted miRNA targets found significantly reduced expression of genes with target regions compared with those without: hsa-miR-140-3p (P=0.002), hsa-miR-182 (P=0.001), hsa-miR-92a and hsa-miR-92b (P=2.2×10−16). In conclusion, using whole blood as a ‘surrogate tissue’ in patients with CAD, we have identified differentially expressed miRNAs, differentially regulated genes and modulated pathways which warrant further investigation in the setting of cardiovascular function. This approach may represent a novel non-invasive strategy to unravel potentially modifiable pathways and possible therapeutic targets in cardiovascular disease

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo

Cortactin is required for endothelial barrier function and leukocyte recruitment in vivo