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Soil performs numerous functions, which allow us to produce food and feed and provide us with clean freshwater. These functions rely on the high diversity of organisms residing in soils. Within the high complexity of the soil food web, nematodes, worm-shaped animals belonging to the phylum Nematoda, are an informative group for assessing the status of a soil-dwelling community due to their ubiquity, abundance and trophic diversity. Although nematodes also possess several other assets favourable for a biological indicator of soil ecosystems (e.g. easy extractability, differential sensitivities to disturbances, ecological interpretability), their microscopic identification demands a considerable amount of expertise and time because of their relatively conserved morphology. Hence, the use of a molecular method for the identification and quantification of nematode assemblages has the potential to lift practical limitations and allows for more intensive sampling schemes. The aim of the research described in this thesis was to assess the suitability of molecular taxon-specific assays, developed on the basis of a phylum-wide molecular framework of 2,400 full-length small subunit ribosomal DNA sequences, for the monitoring of nematode assemblages in field experiments. The method was applied to monitor the impact of different types of disturbances on the soil food web, i.e. agricultural practices, invasive plant species and the effects of genetically modified crop (potato). The second chapter of this thesis presents the background of the molecular method and the results of its first field application. It demonstrates the suitability of this method for use in extensive field experiments and the results of this study reveal distinct seasonal fluctuations between nematode genera classified to belong to the same feeding type group. A distinct response of nematode genera within trophic groups – taxonomically diverse groups of nematodes having the nature of their main food source as a common denominator – was also observed in the study described in Chapter 3. In this chapter, a study was conducted to investigate the impact of an invasive plant species called Giant goldenrod (Solidago gigantea, native to North America) on the plant community as well as on different trophic levels of the soil food web. In addition to monitoring the nematode community, pH and fungal biomass were measured in plots invaded or uninvaded by Giant goldenrod in two contrasting habitats. The results revealed that, in addition to outcompeting native plant species, this invader also reduced pH and increased fungal biomass in the soil of both habitats. Based on the results concerning the nematode community, the impact on the soil food web seemed to be selective since the local increase of fungal biomass appeared to benefit only one fungivorous nematode lineage of the three present in the field. This suggests that invasion by Giant goldenrod only stimulates one part of the fungal community. Contrary to the preceding chapters, the effects of different disturbances on the nematode community were studied in an arable setting in chapters 4 and 5. Chapter 4 describes a field experiment in which the impact of biofumigation, a pest control measure, on the nematode community was assessed. Biofumigation is considered as an alternative for the use of synthetic fumigants and entails the incorporation of mulched brassicaceous plant material, which, upon de-compartmentalisation, releases general biocides called isothiocyanates. In our experiment, these compounds as well as their precursors could not be related to the effects observed for the nematode community. Therefore, changes in nematode assemblages are more likely to be related to the intense mechanical disturbance and green manure – the addition of a large quantity of fresh plant material to the topsoil – rather than the release of isothiocyanates from the plant material Chapter 5 presents a field experiment in which the possible belowground side effects of a waxy starch GM potato, a genetically modified plant blocked in its amylose biosynthesis, were investigated. The nematode community was monitored during the growing season of this GM variety, its parental line and four other conventional potato cultivars in two experimental fields. Although we observed clear effects of location and time, no GM-related effects were observed on the nematode community. Our results, in line with previous studies concerning the microbial community, indicate there are no observable, non-transient effects related to this particular GM trait on the soil food web during the growing season. Overall, the results presented in this thesis demonstrate that, first of all, the developed molecular approach is suitable as a tool for the quantitative monitoring of nematode assemblages in field experiments, and, secondly, how a molecular monitoring method based on nematode taxon-specific DNA motifs can be exploited to get new insights into the ecology of terrestrial nematodes and – more in general – into the ecological functioning of this obscure, highly biodiverse and poorly understood habitat below our feet.

Soil performs numerous functions, which allow us to produce food and feed and provide us with clean freshwater. These functions rely on the high diversity of organisms residing in soils. Within the high complexity of the soil food web, nematodes, worm-shaped animals belonging to the phylum Nematoda, are an informative group for assessing the status of a soil-dwelling community due to their ubiquity, abundance and trophic diversity. Although nematodes also possess several other assets favourable for a biological indicator of soil ecosystems (e.g. easy extractability, differential sensitivities to disturbances, ecological interpretability), their microscopic identification demands a considerable amount of expertise and time because of their relatively conserved morphology. Hence, the use of a molecular method for the identification and quantification of nematode assemblages has the potential to lift practical limitations and allows for more intensive sampling schemes. The aim of the research described in this thesis was to assess the suitability of molecular taxon-specific assays, developed on the basis of a phylum-wide molecular framework of 2,400 full-length small subunit ribosomal DNA sequences, for the monitoring of nematode assemblages in field experiments. The method was applied to monitor the impact of different types of disturbances on the soil food web, i.e. agricultural practices, invasive plant species and the effects of genetically modified crop (potato). The second chapter of this thesis presents the background of the molecular method and the results of its first field application. It demonstrates the suitability of this method for use in extensive field experiments and the results of this study reveal distinct seasonal fluctuations between nematode genera classified to belong to the same feeding type group. A distinct response of nematode genera within trophic groups – taxonomically diverse groups of nematodes having the nature of their main food source as a common denominator – was also observed in the study described in Chapter 3. In this chapter, a study was conducted to investigate the impact of an invasive plant species called Giant goldenrod (Solidago gigantea, native to North America) on the plant community as well as on different trophic levels of the soil food web. In addition to monitoring the nematode community, pH and fungal biomass were measured in plots invaded or uninvaded by Giant goldenrod in two contrasting habitats. The results revealed that, in addition to outcompeting native plant species, this invader also reduced pH and increased fungal biomass in the soil of both habitats. Based on the results concerning the nematode community, the impact on the soil food web seemed to be selective since the local increase of fungal biomass appeared to benefit only one fungivorous nematode lineage of the three present in the field. This suggests that invasion by Giant goldenrod only stimulates one part of the fungal community. Contrary to the preceding chapters, the effects of different disturbances on the nematode community were studied in an arable setting in chapters 4 and 5. Chapter 4 describes a field experiment in which the impact of biofumigation, a pest control measure, on the nematode community was assessed. Biofumigation is considered as an alternative for the use of synthetic fumigants and entails the incorporation of mulched brassicaceous plant material, which, upon de-compartmentalisation, releases general biocides called isothiocyanates. In our experiment, these compounds as well as their precursors could not be related to the effects observed for the nematode community. Therefore, changes in nematode assemblages are more likely to be related to the intense mechanical disturbance and green manure – the addition of a large quantity of fresh plant material to the topsoil – rather than the release of isothiocyanates from the plant material Chapter 5 presents a field experiment in which the possible belowground side effects of a waxy starch GM potato, a genetically modified plant blocked in its amylose biosynthesis, were investigated. The nematode community was monitored during the growing season of this GM variety, its parental line and four other conventional potato cultivars in two experimental fields. Although we observed clear effects of location and time, no GM-related effects were observed on the nematode community. Our results, in line with previous studies concerning the microbial community, indicate there are no observable, non-transient effects related to this particular GM trait on the soil food web during the growing season. Overall, the results presented in this thesis demonstrate that, first of all, the developed molecular approach is suitable as a tool for the quantitative monitoring of nematode assemblages in field experiments, and, secondly, how a molecular monitoring method based on nematode taxon-specific DNA motifs can be exploited to get new insights into the ecology of terrestrial nematodes and – more in general – into the ecological functioning of this obscure, highly biodiverse and poorly understood habitat below our feet.

Nematodes are probably the most abundant Metazoans on our planet. They are present in densities of millions of individuals per square meter in soil and sediments. However, these inconspicuous animals are hardly known to the general public as most individuals are colorless and smaller than 1 mm. If people are aware of nematodes, it is because of the damage they inflict on humans and animals such as elephantiasis (Wucheria bancrofti) and ascariasis (in humans Ascaris lubricoides; in pigs Ascaris suum), or on plants such as potato (potato cyst nematode) and tomato (root-knot nematodes). To a far lesser extent it is known that majority of nematodes are key players in the soil food web, and as such they can be used as indicators for the biological condition of the environment they live in. This PhD thesis focuses on terrestrial nematodes belonging to four orders: Dorylaimida, Mononchida, Aphelenchida and Tylenchida, which represent animals of ecological and economical relevance. Members of families belonging to the first two are highly sensitive to the environmental disturbances, and are informative as biological indicators. The order Aphelenchida harbors numerous facultative plant-parasitic species. In the absence of a host plant, most of them are able to feed on fungi as an alternative food source. This is in contrast to the distal representatives of the order Tylenchida that are invariably obligate parasites of higher plants. Stress-sensitive nematode orders Dorylaimida and Mononchida have a high potential for soil health assessment. The SSU rDNA-based analysis of these two orders resulted in two highly distinct phylogenies. Relationships among the Mononchida, an order dominated by carnivorous nematodes, were to some extent in accordance with the classical nematode systematics. It is noted that the families Mylonchulidae, Mononchidae and Anatonchidae are not monophyletic. Nevertheless, it was possible to design family-specific primers for rDNA-based molecular detection. On the contrary, resolution of the SSU rDNA tree of the Dorylaimida was extremely poor, except for the plant-parasitic family Longidoridae and the mainly predaceous family Nygolaimidae. Analysis of a 1,000 bp fragment of the 5’ region of large subunit (LSU) rDNA resulted in an improved resolution. Twelve subclades were distinguished and this topology was only in slight agreement with the classical systematics of the suborder Dorylaimina. The poor resolution generated by SSU rDNA sequence analysis within this species-rich suborder is remarkable; it has not been observed in any other suborder in the phylum Nematoda. Possibly, Dorylaimina diversification is the result of rapid speciation events. A plant-parasitic lifestyle apparently accelerates the rate of change of rDNA genes. This was not only true for the obligate plant-parasitic Longidoridae, but also for the facultative plant-parasitic Aphelenchoididae. Most members of the genus Aphelenchoides are fungivores, but a few of them feed on higher plants as well. As they feed on aboveground parts of higher plants they are usually called ‘foliar nematodes’. Species such as Aphelenchoides besseyi, A. fragariae and A. ritzemabosi parasitize on ornamental plants in greenhouses and nurseries, and some field crops such as rice or strawberry. Moreover, A. subtenuis causes serious damage infecting flower bulbs. Identification of foliar nematode species, and the distinction between plant- parasitic species and other, mostly harmless, fungal feeding representatives of the genus Aphelenchoides is hampered by the scarcity of informative morphological characters and lack of well-established systematics. Based on nearly full-length SSU rDNA sequences, a phylogenetic tree was generated, where the four target species appeared as distinct, well-supported, monophyletic groups. The presence of species-specific DNA motifs made it possible to design PCR primers for the detection and quantification of the foliar nematode species in complex DNA backgrounds such as plant material and soil samples. The order Tylenchida is dominated by obligatory plant-parasitic nematode taxa and includes economically high-impact species such as the lesion (Pratylenchus spp.), root-knot (Meloidgyne spp.) and cyst (Heterodera, Globodera) nematodes. The sequence diversification of cellulases, a non-neutral, plant pathogenicity-related genes, was investigated. Unlike by far most other animals, nematodes do not depend on endosymbionts for the production of cell wall-degrading enzymes. Among a repertoire of these proteins, glycoside hydrolase family 5 (GHF5) cellulases are best studied. It is hypothesized that they were acquired by one or multiple horizontal gene transfer (HGT) events. Moreover, the nature of the donor - hypothesized to be a plant-parasitic soil bacterium - and the recipient, possibly a bacterivorous nematode is fully unclear. Using a range of primers, partial GHF5 cellulase sequences spanning the core catalytic domain were amplified and sequenced from basal Meloidogyne, and a range of Pratylenchus and Hirschmanniella species. Phylogenetic analysis of more than 100 partial GHF5 cellulase sequences resulted in a division of the enzymes’ catalytic domains into three types (A, B, C). Type B was numerically dominant, and notably the P. thornei cellulase was positioned sister to all type B root-knot nematode cellulases. Moreover, the overall topology of the catalytic domain B-type showed remarkable resemblance with trees based on rDNA sequences. This analysis suggests that most likely the cellulases were passed on by ancestors of a family nowadays known as the Pratylenchidae, and root-knot and cyst nematodes did not acquire these genes directly by lateral genes transfer. To further elucidate the relationship between a part of the family Pratylenchidae (namely the subfamilies Pratylenchinae and Hirschmanniellinae) and the Meloidogynidae, two neutral (= pathogenicity-unrelated) genes were taken into consideration: SSU rDNA and a part of the largest subunit of the RNA polymerase II gene (rpb1). Both morphological and molecular data seem to point at root-knot nematodes being a subclade branching from the migratory endoparasites Pratylenchidae. Extension of the SSU rDNA data set - more sequences from a broader range of species – did not result in a well- resolved relationship between the Pratylenchidae and the Meloidogynidae. A switch to another gene that was previously exploited to investigate relationships within the genus Meloidogyne, a fragment of the largest subunit of RNA polymerase II sequences, did not provide us with more robust information about the evolutionary transition between lesion and root-knot nematodes. The genus Pratylenchus comprises more than 100 species. It is referred to as a stenomorphic genus since only a few subtle characteristics are used for species identification. In the current study, only a subset of these species was taken to consideration; predominantly species that are relatively well-characterized as pathogens in agro-ecosystems. It was postulated that the Pratylenchus species closest to the basal root-knot nematodes should be sought among the less well-known and agronomically less relevant lesion nematode species.

Burning of fossil fuels has raised the level of atmospheric carbon dioxide, which contributes to global climate warming. As a result the mean earth surface temperature has increased faster in the past decades than in the previous thousands of years before. This rapid climate warming together with habitat fragmentation and other land use changes puts a major pressure on many plants and animals. They should either adapt to the warmer climate conditions or disperse in order to keep up with their optimal climatic conditions. Range expansion brings new interactions within the ecosystem in the new range. This can lead to potential benefits, for example range shifting species that do not encounter natural enemies in the new range might become invasive. Although invasive species are a well-studied phenomenon, there is relatively little known about the general mechanisms of biological invasions under climate change. In this thesis I focus on plant species that expand range due to current climate warming. I examined how these range-expanding plants interact with aboveground herbivorous insects and - mostly - how they establish belowground interactions with components of the soil food web. I examined how these interactions in the new range may play a role in the successful establishment of climate change induced range-expanding plants in plant communities of the new range. The focus of my study was on riverine (riparian) areas along the great rivers in the Netherlands, which are well connected with southern Europe by the Rhine and Rhine-Danube canal. In the first experiment we examined exotic plant exposure to aboveground and belowground enemies. We used plants that originated from Eurasia (intra-continental range expanders) and plants that originated from other continents (inter-continental range expanders). We compared these exotic plants with phylogenetically related natives. We grew the plants with and without non-coevolved polyphagous (generalist) herbivores, a locust Schistocerca gregaria and an aphid Myzus persicae. We also exposed all plants to a general soil community from the invaded range and compared their plant-soil feedback responses. Then I tested how individual plants responded to aboveground and belowground plant enemies and I compared this to their combined effects. I also tested whether the strength of aboveground control by generalist shoot-feeding insects was indicative of the strength of belowground control by plant-soil feedback. In the next study I examined how the soil nematode community from the new range responds to exotic plant species compared to related native plants species. As a follow up, I determined the rhizosphere community composition of bacteria, fungi, arbuscular mycorrhizal fungi (AMF) and fusaria. All groups of microbes were analyzed qualitatively and the non-mycorrhizal fungal biomass and fusaria were also analyzed quantitatively. I tested the hypothesis that range-expanding plant species have a different rhizosphere microbial community composition than natives. Finally, I compared the early establishment of range-expanding exotics and phylogenetically related plant species that are native in the invaded habitats. In a greenhouse I grew five range-expanding plant species and five related natives in sterilized and non-sterile inoculated soils from the new range, both alone and with a background community of plant species present in the invaded habitat. In the field, I grew the same plants species in artificially created sparse and dense plant communities. I tested whether range-expanding exotic plant species establish better under competition with native vegetation than phylogenetically related natives, because exotics may benefit from less negative interactions with the soil community compared to natives.

Burning of fossil fuels has raised the level of atmospheric carbon dioxide, which contributes to global climate warming. As a result the mean earth surface temperature has increased faster in the past decades than in the previous thousands of years before. This rapid climate warming together with habitat fragmentation and other land use changes puts a major pressure on many plants and animals. They should either adapt to the warmer climate conditions or disperse in order to keep up with their optimal climatic conditions. Range expansion brings new interactions within the ecosystem in the new range. This can lead to potential benefits, for example range shifting species that do not encounter natural enemies in the new range might become invasive. Although invasive species are a well-studied phenomenon, there is relatively little known about the general mechanisms of biological invasions under climate change. In this thesis I focus on plant species that expand range due to current climate warming. I examined how these range-expanding plants interact with aboveground herbivorous insects and - mostly - how they establish belowground interactions with components of the soil food web. I examined how these interactions in the new range may play a role in the successful establishment of climate change induced range-expanding plants in plant communities of the new range. The focus of my study was on riverine (riparian) areas along the great rivers in the Netherlands, which are well connected with southern Europe by the Rhine and Rhine-Danube canal. In the first experiment we examined exotic plant exposure to aboveground and belowground enemies. We used plants that originated from Eurasia (intra-continental range expanders) and plants that originated from other continents (inter-continental range expanders). We compared these exotic plants with phylogenetically related natives. We grew the plants with and without non-coevolved polyphagous (generalist) herbivores, a locust Schistocerca gregaria and an aphid Myzus persicae. We also exposed all plants to a general soil community from the invaded range and compared their plant-soil feedback responses. Then I tested how individual plants responded to aboveground and belowground plant enemies and I compared this to their combined effects. I also tested whether the strength of aboveground control by generalist shoot-feeding insects was indicative of the strength of belowground control by plant-soil feedback. In the next study I examined how the soil nematode community from the new range responds to exotic plant species compared to related native plants species. As a follow up, I determined the rhizosphere community composition of bacteria, fungi, arbuscular mycorrhizal fungi (AMF) and fusaria. All groups of microbes were analyzed qualitatively and the non-mycorrhizal fungal biomass and fusaria were also analyzed quantitatively. I tested the hypothesis that range-expanding plant species have a different rhizosphere microbial community composition than natives. Finally, I compared the early establishment of range-expanding exotics and phylogenetically related plant species that are native in the invaded habitats. In a greenhouse I grew five range-expanding plant species and five related natives in sterilized and non-sterile inoculated soils from the new range, both alone and with a background community of plant species present in the invaded habitat. In the field, I grew the same plants species in artificially created sparse and dense plant communities. I tested whether range-expanding exotic plant species establish better under competition with native vegetation than phylogenetically related natives, because exotics may benefit from less negative interactions with the soil community compared to natives.

Nematodes – “eel worms”; members of the phylum Nematoda – can be considered as a success story within the Metazoa (multicellular, heterotrophic eukaryotes in which cells lack cell walls): they are speciose and – probably - the most numerous group of multicellular animals on our planet. Nematodes are present in virtually all terrestrial, freshwater and marine habitats. Nematodes are trophically diverse; they may feed on bacteria, fungi/oomycetes, algae and protozoa, other nematodes or on a combination of these (omnivores), or live as facultative or obligatory parasites of plants or animals. As they are abundant, ubiquitous and occupy several trophic levels, they play an important role in the soil food web. Nematode parasites of animals affect billions of humans and livestock, while plant parasites such as cyst, root knot and lesion nematodes cause large agricultural losses worldwide. Despite their undisputed ecological and economical relevance, the systematics of the phylum Nematoda is far from established. One of the aims of this research was to further elucidate nematode phylogeny using molecular data. First a phylogenetic tree was constructed of 349 taxa, spanning the entire phylum Nematoda, on the basis of full length small subunit ribosomal DNA (SSU rDNA) sequences. A series of mostly well-supported bifurcations defined twelve major clades, whereas the most basal clade was defined by representatives of the Enoplida and Triplonchida. Our analysis confirmed the paraphyly of the Adenophorea. Furthermore it was found that the SSU rDNA from representatives of the distal clades evolved at a higher rate than the SSU rDNA from the basal clades. In the meantime, a substantial number of sequences was added to our overall SSU rDNA nematode alignment - both public data (GenBank) and data generated by ourselves (≈ 1,500 sequences in total; February 2008). It is noted that the clade division as proposed in 2006 on the basis of “only” 349 taxa still seems to be valid. Subsequent research focused on three specific groups; Dorylaimia, Chromadoria and Tylenchomorpha. Within the suborder Dorylaimina, the SSU rDNA provided an exceptionally low phylogenetic signal, and - therefore – a part (≈ 1,000 bp) of the more variable large subunit ribosomal DNA (LSU rDNA) was analyzed. In most cases nematode relationships could be elucidated with good support, although some areas in the trees remained unresolved. Generally speaking the results of molecular phylogenetics corresponded fairly well with classical nematode taxonomy. The main exception was the order Dorylaimida where twelve subclades could be distinguished which bore little resemblance to classical taxonomy. Furthermore the suitability of ribosomal DNA for a (semi-) quantative molecular identification method was demonstrated using quantitative PCR (q-PCR) and primers designed to specifically amplify members of the order Mononchida and the potato cyst nematodes Globodera pallida and G. rostochiensis. Plant parasitism has arisen several times within the phylum Nematoda (once in the Triplonchida, at least three times in the Dorylaimida and at least twice in the Tylenchomorpha). The long-standing and generally accepted hypothesis states that plant parasites evolved from fungal feeding ancestors. However, while in most cases plant parasites were associated with fungal feeding nematodes, this hypothesis could neither be confirmed nor denied with the results of our phylogenetic analyses. In the case of two Dorylaimida (Pungentus and Longidorella), however, the ancestor was probably an omnivore. The analysis of this problem was substantially hampered by the lack of knowledge on feeding behavior of basal Tylenchomorpha. Presumably, the common ancestor of the nematodes lived in a marine environment and - if this assumption is correct - the transition to a limnoterrestrial environment must have taken place at least once. Surprisingly, analysis of the Chromadoria (minus the Rhabditida) revealed that transitions from a thalassic to a limnoterrestrial habitat (and vice versa) have taken place at least 11 times in the Chromadoria. Given their frequency these transitions are apparently fairly easy to achieve for nematodes and the possible adaptations involved were discussed. Nematodes vary widely in their responses to environmental disturbance, making them good bio-indicators of soil health. Yet it is not known with certainty which traits are responsible for tolerance to stress in nematodes. A framework was laid out to study correlations between nematode traits and stress tolerance. Furthermore the importance of accounting for the confounding effects of phylogeny was demonstrated. This is a first step towards a transparent, ecological grouping of free-living nematodes. It is worthwhile mentioning that - on the basis of the rDNA-based molecular framework described in this PhD thesis - DNA sequences signatures were identified for nearly all North-West European terrestrial and freshwater nematodes families. The relationship between quantitative PCR signal and numbers of individuals has been established for nearly all families and a first testing of DNA barcode-based community analysis is planned for spring 2008.

Nematodes – “eel worms”; members of the phylum Nematoda – can be considered as a success story within the Metazoa (multicellular, heterotrophic eukaryotes in which cells lack cell walls): they are speciose and – probably - the most numerous group of multicellular animals on our planet. Nematodes are present in virtually all terrestrial, freshwater and marine habitats. Nematodes are trophically diverse; they may feed on bacteria, fungi/oomycetes, algae and protozoa, other nematodes or on a combination of these (omnivores), or live as facultative or obligatory parasites of plants or animals. As they are abundant, ubiquitous and occupy several trophic levels, they play an important role in the soil food web. Nematode parasites of animals affect billions of humans and livestock, while plant parasites such as cyst, root knot and lesion nematodes cause large agricultural losses worldwide. Despite their undisputed ecological and economical relevance, the systematics of the phylum Nematoda is far from established. One of the aims of this research was to further elucidate nematode phylogeny using molecular data. First a phylogenetic tree was constructed of 349 taxa, spanning the entire phylum Nematoda, on the basis of full length small subunit ribosomal DNA (SSU rDNA) sequences. A series of mostly well-supported bifurcations defined twelve major clades, whereas the most basal clade was defined by representatives of the Enoplida and Triplonchida. Our analysis confirmed the paraphyly of the Adenophorea. Furthermore it was found that the SSU rDNA from representatives of the distal clades evolved at a higher rate than the SSU rDNA from the basal clades. In the meantime, a substantial number of sequences was added to our overall SSU rDNA nematode alignment - both public data (GenBank) and data generated by ourselves (≈ 1,500 sequences in total; February 2008). It is noted that the clade division as proposed in 2006 on the basis of “only” 349 taxa still seems to be valid. Subsequent research focused on three specific groups; Dorylaimia, Chromadoria and Tylenchomorpha. Within the suborder Dorylaimina, the SSU rDNA provided an exceptionally low phylogenetic signal, and - therefore – a part (≈ 1,000 bp) of the more variable large subunit ribosomal DNA (LSU rDNA) was analyzed. In most cases nematode relationships could be elucidated with good support, although some areas in the trees remained unresolved. Generally speaking the results of molecular phylogenetics corresponded fairly well with classical nematode taxonomy. The main exception was the order Dorylaimida where twelve subclades could be distinguished which bore little resemblance to classical taxonomy. Furthermore the suitability of ribosomal DNA for a (semi-) quantative molecular identification method was demonstrated using quantitative PCR (q-PCR) and primers designed to specifically amplify members of the order Mononchida and the potato cyst nematodes Globodera pallida and G. rostochiensis. Plant parasitism has arisen several times within the phylum Nematoda (once in the Triplonchida, at least three times in the Dorylaimida and at least twice in the Tylenchomorpha). The long-standing and generally accepted hypothesis states that plant parasites evolved from fungal feeding ancestors. However, while in most cases plant parasites were associated with fungal feeding nematodes, this hypothesis could neither be confirmed nor denied with the results of our phylogenetic analyses. In the case of two Dorylaimida (Pungentus and Longidorella), however, the ancestor was probably an omnivore. The analysis of this problem was substantially hampered by the lack of knowledge on feeding behavior of basal Tylenchomorpha. Presumably, the common ancestor of the nematodes lived in a marine environment and - if this assumption is correct - the transition to a limnoterrestrial environment must have taken place at least once. Surprisingly, analysis of the Chromadoria (minus the Rhabditida) revealed that transitions from a thalassic to a limnoterrestrial habitat (and vice versa) have taken place at least 11 times in the Chromadoria. Given their frequency these transitions are apparently fairly easy to achieve for nematodes and the possible adaptations involved were discussed. Nematodes vary widely in their responses to environmental disturbance, making them good bio-indicators of soil health. Yet it is not known with certainty which traits are responsible for tolerance to stress in nematodes. A framework was laid out to study correlations between nematode traits and stress tolerance. Furthermore the importance of accounting for the confounding effects of phylogeny was demonstrated. This is a first step towards a transparent, ecological grouping of free-living nematodes. It is worthwhile mentioning that - on the basis of the rDNA-based molecular framework described in this PhD thesis - DNA sequences signatures were identified for nearly all North-West European terrestrial and freshwater nematodes families. The relationship between quantitative PCR signal and numbers of individuals has been established for nearly all families and a first testing of DNA barcode-based community analysis is planned for spring 2008.

Terrestrial invertebrates offer meaningful targets for assessing the potential adverse effects of chemicals on soil ecosystems. Invertebrates play a major role in the functioning of the soil ecosystem by enhancing the soil structure, mineralization and the decomposition of organic material, and because of their role in the foodweb. The most dominant group of terrestrial invertebrates, in fact of all multicellular organisms on earth, are nematodes, also called threadworms or roundworms. Nematodes are usually small (0.2-2 mm), transparent and present in almost every habitat on earth. In soil, they usually occur in high abundances and the nematode community comprises a considerable species diversity (Sohlenius, 1980). Nematodes belonging to the terrestrial bacterial feeders inhabit the interstitual water of soil particles (Houx and Aben, 1993). Therefore, they are subjected directly to the dissolved fraction of contaminants in soils, apart from being subjected indirectly via the foodsource. After extraction from the soil, many of these terrestrial bacterivorous nematodes can easily be reared in the laboratory in growth media or on agar plates with bacteria as foodsource.Plectus acuminatus (Nematoda, Torquentia, Plectidae) Bastian 1865 is an example of such a free-living terrestrial bacterivorous nematode species, easy to rear in the laboratory. This species has an egg-to-egg period of approximately 3 weeks and a life span of about 3 months at 20°C. P. acuminatus appeared to be a suitable species for toxicity tests (Kammenga et al. , 1996) and is ubiquitous in the moderate regions of the world (e.g. in soils in the Netherlands (Bongers, 1988) and the UK (Arts, unpublished)).Caenorhabditis elegans (Nematoda, Secernentea, Rhabditidae) Maupas 1899 is another example of a free-living terrestrial bacterivorous nematode species. It is the most investigated nematode species in laboratory experiments that exists. C. elegans strain N2 has originally been extracted from soil from the area of Bristol, UK, but has not been found in Dutch soils yet. It has been reared and maintained in the laboratory for decades. C. elegans is homozygous and doesn't suffer from any inbreed depression. Its life-cycle is very short with an egg-to-egg period of nearly 3 days and a total life span of about 20 days at 20°C (Wood, 1988). Therefore, this nematode species is very suitable to study life-cycle traits and to perform multi-generation experiments.The purpose of this thesis is to evaluate metal stress in free-living terrestrial bacterivorous nematodes by measuring the response on the one hand at a very low organisational level (biomarker response) and on the other hand at a high organisational level (fitness consequences at the population level after consecutive generations). The biomarker response can predict effects of toxicants on cellular function, which might lead to changes in the physiology and/or histology of an organism. These changes possibly exert effects on life-cycle traits, which could lead to changes in fitness, which might eventually lead to extinction of the population.Biomarkers are changes at the molecular, biochemical or cellular level in organisms following exposure to pollutants (Peakall and Shugart, 1992; Deplegde and Fossi, 1994) and are usually the first detectable responses to environmental perturbation. Because these alterations underlie all effects at higher organisational levels, they can be helpful tools in ecotoxicological risk assessment.If we select as biomarker cellular and biochemical events which are intimately involved in protecting and defending the cell from environmental insults, we have ideal candidates for biomarkers of exposure and possibly of effect (Sanders, 1990). Cells dramatically alter their gene expression in response to environmental stress, attempting to protect themselves from damage and to repair existing damage (Schlesinger et al. , 1982). This response is called the cellular stress response. Changes in gene expression associated with the stress response are extremely rapid and result in the induced synthesis and accumulation of stress proteins. One group of stress proteins are the heat shock proteins (hsps), first discovered upon heat exposure but later found to be induced by a wide variety of chemical, physical and biological stressors (e.g. listed in Nover, 1991 and Sanders, 1993).Hsps possibly all function as molecular chaperones (Ellis, 1987), for one, facilitating the synthesis, folding, assembly and intracellular transport of many proteins, reducing protein denaturation and aggregation and aiding in protein renaturation (e.g. Ellis and van der Vies, 1991; Parsell and Lindquist, 1993). The common signal elicited by all hsp-inducing stressors involves an abnormally high concentration of damaged/aggregated proteins within cells, a phenomenon generally referred to as 'proteotoxicity' (Hightower, 1993). Hsp biomarkers give an integrated response summarizing the total proteotoxic damage caused within the target organism or organism tissue.Each hsp is the member of a multigene family, regulated by different promotors and coding for closely related protein isoforms (Lindquist, 1986). Based on their molecular weight, hsps can be classified into different families (Sanders, 1993). The family of 55-65 kDa is called chaperonin. The members of this family have thus far been found in eubacteria and in eukaryotic cells, almost exclusively in organelles which are probably of endosymbiotic origin (mitochondria, chloroplasts) (Hemmingsen et al. , 1988) designated hsp60, stress-60, cpn60, GroEL ( E. coli ) or RuSBP (Rubisco Subunit Binding Protein (chloroplast)). Hsp60 is a nucleus-encoded, constitutively expressed protein. Under stressfull conditions, the hsp60 expression can be dramatically increased. Together with the ubiquitous hsp70 family, which is the most highly conserved and the largest of all the hsp families, the hsp60 family has great potential as a biomarker for general stress (Sanders, 1990).Therefore, both the hsp70 and hsp60 response were qualitatively analyzed in the nematode P. acuminatus (see chapter 2) in order to select the most sensitive hsp-biomarker to increasing metal concentrations. The hsp70 and hsp60 responses were studied following exposure to heat, to copper chloride and to cadmium chloride. Mini two-dimensional polyacrylamide gel electrophoresis was used for protein separation. Poly- and monoclonal antibodies raised against hsp70 or hsp60 in various organisms were used to detect the respective hsps by immunoblotting. Both hsp60 and hsp70 could be identified after exposure of the nematodes to heat, indicating the broad cross reactivity among species to the antibodies used. The induction of hsp60 in P. acuminatus was related to increased concentrations of cadmium and copper chloride. For copper chloride, the induction of hsp60 was 3 orders of magnitude more sensitive than was the EC20 for reproduction; for cadmium chloride, the hsp60 induction was 2 orders of magnitude more sensitive. The hsp70 response in P. acuminatus was also elevated after exposure of the nematodes to cadmium and copper chloride, but this response was relatively weak compared to the hsp60 response. Therefore, it was concluded that the hsp60 response may be suitable as a potential biomarker to metal stress in P. acuminatus .The hsp60 response in P. acuminatus has been further investigated quantitatively in the laboratory, at the protein level as well as at the mRNA level after exposure to various metals (see chapter 3). The mRNA response may be more sensitive and reproducible compared to the protein response and was therefore worth considering. Both the hsp60 protein and mRNA response were measured after 24 hours of exposure to either zinc chloride (0-550mM) or copper chloride (0-59mM), the protein response also after 24 hours of exposure to cadmium chloride (0-109mM). Furthermore, we identified hsp60 in P. acuminatus by elucidating its full-length mRNA sequence and deduced amino acid translation and comparing this to other known sequences. After exposure of the nematodes to zinc chloride, a significant optimum curve was found for the hsp60 response at the protein level, with a maximum induction of over 8 fold the control response at a concentration of 291mM zinc chloride. Most likely, the hsp60 response increased until the ability of the heat shock system to react to increasing metal concentrations reached its climax, after which a further increase in metal concentrations resulted in a decline of the hsp60 level, which might be interpreted as a result of pathological tissue damage as described by Eckwert et al . (1997) concerning the hsp70 response in the isopod species Oniscus asellus . A significant hsp60 increase at the protein level was also detected with increasing copper chloride concentrations, but the maximum hsp60 induction was not reached within the investigated copper concentration range. When the nematodes were exposed to cadmium chloride, no significant trend was observed. At the mRNA level, in P. acuminatus no considerable hsp60 induction was obtained when compared to control levels and to the protein levels at the investigated metal concentration range after 24 hours of exposure. Though the variability at the hsp60 protein level in P. acuminatus was much higher compared to the hsp60 mRNA level, the increase upon metal exposure was much higher at the protein level and occurred at higher metal concentrations. Therefore, the hsp60 protein response in P. acuminatus may have more potential as a biomarker for metal stress than the hsp60 mRNA response.In chapter 4, the application of the hsp60 protein response in P. acuminatus as a biomarker for metal pollution is evaluated in an in situ bio-assay in a field experiment along a metal gradient near Avonmouth, UK. Because it is impossible to determine nematodes to the species level without killing or at least heavily stressing them, P. acuminatus specimens were transplanted into six field sites along the metal gradient and the hsp60 protein response was measured. The response appeared to be significantly higher in the nematodes transplanted into the field site with the lowest metal concentrations compared to the other field sites. The responses of the nematodes in the other field sites did not significantly differ from each other. It can be concluded that the hsp60 response in P. acuminatus alone was not a suitable biomarker for heavily contaminated soils. However, this biomarker had indicative value when related to other biomarker responses measured simultaneously in the same field sites (e.g. the hsp70 response in the isopod species O. asellus and Porcellio scaber ). Furthermore, it might be a suitable biomarker for less heavily contaminated soils. This would have to be investigated in field experiments, because laboratory experiments provide no alternative.From this thesis it can be concluded that the hsp60 response in P. acuminatus could be used as a biomarker for metal exposure, but with the following limitations:An increase of the hsp60 response in the nematode indicates the presence of a proteotoxic stress factor. To identify this stress factor, additional measurements would have to be performed, e.g. chemical analyses, the application of biomarkers which identify specific stressors.The hsp60 response in P. acuminatus is only elevated within a restricted and relatively low concentration range of the metal(s). No elevation of the response means that the stressor level is either too low or too high. The hsp60 response should always be related to other biomarker responses in order to interpret the response. This is not only important in case no elevation is measured, but also to determine where the hsp60 response should be located compared to the maximum induction value, indicating whether the response is increasing or already quenching.The outcome of short-term toxicity studies, such as the hsp responses described in this thesis, may not be used for predicting long-term demographic effects. Because effects at the population level are mediated through effects on fitness, the change in fitness under metal stress in a multi-generation experiment is studied using the nematode C. elegans (chapter 5). The strong advantage of testing multiple generations instead of one generation is the detection of possible trade-off mechanisms among life-history traits and fitness consequences, thus eliciting the probable course of the final consequences of chronic metal stress on the existence of the population. A life-history model of C. elegans was developed to calculate fitness maximisation in populations exposed to cadmium chloride during multiple generations. It was shown that the maximum fitness of C. elegans depended strongly on the trade-off between sperm maturation time and juvenile development. Once C. elegans was exposed to cadmium chloride, fitness decreased during the first generation. After exposure of consecutive generations, fitness increased slightly but significantly compared to the first exposed generation, while various life-cycle traits were strongly affected. The life-history modelling of C. elegans showed that cadmium chloride decreased fitness by impairment of juvenile development. The sperm maturation time remained constant. After long-term exposure of multiple generations, C. elegans counteracted the effect on juveniles by growing faster and increasing reproduction and fitness. This chapter illustrates that the combination of detailed knowledge of the life-cycle and life-history modelling provides insight into the underlying mechanisms of toxicant induced life-cycle changes and fitness consequences.

Terrestrial invertebrates offer meaningful targets for assessing the potential adverse effects of chemicals on soil ecosystems. Invertebrates play a major role in the functioning of the soil ecosystem by enhancing the soil structure, mineralization and the decomposition of organic material, and because of their role in the foodweb. The most dominant group of terrestrial invertebrates, in fact of all multicellular organisms on earth, are nematodes, also called threadworms or roundworms. Nematodes are usually small (0.2-2 mm), transparent and present in almost every habitat on earth. In soil, they usually occur in high abundances and the nematode community comprises a considerable species diversity (Sohlenius, 1980). Nematodes belonging to the terrestrial bacterial feeders inhabit the interstitual water of soil particles (Houx and Aben, 1993). Therefore, they are subjected directly to the dissolved fraction of contaminants in soils, apart from being subjected indirectly via the foodsource. After extraction from the soil, many of these terrestrial bacterivorous nematodes can easily be reared in the laboratory in growth media or on agar plates with bacteria as foodsource.Plectus acuminatus (Nematoda, Torquentia, Plectidae) Bastian 1865 is an example of such a free-living terrestrial bacterivorous nematode species, easy to rear in the laboratory. This species has an egg-to-egg period of approximately 3 weeks and a life span of about 3 months at 20°C. P. acuminatus appeared to be a suitable species for toxicity tests (Kammenga et al. , 1996) and is ubiquitous in the moderate regions of the world (e.g. in soils in the Netherlands (Bongers, 1988) and the UK (Arts, unpublished)).Caenorhabditis elegans (Nematoda, Secernentea, Rhabditidae) Maupas 1899 is another example of a free-living terrestrial bacterivorous nematode species. It is the most investigated nematode species in laboratory experiments that exists. C. elegans strain N2 has originally been extracted from soil from the area of Bristol, UK, but has not been found in Dutch soils yet. It has been reared and maintained in the laboratory for decades. C. elegans is homozygous and doesn't suffer from any inbreed depression. Its life-cycle is very short with an egg-to-egg period of nearly 3 days and a total life span of about 20 days at 20°C (Wood, 1988). Therefore, this nematode species is very suitable to study life-cycle traits and to perform multi-generation experiments.The purpose of this thesis is to evaluate metal stress in free-living terrestrial bacterivorous nematodes by measuring the response on the one hand at a very low organisational level (biomarker response) and on the other hand at a high organisational level (fitness consequences at the population level after consecutive generations). The biomarker response can predict effects of toxicants on cellular function, which might lead to changes in the physiology and/or histology of an organism. These changes possibly exert effects on life-cycle traits, which could lead to changes in fitness, which might eventually lead to extinction of the population.Biomarkers are changes at the molecular, biochemical or cellular level in organisms following exposure to pollutants (Peakall and Shugart, 1992; Deplegde and Fossi, 1994) and are usually the first detectable responses to environmental perturbation. Because these alterations underlie all effects at higher organisational levels, they can be helpful tools in ecotoxicological risk assessment.If we select as biomarker cellular and biochemical events which are intimately involved in protecting and defending the cell from environmental insults, we have ideal candidates for biomarkers of exposure and possibly of effect (Sanders, 1990). Cells dramatically alter their gene expression in response to environmental stress, attempting to protect themselves from damage and to repair existing damage (Schlesinger et al. , 1982). This response is called the cellular stress response. Changes in gene expression associated with the stress response are extremely rapid and result in the induced synthesis and accumulation of stress proteins. One group of stress proteins are the heat shock proteins (hsps), first discovered upon heat exposure but later found to be induced by a wide variety of chemical, physical and biological stressors (e.g. listed in Nover, 1991 and Sanders, 1993).Hsps possibly all function as molecular chaperones (Ellis, 1987), for one, facilitating the synthesis, folding, assembly and intracellular transport of many proteins, reducing protein denaturation and aggregation and aiding in protein renaturation (e.g. Ellis and van der Vies, 1991; Parsell and Lindquist, 1993). The common signal elicited by all hsp-inducing stressors involves an abnormally high concentration of damaged/aggregated proteins within cells, a phenomenon generally referred to as 'proteotoxicity' (Hightower, 1993). Hsp biomarkers give an integrated response summarizing the total proteotoxic damage caused within the target organism or organism tissue.Each hsp is the member of a multigene family, regulated by different promotors and coding for closely related protein isoforms (Lindquist, 1986). Based on their molecular weight, hsps can be classified into different families (Sanders, 1993). The family of 55-65 kDa is called chaperonin. The members of this family have thus far been found in eubacteria and in eukaryotic cells, almost exclusively in organelles which are probably of endosymbiotic origin (mitochondria, chloroplasts) (Hemmingsen et al. , 1988) designated hsp60, stress-60, cpn60, GroEL ( E. coli ) or RuSBP (Rubisco Subunit Binding Protein (chloroplast)). Hsp60 is a nucleus-encoded, constitutively expressed protein. Under stressfull conditions, the hsp60 expression can be dramatically increased. Together with the ubiquitous hsp70 family, which is the most highly conserved and the largest of all the hsp families, the hsp60 family has great potential as a biomarker for general stress (Sanders, 1990).Therefore, both the hsp70 and hsp60 response were qualitatively analyzed in the nematode P. acuminatus (see chapter 2) in order to select the most sensitive hsp-biomarker to increasing metal concentrations. The hsp70 and hsp60 responses were studied following exposure to heat, to copper chloride and to cadmium chloride. Mini two-dimensional polyacrylamide gel electrophoresis was used for protein separation. Poly- and monoclonal antibodies raised against hsp70 or hsp60 in various organisms were used to detect the respective hsps by immunoblotting. Both hsp60 and hsp70 could be identified after exposure of the nematodes to heat, indicating the broad cross reactivity among species to the antibodies used. The induction of hsp60 in P. acuminatus was related to increased concentrations of cadmium and copper chloride. For copper chloride, the induction of hsp60 was 3 orders of magnitude more sensitive than was the EC20 for reproduction; for cadmium chloride, the hsp60 induction was 2 orders of magnitude more sensitive. The hsp70 response in P. acuminatus was also elevated after exposure of the nematodes to cadmium and copper chloride, but this response was relatively weak compared to the hsp60 response. Therefore, it was concluded that the hsp60 response may be suitable as a potential biomarker to metal stress in P. acuminatus .The hsp60 response in P. acuminatus has been further investigated quantitatively in the laboratory, at the protein level as well as at the mRNA level after exposure to various metals (see chapter 3). The mRNA response may be more sensitive and reproducible compared to the protein response and was therefore worth considering. Both the hsp60 protein and mRNA response were measured after 24 hours of exposure to either zinc chloride (0-550mM) or copper chloride (0-59mM), the protein response also after 24 hours of exposure to cadmium chloride (0-109mM). Furthermore, we identified hsp60 in P. acuminatus by elucidating its full-length mRNA sequence and deduced amino acid translation and comparing this to other known sequences. After exposure of the nematodes to zinc chloride, a significant optimum curve was found for the hsp60 response at the protein level, with a maximum induction of over 8 fold the control response at a concentration of 291mM zinc chloride. Most likely, the hsp60 response increased until the ability of the heat shock system to react to increasing metal concentrations reached its climax, after which a further increase in metal concentrations resulted in a decline of the hsp60 level, which might be interpreted as a result of pathological tissue damage as described by Eckwert et al . (1997) concerning the hsp70 response in the isopod species Oniscus asellus . A significant hsp60 increase at the protein level was also detected with increasing copper chloride concentrations, but the maximum hsp60 induction was not reached within the investigated copper concentration range. When the nematodes were exposed to cadmium chloride, no significant trend was observed. At the mRNA level, in P. acuminatus no considerable hsp60 induction was obtained when compared to control levels and to the protein levels at the investigated metal concentration range after 24 hours of exposure. Though the variability at the hsp60 protein level in P. acuminatus was much higher compared to the hsp60 mRNA level, the increase upon metal exposure was much higher at the protein level and occurred at higher metal concentrations. Therefore, the hsp60 protein response in P. acuminatus may have more potential as a biomarker for metal stress than the hsp60 mRNA response.In chapter 4, the application of the hsp60 protein response in P. acuminatus as a biomarker for metal pollution is evaluated in an in situ bio-assay in a field experiment along a metal gradient near Avonmouth, UK. Because it is impossible to determine nematodes to the species level without killing or at least heavily stressing them, P. acuminatus specimens were transplanted into six field sites along the metal gradient and the hsp60 protein response was measured. The response appeared to be significantly higher in the nematodes transplanted into the field site with the lowest metal concentrations compared to the other field sites. The responses of the nematodes in the other field sites did not significantly differ from each other. It can be concluded that the hsp60 response in P. acuminatus alone was not a suitable biomarker for heavily contaminated soils. However, this biomarker had indicative value when related to other biomarker responses measured simultaneously in the same field sites (e.g. the hsp70 response in the isopod species O. asellus and Porcellio scaber ). Furthermore, it might be a suitable biomarker for less heavily contaminated soils. This would have to be investigated in field experiments, because laboratory experiments provide no alternative.From this thesis it can be concluded that the hsp60 response in P. acuminatus could be used as a biomarker for metal exposure, but with the following limitations:An increase of the hsp60 response in the nematode indicates the presence of a proteotoxic stress factor. To identify this stress factor, additional measurements would have to be performed, e.g. chemical analyses, the application of biomarkers which identify specific stressors.The hsp60 response in P. acuminatus is only elevated within a restricted and relatively low concentration range of the metal(s). No elevation of the response means that the stressor level is either too low or too high. The hsp60 response should always be related to other biomarker responses in order to interpret the response. This is not only important in case no elevation is measured, but also to determine where the hsp60 response should be located compared to the maximum induction value, indicating whether the response is increasing or already quenching.The outcome of short-term toxicity studies, such as the hsp responses described in this thesis, may not be used for predicting long-term demographic effects. Because effects at the population level are mediated through effects on fitness, the change in fitness under metal stress in a multi-generation experiment is studied using the nematode C. elegans (chapter 5). The strong advantage of testing multiple generations instead of one generation is the detection of possible trade-off mechanisms among life-history traits and fitness consequences, thus eliciting the probable course of the final consequences of chronic metal stress on the existence of the population. A life-history model of C. elegans was developed to calculate fitness maximisation in populations exposed to cadmium chloride during multiple generations. It was shown that the maximum fitness of C. elegans depended strongly on the trade-off between sperm maturation time and juvenile development. Once C. elegans was exposed to cadmium chloride, fitness decreased during the first generation. After exposure of consecutive generations, fitness increased slightly but significantly compared to the first exposed generation, while various life-cycle traits were strongly affected. The life-history modelling of C. elegans showed that cadmium chloride decreased fitness by impairment of juvenile development. The sperm maturation time remained constant. After long-term exposure of multiple generations, C. elegans counteracted the effect on juveniles by growing faster and increasing reproduction and fitness. This chapter illustrates that the combination of detailed knowledge of the life-cycle and life-history modelling provides insight into the underlying mechanisms of toxicant induced life-cycle changes and fitness consequences.