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The impact of diseases caused by a wide range of pathogens (viruses, bacteria and parasites) is the most important problem in aquaculture of common carp (Cyprinus carpio L.). Genetic selection aimed at obtaining population of more resistant common carp is potential and sustainable approach to disease control in semi-intensive carp pond farming. Genes of the major histocompatibility complex (MHC) are candidate marker genes for studies on association with disease resistance. The MHC contains some of the most polymorphic genes known to date and are considered crucial to adaptive immunity. MHC molecules bind both self and foreign peptides and present them to T lymphocytes (T cells). MHC class I molecules present endogenously derived peptides to CD8+ T cells, while MHC class II molecules present exogenously derived peptides to CD4+ T cells. Each MHC molecule has the ability to bind and present different groups of peptides in more or less successful ways. This can influence the immune response of an organism since the peptides derived from a certain pathogen may either not be presented by specific MHC molecules, which can result in higher susceptibility or, may be bound with a high affinity by specific MHC molecules which could lead to increased resistance to the pathogenic organism. In teleosts, unlike to humans, tetrapods and cartilaginous fish, class I and class II genes are not linked and segregate independently, which allows for association studies of only class I or only class II MH genes with disease resistance. MHC class II molecules generally have a broader spectrum of action in the immune system than the MHC class I genes. There are also observations that suggest a more intense selection pressure and a more rapid evolution of MH class II than class I alleles in fish. Although the expression of both MH class II chains is equivalent, the beta chain generally has a higher degree of polymorphism than the alpha chain. This thesis addressed possible implementations of MH class II B genes for selection aimed at improving of a common carp resistance in semi-intensive pond farming. In common carp there are two paralogous groups of MH class II B genes, Cyca- DAB1-like and Cyca-DAB3-like genes. In a preliminary study, we examined the polymorphism for the Cyca-DAB1-like and Cyca-DAB3-like genes in different European common carp lines (chapter 2). These carp lines were of various geographical origins and part of carp live gene bank, which is maintained at the Institute of Ichthyobiology and Aquaculture in Gołysz. Previous observations over a period of at least 15 years revealed significant differences between lines in survival rate and parasite load under natural conditions. Also, differences in resistance to atypical Aeromonas salmonicida in laboratory based challenge tests was observed, suggesting genetic differences in resistance between the carp lines. Analysis of polymorphism of MH class II B genes in selected carp lines revealed a ubiquitous presence and high polymorphism of Cyca-DAB1-like but not Cyca-DAB3-like genes. The observed allelic polymorphism for Cyca-DAB1-like genes rather than Cyca-DAB3-like genes stimulated further studies into the association of Cyca-DAB1-like allelic polymorphism and disease resistance of common carp. In order to study association between Cyca-DAB1-like gene polymorphism and resistance of common carp we optimized a technique designated polymerase chain reaction -restriction fragments- single strand conformation polymorphism (PCR-RFSSCP) to be able to screen and type large numbers of individual carp (chapter 3). The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RFSSCP analysis of n = 79 carp individuals from 8 lines challenged with Aeromonas hydrophila revealed the presence of different genotypes consisting of unique combinations of Cyca-DAB1 and Cyca-DAB2 sequences. We found four alleles for the Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02). We noted that the Cyca-DAB2 gene was either homozygous or absent. The degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes clearly correlated with the number of SSCP bands. Thus, we proved that PCR-RF-SSCP is a reliable technique that can be used for screening large number of individuals for investigating the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. Previously, we performed a long-term divergent selection of common carp for antibody production, which successfully resulted in carp lines with a different immune response. We studied the segregation of Cyca-DAB genes with the DNP-specific antibody response and we showed that the presence of Cyca-DAB1-like, but not Cyca- DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp (chapter 4). Background genes other than Cyca-DAB genes also influenced the level of antibody response. We also studied the transcription of both Cyca-DAB1-like and Cyca- DAB3-like genes in different organs of carp. The constitutive transcription for both Cyca-DAB1-like and Cyca-DAB3-like genes was high, although Cyca-DAB1-like genes consistently showed slightly higher mRNA transcription than Cyca-DAB3-like genes in some immunological relevant organs. Sequence information, constitutive transcription levels and co-segregation data indicated that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. We then proceeded to study association of Cyca-DAB1-like genotypes with resistance to four different pathogens; the bacterium Aeromonas hydrophila, the ectoparasite Argulus japonicus, and the blood parasite Trypanoplasma borreli (chapter 5) and the viral pathogen Cyprinid herpesvirus-3 (CyHV-3) (chapter 6). We used a large number of individuals of different carp lines and revealed, using PCR-RF-SSCP, the presence of n = 9 unique Cyca-DAB1-like genotypes, of which three genotypes (B, D, and E) were most common (chapter 5). In general, Cyca-DAB2 was often homozygous or absent while allelic polymorphism was detected in Cyca-DAB1 gene. We could detect significant associations between genotype E and abundance of A. japonicus and between genotype D and higher level of parasitaemia after T. borreli infection. We also observed a significant association between Cyca-DAB1 heterozygosity and lower level of parasitaemia after T. borreli infection. In chapter 6, we showed a strong association between Cyca-DAB1-like genotypes and resistance or susceptibility to CyHV-3. One genotype (E) performed significantly better, resulting in carp more resistant to CyHV-3, while three other genotypes (B, H and J) could be linked to higher susceptibility to the virus. Subsequent analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. The resistant genotype E did not comprise the Cyca-DAB2 gene and consisted of a homozygous Cyca-DAB1*05 allele. Phylogenetic analysis of all Cyca-DAB1 alleles showed that the Cyca-DAB1*05 allele represents the oldest allele in our study (chapter 7). We discussed the possibility for using Cyca-DAB1 allelic polymorphism as a potential genetic marker in future breeding programs of common carp (chapter 7). We expect that selection of carp for particular MH class II B genotypes or alleles could allow for an increased survival upon challenge with selected pathogens and possibly, increased survival rate under pond conditions.

Like many other plants, chemical defence compounds are involved in the defense of Barbarea vulgaris against natural enemies. Barbarea vulgaris produces glucosinolates, which are present in most crucifers such as cabbage, mustard, and the scientific model species Arabidopsis thaliana. Glucosinolates form, together with an enzyme (myrosinase), a two-component system: the enzyme and glucosinolates are stored in spatially separated compartments. Upon cell disruption they come into contact, and the glucosinolates are catabolised by the enzyme. For generalist herbivores the glucosinolates, and especially their breakdown products, are often toxic and protect the plants against herbivory. However, specialist herbivores often use these same compounds to recognize suitable host plants. The breakdown products are also responsible for the specific taste to many cabbage and mustard species There are over 100 different glucosinolates, each with a different chemical structure. The structure determines, amongst other factors, which breakdown product will be formed upon damage. Every plant species has its own typical composition of glucosinolates. I studied how these glucosinolate profiles may affect plant resistance against herbivores. In a screening of several Dutch populations it was found that Barbarea vulgaris plants differed in glucosinolate profile. The majority of the plants contained mainly glucobarbarin, a glucosinolate typical for this species and named after the genus Barbarea. Also populations sampled in Germany, Belgium, France, and Switserland, consisted completely of plants with mainly glucobarbarin. In half of the Dutch populations I found that a minority of the plants (2-22%) produced another glucosinolate, named gluconasturtiin. The difference in the chemical structure between these two glucosinolates is very small; glucobarbarin has only one hydroxyl group more than gluconasturtiin. However, this small structural difference may be of large biological relevance. When gluconasturtiin reacts with myrosinase a toxic and unpalatable isothiocyanate is formed, whereas glucobarbarin, due to the position of the hydroxyl group, produces oxazolidinethions. It is unknown whether these oxazolidinethiones are toxic, but in mammals they can inhibit the iodine intake, thereby causing thyroid problems. The Barbarea vulgaris glucosinolate polymorphism thus has two chemotypes. I characterized these chemotypes and used them to study the effects of different glucosinolates on herbivores. The difference in glucosinolate profile is consistently present in all plant organs of B. vulgaris, but it is larger in the aboveground organs than in the roots. The glucosinolate profile does not change upon induction by insects nor upon artificial induction by addition of jasmonic acid. I crossed plants, analysed the chemotype of the offspring and showed that the ability to produce glucobarbarin is heritable and regulated by a dominant gene. Based on the assumption that there is a specific enzyme that converts gluconasturtiin to glucobarbarin by a single hydroxylation step, I identified some candidate genes. Further research is needed to determine whether one of these candidate genes is indeed responsible the difference between the chemotypes. Subsequently, I studied the effect of chemotype on leaf and root herbivores. Plants with mainly glucobarbarin turned out to be very resistant to the generalist leaf-eating larvae of the Mamestra brassicae moth. Almost none of the larvae survived on plants with mainly glucobarbarin. If the larvae were given the choice, they strongly preferred to feed on plants with mainly gluconasturtiin. However, female moths deposited approximately the same number of eggs on each chemotype. Larvae of the specialist small cabbage white grew equally well on each chemotype and did not distinguish between the chemotypes in choice experiments. Larvae of the specialist cabbage root fly, however, performed worse on roots of the gluconasturtiin type than on roots of the glucobarbarin type. Plants responded to the root fly infection by a reduction of the root and shoot biomass with 50%, and decreased levels of nutrients such as sugars and amino acids. In order to translate the results obtained in the greenhouse to the natural situation, I planted plants of both chemotypes in an experimental garden. Over a period of two years, the numbers of several herbivores were counted on each chemotype every week during the growth season. This revealed that some aboveground insects had a preference for a certain chemotype, and others did not. Butterflies of the small cabbage white preferred to oviposit on plants with gluconasturtiin, but flea beetles and gall midges were more abundant on plants with glucobarbarin. The cabbage aphid and the green peach aphid did not show any preference and were equally abundant on both chemotypes. Three to four times a year, I dug up a subset of the plants to analyse the root herbivores and the soil nematode community. The numbers and community structure of these belowground organisms did not differ between the chemotypes. Additionally, I investigated whether there are other chemical differences between the chemotypes, apart from glucosinolate profile, that could explain the preference of the above insect species. Therefore, I performed extensive metabolomics analyses, using LC-TOF-MS. Multivariate analyses showed that the major chemical differences between the chemotypes was due to differences in glucosinolates. These differences were larger in shoots than in roots. Apart from glucosinolates only eight unidentified compounds differed between the chemotypes. Known defense compounds such as flavonoids and saponins were identified but did not differ between the chemotypes. Therefore, it is very likely that the differences in herbivore performance and preference are predominantly caused by the differences in glucosinolate profile. Based on my results, I conclude that the structure of glucosinolates can cause significant differences in resistance against several herbivores. However, in the case of B. vulgaris there is no supreme chemotype that is more resistant in all cases. Which of the two chemotypes accrues the largest benefit in a natural environment thus will depend on the herbivore community in their population. In this way the B. vulgaris glucosinolate polymorphism can be maintained in natural populations.

Bij vrijwel ieder mens neemt de longfunctie met het ouder worden af, gemiddeld met 30 ml per jaar. Mensen die roken hebben vaak een versnelde achteruitgang van de longfunctie wat aanleiding kan geven tot klachten van de luchtwegen en longziekte. Een van de ziekten die kan ontstaan door roken is Chronic Obstructive Pulmonary Disease (COPD). COPD is de verzamelnaam voor chronische bronchitis en longemfyseem, aandoeningen die worden gekarakteriseerd door luchtwegobstructie, I uc htwegontste king en afbraak van longweefsel. Bij chronische bronchitis zijn er Meer slijmproducerende cellen, waardoor er Meer slijm in de luchtwegen komt en Mensen benauwd worden en moeten hoesten. Bij emfyseem vindt afbraak plaats van de longblaasjes, zodat er minder zuurstof uit de ingeademde lucht kan worden opgenomen door het lichaam. Dit leidt tot kortademigheid en in ernstige gevallen van COPD moeten patienten extra zuurstof toegediend krijgen. Veel COPD patienten hebben zowel kenmerken van chronische bronchitis als van longemfyseem. Ongeveer 10% van de Nederlandse bevolking leidt aan COPD. De ziekte is chronisch en er zijn tot op heden nog geen goede medicijnen om de ziekte tot stilstand te kunnen brengen.

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.

Like many other plants, chemical defence compounds are involved in the defense of Barbarea vulgaris against natural enemies. Barbarea vulgaris produces glucosinolates, which are present in most crucifers such as cabbage, mustard, and the scientific model species Arabidopsis thaliana. Glucosinolates form, together with an enzyme (myrosinase), a two-component system: the enzyme and glucosinolates are stored in spatially separated compartments. Upon cell disruption they come into contact, and the glucosinolates are catabolised by the enzyme. For generalist herbivores the glucosinolates, and especially their breakdown products, are often toxic and protect the plants against herbivory. However, specialist herbivores often use these same compounds to recognize suitable host plants. The breakdown products are also responsible for the specific taste to many cabbage and mustard species There are over 100 different glucosinolates, each with a different chemical structure. The structure determines, amongst other factors, which breakdown product will be formed upon damage. Every plant species has its own typical composition of glucosinolates. I studied how these glucosinolate profiles may affect plant resistance against herbivores. In a screening of several Dutch populations it was found that Barbarea vulgaris plants differed in glucosinolate profile. The majority of the plants contained mainly glucobarbarin, a glucosinolate typical for this species and named after the genus Barbarea. Also populations sampled in Germany, Belgium, France, and Switserland, consisted completely of plants with mainly glucobarbarin. In half of the Dutch populations I found that a minority of the plants (2-22%) produced another glucosinolate, named gluconasturtiin. The difference in the chemical structure between these two glucosinolates is very small; glucobarbarin has only one hydroxyl group more than gluconasturtiin. However, this small structural difference may be of large biological relevance. When gluconasturtiin reacts with myrosinase a toxic and unpalatable isothiocyanate is formed, whereas glucobarbarin, due to the position of the hydroxyl group, produces oxazolidinethions. It is unknown whether these oxazolidinethiones are toxic, but in mammals they can inhibit the iodine intake, thereby causing thyroid problems. The Barbarea vulgaris glucosinolate polymorphism thus has two chemotypes. I characterized these chemotypes and used them to study the effects of different glucosinolates on herbivores. The difference in glucosinolate profile is consistently present in all plant organs of B. vulgaris, but it is larger in the aboveground organs than in the roots. The glucosinolate profile does not change upon induction by insects nor upon artificial induction by addition of jasmonic acid. I crossed plants, analysed the chemotype of the offspring and showed that the ability to produce glucobarbarin is heritable and regulated by a dominant gene. Based on the assumption that there is a specific enzyme that converts gluconasturtiin to glucobarbarin by a single hydroxylation step, I identified some candidate genes. Further research is needed to determine whether one of these candidate genes is indeed responsible the difference between the chemotypes. Subsequently, I studied the effect of chemotype on leaf and root herbivores. Plants with mainly glucobarbarin turned out to be very resistant to the generalist leaf-eating larvae of the Mamestra brassicae moth. Almost none of the larvae survived on plants with mainly glucobarbarin. If the larvae were given the choice, they strongly preferred to feed on plants with mainly gluconasturtiin. However, female moths deposited approximately the same number of eggs on each chemotype. Larvae of the specialist small cabbage white grew equally well on each chemotype and did not distinguish between the chemotypes in choice experiments. Larvae of the specialist cabbage root fly, however, performed worse on roots of the gluconasturtiin type than on roots of the glucobarbarin type. Plants responded to the root fly infection by a reduction of the root and shoot biomass with 50%, and decreased levels of nutrients such as sugars and amino acids. In order to translate the results obtained in the greenhouse to the natural situation, I planted plants of both chemotypes in an experimental garden. Over a period of two years, the numbers of several herbivores were counted on each chemotype every week during the growth season. This revealed that some aboveground insects had a preference for a certain chemotype, and others did not. Butterflies of the small cabbage white preferred to oviposit on plants with gluconasturtiin, but flea beetles and gall midges were more abundant on plants with glucobarbarin. The cabbage aphid and the green peach aphid did not show any preference and were equally abundant on both chemotypes. Three to four times a year, I dug up a subset of the plants to analyse the root herbivores and the soil nematode community. The numbers and community structure of these belowground organisms did not differ between the chemotypes. Additionally, I investigated whether there are other chemical differences between the chemotypes, apart from glucosinolate profile, that could explain the preference of the above insect species. Therefore, I performed extensive metabolomics analyses, using LC-TOF-MS. Multivariate analyses showed that the major chemical differences between the chemotypes was due to differences in glucosinolates. These differences were larger in shoots than in roots. Apart from glucosinolates only eight unidentified compounds differed between the chemotypes. Known defense compounds such as flavonoids and saponins were identified but did not differ between the chemotypes. Therefore, it is very likely that the differences in herbivore performance and preference are predominantly caused by the differences in glucosinolate profile. Based on my results, I conclude that the structure of glucosinolates can cause significant differences in resistance against several herbivores. However, in the case of B. vulgaris there is no supreme chemotype that is more resistant in all cases. Which of the two chemotypes accrues the largest benefit in a natural environment thus will depend on the herbivore community in their population. In this way the B. vulgaris glucosinolate polymorphism can be maintained in natural populations.

The impact of diseases caused by a wide range of pathogens (viruses, bacteria and parasites) is the most important problem in aquaculture of common carp (Cyprinus carpio L.). Genetic selection aimed at obtaining population of more resistant common carp is potential and sustainable approach to disease control in semi-intensive carp pond farming. Genes of the major histocompatibility complex (MHC) are candidate marker genes for studies on association with disease resistance. The MHC contains some of the most polymorphic genes known to date and are considered crucial to adaptive immunity. MHC molecules bind both self and foreign peptides and present them to T lymphocytes (T cells). MHC class I molecules present endogenously derived peptides to CD8+ T cells, while MHC class II molecules present exogenously derived peptides to CD4+ T cells. Each MHC molecule has the ability to bind and present different groups of peptides in more or less successful ways. This can influence the immune response of an organism since the peptides derived from a certain pathogen may either not be presented by specific MHC molecules, which can result in higher susceptibility or, may be bound with a high affinity by specific MHC molecules which could lead to increased resistance to the pathogenic organism. In teleosts, unlike to humans, tetrapods and cartilaginous fish, class I and class II genes are not linked and segregate independently, which allows for association studies of only class I or only class II MH genes with disease resistance. MHC class II molecules generally have a broader spectrum of action in the immune system than the MHC class I genes. There are also observations that suggest a more intense selection pressure and a more rapid evolution of MH class II than class I alleles in fish. Although the expression of both MH class II chains is equivalent, the beta chain generally has a higher degree of polymorphism than the alpha chain. This thesis addressed possible implementations of MH class II B genes for selection aimed at improving of a common carp resistance in semi-intensive pond farming. In common carp there are two paralogous groups of MH class II B genes, Cyca- DAB1-like and Cyca-DAB3-like genes. In a preliminary study, we examined the polymorphism for the Cyca-DAB1-like and Cyca-DAB3-like genes in different European common carp lines (chapter 2). These carp lines were of various geographical origins and part of carp live gene bank, which is maintained at the Institute of Ichthyobiology and Aquaculture in Gołysz. Previous observations over a period of at least 15 years revealed significant differences between lines in survival rate and parasite load under natural conditions. Also, differences in resistance to atypical Aeromonas salmonicida in laboratory based challenge tests was observed, suggesting genetic differences in resistance between the carp lines. Analysis of polymorphism of MH class II B genes in selected carp lines revealed a ubiquitous presence and high polymorphism of Cyca-DAB1-like but not Cyca-DAB3-like genes. The observed allelic polymorphism for Cyca-DAB1-like genes rather than Cyca-DAB3-like genes stimulated further studies into the association of Cyca-DAB1-like allelic polymorphism and disease resistance of common carp. In order to study association between Cyca-DAB1-like gene polymorphism and resistance of common carp we optimized a technique designated polymerase chain reaction -restriction fragments- single strand conformation polymorphism (PCR-RFSSCP) to be able to screen and type large numbers of individual carp (chapter 3). The advantages of this technique are simplicity, high sensitivity and low costs. PCR-RFSSCP analysis of n = 79 carp individuals from 8 lines challenged with Aeromonas hydrophila revealed the presence of different genotypes consisting of unique combinations of Cyca-DAB1 and Cyca-DAB2 sequences. We found four alleles for the Cyca-DAB1 (*02-*05) gene but only one allele for Cyca-DAB2 (*02). We noted that the Cyca-DAB2 gene was either homozygous or absent. The degree of heterozygosity of the Cyca-DAB1 and Cyca-DAB2 genes clearly correlated with the number of SSCP bands. Thus, we proved that PCR-RF-SSCP is a reliable technique that can be used for screening large number of individuals for investigating the Cyca-DAB1 and Cyca-DAB2 genes polymorphism in common carp. Previously, we performed a long-term divergent selection of common carp for antibody production, which successfully resulted in carp lines with a different immune response. We studied the segregation of Cyca-DAB genes with the DNP-specific antibody response and we showed that the presence of Cyca-DAB1-like, but not Cyca- DAB3-like genes, preferentially leads to a high DNP-specific antibody response in carp (chapter 4). Background genes other than Cyca-DAB genes also influenced the level of antibody response. We also studied the transcription of both Cyca-DAB1-like and Cyca- DAB3-like genes in different organs of carp. The constitutive transcription for both Cyca-DAB1-like and Cyca-DAB3-like genes was high, although Cyca-DAB1-like genes consistently showed slightly higher mRNA transcription than Cyca-DAB3-like genes in some immunological relevant organs. Sequence information, constitutive transcription levels and co-segregation data indicated that both paralogous Cyca-DAB1-like and Cyca-DAB3-like groups represent functional MH class II B genes. We then proceeded to study association of Cyca-DAB1-like genotypes with resistance to four different pathogens; the bacterium Aeromonas hydrophila, the ectoparasite Argulus japonicus, and the blood parasite Trypanoplasma borreli (chapter 5) and the viral pathogen Cyprinid herpesvirus-3 (CyHV-3) (chapter 6). We used a large number of individuals of different carp lines and revealed, using PCR-RF-SSCP, the presence of n = 9 unique Cyca-DAB1-like genotypes, of which three genotypes (B, D, and E) were most common (chapter 5). In general, Cyca-DAB2 was often homozygous or absent while allelic polymorphism was detected in Cyca-DAB1 gene. We could detect significant associations between genotype E and abundance of A. japonicus and between genotype D and higher level of parasitaemia after T. borreli infection. We also observed a significant association between Cyca-DAB1 heterozygosity and lower level of parasitaemia after T. borreli infection. In chapter 6, we showed a strong association between Cyca-DAB1-like genotypes and resistance or susceptibility to CyHV-3. One genotype (E) performed significantly better, resulting in carp more resistant to CyHV-3, while three other genotypes (B, H and J) could be linked to higher susceptibility to the virus. Subsequent analysis of the alleles that compose the Cyca-DAB1-like genotypes linked one particular allele (Cyca-DAB1*05) to significantly increased, and two alleles (Cyca-DAB1*02 and Cyca-DAB1*06) to significantly decreased resistance to CyHV-3. The resistant genotype E did not comprise the Cyca-DAB2 gene and consisted of a homozygous Cyca-DAB1*05 allele. Phylogenetic analysis of all Cyca-DAB1 alleles showed that the Cyca-DAB1*05 allele represents the oldest allele in our study (chapter 7). We discussed the possibility for using Cyca-DAB1 allelic polymorphism as a potential genetic marker in future breeding programs of common carp (chapter 7). We expect that selection of carp for particular MH class II B genotypes or alleles could allow for an increased survival upon challenge with selected pathogens and possibly, increased survival rate under pond conditions.

Bij vrijwel ieder mens neemt de longfunctie met het ouder worden af, gemiddeld met 30 ml per jaar. Mensen die roken hebben vaak een versnelde achteruitgang van de longfunctie wat aanleiding kan geven tot klachten van de luchtwegen en longziekte. Een van de ziekten die kan ontstaan door roken is Chronic Obstructive Pulmonary Disease (COPD). COPD is de verzamelnaam voor chronische bronchitis en longemfyseem, aandoeningen die worden gekarakteriseerd door luchtwegobstructie, I uc htwegontste king en afbraak van longweefsel. Bij chronische bronchitis zijn er Meer slijmproducerende cellen, waardoor er Meer slijm in de luchtwegen komt en Mensen benauwd worden en moeten hoesten. Bij emfyseem vindt afbraak plaats van de longblaasjes, zodat er minder zuurstof uit de ingeademde lucht kan worden opgenomen door het lichaam. Dit leidt tot kortademigheid en in ernstige gevallen van COPD moeten patienten extra zuurstof toegediend krijgen. Veel COPD patienten hebben zowel kenmerken van chronische bronchitis als van longemfyseem. Ongeveer 10% van de Nederlandse bevolking leidt aan COPD. De ziekte is chronisch en er zijn tot op heden nog geen goede medicijnen om de ziekte tot stilstand te kunnen brengen.

Iron is fundamental to the biology of eukaryotic cells since it plays a key role in many metabolic functions. Iron concentrations are tightly regulated, for example by ferritin, because excessive iron leads to tissue damage. Iron cannot cross cellular membranes directly and most cells acquire iron from the iron transporting protein transferrin (Tf), via transferrin receptors. During nutritional immunity the body reacts with a metabolic adjustment in order to render important nutrients unavailable to invading microorganisms. However, pathogens also have evolved a range of mechanisms to acquire iron from the host (chapter 1). In the study described in this thesis, we used a natural host-parasite model of common carp (Cyprinus carpio L.) infected with Trypanoplasma borreli, a protozoan kinetoplastid, extracellular blood parasite of carp to get more insight in the competition for iron between host and parasite. Transferrin of common carp is highly polymorphic with several alleles identified according to differences in electrophoretic mobility. We studied the implications of Tf polymorphisms for iron binding and modulation of immune function. We performed a series of challenge experiments infecting five genetically different, commercially exploited carp lines with T. borreli. Our results indicated that Tf genotype may influence the susceptibility to pathogens. We observed a significant association of the DD genotype of Tf with low parasitaemia in two resistant carp lines (Polish ‘R2’ and ‘K’), but a reverse association in the most susceptible carp line ‘D’ (chapter 2). We also showed that variation in resistance to T. borreli could be controlled by sex-related genetic factors. Examination of parasite growth in vitro, in culture media supplemented with 3% serum taken from fish with different Tf genotypes, showed a faster decrease in number of parasites in media supplemented with serum from DD-typed animals (chapter 2). In general, pathogens also have mechanisms to acquire iron from the host. We developed a method for Tf depletion of carp serum using specific antibodies to carp Tf, and compared T. borreli multiplication and survival in the presence or absence of Tf in vitro. Parasites were dying in medium containing Tf-depleted serum, which clearly showed that Tf is essential for parasite growth and multiplication (chapter 3). We isolated two allelic forms of carp Tf (alleles D and G) to purity using rivanol precipitation and ion-exchange chromatography (chapter 5). We showed that parasite growth in vitro could be reconstituted by the addition of purified Tf to Tf-depleted serum (chapter 3). We observed differences in T. borreli multiplication and survival in culture media containing different sera typed differently for Tf genotypes (chapter 3). We identified four complete coding sequences for common carp Tf alleles C, D, F and G, and confirmed the overall similarity of the carp Tf three-dimensional structure to Tfs of other species. We could show that carp Tf differs significantly in critical iron-binding sites in the N-lobe of the molecule, as compared to other non-cyprinid fish species (chapter 4). The substitution of a majority of the iron-coordinating residues in the Nlobe indeed seems to affect the ability to bind iron, which may be compensated for by higher serum concentrations of Tf (chapter 7). Comparison of constitutive gene expression of two Tf alleles D and G showed a comparably high gene expression level in liver and small but consistent differences in gene expression for allele D over allele G in other immunologically important organs (chapter 4). Our data suggest that the allelic polymorphism is not related to differences in iron binding and/or binding to the host Tf receptor but could be linked with other factors, such as competition for iron with pathogens (chapter 4). Transferrin itself may also exert effects that are not directly linked with maintaining iron levels and Tf cleavage products have been shown to stimulate macrophages to produce large amounts of nitric oxide (NO). To study the induction of NO in carp head kidney-derived macrophages, we isolated two allelic forms of carp Tf (alleles D and G) to purity and showed that the level of activation of macrophages by Tf was different for the D and G allele (chapter 5). Differences in NO levels induced could be related to different cleavage forms of the two alleles D and G, as shown by Western blot, confirming that full-length Tf cannot induce NO. The D-type Tf cleavage products induced significantly higher nitric oxide (NO) production than cleavage products of Gtype Tf. (chapter 5). Transferrin uptake by trypanosome parasites involves Tf binding to a receptor. The TfR-Tf complex then is internalised and transported to lysosomes, where Tf is proteolytically degraded. We described the cloning and sequencing of a cathepsin L-like cysteine proteinase from T. borreli and production of a recombinant and biologically active enzyme (chapter 6). We demonstrated that the T. borreli cysteine proteinase is able to digest host transferrin. Likely, Tf cleavage fragments are released from the trypanosomes while iron would remain parasite-associated, possibly contributing to thepathogenicity of the parasite by inducing high amounts of NO in carp macrophages (chapter 7). Our study dealt with different aspects of Tf polymorphism, discussing the role of Tf in immunity of common carp and the influence of allelic polymorphism on competition for iron between host and pathogen (chapter 7). Further investigations should shed more light on the selective advantage of particular alleles to provide a basis for incorporating Tf as a genetic marker in marker-assisted selection programmes for increased resistance to diseases. This could contribute to improved survival of carp kept under semiintensive farming systems in ponds.

The aim of this thesis was the implementation and application of SNP markers in animal breeding and genetics. The emphasis was on the analysis of fatness traits in pigs, in particular of the imprinted QTL region on SSC2p. Several aspects of the implemetation of SNP markers in genetic analysis oflivestockwereaddressed, including SNP discovery methods and tools for enhancing sequencing and genotyping data analysis. The QTL on SSC2p was reanalysed after the identification of a SNP in the IGF2 gene that had high impact on lean muscle growth and back fat thickness. This re-analysis showed that the QTL region is narrowed down to a single base pair: the quantitative trait nucleotide (QTN). For research in other genomic regions, the SNP marker density needed in association studies was an issue of discussion. To address this question, the extent of LD in the porcine genome was studied. It was estimated that the majority of marker pairs separated by less than 50 kb show levels of LD that might be useful in association studies. Further investigation of LD and the extent of LD,however, is crucial in order to develop a map of the patterns of sequence variation in the porcine genome.

Excessive fat is a well known problem in poultry. It depresses feed efficiency, has no commercial value, and is less appreciated by consumers. Therefore, considerable research effort has been applied around the world to study factors associated with fat deposition and methods of decreasing it.his dissertation concerns the identification of genes controlling fat deposition in broilers. The strategy used to identify these genes is the so-called positional candidate gene approach. This approach moves from mapping quantitative trait loci (QTL) towards the identification of candidate genes. A total genome scan of a large three generation broiler population has resulted in the localization of QTL for fat deposition. Several of these QTL are confirmed as well as new QTL are identified in an advanced intercross line produced from this broiler population. The construction of high resolution comparative maps has helped to identify potential candidate genes for fat deposition mapped in the QTL regions. These genes and the polymorphisms within these genes will be of interest for future research. Identified SNPs can be used to refine the map position of the QTL by analysing linkage and linkage disequilibrium.Finally, a causal link between the genetic trait and the candidate genes can be established from functional evidence.

Excessive fat is a well known problem in poultry. It depresses feed efficiency, has no commercial value, and is less appreciated by consumers. Therefore, considerable research effort has been applied around the world to study factors associated with fat deposition and methods of decreasing it.his dissertation concerns the identification of genes controlling fat deposition in broilers. The strategy used to identify these genes is the so-called positional candidate gene approach. This approach moves from mapping quantitative trait loci (QTL) towards the identification of candidate genes. A total genome scan of a large three generation broiler population has resulted in the localization of QTL for fat deposition. Several of these QTL are confirmed as well as new QTL are identified in an advanced intercross line produced from this broiler population. The construction of high resolution comparative maps has helped to identify potential candidate genes for fat deposition mapped in the QTL regions. These genes and the polymorphisms within these genes will be of interest for future research. Identified SNPs can be used to refine the map position of the QTL by analysing linkage and linkage disequilibrium.Finally, a causal link between the genetic trait and the candidate genes can be established from functional evidence.

The aim of this thesis was the implementation and application of SNP markers in animal breeding and genetics. The emphasis was on the analysis of fatness traits in pigs, in particular of the imprinted QTL region on SSC2p. Several aspects of the implemetation of SNP markers in genetic analysis oflivestockwereaddressed, including SNP discovery methods and tools for enhancing sequencing and genotyping data analysis. The QTL on SSC2p was reanalysed after the identification of a SNP in the IGF2 gene that had high impact on lean muscle growth and back fat thickness. This re-analysis showed that the QTL region is narrowed down to a single base pair: the quantitative trait nucleotide (QTN). For research in other genomic regions, the SNP marker density needed in association studies was an issue of discussion. To address this question, the extent of LD in the porcine genome was studied. It was estimated that the majority of marker pairs separated by less than 50 kb show levels of LD that might be useful in association studies. Further investigation of LD and the extent of LD,however, is crucial in order to develop a map of the patterns of sequence variation in the porcine genome.

Drug effects (pharmacodynamics) are determined by drug concentration at target site and the affinity of the drug for a target. Pharmacogenetics describes inherited differences in drug metabolising enzyme activities and differences in drug transporters and receptors.Answers were sought on the following questions:1. What is the value of genotyping on polymorphic drug metabolising enzymes compared to the classical phenotyping stategies?2. What is the situation concerning CYP2D6 and CYP2C19 polymorphism in the Dutch population?3. Is knowledge of the individual metaboliser status of CYP2D6 or CYP2C19 valuable in clinical pharmacological research and practice of pharmacotherapy

A new strategy to identify pathogenicity factors from the potato cyst nematode Globodera rostochiensis is developed. cDNA-AFLP technology and in situ hybridization allowed us to efficiently select putative pathogenicity factors among thousands of expressed genes. As a result, an unprecedented number of putative pathogenicity factors from this obligatory plant parasite were cloned.A powerful bioinformatics tool named GenEST to link expression data generated from cDNA-AFLP directly to cDNA sequence data was developed. This computer program would be very useful for functional genomics studies in other systems as well.A RanBPM (Ran-Binding Protein in Microtubule organization)-like gene family was identified. The proteins encoded by these genes are probably secreted by the nematode into plant cells and might manipulate the host cell development by changing the dynamic instability of microtubules.A promoter region from the potato cyst nematode was shown to be functional in a distantly related nematode species Caenorhabditis elegans . This promoter could be a valuable tool to drive gene expression in transgenic plant-parasitic nematode species to assess the relative importance of putative pathogenicity factors.A paper based from one chapter of this thesis will be submitted to Science. In view of their press embargo policy, I will not discussed the detail of this particular finding here.

Drug effects (pharmacodynamics) are determined by drug concentration at target site and the affinity of the drug for a target. Pharmacogenetics describes inherited differences in drug metabolising enzyme activities and differences in drug transporters and receptors.Answers were sought on the following questions:1. What is the value of genotyping on polymorphic drug metabolising enzymes compared to the classical phenotyping stategies?2. What is the situation concerning CYP2D6 and CYP2C19 polymorphism in the Dutch population?3. Is knowledge of the individual metaboliser status of CYP2D6 or CYP2C19 valuable in clinical pharmacological research and practice of pharmacotherapy

A new strategy to identify pathogenicity factors from the potato cyst nematode Globodera rostochiensis is developed. cDNA-AFLP technology and in situ hybridization allowed us to efficiently select putative pathogenicity factors among thousands of expressed genes. As a result, an unprecedented number of putative pathogenicity factors from this obligatory plant parasite were cloned.A powerful bioinformatics tool named GenEST to link expression data generated from cDNA-AFLP directly to cDNA sequence data was developed. This computer program would be very useful for functional genomics studies in other systems as well.A RanBPM (Ran-Binding Protein in Microtubule organization)-like gene family was identified. The proteins encoded by these genes are probably secreted by the nematode into plant cells and might manipulate the host cell development by changing the dynamic instability of microtubules.A promoter region from the potato cyst nematode was shown to be functional in a distantly related nematode species Caenorhabditis elegans . This promoter could be a valuable tool to drive gene expression in transgenic plant-parasitic nematode species to assess the relative importance of putative pathogenicity factors.A paper based from one chapter of this thesis will be submitted to Science. In view of their press embargo policy, I will not discussed the detail of this particular finding here.

Drug effects (pharmacodynamics) are determined by drug concentration at target site and the affinity of the drug for a target. Pharmacogenetics describes inherited differences in drug metabolising enzyme activities and differences in drug transporters and receptors.Answers were sought on the following questions:1. What is the value of genotyping on polymorphic drug metabolising enzymes compared to the classical phenotyping stategies?2. What is the situation concerning CYP2D6 and CYP2C19 polymorphism in the Dutch population?3. Is knowledge of the individual metaboliser status of CYP2D6 or CYP2C19 valuable in clinical pharmacological research and practice of pharmacotherapy

Factors governing wing dimorphism in ground beetles (Coleoptera: Carabidae) of the Calathus melanocephalus complex have been studied in relation to dispersal by flight. In Western Europe this complex consists of three well-defined species, as was shown by cross-breeding experiments and morphological studies: Calathus cinctus Motschulsky (= C. erythroderus Gemminger & Harold), Calathus melanocephalus (Linnaeus) and Calathus mollis (Marsham). The synonymy of Calathus erythroderus with C.cinctus was established. To stabilize the nomenclature of the species involved lectotypes were designated for Carabus melanocephalus Linnaeus and Carabus mollis Marsham. Action undertaken to conserve the specific name of Carabus mollis Marsham was approved by the International Commission on Zoological Nomenclature. Data on morphology, life history and distribution of the three species involved were listed and an identification key is included.Calathus cinctus and C. melanocephalus are wing-dimorphic with either strongly reduced wings (short winged or brachypterous) or fully developed wings (long winged or macropterous), whereas C . mollis is always macropterous. Long winged beetles are considered potential flyers, representing the dispersal morph of these species. Populations of C. cinctus and C. melanocephalus showed different proportions of long winged beetles.Wing dimorphism in Calathus cinctus and C . melanocephalus was found to be genetically determined and to be inherited in a simple Mendelian fashion with brachyptery dominant to macroptery. In C.cinctus the long winged genotype is always expressed, whereas in C . melanocephalus the expression of the long winged genotype is under environmental control. It was shown that at least temperature and food-supply affected the expression of the long winged genotype in C.melanocephalus: more favourable temperatures (i.e. relatively high temperatures) and a higher food-supply resulted in a higher number of long winged beetles compared with less favourable temperatures (i.e. both low and extremely high temperatures) and a lower food-supply.Wing-morph frequencies in populations of wing-dimorphic species may also be affected by differences in fitness between short and long winged beetles. Fecundity (egg production and oviposition period), development time, and growth were studied in this respect.In both Calathus cinctus and C.melanocephalus long winged females showed a significantly higher egg production than short winged females, and they also tended to produce eggs during a longer period. Considerable differences in fecundity were observed between the species: the highest egg production was found in Calathus cinctus, a species with a late start and a long oviposition period, and the lowest egg production was found in C . mollis, a species with an early start and a comparably long oviposition period. Calathus melanocephalus showed an egg production intermediate between those of C . cinctus and C.mollis, with an early start and a relatively short oviposition period.In all three species development times were generally shorter at higher temperatures and longer at lower temperatures, but in both Calathus cinctus and C . melanocephalus a general relationship between temperature and development time was found neither for wing-morph nor for sex. Development time and growth of long winged genotypes of Calathus melanocephalus were strongly influenced by food- supply: with a higher food-supply shorter development times and higher hatching weights were found. Long winged beetles of both sexes developed faster and were heavier than short winged ones reared under similar conditions, and females weighed more than males.Under laboratory conditions both long winged males and females of the three species studied built up functional flight muscles and were capable of flight. Flight activity in the field, however, was only observed in Calathus cinctus. Building up flight muscles, flight, and subsequent resorption of flight muscles only occurred in the pre- oviposition period, but mating and even ovarian development may occur during the flight period too.The dispersal abilities of the three species studied are supposed to be different because there are large differences in flight potential between the species. In Calathus cinctus and C.melanocephalus only part of the individuals is long winged and has flight potential, whereas in Calathus mollis all individuals have this ability. Moreover, in Calathus cinctus the long winged genotype, and so the potential for flight, is always expressed, whereas in C . melanocephalus the expression of the long winged genotype depends on environmental conditions, such as temperature and food- supply, and therefore the flight potential for flight is partly expressed only.Differences in flight potential were clearly associated with differences in life histories and habitat selection. Calathus cinctus occupies temporary habitats, showing a high potential for flight and a high turnover of populations, whereas C . melanocephalus occurs in less temporary habitats, showing a much lower potential for flight and a relatively low turnover of populations. Calathus mollis inhabits coastal dunes and blowing sands, showing a maximum potential for flight, obviously to keep pace with changes in this extreme environment.The higher fecundity of long winged females, closely linked with larger body size and higher weight, is considered to benefit the colonization abilities of the species.The wing-morph determination found in Calathus cinctus and C . melanocephalus fitted the current model, but a trade-off between flightlessness and fecundity was not found. Both flight capability and high fecundity are considered to represent closely linked characters of species adapted to unstable, temporary habitats.Wing dimorphism is supposed to be maintained by a 'balance' between the loss of long winged genotypes by flight activities and the frequency of successful (re)colonizations. In Calathus cinctus both the loss of long winged genotypes and the colonization success is supposed to be high (a short term, opportunistic 'strategy'), whereas in C . melanocephalus both the loss of long winged genotypes and the colonization success is supposed to be low. However, in the latter species the maintenance of wing dimorphism is favoured by the environmental control of the expression of long winged genotypes (a long term, more ensuring 'strategy').

Factors governing wing dimorphism in ground beetles (Coleoptera: Carabidae) of the Calathus melanocephalus complex have been studied in relation to dispersal by flight. In Western Europe this complex consists of three well-defined species, as was shown by cross-breeding experiments and morphological studies: Calathus cinctus Motschulsky (= C. erythroderus Gemminger & Harold), Calathus melanocephalus (Linnaeus) and Calathus mollis (Marsham). The synonymy of Calathus erythroderus with C.cinctus was established. To stabilize the nomenclature of the species involved lectotypes were designated for Carabus melanocephalus Linnaeus and Carabus mollis Marsham. Action undertaken to conserve the specific name of Carabus mollis Marsham was approved by the International Commission on Zoological Nomenclature. Data on morphology, life history and distribution of the three species involved were listed and an identification key is included.Calathus cinctus and C. melanocephalus are wing-dimorphic with either strongly reduced wings (short winged or brachypterous) or fully developed wings (long winged or macropterous), whereas C . mollis is always macropterous. Long winged beetles are considered potential flyers, representing the dispersal morph of these species. Populations of C. cinctus and C. melanocephalus showed different proportions of long winged beetles.Wing dimorphism in Calathus cinctus and C . melanocephalus was found to be genetically determined and to be inherited in a simple Mendelian fashion with brachyptery dominant to macroptery. In C.cinctus the long winged genotype is always expressed, whereas in C . melanocephalus the expression of the long winged genotype is under environmental control. It was shown that at least temperature and food-supply affected the expression of the long winged genotype in C.melanocephalus: more favourable temperatures (i.e. relatively high temperatures) and a higher food-supply resulted in a higher number of long winged beetles compared with less favourable temperatures (i.e. both low and extremely high temperatures) and a lower food-supply.Wing-morph frequencies in populations of wing-dimorphic species may also be affected by differences in fitness between short and long winged beetles. Fecundity (egg production and oviposition period), development time, and growth were studied in this respect.In both Calathus cinctus and C.melanocephalus long winged females showed a significantly higher egg production than short winged females, and they also tended to produce eggs during a longer period. Considerable differences in fecundity were observed between the species: the highest egg production was found in Calathus cinctus, a species with a late start and a long oviposition period, and the lowest egg production was found in C . mollis, a species with an early start and a comparably long oviposition period. Calathus melanocephalus showed an egg production intermediate between those of C . cinctus and C.mollis, with an early start and a relatively short oviposition period.In all three species development times were generally shorter at higher temperatures and longer at lower temperatures, but in both Calathus cinctus and C . melanocephalus a general relationship between temperature and development time was found neither for wing-morph nor for sex. Development time and growth of long winged genotypes of Calathus melanocephalus were strongly influenced by food- supply: with a higher food-supply shorter development times and higher hatching weights were found. Long winged beetles of both sexes developed faster and were heavier than short winged ones reared under similar conditions, and females weighed more than males.Under laboratory conditions both long winged males and females of the three species studied built up functional flight muscles and were capable of flight. Flight activity in the field, however, was only observed in Calathus cinctus. Building up flight muscles, flight, and subsequent resorption of flight muscles only occurred in the pre- oviposition period, but mating and even ovarian development may occur during the flight period too.The dispersal abilities of the three species studied are supposed to be different because there are large differences in flight potential between the species. In Calathus cinctus and C.melanocephalus only part of the individuals is long winged and has flight potential, whereas in Calathus mollis all individuals have this ability. Moreover, in Calathus cinctus the long winged genotype, and so the potential for flight, is always expressed, whereas in C . melanocephalus the expression of the long winged genotype depends on environmental conditions, such as temperature and food- supply, and therefore the flight potential for flight is partly expressed only.Differences in flight potential were clearly associated with differences in life histories and habitat selection. Calathus cinctus occupies temporary habitats, showing a high potential for flight and a high turnover of populations, whereas C . melanocephalus occurs in less temporary habitats, showing a much lower potential for flight and a relatively low turnover of populations. Calathus mollis inhabits coastal dunes and blowing sands, showing a maximum potential for flight, obviously to keep pace with changes in this extreme environment.The higher fecundity of long winged females, closely linked with larger body size and higher weight, is considered to benefit the colonization abilities of the species.The wing-morph determination found in Calathus cinctus and C . melanocephalus fitted the current model, but a trade-off between flightlessness and fecundity was not found. Both flight capability and high fecundity are considered to represent closely linked characters of species adapted to unstable, temporary habitats.Wing dimorphism is supposed to be maintained by a 'balance' between the loss of long winged genotypes by flight activities and the frequency of successful (re)colonizations. In Calathus cinctus both the loss of long winged genotypes and the colonization success is supposed to be high (a short term, opportunistic 'strategy'), whereas in C . melanocephalus both the loss of long winged genotypes and the colonization success is supposed to be low. However, in the latter species the maintenance of wing dimorphism is favoured by the environmental control of the expression of long winged genotypes (a long term, more ensuring 'strategy').