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Naast het bestaande rekenmodel voor de schatting van het deel van de aalvangst met gehalten som-TEQ onder de voedselveiligheidsnorm (“percentage aal onder de norm”) is nu een rekenmodel gemaakt zodat ook voor de som van niet-dioxine achtige PCBs (som-ndl-PCB) het percentage van de vangst onder de voedselveiligheidsnorm kan worden geschat. De gehalten som-TEQ en som-ndl-PCB in aal zijn aan elkaar gerelateerd, maar een eventuele overschrijding van de norm in aal kan van de som-TEQ, van de somndl-PCB óf van beide normen zijn. Dit kan per locatie verschillen. De norm voor som-ndl-PCBs wordt in de aal uit de onderzochte locaties het vaakst overschreden. Beide normen wegen wettelijk even zwaar; het laagste geschatte percentage aal onder de norm uit beide modellen wordt genomen als hét “percentage aal onder de norm”. Door het gebruik van de norm voor som-TEQ én som-ndl-PCBs wordt het geschatte percentage aal onder de norm in het gesloten gebied lager. Daarnaast zijn de beide modellen aangepast zodat de gemeten man-vrouw verhouding in het gebruikte mengmonster aal in de berekening wordt meegenomen.

Naast het bestaande rekenmodel voor de schatting van het deel van de aalvangst met gehalten som-TEQ onder de voedselveiligheidsnorm (“percentage aal onder de norm”) is nu een rekenmodel gemaakt zodat ook voor de som van niet-dioxine achtige PCBs (som-ndl-PCB) het percentage van de vangst onder de voedselveiligheidsnorm kan worden geschat. De gehalten som-TEQ en som-ndl-PCB in aal zijn aan elkaar gerelateerd, maar een eventuele overschrijding van de norm in aal kan van de som-TEQ, van de somndl-PCB óf van beide normen zijn. Dit kan per locatie verschillen. De norm voor som-ndl-PCBs wordt in de aal uit de onderzochte locaties het vaakst overschreden. Beide normen wegen wettelijk even zwaar; het laagste geschatte percentage aal onder de norm uit beide modellen wordt genomen als hét “percentage aal onder de norm”. Door het gebruik van de norm voor som-TEQ én som-ndl-PCBs wordt het geschatte percentage aal onder de norm in het gesloten gebied lager. Daarnaast zijn de beide modellen aangepast zodat de gemeten man-vrouw verhouding in het gebruikte mengmonster aal in de berekening wordt meegenomen.

Understanding the factors influencing sex of egg and sex ratio in laying chicken may lead to finding potential solutions for the problem of killing of day old male chicks, which is the current practice in breeding of laying hens. In studies described in this thesis, it was investigated if the sex of the chicken egg can be predicted by measurable differences in male and female eggs at unincubated stage and if the female primary sex ratio can be induced in laying chicken using different experimental conditions such as feed restriction and corticosterone feeding. The method of sex determination in unincubated chicken eggs using PCR targeted to CHD1 gene was first developed. This method was subsequently used to study the primary sex ratio bias as well as relationship between egg sex and yolk hormones. No significant relationship of the sex of egg with concentrations of several hormones (testosterone, estradiol, androstenedione, progesterone, dihydrotestosterone) and glucose in yolk as well as of egg parameters (mass, width and length) was found. Effect of feed availability on sex ratio was tested in two separate studies. In one study, the rate of change of hen body mass between day of laying and day of laying minus 2 days (encompass time of meiosis completion) was a significant predictor for the sex of that egg, suggesting meiotic drive as mechanism of sex ratio bias. This relationship was not found in the later study. The difference in results could be due to the reason that hens decreased in body mass much less in the later study as compared to earlier study. Blood corticosterone concentrations were associated with sex ratio per hen in the earlier study. Effect of egg mass on egg sex was studied during the later experiment of feed restriction. The egg sex ratio per hen was negatively associated with the average egg mass per hen in the feed restriction group. Two groups of hens were selected from the feed restriction group i.e. male biased hens with low egg mass and female biased hens with high egg mass for microarray analysis of gene expression in the germinal disc of collected F1 follicle. The results did not show differential expression of genes between the groups. However, gene set enrichment analysis showed that a number of processes related to cell cycle progression, mitotic/meiotic apparatus and chromosomal movement were differently enriched between the groups, supporting meiotic drive as potential mechanisms underlying sex ratio determination. In another experiment, blood circulating levels of corticosterone in hens were increased by feeding corticosterone mixed feed under ad libitum. The blood levels of corticosterone were significantly higher in treated hens but these levels were not associated with sex ratio. Treatment did not affect the overall sex ratio, but affected the sex ratio in interaction with hen body mass. In the corticosterone group, sex ratio, laying rate, and fertility rate per hen were decreased in heavy hens. These results suggest that three parameters (sex ratio, laying rate and fertility rate) are connected at the level of ovarian physiology. Interference with meiosis have been shown to affect these three parameters, suggesting the involvement of meiotic drive as mechanism of sex ratio bias.

Bram Kuijper deed onderzoek naar de relatie tussen seksuele selectie en sekse-allocatie en de onderliggende mechanismen. Hij concludeert onder meer: • Het maken van voorspellingen gebaseerd op slechts één modelleertechniek is vragen om problemen. Een pluralistische aanpak waarbij meerdere technieken met elkaar worden vergeleken geeft de meest robuuste voorspellingen met betrekking tot evolutionaire processen. • Nieuwe theoretische modellen zijn nodig om te onderzoeken of conclusies van bestaande modellen over seksuele selectie inderdaad zo algemeen geldend zijn als wordt gedacht: hierbij moet vooral worden gedacht aan mechanistische processen met betrekking tot paringskeuze, overerving en interacties met andere evolutionaire processen (zorg voor nakomelingen, sekse-allocatie). • Seksuele selectie in hermafrodieten (beide seksen aanwezig in hetzelfde individu) komt over het algemeen moeilijker van de grond dan in gonochoristen (seksen in aparte individuen). Dit geldt echter niet wanneer beide partners elkaar kiezen (in plaats van één partner de ander, welke dan altijd instemt): dan is seksuele selectie even plausibel in gonochoristen als in hermafrodieten. • De evolutie van haplodiploïdie door toedoen van ‘male-killers’ lijkt veel specifieker te zijn dan eerder werd voorspeld (bijvoorbeeld Normark 2004a). Haplodiploïdie komt daarom alleen onder specifieke omstandigheden van de grond, bijvoorbeeld wanneer ‘male-killers’ mutualistisch zijn of wanneer populaties gekenmerkt worden door een hoge mate van inteelt. • Twee of meerdere CSD loci liggen waarschijnlijk ten grondslag aan de geslachtsbepaling van de wesp Cotesia glomerata, waardoor het één van de weinige bekende soorten binnen de Hymenoptera is met multi-locus CSD. • Conflict tussen ouders en nakomelingen over sekse-allocatie kan leiden tot een stabiele mix van verschillende seksedeterminatiesystemen. Een dergelijk conflict kan bijvoorbeeld verklaren waarom sekschromosomen aanwezig zijn in sommige soorten met temperatuurafhankelijke geslachtsbepaling. • Flexibele seksratio’s gebaseerd op de aantrekkelijkheid van de partner evolueren vaak naar een patroon waarbij vrouwtjes gepaard met aantrekkelijke mannetjes zonen produceren, en vrouwtjes met onaantrekkelijke mannetjes dochters. Desalniettemin is selectie voor flexibele seksratios gebaseerd op de aantrekkelijkheid van de partner vaak zwak en afhankelijk van het mechanisme dat seksuele selectie veroorzaakt. • De evolutie van sekse-allocatie gebaseerd op aantrekkelijkheid van partners samen met de evolutie van een vrouwelijke voorkeur voor aantrekkelijke mannetjes leidt uiteindelijk tot het verlies van deze voorkeur. Daarmee gaat ook mannelijke aantrekkelijkheid verloren, waarmee sekseallocatie zichzelf uiteindelijk ondermijnt.

Bram Kuijper deed onderzoek naar de relatie tussen seksuele selectie en sekse-allocatie en de onderliggende mechanismen. Hij concludeert onder meer: • Het maken van voorspellingen gebaseerd op slechts één modelleertechniek is vragen om problemen. Een pluralistische aanpak waarbij meerdere technieken met elkaar worden vergeleken geeft de meest robuuste voorspellingen met betrekking tot evolutionaire processen. • Nieuwe theoretische modellen zijn nodig om te onderzoeken of conclusies van bestaande modellen over seksuele selectie inderdaad zo algemeen geldend zijn als wordt gedacht: hierbij moet vooral worden gedacht aan mechanistische processen met betrekking tot paringskeuze, overerving en interacties met andere evolutionaire processen (zorg voor nakomelingen, sekse-allocatie). • Seksuele selectie in hermafrodieten (beide seksen aanwezig in hetzelfde individu) komt over het algemeen moeilijker van de grond dan in gonochoristen (seksen in aparte individuen). Dit geldt echter niet wanneer beide partners elkaar kiezen (in plaats van één partner de ander, welke dan altijd instemt): dan is seksuele selectie even plausibel in gonochoristen als in hermafrodieten. • De evolutie van haplodiploïdie door toedoen van ‘male-killers’ lijkt veel specifieker te zijn dan eerder werd voorspeld (bijvoorbeeld Normark 2004a). Haplodiploïdie komt daarom alleen onder specifieke omstandigheden van de grond, bijvoorbeeld wanneer ‘male-killers’ mutualistisch zijn of wanneer populaties gekenmerkt worden door een hoge mate van inteelt. • Twee of meerdere CSD loci liggen waarschijnlijk ten grondslag aan de geslachtsbepaling van de wesp Cotesia glomerata, waardoor het één van de weinige bekende soorten binnen de Hymenoptera is met multi-locus CSD. • Conflict tussen ouders en nakomelingen over sekse-allocatie kan leiden tot een stabiele mix van verschillende seksedeterminatiesystemen. Een dergelijk conflict kan bijvoorbeeld verklaren waarom sekschromosomen aanwezig zijn in sommige soorten met temperatuurafhankelijke geslachtsbepaling. • Flexibele seksratio’s gebaseerd op de aantrekkelijkheid van de partner evolueren vaak naar een patroon waarbij vrouwtjes gepaard met aantrekkelijke mannetjes zonen produceren, en vrouwtjes met onaantrekkelijke mannetjes dochters. Desalniettemin is selectie voor flexibele seksratios gebaseerd op de aantrekkelijkheid van de partner vaak zwak en afhankelijk van het mechanisme dat seksuele selectie veroorzaakt. • De evolutie van sekse-allocatie gebaseerd op aantrekkelijkheid van partners samen met de evolutie van een vrouwelijke voorkeur voor aantrekkelijke mannetjes leidt uiteindelijk tot het verlies van deze voorkeur. Daarmee gaat ook mannelijke aantrekkelijkheid verloren, waarmee sekseallocatie zichzelf uiteindelijk ondermijnt.

Een van de belangrijkste concepten in de evolutionaire biologie is het streven van individuen naar een zo groot mogelijke fitness. Fitness is de relatieve bijdrage van een individu aan de toekomstige genenpool van de soort waartoe het behoord. Naast overleven, waarmee de genen voortbestaan in het individu zelf, is voortplanting de manier om fitness te vergroten. Het streven naar een zo hoog mogelijke fitness resulteert in selectie. Individuen die het best zijn aangepast aan hun omgeving zullen de hoogste fitness behalen en hun genen zullen oververtegenwoordigd zijn in de toekomstige genenpool. Er is dus natuurlijke selectie voor het "type" individu dat het beste aan zijn of haar omgeving is aangepast. Om een zo hoog mogelijke fitness te bereiken moeten individuen keuzes maken. De hoeveelheid energie en tijd die zij ter beschikking hebben is beperkt en dus moeten de kosten en baten van een keuze afgewogen worden. Individuen moeten bijvoorbeeld afwegen met welke partner te reproduceren en hoeveel nakomelingen zij zouden moeten produceren. Binnen deze keuze is de verhouding tussen het aantal zonen en doch ters ook van belang. Met deze laatste afweging, geslachtsallocatie, houd ik mij in deze dissertatie bezig. Geslachtsallocatie is gedefinieerd als de relatieve investering van ouders in de productie van zonen ten opzichte van hun totale nageslacht. Geslachtsallocatie wordt gestuurd door twee processen. Ten eerste door natuurlijke selectie die de relatieve investering in zonen en dochters evolutionair beïnvloedt. Dit proces duidt men meestal aan met geslachtsallocatie. Bijvoorbeeld: als zonen een lagere overlevingskans hebben dan dochters zouden er in de hele populatie meer zonen geboren moeten worden om er voor te zorgen dat er op het moment van voortplanten evenveel mannen als vrouwen zijn.

Een van de belangrijkste concepten in de evolutionaire biologie is het streven van individuen naar een zo groot mogelijke fitness. Fitness is de relatieve bijdrage van een individu aan de toekomstige genenpool van de soort waartoe het behoord. Naast overleven, waarmee de genen voortbestaan in het individu zelf, is voortplanting de manier om fitness te vergroten. Het streven naar een zo hoog mogelijke fitness resulteert in selectie. Individuen die het best zijn aangepast aan hun omgeving zullen de hoogste fitness behalen en hun genen zullen oververtegenwoordigd zijn in de toekomstige genenpool. Er is dus natuurlijke selectie voor het "type" individu dat het beste aan zijn of haar omgeving is aangepast. Om een zo hoog mogelijke fitness te bereiken moeten individuen keuzes maken. De hoeveelheid energie en tijd die zij ter beschikking hebben is beperkt en dus moeten de kosten en baten van een keuze afgewogen worden. Individuen moeten bijvoorbeeld afwegen met welke partner te reproduceren en hoeveel nakomelingen zij zouden moeten produceren. Binnen deze keuze is de verhouding tussen het aantal zonen en doch ters ook van belang. Met deze laatste afweging, geslachtsallocatie, houd ik mij in deze dissertatie bezig. Geslachtsallocatie is gedefinieerd als de relatieve investering van ouders in de productie van zonen ten opzichte van hun totale nageslacht. Geslachtsallocatie wordt gestuurd door twee processen. Ten eerste door natuurlijke selectie die de relatieve investering in zonen en dochters evolutionair beïnvloedt. Dit proces duidt men meestal aan met geslachtsallocatie. Bijvoorbeeld: als zonen een lagere overlevingskans hebben dan dochters zouden er in de hele populatie meer zonen geboren moeten worden om er voor te zorgen dat er op het moment van voortplanten evenveel mannen als vrouwen zijn., Sex allocation theory is successful in predicting sex ratio variation in some taxa, but often not in birds and mammals. An important reason may be that most theoretical models do not account for the complexities of avian and mammalian life-history. Radersma investigated the effect of manipulated brood sex ratios on long-term fitness benefits. Since offspring recruitment, parental survival and parental future fecundity were unaffected, there was stabilizing selection for producing brood sex ratios at parity. To gain insight in the underlying mechanism he focused on different aspects of the offspring’s first year. Brood sex ratio did not affect fledging behaviour, but there were some effects on social behaviour in winter, which did not correlate to fitness. As adults, individuals raised as the rare sex in a brood had larger tarsi than individuals of the abundant sex. This relationship was neither present in the nestling phase nor caused by selective disappearance. This suggests a sex-specific directional effect of brood sex ratio on the late development of the offspring, which potentially links brood sex ratio to fecundity. Stabilizing selection for brood sex ratios at parity might provide an explanation why sex allocation in birds and mammals is often subtle. Higher benefits for equal brood sex ratios counteract selection for facultative sex allocation to extreme values.

Een van de belangrijkste concepten in de evolutionaire biologie is het streven van individuen naar een zo groot mogelijke fitness. Fitness is de relatieve bijdrage van een individu aan de toekomstige genenpool van de soort waartoe het behoord. Naast overleven, waarmee de genen voortbestaan in het individu zelf, is voortplanting de manier om fitness te vergroten. Het streven naar een zo hoog mogelijke fitness resulteert in selectie. Individuen die het best zijn aangepast aan hun omgeving zullen de hoogste fitness behalen en hun genen zullen oververtegenwoordigd zijn in de toekomstige genenpool. Er is dus natuurlijke selectie voor het "type" individu dat het beste aan zijn of haar omgeving is aangepast. Om een zo hoog mogelijke fitness te bereiken moeten individuen keuzes maken. De hoeveelheid energie en tijd die zij ter beschikking hebben is beperkt en dus moeten de kosten en baten van een keuze afgewogen worden. Individuen moeten bijvoorbeeld afwegen met welke partner te reproduceren en hoeveel nakomelingen zij zouden moeten produceren. Binnen deze keuze is de verhouding tussen het aantal zonen en doch ters ook van belang. Met deze laatste afweging, geslachtsallocatie, houd ik mij in deze dissertatie bezig. Geslachtsallocatie is gedefinieerd als de relatieve investering van ouders in de productie van zonen ten opzichte van hun totale nageslacht. Geslachtsallocatie wordt gestuurd door twee processen. Ten eerste door natuurlijke selectie die de relatieve investering in zonen en dochters evolutionair beïnvloedt. Dit proces duidt men meestal aan met geslachtsallocatie. Bijvoorbeeld: als zonen een lagere overlevingskans hebben dan dochters zouden er in de hele populatie meer zonen geboren moeten worden om er voor te zorgen dat er op het moment van voortplanten evenveel mannen als vrouwen zijn.

Keywords: dairy cattle; oestrus; behaviour; pedometer; reproductive hormones; ovulation time; insemination strategyIn dairy practice, calving rates after first insemination are often less than 50%. Part of this low percentage might be explained by wrongly timed inseminations. The aim was to establish the relationship between various oestrus characteristics and ovulation time in order to investigate whether these oestrus characteristics could predict ovulation time and to study the consequences of variation in the interval between insemination and ovulation on the success of fertilization and embryonic characteristics. The ultimate goal of the project was to come to an optimal insemination strategy that can be used in practice. The relationship between behavioural oestrous signs, changes in activity (measured by pedometers) and progesterone profiles and time of ovulation were. It was shown that monitoring progesterone profiles was not suitable to predict time of ovulation because of the large variation found in the decrease of progesterone relative to ovulation. Although the prediction of ovation time using behavioural oestrus signs was quite accurate, it seems not suitable for practice because of the high labour requirements. The increase in the number of steps measured by pedometers seems useful to predict time of ovulation accurately and could be easily implemented in practice. Effects of the interval between insemination and ovulation on success of fertilization and embryonic characteristics were also studied. It was shown that the insemination-ovulation interval in which high fertilization rates were observed was quite long (insemination from 36 to 12h before ovulation), while the interval in which the majority of fertilized ovum developed into a good quality embryo was considerably shorter (insemination from 24 to 12h before ovulation); the interval between insemination and ovulation did not affect the sex ratio of the embryos.In conclusion, the best chance to increase calving rates seems to be to use the insemination strategy, in which cows are inseminated between 5 to 17h after the first increase in the number of steps.

Selfish genetic elements are defined as genetic elements that have a replication advantage relative to the rest of the genome. They are ubiquitous in nature and were extensively reported for almost all species studied so far. A special type of selfish genetic element, the sex ratio distorter, is most frequent in arthropods and changes the offspring sex ratio of its host. An example of an extremely selfish male biasing sex ratio distorter is the paternal sex ratio (PSR) chromosome in the parasitoid wasps Trichogramma kaykai and Nasonia vitripennis . These wasps have an arrhenotokous sex determination in which fertilized diploid eggs develop into females and males develop from unfertilized haploid eggs. Only part of the male wasps contains this additional B chromosome, which upon fertilizationeliminates the paternal genome, while keeping itself and the maternal chromosomes intact. The resulting haploid embryo develops into a B chromosome-carrying male. This extremely selfish B chromosome was first discovered in N. vitripennis . The recent discovery of a second PSR chromosome in the unrelated wasp T. kaykai provided an opportunity for a comparative study on PSR chromosomes. In this study I determined the mechanism, the origin and structure of the PSR chromosome in T. kaykai and compared my results with previous studies on the PSR chromosome in N. vitripennis .The mode of action of the Trichogramma PSR chromosome was revealed by examining microscopic preparations of freshly fertilized eggs. This chromosome modifies the paternal genome into a dense chromatin mass at the beginning of the first mitotic division, while the PSR chromosome itself escapes its own destructive effect and continues embryo development with the maternal chromosomes. Comparing the modes of action of the Trichogramma and Nasonia PSR chromosomes suggests that both systems are identical, except for the diameter of the paternal chromatin mass (PCM) and the occurrence of PCM-associated nuclei. However, their molecular mechanism remains unknown. Furthermore, both PSR chromosomes share the main structural characteristics of B chromosomes: They are much smaller than the normal chromosomes and contain high amounts of transposable elements and tandem repetitive DNA. B chromosomes are thought to accumulate transposable elements because they do not recombine meiotically with the normal chromosomes. Repeats have proven to be an important factor for the transmission efficiency of the Nasonia PSR chromosomes and are also thought to correlate with the B chromosome size in general. However, none of the DNA sequences found on the Trichogramma PSR chromosome were found on the Nasonia PSR chromosome or visa versa. About two thirds of the Trichogramma PSR chromosome comprises tandem arrays of 45S ribosomal DNA (rDNA), while on the Nasonia PSR chromosome three B chromosome specific repeat families are located and one repeat family that is also present on the Nasonia genome. 45S rDNA consists of three conserved genes essential for protein processing separated by three spacer sequences that are hypervariable between species but conserved within species. These spacer sequences are therefore often used for taxonomic purposes. One of the spacer sequences of the 45S rDNA on the Trichogramma PSR chromosome, i.e. ITS2, contains at least five different sequences that resemble either the ITS2 of T. kaykai or the related T. oleae . We therefore concluded that this B chromosome originated from T. oleae or a T. oleae -like species. Retrotransposon analysis revealed that the Nasonia PSR chromosome most likely originated from the Nasonia related wasp genus Trichomalopsis . Though both PSR chromosomes have a similar mode of action, the absence of any sequence homology between both chromosomes implies different PSR chromosome ancestors. This again makes it less likely that the molecular mechanism of paternal genome loss is identical. Future studies should focus on comparing the molecular mode of action and DNA sequence homology of both PSR chromosomes and revealing the incidence of more PSR chromosomes in other haplo-diploid organisms. This will not only provide more knowledge on the mechanism of early embryogenesis and in particular on the paternal chromosome processing following fertilization, but also on the origin and evolution of PSR chromosomes. Extending our knowledge on PSR chromosomes is expected most useful for the control of pest insects with haplo-diploid sex determination systems like the Argentine ant. Without females such insect populations will quickly perish.

Selfish genetic elements are defined as genetic elements that have a replication advantage relative to the rest of the genome. They are ubiquitous in nature and were extensively reported for almost all species studied so far. A special type of selfish genetic element, the sex ratio distorter, is most frequent in arthropods and changes the offspring sex ratio of its host. An example of an extremely selfish male biasing sex ratio distorter is the paternal sex ratio (PSR) chromosome in the parasitoid wasps Trichogramma kaykai and Nasonia vitripennis . These wasps have an arrhenotokous sex determination in which fertilized diploid eggs develop into females and males develop from unfertilized haploid eggs. Only part of the male wasps contains this additional B chromosome, which upon fertilizationeliminates the paternal genome, while keeping itself and the maternal chromosomes intact. The resulting haploid embryo develops into a B chromosome-carrying male. This extremely selfish B chromosome was first discovered in N. vitripennis . The recent discovery of a second PSR chromosome in the unrelated wasp T. kaykai provided an opportunity for a comparative study on PSR chromosomes. In this study I determined the mechanism, the origin and structure of the PSR chromosome in T. kaykai and compared my results with previous studies on the PSR chromosome in N. vitripennis .The mode of action of the Trichogramma PSR chromosome was revealed by examining microscopic preparations of freshly fertilized eggs. This chromosome modifies the paternal genome into a dense chromatin mass at the beginning of the first mitotic division, while the PSR chromosome itself escapes its own destructive effect and continues embryo development with the maternal chromosomes. Comparing the modes of action of the Trichogramma and Nasonia PSR chromosomes suggests that both systems are identical, except for the diameter of the paternal chromatin mass (PCM) and the occurrence of PCM-associated nuclei. However, their molecular mechanism remains unknown. Furthermore, both PSR chromosomes share the main structural characteristics of B chromosomes: They are much smaller than the normal chromosomes and contain high amounts of transposable elements and tandem repetitive DNA. B chromosomes are thought to accumulate transposable elements because they do not recombine meiotically with the normal chromosomes. Repeats have proven to be an important factor for the transmission efficiency of the Nasonia PSR chromosomes and are also thought to correlate with the B chromosome size in general. However, none of the DNA sequences found on the Trichogramma PSR chromosome were found on the Nasonia PSR chromosome or visa versa. About two thirds of the Trichogramma PSR chromosome comprises tandem arrays of 45S ribosomal DNA (rDNA), while on the Nasonia PSR chromosome three B chromosome specific repeat families are located and one repeat family that is also present on the Nasonia genome. 45S rDNA consists of three conserved genes essential for protein processing separated by three spacer sequences that are hypervariable between species but conserved within species. These spacer sequences are therefore often used for taxonomic purposes. One of the spacer sequences of the 45S rDNA on the Trichogramma PSR chromosome, i.e. ITS2, contains at least five different sequences that resemble either the ITS2 of T. kaykai or the related T. oleae . We therefore concluded that this B chromosome originated from T. oleae or a T. oleae -like species. Retrotransposon analysis revealed that the Nasonia PSR chromosome most likely originated from the Nasonia related wasp genus Trichomalopsis . Though both PSR chromosomes have a similar mode of action, the absence of any sequence homology between both chromosomes implies different PSR chromosome ancestors. This again makes it less likely that the molecular mechanism of paternal genome loss is identical. Future studies should focus on comparing the molecular mode of action and DNA sequence homology of both PSR chromosomes and revealing the incidence of more PSR chromosomes in other haplo-diploid organisms. This will not only provide more knowledge on the mechanism of early embryogenesis and in particular on the paternal chromosome processing following fertilization, but also on the origin and evolution of PSR chromosomes. Extending our knowledge on PSR chromosomes is expected most useful for the control of pest insects with haplo-diploid sex determination systems like the Argentine ant. Without females such insect populations will quickly perish.

Organisms display a great variety of sex ratios (ratios of females vs. males), ranging from 100% females to a male bias. These sex ratios are not always only determined by the genes of the organism itself but may actually often be manipulated or distorted by "sex ratio distorters". One sex ratio distorter, the bacterium Wolbachia that lives in the cytoplasm of the cells of its host organism, has received much attention by biologists all over the world. This interest mainly arises from the fact that it manipulates arthropod or nematode reproduction in several ways - feminization, induction of cytoplasmic incompatibility, male-killing and parthenogenesis-induction - to enhance its own inheritance from mother to daughter. Because sperm cells do not contain enough cytoplasm, they cannot transmit Wolbachia and males are a dead end for the bacterium. Recent estimates of Wolbachia 's prevalence range from 17 to even 76% of the insect species.In many wasp, thrips and mite species Wolbachia has switched the mode of reproduction from sexuality to complete parthenogenesis (±100% females). However, in minute parasitoid wasps of the genus Trichogramma , which are used worldwide as natural enemies in biological control of lepidopteran pests, only a part of the females in a population is infected with Wolbachia and can therefore reproduce through parthenogenesis. My aim in this thesis is to gain more insight in the dynamics of parthenogenesis-inducing (PI) Wolbachia and to explain the coexistence of infected and uninfected forms in Trichogramma wasps. After first reviewing the literature on PI Wolbachia in chapter 2 , I tried to further our understanding of the coexistence of the two reproductive forms in natural Trichogramma kaykai and T. deion populations by combining fieldwork, molecular techniques, behavioural- and crossing experiments with model studies.Vertical transmission of Wolbachia from mother to daughter has been viewed as the main mode of transmission but in chapter 3 & 4 we show an unexpectedly frequent natural inter- and intraspecific horizontal transmission between and within Trichogrammakaykai and T. deion larvae sharing a common food source, a butterfly egg. Originally uninfected immature wasps could acquire Wolbachia inside the host egg but not all newly infected females exhibit parthenogenesis. In T. kaykai, intraspecific horizontal transfer was followed by complete parthenogenesis in future generations but when T. kaykai females received Wolbachia from T. deion , the infection tended to be lost several generations after interspecific horizontal transfer. Our results largely explain the discordance between Wolbachia - and (Trichogrammatid) host phylogenies. Frequent horizontal transfer might select for high virulence in these bacteria .Because of a nuclear-cytoplasmic conflict between Wolbachia and the nuclear genes of Trichogramma and the previously described horizontal transfer of Wolbachia , the infection is most likely associated with fitness costs in populations where infected and uninfected individuals coexist. In chapter 5 we show that infected T. kaykai suffer a reduced survival compared to uninfected conspecifics when they shared the same host. The survival rate of infected immatures was higher when they competed with other infected immatures from a different infected parent than in competition with uninfected immatures. This shows that PI Wolbachia -infected Trichogramma can suffer a substantial fitness cost. Because of this reduced competitive ability of infected larvae, horizontal transfer that occurs under the same superparasitism circumstances does not contribute much to an increase in the infection rate in the population.Previous work showed that the presence of another sex ratio distorter in males, a B chromosome called PSR (Paternal Sex Ratio) that destroys the paternal chromosomes after fertilization thereby causing an all-male or a male-biased offspring sex ratio, contributes to a low infection frequency in T. kaykai . In chapter 6 we determined if a PSR factor causes low infection frequencies in other species as well. Therefore, we studied natural populations of three Trichogramma species - T. kaykai , T. deion and T. pratti - from the Mojave Desert. Our data showed that all the male-biased and all-male Trichogramma broods collected from the butterfly Apodemia mormo deserti that contained males expressing the PSR phenotype, belonged to T. kaykai. In laboratory tests, 71.4% of the T. kaykaiPSR males horizontally transmitted the PSR phenotype to T. deion . This percentage is comparable to the transmission rate of PSR to T. kaykai females, namely 81.6%. Consequently, the PSR can be transmitted to T. deion and we expect this to happen in the field because T. kaykai and T. deion sometimes emerge from the same butterfly egg. Despite this, we cannot find PSR in T. deion . Modeling shows that low Wolbachia infection frequencies can only be attained when the PSR rates are very high. Therefore, other factors should keep the PI Wolbachia -infection from spreading to fixation in this species, e.g. nuclear suppressor genes.The mating structure in the host population plays a major role in the dynamics of PI Wolbachia and PSR . A PSR factor prevents the Wolbachia infection from spreading to all the females in T. kaykai because uninfected T. kaykai females show a high level of sib (brother-sister) mating. Sib mating is a barrier against the destructive effect of mating with a PSR -carrying male. Infected females do not have this advantage. Using a population genetic model with microsatellites as genetic markers in chapter 7 , we estimated high levels of sib-mating of 70% and an off-patch mating of 15%. Thirty-five percent of the patches were estimated to be parasitized by two T. kaykai females. Incorporating such levels of sib mating in a previously developed model describing the dynamics of PI Wolbachia and PSR in a Trichogramma population, resulted in stable low frequencies of infection, i.e., a coexistence between infected and uninfected individuals, and of the PSR chromosome. Our results show how mating structure allows the two sex ratio distorters to coexist in the population.The main conclusion from this thesis is that, despite the high vertical transmission and regular horizontal transfer of Wolbachia , a PI Wolbachia -infection can be attained at low frequencies in Trichogramma , due to the presence of a non-mendelian suppressor, like the male-biasing PSR factor in T. kaykai, but also due to other factors. In T. deion, for example, PSR does not keep the infection frequency at low levels but a nuclear mendelian suppressor against the PI Wolbachia might have evolved.Next to their significance for the understanding of the evolutionary pathways of Wolbachia -host interactions, the results reported in this thesis may also have important implications for future use of natural enemies, and more specifically Trichogramma wasps, in inundative biological control. We may now have a good method to render wasps parthenogenetic, via super- or multiparasitism by infected and uninfected females, thereby increasing the efficacy of parasitoid releases against lepidopteran pests.

Organisms display a great variety of sex ratios (ratios of females vs. males), ranging from 100% females to a male bias. These sex ratios are not always only determined by the genes of the organism itself but may actually often be manipulated or distorted by "sex ratio distorters". One sex ratio distorter, the bacterium Wolbachia that lives in the cytoplasm of the cells of its host organism, has received much attention by biologists all over the world. This interest mainly arises from the fact that it manipulates arthropod or nematode reproduction in several ways - feminization, induction of cytoplasmic incompatibility, male-killing and parthenogenesis-induction - to enhance its own inheritance from mother to daughter. Because sperm cells do not contain enough cytoplasm, they cannot transmit Wolbachia and males are a dead end for the bacterium. Recent estimates of Wolbachia 's prevalence range from 17 to even 76% of the insect species.In many wasp, thrips and mite species Wolbachia has switched the mode of reproduction from sexuality to complete parthenogenesis (±100% females). However, in minute parasitoid wasps of the genus Trichogramma , which are used worldwide as natural enemies in biological control of lepidopteran pests, only a part of the females in a population is infected with Wolbachia and can therefore reproduce through parthenogenesis. My aim in this thesis is to gain more insight in the dynamics of parthenogenesis-inducing (PI) Wolbachia and to explain the coexistence of infected and uninfected forms in Trichogramma wasps. After first reviewing the literature on PI Wolbachia in chapter 2 , I tried to further our understanding of the coexistence of the two reproductive forms in natural Trichogramma kaykai and T. deion populations by combining fieldwork, molecular techniques, behavioural- and crossing experiments with model studies.Vertical transmission of Wolbachia from mother to daughter has been viewed as the main mode of transmission but in chapter 3 & 4 we show an unexpectedly frequent natural inter- and intraspecific horizontal transmission between and within Trichogrammakaykai and T. deion larvae sharing a common food source, a butterfly egg. Originally uninfected immature wasps could acquire Wolbachia inside the host egg but not all newly infected females exhibit parthenogenesis. In T. kaykai, intraspecific horizontal transfer was followed by complete parthenogenesis in future generations but when T. kaykai females received Wolbachia from T. deion , the infection tended to be lost several generations after interspecific horizontal transfer. Our results largely explain the discordance between Wolbachia - and (Trichogrammatid) host phylogenies. Frequent horizontal transfer might select for high virulence in these bacteria .Because of a nuclear-cytoplasmic conflict between Wolbachia and the nuclear genes of Trichogramma and the previously described horizontal transfer of Wolbachia , the infection is most likely associated with fitness costs in populations where infected and uninfected individuals coexist. In chapter 5 we show that infected T. kaykai suffer a reduced survival compared to uninfected conspecifics when they shared the same host. The survival rate of infected immatures was higher when they competed with other infected immatures from a different infected parent than in competition with uninfected immatures. This shows that PI Wolbachia -infected Trichogramma can suffer a substantial fitness cost. Because of this reduced competitive ability of infected larvae, horizontal transfer that occurs under the same superparasitism circumstances does not contribute much to an increase in the infection rate in the population.Previous work showed that the presence of another sex ratio distorter in males, a B chromosome called PSR (Paternal Sex Ratio) that destroys the paternal chromosomes after fertilization thereby causing an all-male or a male-biased offspring sex ratio, contributes to a low infection frequency in T. kaykai . In chapter 6 we determined if a PSR factor causes low infection frequencies in other species as well. Therefore, we studied natural populations of three Trichogramma species - T. kaykai , T. deion and T. pratti - from the Mojave Desert. Our data showed that all the male-biased and all-male Trichogramma broods collected from the butterfly Apodemia mormo deserti that contained males expressing the PSR phenotype, belonged to T. kaykai. In laboratory tests, 71.4% of the T. kaykaiPSR males horizontally transmitted the PSR phenotype to T. deion . This percentage is comparable to the transmission rate of PSR to T. kaykai females, namely 81.6%. Consequently, the PSR can be transmitted to T. deion and we expect this to happen in the field because T. kaykai and T. deion sometimes emerge from the same butterfly egg. Despite this, we cannot find PSR in T. deion . Modeling shows that low Wolbachia infection frequencies can only be attained when the PSR rates are very high. Therefore, other factors should keep the PI Wolbachia -infection from spreading to fixation in this species, e.g. nuclear suppressor genes.The mating structure in the host population plays a major role in the dynamics of PI Wolbachia and PSR . A PSR factor prevents the Wolbachia infection from spreading to all the females in T. kaykai because uninfected T. kaykai females show a high level of sib (brother-sister) mating. Sib mating is a barrier against the destructive effect of mating with a PSR -carrying male. Infected females do not have this advantage. Using a population genetic model with microsatellites as genetic markers in chapter 7 , we estimated high levels of sib-mating of 70% and an off-patch mating of 15%. Thirty-five percent of the patches were estimated to be parasitized by two T. kaykai females. Incorporating such levels of sib mating in a previously developed model describing the dynamics of PI Wolbachia and PSR in a Trichogramma population, resulted in stable low frequencies of infection, i.e., a coexistence between infected and uninfected individuals, and of the PSR chromosome. Our results show how mating structure allows the two sex ratio distorters to coexist in the population.The main conclusion from this thesis is that, despite the high vertical transmission and regular horizontal transfer of Wolbachia , a PI Wolbachia -infection can be attained at low frequencies in Trichogramma , due to the presence of a non-mendelian suppressor, like the male-biasing PSR factor in T. kaykai, but also due to other factors. In T. deion, for example, PSR does not keep the infection frequency at low levels but a nuclear mendelian suppressor against the PI Wolbachia might have evolved.Next to their significance for the understanding of the evolutionary pathways of Wolbachia -host interactions, the results reported in this thesis may also have important implications for future use of natural enemies, and more specifically Trichogramma wasps, in inundative biological control. We may now have a good method to render wasps parthenogenetic, via super- or multiparasitism by infected and uninfected females, thereby increasing the efficacy of parasitoid releases against lepidopteran pests.