Search

Life history theory explains how an organism’s reproductive success is driven by trade-offs among life-history traits. Organisms can have contrasting life-history strategies. For example, Chinook salmons reproduce once and then they die (semelparity), whereas Atlantic salmons reproduce several times in their lifetime (iteroparity). What is an optimal life-history strategy for a filamentous fungus? Theory predicts that based on the quality and quantity of resources and the presence of competition, a fungus can be semelparous or iteroparous.In the first part of this thesis, adaptive constraints experienced by fifteen natural isolates of A. nidulans during evolution in their natural environment have been tested. No significant correlation between growth rate and asexual spore yield was observed. Instead, there was a clear dependence of the two traits on sugar concentration and nutrient richness. Further, six natural isolates were selected for a short evolution experiment in one environment where the isolates showed most variation in growth and asexual reproduction. A negative correlation between growth rate and spore density was observed among independently evolved replicate populations that approached significance, but not between growth rate and spore yield. All changes occurred in an antagonistic fashion: increases in growth rate were associated with decreases in spore yield/density, and vice versa, indicating short-term adaptive constraints from a growth-reproduction trade-off.In the remainder of the thesis, the scope and mechanisms of adaptation in an Aspergillus Short-term Evolution eXperiment (ASEX) were tested including the role of trade-offs between growth rate and asexual reproduction. Surprisingly, asexual spore yield consistently decreased in all the populations and changes in growth rate and spore yield approached significance. The competitive fitness of the evolved populations relative to their ancestor had not improved either. However, fitness measurements for two populations using more recent predecessors as competitors suggested that non-transitive fitness interactions explained the lack of fitness increases relative to the ancestor. Upon closer inspection, all populations had evolved morphological diversity as early as in week 20.Competition experiments between the two morphotypes ancestor-like (AL) and fluffy-like (FL) in head-to-head competitions at low and high frequencies showed that the two types were engaged in negative frequency-dependent fitness interactions. The two types engaged in resource competition in which FL was a superior competitor for the limiting nutrient (glucose).The genomic basis of adaptation of the ASEX populations, based on genome-sequence analysis of clones from both morphotypes of all populations at week 10 and 52 of the experiment, showed that genes that were mutated in two or more populations, occurred more frequently than expected by chance, indicating that they were under positive selection. Except in one population, in all other tested populations, the two morphs from the final time point derived from unrelated genetic backgrounds, suggesting their frequent extinction and re-emergence.In conclusion, there was little evidence for adaptive constraints from a trade-off between growth and reproduction in A. nidulans. In both laboratory evolution studies, I found a reduction in asexual spore yield and variable changes in mycelial growth rate, questioning their general role during adaptation in this filamentous fungus. Fungi occupy a wide range of niches and are essential players in recycling carbon and nitrogen in the biosphere. It is crucial to understand which components of fitness contribute to adaptation to these different ecological conditions. For this, we need to study the adaptive role of fitness components in fungal isolates from different niches and develop fungal-specific life-history models.

Mutualisms are abundant in nature despite our common understanding that evolution by natural selection is driven by competition. To understand how mutualisms can arise and what maintains their long-term stability, in this thesis I explore mutualisms at three levels of selection in the basidiomycete genus Termitomyces: the domestication of the fungus itself by a termite host, the inheritance and genetic variation of mitochondria in the fungal cell following sexual reproduction, and the transfer of tRNA genes from mitochondria to linear plasmids that results in co-dependency. In addition, I reconstruct the mitochondrial genomes of several Termitomyces and related species to analyze mtDNA variation and evolution in this diverse group of fungi.The mitochondrial genomes of Termitomyces are characterized by a large inverted repeat and a relatively high G4DNA motif content (Chapter 2). The significant enrichment of G4DNA in the repeats compared to the single copy regions suggests there may be reduced selection against G4DNA within the repeat regions, perhaps due to enhanced homologous recombination-driven repair of double-stranded breaks. G4DNA in fungal mtDNA is highly reduced in coding regions compared to human mtDNA, which may be the result of compensatory evolution in vertebrates to resolve G4DNA formation in transcribed RNA.The common ancestor of Termitomyces was likely reliant on insect faeces as a growth substrate prior to its domestication by termites (Chapter 3). A phylogenomic reconstruction of Termitomyces and other Lyophyllaceae reveals that the free-living, insect-associated coprophiles Arthromyces and Blastosporella are close relatives of Termitomyces. Several key traits likely predate the fungus-termite mutualism, including conidiospores, a pseudorhiza, a perforatorium, and a repertoire of carbohydrate-active enzymes. The combination of these traits probably facilitated the transition of Termitomyces’ free-living ancestor to a termite-domesticated lifestyle.The mitochondria of several Termitomyces species seem to be dependent on linear mitochondrial plasmids for their supply of an essential tRNA gene (Chapter 4). Phylogenetic reconstruction of plasmids, mostly derived from Termitomyces species, reveals that transfer of mitochondrial tRNA genes from mtDNA to plasmids occurred independently in several Termitomyces species. In two cases, the tRNA gene was subsequently lost from the mtDNA of Termitomyces, the only known occurrence of complete loss of a tRNA function from Termitomyces mtDNA. This suggests an abrupt emergence of genetic addiction of mtDNA to a plasmid.The mtDNA of Termitomyces shows limited recombination near regions containing mobile homing endonucleases (Chapter 5). Experimental matings of two Termitomyces homokaryons, along with complete mtDNA sequence data for six wild populations, suggest the inheritance of mtDNA proceeds without extensive heteroplasmy or genome-wide recombination. Previous studies noted Termitomyces appears to lack nuclear migration during mating, which would imply heterokaryon formation occurs in cells containing mixed cytoplasm. My results indicate that nuclear migration is either present but limited, or heteroplasmic cells quickly converge to a homoplasmic state. In either case, physical recombination of mtDNA is constrained but self-splicing endonucleases are able to transfer between different parental mitochondria during sex.

In this thesis I focussed on a mutualistic symbiosis; the fungus-growing termites and their fungal symbiont Termitomyces. The origin of this symbiosis between fungus-growing termites (Macrotermitinae) and Basidiomycete fungi of the genus Termitomyces has been estimated at approximately 30 million years ago in the rainforests of central Africa. The symbiosis has a single origin with no known reversals of both partners to a non-symbiotic state. The single origin of fungiculture and the absence of any reversals of the fungus to a free-living state are puzzling as both parties have retained independent reproduction and dispersal, which could potentially de-stabilize the symbiosis by leaving room for the fungus to escape or for the termite to domesticate more than one symbiont. For this thesis I studied the biology of the closest relatives of Termitomyces in order to reconstruct the biology of its ancestor. Furthermore, I studied the phylogeny and biology of the genus Termitomyces in more detail to understand what made the genus so diverse and successful.In order to make inferences on the events leading to domestication of Termitomyces a well-supported phylogeny is needed. In chapter 2 I performed a phylogenomic analysis of 25 species of Termitomyces and 21 related non-domesticated species. To reconstruct the biology of the ancestor of Termitomyces I also studied the biology of the non-domesticated species. The phylogenomic analysis recovered the insect-faecal associated genus Arthromyces as the sister group of Termitomyces. I found that Arthromyces shares a suite of traits with Termitomyces, indicating that their common ancestor also possessed these traits. I hypothesize that this set of traits predisposed the ancestor of Termitomyces towards domestication.One of traits found in many species related to Termitomyces is the production of asexual spores (conidia). I hypothesized that these conidia play a role in local dispersal of the fungus and potentially in faster substrate colonisation. In chapter 3 I conducted a population study on the insect-faecal associated and conidia-producing fungus Blastosporella zonata. I sequenced two highly variable genetic markers of 21 collections of B. zonata mushrooms at three collection sites in cloud forests near Murillo, Colombia. I found signatures of clonality within a collection site but usually not between sites, which indicates that conidia are mainly used for local dispersal. There was genetic diversity as well, particularly between collection sites, indicating an important role for sexual spores (basidiospores) and resulting in unique genotypes of most collections. Unexpectedly. by reconstructing fungal haplotypes and subsequent phylogenetic analysis on these, I found evidence for the existence of two cryptic biological species in B. zonata.In the studies I conducted in chapter 2 I used five unidentified species of Tephrocybe. As the morphological descriptions as well as the DNA sequences of these species did not match any species already known, I chose to describe those species in chapter 4. The taxonomy for these five new species was challenging as morphological traits and biogeography did not align with the molecular phylogeny. Therefore, I discussed several alternative options to keep genera monophyletic. This ultimately led to the erection of four new genera: Australocybe, Nigrocarnea, Phaeotephrocybe and Praearthromyces.The genus Termitomyces contains about 40 described species and harbours a lot of morphological diversity. Probably many species of Termitomyces remain undescribed as there is a strong bias towards the description of species that regularly produce mushrooms. In chapter 5 I assembled a large dataset of over 1500 DNA sequences of Termitomyces and used species delimitation software to sort sequences into hypothetical species based on the barcoding gap within the dataset. Using this approach, I recovered 87 phylogenetic species and for these species I collected specimen metadata such as continent of origin and termite host genus. A phylogenetic reconstruction of the 87 species revealed five main symbiont groups which corresponded with the five largest fungus-growing termite genera. I identified several factors which could be involved in speciation: fruiting mode (underground or aboveground), geographic separation, symbiont transmission mode and suppression of mushroom formation by the host termite. Finally, I demonstrated that mushroom morphology does not correlate with phylogeny and therefore, without additional support of molecular data, is not useful for taxonomy in this genus.Uniparental vertical transmission of symbionts is an important factor in aligning reproductive interests of host and symbiont and thus stabilizing the symbiosis. In fungus-growing termites, horizontal transmission is the predominant transmission mode, but uniparental vertical transmission has evolved via the female reproductives of the genus Microtermes and via the male reproductives of the species M. bellicosus. In chapter 6 I studied populations of Microtermes and their symbionts in South Africa. I found high genetic diversity in symbiont populations and low host-specificity, which indicates that frequent horizontal symbiont exchange occurs as well as sexual reproduction, despite the absence of mushrooms of these species. I therefore argue that Microtermes species regularly acquire symbionts associated with species of other genera that do not suppress mushroom formation, such as species of the genus Ancistrotermes.New species of mushroom-forming fungi are often described based on morphological features of the mushroom. In the genus Termitomyces this has caused a heavy bias towards the description of species, which regularly produce mushrooms, leaving species that rarely or infrequently fruit undescribed. In chapter 7 I describe, for the first time in this genus, a species of Termitomyces with no reported naturally produced mushrooms, based on DNA sequence evidence, biological data and asexual features.In chapter 8, I discuss how investigating and understanding the biology of species closely related to fungal symbionts can assist in reconstructing the biology of the ancestor and thereby determine conditions that facilitated fungal domestication by an insect. Although my research focused on a specific group of fungus-growing insects, the fungus-growing termites, I discuss how my approach can be applied to different unrelated groups of fungus-growing insects as well. Finally I discuss how my work not only offers significant contributions to the knowledge on the origin and subsequent evolution of fungiculture in termites, but also contributed to general fundamental knowledge of mutualistic symbioses in general. &nbsp

Life is organized in a hierarchical fashion; smaller replicating entities cooperate to make more complex organizational forms. From an evolutionary perspective there is a tension between lower-level selection and higher-level organization; natural selection at the lower level can oppose the higher-level organization, if reproductive interests between the two levels are not aligned. In this thesis I explored this tension and the stabilizing mechanisms that align the interests of different organizational levels at different levels of selection in the termite-fungus symbiosis.The symbiosis between fungus-growing termites (Macrotermitinae) and Termitomyces fungi (Basidiomycota) evolved once, approximately 30 million years ago, without subsequent reversals to non-symbiotic states. The symbiosis has often been described as a farming system in which the termite farmers cultivate their domesticated fungus. Over time both the termites and their fungi have become mutually and obligately dependent on each other, even though in most cases the termites and fungi have retained independent reproduction and dispersal. Independent reproduction implies that the reproductive interests of the termites and their symbionts are not completely aligned, leaving room for conflict between the partners. Since the symbiosis has remained stable over evolutionary time, it is likely that there are mechanisms that have stabilized this level of organization.One of the major questions in the termite-fungus symbiosis is how sexual reproduction in the partners is correlated in time. Even though the termites and their fungal symbionts reproduce and disperse independently to establish new colonies, the fungal symbiont typically forms mushrooms a few weeks after the colony has produced reproductive termites. It has been hypothesized that this timing of mushroom formation is due to a trade-off between alate and worker production by the queen of the termite colony. Under the assumption of a maximal rate of termite reproduction, investment in the production of alates leads to a reduction in the production of workers. Because workers consume the fungus, reduced numbers of workers will allow mushrooms to ‘escape’ from the host colony. In chapter 2 we tested a specific version of this hypothesis, viz. that the typical asexual structures found in all species of Termitomyces – nodules – are immature stages of mushrooms that are normally harvested in a primordial stage, except when there are too few workers. We refuted this version of the hypothesis by showing that nodules and mushrooms are completely different structures from the earliest developmental stages that we could sample.Due to the independent reproduction and dispersal of the termites and their fungi, the interaction between host and symbiont needs to be re-established at the start of each termite colony. It is known that there is a certain interaction specificity between termites and Termitomyces fungi, but it is unknown what factors contribute to the observed combinations of termite and fungus. It has been hypothesized that substrate provisioning by termite farmers could explain the observed interaction specificity. In chapter 3 we explored whether differences in nutrient requirement between fungi from different termite species can be found.In the termite-fungus symbiosis, horizontal symbiont transmission is also associated with sexual reproduction of the fungus. The dispersing fungal spores are sexual spores produced in the mushrooms. It has been shown that for inhabitant symbionts, like Termitomyces, those that undergo little genetic change should be selected as they live in a stable biotic environment to which they have become adapted. Following from this observation, there should have been selection for a low recombination rate in Termitomyces fungi. In chapter 4 we constructed a new, more contiguous reference assembly of the Termitomyces symbiont of M. natalensis that allows for the study of recombinational landscapes. Also, we isolated a full-sibling mapping population of this Termitomyces species and used these to create the first linkage map of a Termitomyces fungus using a Genotyping-by-Sequencing approach. Finally, we performed an initial study into the recombination landscape of this Termitomyces species and showed that its recombination rate varies substantially across the genome. To be able to answer whether Termitomyces fungi indeed have evolved a low recombination rate, the recombination landscapes of more Termitomyces species as well as those of its close, free-living relatives should be studied.In chapter 5 we zoomed in on the basidiomycete life cycle and explored how the peculiarities of basidiomycete life cycle open possibilities for lower-level selection that conflicts with the higher-level organization (the fungal mycelium). The first difference between basidiomycetes and the vast majority of sexual life cycles is that after gamete fusion, the nuclei remain separate for almost the whole life cycle. The second difference is that the nuclei of two fusing gametes can move through the whole body – the whole mycelium - of their mating partner. We show that by remaining separate, the fates of these two separate nuclei are not fully aligned, which means that selection can act on the individual nuclei at the cost of the dikaryon. Also, we show that these life cycle peculiarities could enhance the conflict of interest between nuclei and mitochondria, possibly leading to reduced fitness of the dikaryon.Although the stability of the termite-fungus symbiosis has attracted the interest of many evolutionary biologists, the interest in the termite-fungus symbiosis is not for fundamental questions only. All mushrooms of Termitomyces fungi are edible and considered delicacies in the areas where they are found. The work described in this thesis concerning Termitomyces will also aid the search for Termitomyces mushroom cultivation methods. The work in chapter 2 brings us closer to find the factors that promote mushroom formation. The work in chapter 3 will aid the optimization of Termitomyces growth substrate. Finally, the work in chapter 4 could in the future help for breeding and analysing desirable traits for the cultivation of Termitomyces mushrooms, so that in future we will be able to re-domesticate the fungus that was domesticated by termites 30 million years ago.

Biological control is the use of an organism, the biological control agent (BCA), to control the population of another organism, the pest. While BCAs are used around the world and in a variety of agricultural contexts, increasing the uptake would be beneficial to key goals of sustainable agriculture. To do so, there is the desire for improving BCAs, either in terms of their production, their performance, or the reduction of non-target effects after release. One way to improve BCAs is to use a genetics approach with next-generation sequencing and genomics. The study of genetics is essentially about evolution and inheritance, and its application on BCAs is straightforward: are the traits that we are interested in, such as parasitism rate for parasitoid wasps or starvation resistance for predatory bugs, 1) heritable, and 2) able to be improved without deleterious (side-)effects? Is there enough genetic variation within a population to select for improvement of these traits? While these scenarios can be used for improving the BCAs themselves, the way that BCAs are monitored, stored, and assessed for non-target effects can also be improved using genetics and/or genomics. This thesis and the work within involved generating genomic results and resources for five biological control agents (including a predatory mite, two parasitoid wasps, and a predatory mirid bug), complete with context and suggestions for future directions. This thesis is also intended to act as a sort of guidebook for biological control practitioners considering genome projects. The introduction (Chapter 1) begins with defining an anthology, and the intention to treat this thesis as such. After introducing concepts of biological control, genetics and genomics, and the international project behind this thesis (BINGO-ITN), the research chapters begin. Chapter 2 contains a systematic review of biological control literature centered on documenting the amount of genetic variability research on BCAs, ending with a call for more rigorous reporting of variables such as heritability and evolvability. Chapter 3 contains a previously published work on whole genome sequencing via nanopore MinION technology and resulting microsatellite generation and population genetics of the predatory mite, Amblyseius swirskii. Chapter 4 and Chapter 5 are both in the style of genome reports, reduced-length manuscripts that, while destined for peer-review, are more straight to the point of describing genomes and any resulting analyses. Chapter 4 is the linked-read de novo annotated genome of parasitoid wasp Bracon brevicornis and resulting analyses into a putative region related to complementary sex determination, a defining feature of this parasitoid. Chapter 5 contains the hybrid de novo annotated genome of parasitoid wasp Trichogramma brassicae, where long- and short-read technology were used on a strain that was aided in its homozygosity by a Wolbachia infection. Chapter 6 features population studies and builds upon the reads generated in Chapter 5 by mining microsatellites from the short-reads to be used upon wild-caught lines of sister species Trichogramma evanescens. These German-origin lines were compared in a variety of ways using population genetics (the aforementioned microsatellites) as well as population genomics via pooled sequencing analyses and a de novo (unannotated) genome. Chapter 7, the final research chapter, contains the linked-read de novo annotated genome of predatory mirid Nesidiocoris tenuis, an important BCA used throughout the Mediterranean that is also considered a pest in other areas of the world. In addition to the genome, additional analyses include bacterial decontamination (leading to putative symbiont identification), potential lateral gene transfer events (though it is difficult to qualify initially), population genomics, and cytogenetic investigations into karyotype, sex determination system, unique satellites, and the presence/absence of the insect ancestral telomeric motif. This variety of investigations offer a taste of what is possible with a genome, acting as inspiration for future research. The thesis/anthology is wrapped up in Chapter 8, the synthesis, where the preceding research chapters are examined in the light of themes presented in the introduction, and more general notes over the success (or failure?) of these various projects. Appendices contain four summaries (English, Dutch, German, and Spanish) as well as Acknowledgements, About the Author, and About the Artwork found within the thesis (each chapter title has a unique accompanying illustration).

Insects have gained increasing interests in diverse agricultural applications, ranging from the use of insect-derived protein in animal feed to the use of insects as natural enemies of agricultural pests. Using the knowledge of genetics is a powerful way to improve the application of insects in agriculture. The exploitation of genetic variation in insects has been discussed for long time, however, the nature of genetic variation is still largely unexplored for insects outside of well-studies species. The main aim of this thesis was to explore the potential of genetic improvement of insects. To this end, I investigated the genetic basis of wing morphology and associated phenotypic traits in the parasitoid wasp Nasonia vitripennis as a case study. First, I used a modified Animal Model with a pedigree relationship matrix, and the Genomic Restricted Maximum Likelihood method to quantify the amount of genetic variation and variation due to host effects for these traits. Host effects occur because Nasonia species are gregarious wasps, which lay more than one egg into a single host, so that the offspring share a common developmental environment. The results have shown substantial host effects on wing morphology traits, especially for size traits: hosts explained up to 64% of the total phenotypic variation. Significant additive genetic variation were also observed for all traits. Second, I conducted genome-wide association study (GWAS) to further reveal the genetic basis of these traits. Several genomic regions that significantly associated with wing morphology variation were identified. Third, I implemented genomic prediction for wing morphology traits in N. vitripennis to explore its potential in insects. The results have showed promising accuracies based on a cross-validation strategy, which suggests that genomic prediction is feasible in insects. Furthermore, I investigated the relationship between the level of inbreeding and inbreeding depression for wing morphology traits using sex-biased gene expression. I found no evidence for a relationship between inbreeding and sex-biased gene expression, and I did not observe any inbreeding depression for wing morphology traits. Finally, I discussed the potential of improvement of insect parasitoids by selecting better hosts and the implications of the exploration of genetic variation for potential applications in insects selective breeding. Together, this thesis provides important insights into the genetic architecture of wing morphology in the parasitoid wasp Nasonia vitripennis. The findings contribute to the knowledge of the genetic basis of wing morphology traits in insects, and also show the promising potential of using genetic tools to improve insects.

The widespread societal, development and health problem of undernutrition in many developing countries motivated my research described in this thesis. Despite past interventions and economic developments, many developing countries still face high levels of undernutrition; especially stunting (linear growth failure), deficiencies in zinc, vitamins A and B12, and folate, mainly in children under the age of five years. Some of the current and proposed interventions to improve nutritional status include dietary diversification, sugar fortification with vitamin A, high dose vitamin A capsule distribution to under five children twice every year, maize meal fortification, and others. Dietary diversification and improvement in the food preparation methods such as fermentation are potential means with potential to improve availability of nutrients especially for vulnerable populations. For this thesis, the focus is on Zambia, which is one of the most undernourished countries in the world, with 48% of the population considered undernourished. As Zambia is dependent on mainly maize as a staple food with lower consumption of animal food sources, diversification of diets presents an opportunity to combat undernutrition in this population. Within this, the traditional use of fermented foods presents an opportunity for improvement in nutrient intake in the population especially of children below the age of five years. My thesis explores this potential. My research has three central aims. (1) Elucidating the nutritional potential of the traditional fermented foods Mabisi and Munkoyo to complement the diet of vulnerable groups such as children under five years old (Chapter 2). (2) Determining the nutritional aspects and microbial composition of the target traditional fermented beverages Mabisi and Munkoyo and whether microbes influence the level of nutrients in the two products (Chapter 3). (3) Understanding the influence of the favourable bacteria in the products in shaping human gut microbiota towards more favourable composition and its impact on nutritional status (Chapters 4 and 5). In Chapter 2 I describe the potential contribution of Mabisi and Munkoyo to nutrient intake in children under the age of five years using Optifood, a linear programming software that was developed by WHO and partners. Secondary dietary intake data collected using 24-hour recall method was modeled to develop food based recommendations (FBRs), for children aged 1-3 and 4-5 years in Mkushi district of Zambia. Three scenarios per age group were modeled to determine weekly food-based recommendations based on: (1) food based recommendations with the local available foods, (2) food based recommendations with inclusion of Mabisi, a fermented milk beverage, and (3) food based recommendations with inclusion of Munkoyo, a cereal fermented beverage. The scenarios were compared to assess whether food based recommendations with the addition of Mabisi and/or Munkoyo achieved better nutrient intake. FBRs based on only locally available non-fermented foods did not meet ≥70% of recommended nutrient intake (RNI) for calcium, fat, iron and zinc. The addition of Munkoyo to the FBRs did not reduce the number of problem nutrients, but after adding Mabisi to the FBR’s only iron (67% of RNI) remained a problem nutrient in the 1-3 year age group and only zinc (67% of RNI) remained a problem nutrient in the 4-5 year age group. Mabisi, a fermented milk product in combination with the local food pattern is a good additional source of nutrients for these age groups. However, additional nutrition sensitive and cost-effective measures would still be needed to improve nutrient intake, especially that of iron and zinc. Chapter 3 describes the results obtained from laboratory analysis of Mabisi and Munkoyo samples collected during a cross sectional study in Mkushi to determine their nutritional composition and microbial communities. It was hypothesized that Mabisi and Munkoyo each contain distinct microbial communities and that nutritional composition depends on microbial composition. With respect to the variation in microbial community structure, I therefore addressed whether the use of different raw materials can explain the variation in the structure of microbial communities, and if variation in nutritional composition can be explained by differences in microbial community structure. Here, we characterized the nutritional composition and microbial community composition of Mabisi and Munkoyo. We found that the two products are different with respect to the nutritional and the microbial composition. Mabisi was found to have higher crude protein, fat, and carbohydrates than Munkoyo. The microbial community composition was also different for the two products, while both are dominated by lactic acid bacteria. Our analyses showed that variation in nutritional composition, defined as how much consumption would contribute to estimated average requirement (EAR), might be explained by variation in microbial community composition. Consumption of Mabisi appeared to contribute more to the estimated average requirement (% of EAR) and its inclusion in food based recommendations is warranted. We further found evidence that through fermentation of the raw materials (raw milk for Mabisi and cereal for Munkoyo), the levels of B-vitamins can increase. Levels of increase likely depend on the exact composition of the microbial community used for fermentation. Our results show the potential of Mabisi to add value to current diets and suggests that variations in microbial composition between specific product samples can result in variations in nutritional composition. The in vitro experiment in Chapter 4 of this thesis assesses the impact of Mabisi and Munkoyo on the gut microbiota focusing on the potential changes in metabolite profiles of gut microbiota taken from stool samples upon exposure to Mabisi and Munkoyo. The shifts in metabolite profiles were correlated to changes in abundance of a key indicator bacterium (Lactobacillus) for healthy gut microbiota composition. We exposed stool samples to these products and various controls and measured concentrations of short chain fatty acids (SCFAs) and used these as an indicator of beneficial bacteria activity and measured shifts in levels of the indicator beneficial bacterium Lactobacillus. Results show that exposure of the gut microbiota from stool to Mabisi and Munkoyo results in higher levels of SCFAs and also higher levels of Lactobacillus. These results support the idea that consumption of fermented foods can result in healthier metabolism of the gut microbiota as measured with SCFA concentrations. These results can inform further more complex in-vitro as well as in vivo studies on the effects of the traditional fermented foods on gut microbiota metabolism and composition. Chapter 5 describes a survey that was undertaken in Namwala and Mkushi to determine the effect of consuming traditional fermented foods on gut microbiota composition and nutritional status in children 6-24 months old in rural Zambia. Data on food consumption, morbidity and socio-demographic together with stool samples were collected from children aged 6-24 months residing in Namwala and Mkushi in Zambia. The stool samples were analysed for the composition of gut microbiota and for concentrations of short chain fatty acids (SCFAs) as a proxy for microbial metabolism. This data was then linked to data on intake of traditional fermented foods using multivariate analysis of variance. Gut microbiota of children who are fermented beverages consumers was associated with higher relative abundance of Bacteroides and Lactobacillus than the non-consumers. Higher levels of these bacterial groups have been associated with benefits to the host. There was no difference for the SCFA concentrations between the fermented foods consumers and non-consumers, which may be because other parts of the diets that we did not control for also contribute to SCFA production. The results imply that consumption of the two traditional fermented foods used in this study promotes a healthier gut microbiota composition in the children in Zambia. Our results warrant future more formal tests such as controlled human feeding trials to further validate our findings. In the framework of the work in this Thesis it is evident the two products are good sources of nutrients and have potential probiotic effects to confer better health and nutritional status to its consumers. The goal of contributing to improvement of people’s lives through dietary intake improvements using traditional fermented foods could be achieved as shown in this thesis. The work highlights the need to explore locally available and culturally accepted foods, more so the fermented foods such as Mabisi and Munkoyo in the fight against undernutrition. However further work to generate more evidence for the formalization of our products Mabisi and Munkoyo with key messages on benefits formulated for dissemination to the current and potential consumers.

The precise manipulation of plant meiosis at will is one of the most valuable tools for plant breeding, as it allows to ultimately control genetic diversity generated in a population. In this thesis we have used a transient silencing approach, virus-induced gene silencing (VIGS) to modify meiosis in the model organism Arabidopsis thaliana. We have optimized this technique to virtually target any gene in meiosis to induce diverse meiotic phenotypes and to generate offspring with a desired genetic make-up. Using VIGS, we were able to both, downregulate and upregulate meiotic recombination and modify the number of meiotic divisions to induce diploid gamete production. Furthermore, we have shown that VIGS can be used to develop breeding applications such as efficient reverse breeding and controlled generation of polyploids. The results obtained in this thesis illustrate that the use of VIGS can significantly speed up the production of valuable lines for breeding, compared to other techniques available used to modify gene expression or gene structure.

Traditional fermented products are an important part of the diet in many African countries. These products can provide much of the needed proteins, vitamins and other micro-nutrients to its consumers, which include young children. Also, traditional fermented products are rooted in the local context and have high social and cultural value. Increasing our understanding of these spontaneously fermented products can be used to increase the value chain of these products in order to increase economic stability of producers as well as nutritional intake in rural and urban areas. Most traditional fermented products are produced by spontaneous fermentation, meaning that there is no starter culture added to initiate fermentation. As a consequence these products contain a diverse microbial community, which are responsible for the characteristics of the product. Often a back-slopping method is used, where a portion of previously fermented product inoculates fresh raw material. Unknowingly, producers of these products have domesticated these bacterial communities and allowed the species to co-evolve within their community. In this way, these bacterial communities form a model system for ecological and evolutionary research. Historically, experimental evolution was mainly performed with single organisms. In recent years the interest in how individuals evolve in the context of ecosystems is growing. This eco-evolutionary research needs model systems that can represent natural communities in their interactions and complexities, while still being a tangible model system. In this thesis I investigated the microbial communities that are responsible for the fermentation and used the constituting bacteria to learn about bacterial community dynamics over time. This was done by combining three disciplines: 1) Food Microbiology, on the conversion of compounds by bacteria during spontaneous fermentation; 2) Evolution, on the changes in the fermenting community over time under selection pressure; 3) Ecology, on the roles and niches bacteria take within the fermenting community. This thesis had two aims. The first aim was to provide the first step towards the use of microbial communities of spontaneously fermented milk, such as Mabisi and Lait caillé, as model systems for studies on eco-evolutionary dynamics. For this, I performed a series of experiments with an increasing level of control and a decreasing level of complexity. These experiments started with producing Mabisi in traditional ways in the field to allow a study of bacterial dynamics, which resulted in a low level of control of variables. Later, I brought the microbial communities to the laboratory and used controlled environments without changing the diversity of the natural communities. The second aim was to provide ecological and evolutionary insights in Mabisi fermentations in the context of research for development. Here, the focus was on the practical application of bacterial dynamics for dairy fermentation. The potential use of the outcome for producers and consumers of spontaneously fermented foods by taking into account when constructing and interpreting the studies. This thesis starts with a perspective on the use of microbial communities of spontaneously fermented products as model system for eco-evolutionary dynamics (Chapter 2). We outline what model systems are used for experimental testing of evolutionary theory so far, ranging from simple microbial communities in the laboratory and, more recently, to complex (natural) communities. Microbial communities from spontaneously fermented products bear several intrinsic advantages for executing evolution experiments: short generation times, small size and ability to be stored frozen and defrosted to perform competition experiments (fitness tests) between evolved and ancestral lines. Moreover, these natural microbial communities have a limited number of players and form an island of microorganisms that does not have a lot of influx from outside the confined system boundaries. There are several research questions with an evolutionary background that can be addressed using these microbial communities from fermented foods. This includes questions on changes species frequency in space and time, the diversity-stability relationship, niche space, fluctuating environment and community coalescence. Hypotheses on the influence of these factors on community evolution are described as well as a short descriptions of the experimental approach of such studies when microbial communities of spontaneously fermented foods are used. Natural communities of Mabisi were analysed in a field experiment. A method was developed using filter paper disks for the preservation of DNA from diverse microbial communities for later analyses (Chapter 3). The bacterial species composition obtained through DNA extraction via the filter paper method showed sufficient resemblance to the composition obtained via traditional DNA extraction from the liquid milk sample. This method could therefore successfully be used to analyse diverse microbial communities from Mabisi in our remote field sites in rural Zambia. Field experiments were conducted to determine the effect of variations in fermentation vessels and types of back-slopping procedures on acidification and bacterial community composition during fermentation (Chapter 4). Due to high costs and a reduced availability of the traditionally used calabashes, nowadays more and more plastic containers are used for Mabisi production. However, the effect of this change in production practice on the properties of the product is unknown. Together with the local community, I have performed 15 fermentations, using two types of fermentation vessels (calabashes and plastic buckets) and three levels of back-slopping (active back-slopping, passive back-slopping and no back-slopping). In passive back-slopping, the bacteria that start the fermentation are transferred from the previous fermentation round via the fermentation vessel. During active back-slopping, finished product is transferred to raw material to inoculate the fermentation. Overall, bacterial communities decreased in diversity over time, where the drop in pH correlates with a decreased diversity, although the rate of acidification showed variation. In case of active back-slopping, the pH drop started right after inoculation. In the ‘no back-slopping’ and ‘passive back-slopping’ fermentations, there was a clear lag phase before acidification started. No difference was found in bacterial diversity during and at the end of fermentation performed in plastic buckets or previously used calabashes. Besides small differences, all processing methods resulted in a microbial community dominated by Lactococcus lactis. After the natural communities of traditional spontaneous fermentation were analysed, the next step was to bring the natural communities into the laboratory. Here, experiments were performed with lower complexity and an increased control. The species composition and metabolic profile of six different Mabisi samples were analysed before and after repeated propagation cycles (Chapter 5). These communities had similarity in their bacterial species composition and therefore had the potential to converge towards the same final species composition upon repeated propagation in a common environment. Species composition in all replicates propagated from the same original samples changed in a parallel way, yet that groups of communities derived from different original samples did not change in the same way. We observed that communities at the end of the repeated propagation cycles were either dominated by Lactobacillus helveticus or Lactobacillus delbrueckii. By modelling species compositions we tested the influence of four main factors on the species composition: initial species composition, selection imposed by the environment, selection caused by interaction between species and random processes in species dynamics. We found that the final species composition is mostly dependent on initial species composition, followed by random processes. The environment showed to have the least influence on the change in species composition. We had the opportunity to work with a second spontaneously fermented dairy product, called Lait caillé. This traditional product originates from Senegal and is produced in wooden bowls, called lahals (Chapter 6). In terms of complexity and control, this experiment would position in between the field and laboratory experiments with Mabisi. The mode of propagation was traditional, meaning that the bacteria were transferred via the inside of the lahals that were used for repeated fermentations (passive back-slopping). This traditional production method allowed us to study the natural bacterial communities in the lahals and translate our findings towards production practices in Senegal. We analysed the species composition of Lait caillé over time and added a probiotic starter containing Lactobacillus rhamnosus yoba 2012 to the traditional process attempting to enrich the bacterial species community in the final product. We found detectable levels of L. rhamnosus in the final products, which were dominated by Lactobacillus helveticus and Acetobacter species. The abundance of L. rhamnosus ranged between 0.2 and 1 percent of the total bacterial population, which is comparable to the concentration found in probiotic yoghurt. Subsequent rounds of fermentation using passive back-slopping without the addition of new L. rhamnosus led to a loss of this strain from the community of fermenting bacteria. Just as Mabisi it is an important part of the daily diet of men, women and children in rural and urban areas in Senegal. The addition of a health-promoting, probiotic bacterium to these products ensures the delivery of that probiotic activity to consumers. The addition of probiotic strains at every fermentation cycle can enrich the existing complex communities of traditionally fermented Lait caillé while traditional bacterial strains remain dominant in the bacterial communities. In conclusion, the bacterial communities found in the spontaneously fermented products show high diversity independent of production method. Our results show that even in a simple propagation environment and when confronted with new bacteria entering, these communities are able to stay diverse. The next step towards understanding these natural communities would be to construct simple communities based on combinations of individual species, while aiming to generate communities with similar characteristics, for example in terms of stability and aroma formation. The diversity we observed in the bacterial communities leads to functional stability as well as high levels product safety compared to fermented products with a lower species diversity. We found no differences in species diversity between Mabisi resulting from plastic buckets compared to calabashes, suggesting that other fermentation vessels are suitable to replace calabashes. Further, we found that the addition of a probiotic starter can enhance the nutritional value of the fermented product without losing the original fermenting community. These results will be used for improvements in production of traditionally fermented milk to ensure widespread availability of these products thereby improving public health.

Dietary restriction (DR), a moderate reduction in nutrient intake, improves health or extends lifespan across many species. Moreover, recent insights have shown that also the effects of specific nutrients are of importance for the beneficial effects of DR rather than intake alone. However, we still lack much insight through what mechanisms the lifespan increase through diet changes is exactly mediated. To further increase our understanding of the genetic mechanisms of nutrient-dependent lifespan, in Chapter 2, 3, 4, and 5 I employed different methods of genetic interventions (i.e. a genetic knockout, natural genetic variation and experimental evolution) using the model species Drosophila melanogaster and Podospora anserina. To test whether the genetic interventions affected the diet response, a broad range of diets was applied, thereby taking the recent insights of nutritional geometry into account. Furthermore, the response of the fly’s whole-genome transcription to different dietary treatments were assessed in Chapter 6 and 7 to identify and potentially disentangle genetic mechanisms for lifespan from those for reproduction. Chapter 2 addressed the effects of a triple knockout in the insulin-IGF signalling (IIS) pathway, namely for three genes encoding insulin-like peptides in Drosophila (dilp2-3,5). The mutant showed a strong elevation of lifespan that was irrespective of food type, but also a strong reduction of the female fly fecundity. In addition, this assay also revealed that the same knockout can yield different interpretations for its function in the fly’s diet response, which was strongly dependent per diet dimension under consideration (i.e. varying yeast, sugar, or its ratio in the diet). This observation set the stage for other experimental chapters in this thesis, where a broad range of diets was applied to depict the exact genotypic effects that are involved in the lifespan response to diet. For example, in Chapter 2, interactive effects were observed between dilp2-3,5 knockout and the lifespan response to dietary sugar, but however, not for the yeast component of the diet. In Chapter 3, for the same experimental diets, gene expression responses in dilp2-3,5 knockout flies were measured to describe the general dynamics on the pathway level. Interestingly, expression of the remaining fly head-expressed dilp, dilp6, was elevated on higher yeast levels upon dilp2-3,5 knockout. Therefore, compensatory mechanisms within IIS might still partly mediate the lifespan response to yeast. In Chapter 4 the natural genetic variation for the response to DR was explored in wild-derived strains of the fungus Podospora anserina. By applying a broad range of glucose concentrations in a synthetic medium, we constructed reaction norms for 62 natural occurring strains and showed considerable natural variation in the shape of the reaction norms, including the glucose concentration at which lifespan increased and how steeply the fungus’ lifespan responds to diet (the slope S). Furthermore, I identified a significant correlation between a strain’s general lifespan and both parameters, suggesting that the lifespan response to diet partly acts through a mechanism involved in the fungus’ lifespan determination under high nutrient, growth and reproduction permissive, conditions. On moderate glucose restriction levels we showed that a reduced reproduction was not always associated with lifespan extension, which indicates that decoupling of these traits (that often trade-off) can be achieved. An evolutionary perspective on diet response and the connection between reproduction and lifespan, two often interconnected traits in lifespan research, was provided in Chapter 5. Here, experimental evolution (EE) was performed in Drosophila melanogaster to test whether improved reproductive capacity (i.e. local adaptation) to three nutritionally distinct diets directly affected the lifespan response. Adaptation to the distinct nutritional conditions, had no consistent effect on the lifespan response to diet. Other life-history traits that I assessed could more consistently be associated with the evolutionary nutritional treatments, which together suggested that the adaptive genetic mechanisms increasing the fly’s reproduction were not necessarily interconnected singly with a change of lifespan, but rather with a change in the whole life-history strategy. By exploring the fly’s whole-genome transcription response in a continuously changing environment, Chapter 6 continued on the evolutionary relevance of lifespan responses to diet. This type of fluctuations may better reflect the fly’s natural ecological setting than the continuous diets typically applied in whole-genome transcription laboratory studies. This revealed that flies were able to respond quickly to diet fluctuations throughout lifespan by drastically changing their transcription pattern and, moreover, my results indicated that a large part of the whole-genome transcription response could be attributed to the female fly’s reproduction. Because I measured the response of multiple life-history traits to the fluctuating diet changes, I was able to decouple groups of genes associated with lifespan from those associated with reproduction. This is an important step in the direction of unravelling the genetic architecture that specifically mediates the lifespan response to diet, which can be especially useful in whole-genome transcription studies. In Chapter 7, the consistencies between studies for their whole-genome transcription responses upon DR were investigated. This revealed large transcriptomic variations on different regulatory levels, i.e. the level of whole-genome transcription, most significant genes, and also gene ontology. To test whether the observed inconsistent whole-genome transcription responses were primarily a reflection of the fly’s reproduction, such as observed in Chapter 6, a new cohort of flies was subjected to different regimes that resulted in very different age-dependent reproduction patterns. By assessing whole-genome transcription in this cohort at two time points, the gene expression changes reflected the age-dependent reproduction patterns observed, rather than the lifespan phenotypes. Similar to Chapter 6, this again highlighted the importance of measuring multiple life-history traits for associating whole-genome transcription responses to lifespan effects of dietary restriction. In Chapter 8 the acquired insights across the experimental chapters were synthesized, discussing the importance of assessing a broad range of nutrients for the interpretation of any genotypic effect, and in addition discussing the value of measuring multiple life-history traits for genetic associations. In this chapter I also suggested directions for future research in Drosophila and Podospora that may be valuable for further unravelling and understanding the mechanisms of diet responses in other organisms, including in humans.