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Anthropogenic activities are causing air pollution. Amongst air pollutants, tropospheric ozone is a major threat to human health and ecosystem functioning. In this dissertation, I present three studies that aimed at increasing our knowledge on how plant exposure to ozone affects its reproduction and its interactions with insect herbivores and pollinators.
For this purpose, a new fumigation system was built and placed in a greenhouse. The annual plant Sinapis arvensis (wild mustard) was used as the model plant.
Plants were exposed to either 0 ppb (control) or 120 ppb of ozone, for variable amounts of time and at different points of their life cycle. After fumigation, plants were exposed to herbivores or pollinators in the greenhouse, or to both groups of insects in the field.
My research shows that ozone affected reproductive performance differently, depending on the timing of exposure: plants exposed at earlier ages had their reproductive fitness increased, while plants exposed later in their life cycle showed a tendency for reduced reproductive fitness. Plant phenology was a key factor influencing reproductive fitness: ozone accelerated flowering and increased the number of flowers produced by plants exposed at early ages, while plants exposed to ozone at later ages tended to have fewer flowers. On the other hand, the ozone-mediated changes in plant-insect interactions had little impact on plant reproductive success.
The strongest effect of ozone on plant-pollinator interactions was the change in the number of flower visits received per plant, which was strongly linked to the number of open flowers. This means that, as a rule, exposure of plants to ozone early in the life cycle resulted in a higher number of pollinator visits, while exposure later in the life cycle resulted in fewer flower visits by potential pollinators. An exception was observed: the higher number of visits performed by large syrphid flies to young ozone-exposed plants than to the respective control plants went beyond the increase in the number of open flowers in those plants. Also, honeybees spent more time per flower in plants exposed to ozone than on control plants, while other pollinators spent similar amounts of time in control and ozone-exposed plants. This guild-dependent preference for ozone-exposed plants may be due to species-specific preferences related to changes in the quality and quantity of floral rewards.
In the field, ozone-exposed plants showed only a tendency for increased colonization by sucking herbivores and slightly more damage by chewing herbivores than control plants. On the other hand, in the greenhouse experiment, Pieris brassicae butterflies preferred control plants over ozone-exposed plants as oviposition sites. Eggs laid on ozone-exposed plants took longer to hatch, but the chances of survival were higher. Caterpillars performed better in control plants than in ozone-exposed plants, particularly when the temperature was high.
Most of the described effects were dependent on the duration and timing of the ozone exposure and the observed temperature, with the strongest effects being observed for longer exposures and higher temperatures. Furthermore, the timing of exposure altered the direction of the effects.
The expected climate change provides ideal conditions for further increases in tropospheric ozone concentrations, therefore for stronger effects on plants and plant-insect interactions. Acceleration of flowering caused by plant exposure to ozone may put plant-pollinator interactions at risk by promoting desynchronization between plant and pollinator activities. Reduced performance of caterpillars feeding on ozone-exposed plants may weaken herbivore populations. On the other hand, the increased plant reproduction that results from exposing young plants to ozone may be a source of good news in the field of horticulture, when similar results would be achieved in high-value crops. However, plant response to ozone is highly species-specific. In fact, Sinapis arvensis is considered a weed and the advantage conferred by ozone exposure may increase its competitiveness, with negative consequences for crops or plant communities in general. Overall, plant exposure to ozone might constitute a threat for the balance of natural and agro-ecosystems.
In 2020, cancer was the leading cause of death worldwide, accounting for nearly 10 million deaths. Lung cancer was the most common cancer, with 2.21 million cases per year in both sexes. This non-homogeneous disease is further subdivided into small cell lung cancer (SCLC, 15%) and non-small cell lung cancer (NSCLC, 85%). By 2023, the American Cancer Society estimates that NSCLC will account for 13% of all new cancer cases and 21% of all estimated cancer deaths. In recent years, the treatment of patients with NSCLC has improved with the development of new therapeutic interventions and the advent of targeted and personalised therapies. However, these advances have only marginally improved the five-year survival rate, which remains alarmingly low for patients with NSCLC. This observation highlights the importance of having more appropriate experimental and preclinical models to recapitulate, identify and test novel susceptibilities in NSCLC. In recent years, the Trp53fl/fl KRaslsl-G12D/wt mouse model developed by Tuveson, Jacks and Berns has been the main in vivo model used to study NSCLC. This model mimics ADC and SCC to a certain extent. However, it is limited in its ability to reflect the genetic complexity of NSCLC. In this work, we use CRISPR/Cas9 genome editing with targeted mutagenesis and gene deletions to recapitulate the conditional model. By comparing the Trp53fl/fl KRaslsl- G12D/wt with the CRISPR-mediated Trp53mut KRasG12D, we demonstrated that both showed no differences in histopathological features, morphology, and marker expression. Furthermore, next-generation sequencing revealed a very high similarity in their transcriptional profile. Adeno-associated virus-mediated tumour induction and the modular design of the viral vector allow us to introduce additional mutations in a timely manner. CRISPR-mediated mutation of commonly mutated tumour suppressors in NSCLC reliably recapitulated the phenotypes described in patients in the animal model. Lastly, the dual viral approach could induce the formation of lung tumours not only in constitutive Cas9 expressing animals, but also in wildtype animals. Thus, the implementation of CRISPR genome editing can rapidly advance the repertoire of in vivo models for NSCLC research. Furthermore, it can reduce the necessity of extensive breeding.
Cellular growth and proliferation are among the most important processes for cells and
organisms. One of the major determinants of these processes is the amount of proteins
and consequently also the amount of ribosomes. Their synthesis involves several hundred
proteins and four different ribosomal RNA species, is highly coordinated and very
energy-demanding. However, the molecular mechanims of transcriptional regulation of
the protein-coding genes involved, is only poorly understood in mammals.
In this thesis, unbiased genome-wide knockout reporter screens were performed, aiming
to identify previously unknown transcriptional regulators of ribosome biogenesis
factors (RiBis), which are important for the assembly and maturation of ribosomes,
and ribosomal proteins (RPs), which are ribosomal components themself. With that
approach and follow-up (validation) experiments, ALDOA and RBM8A among others,
could be identified as regulators of ribosome biogenesis.
Depletion of the glycolytic enzyme ALDOA led to a downregulation of RiBi- and RPpromoter
driven reporters on protein and transcript level, as well as to a downregulation
of ribosome biogenesis gene transcripts and of mRNAs of other genes important for
proliferation.
Reducing the amount of the exon junction complex protein RBM8A, led to a more prominent
downregulation of one of the fluorescent reporters, but this regulation was independent
of the promoter driving the expression of the reporter. However, acute protein
depletion experiments in combination with nascent RNA sequencing (4sU-Seq)
revealed, that mainly cytosolic ribosomal proteins (CRPs) were downregulated upon
acute RBM8A withdrawal. ChIP experiments showed RBM8A binding to promoters of
RP genes, but also to other chromatin regions. Total POL II or elongating and initiating
POL II levels were not altered upon acute RBM8A depletion.
These data provide a starting point for further research on the mechanisms of transcriptional
regulation of RP and RiBi genes in mammals.
Bees have had an intimate relationship with humans for millennia, as pollinators of fruit, vegetable and other crops and suppliers of honey, wax and other products. This relationship has led to an extensive understanding of their ecology and behavior. One of the most comprehensively understood species is the Western honeybee, Apis mellifera. Our understanding of sex-specific investment in other bees, however, has remained superficial. Signals and cues employed in bee foraging and mating behavior are reasonably well understood in only a handful of species and functional adaptations are described in some species. I explored the variety of sensory adaptations in three model systems within the bees. Females share a similar ecology and similar functional morphologies are to be expected. Males, engage mainly in mating behavior. A variety of male mating strategies has been described which differ in their spatiotemporal features and in the signals and cues involved, and thus selection pressures. As a consequence, males’ sensory systems are more diverse than those of females. In the first part I studied adaptations of the visual system in honeybees. I compared sex and caste-specific eye morphology among 5 species (Apis andreniformis, A. cerana, A. dorsata, A. florea, A. mellifera). I found a strong correlation between body size and eye size in both female castes. Queens have a relatively reduced visual system which is in line with the reduced role of visual perception in their life history. Workers differed in eye size and functional morphology, which corresponds to known foraging differences among species. In males, the eyes are conspicuously enlarged in all species, but a disproportionate enlargement was found in two species (A. dorsata, A. florea). I further demonstrate a correlation between male visual parameters and mating flight time, and propose that light intensities play an important role in the species-specific timing of mating flights. In the second study I investigated eye morphology differences among two phenotypes of drones in the Western honeybee. Besides normal-sized drones, smaller drones are reared in the colony, and suffer from reduced reproductive success. My results suggest that the smaller phenotype does not differ in spatial resolution of its visual system, but suffers from reduced light and contrast sensitivity which may exacerbate the reduction in reproductive success caused by other factors. In the third study I investigated the morphology of the visual system in bumblebees. I explored the association between male eye size and mating behavior and investigated the diversity of compound eye morphology among workers, queens and males in 11 species. I identified adaptations of workers that correlate with distinct foraging differences among species. Bumblebee queens must, in contrast to honeybees, fulfill similar tasks as workers in the first part of their life, and correspondingly visual parameters are similar among both female castes. Enlarged male eyes are found in several subgenera and have evolved several times independently within the genus, which I demonstrate using phylogenetic informed statistics. Males of these species engage in visually guided mating behavior. I find similarities in the functional eye morphology among large-eyed males in four subgenera, suggesting convergent evolution as adaptation to similar visual tasks. In the remaining species, males do not differ significantly from workers in their eye morphology. In the fourth study I investigated the sexual dimorphism of the visual system in a solitary bee species. Males of Eucera berlandi patrol nesting sites and compete for first access to virgin females. Males have enlarged eyes and better spatial resolution in their frontal eye region. In a behavioral study, I tested the effect of target size and speed on male mate catching success. 3-D reconstructions of the chasing flights revealed that angular target size is an important parameter in male chasing behavior. I discuss similarities to other insects that face similar problems in visual target detection. In the fifth study I examined the olfactory system of E. berlandi. Males have extremely long antennae. To investigate the anatomical grounds of this elongation I studied antennal morphology in detail in the periphery and follow the sexual dimorphism into the brain. Functional adaptations were found in males (e.g. longer antennae, a multiplication of olfactory sensilla and receptor neurons, hypertrophied macroglomeruli, a numerical reduction of glomeruli in males and sexually dimorphic investment in higher order processing regions in the brain), which were similar to those observed in honeybee drones. The similarities and differences are discussed in the context of solitary vs. eusocial lifestyle and the corresponding consequences for selection acting on males.
Honeybees are among the few animals that rely on eusociality to survive. While the
task of queen and drones is only reproduction, all other tasks are accomplished by sterile
female worker bees. Different tasks are mostly divided by worker bees of different ages
(temporal polyethism). Young honeybees perform tasks inside the hive like cleaning and
nursing. Older honeybees work at the periphery of the nest and fulfill tasks like guarding
the hive entrance. The oldest honeybees eventually leave the hive to forage for resources
until they die. However, uncontrollable circumstances might force the colony to adapt or
perish. For example, the introduced Varroa destructor mite or the deformed wing virus
might erase a lot of in-hive bees. On the other hand, environmental events might kill a
lot of foragers, leaving the colony with no new food intake. Therefore, adaptability of
task allocation must be a priority for a honeybee colony.
In my dissertation, I employed a wide range of behavioral, molecular biological and analytical techniques to unravel the underlying molecular and physiological mechanisms of
the honeybee division of labor, especially in conjunction with honeybee malnourishment.
The genes AmOARα1, AmTAR1, Amfor and vitellogenin have long been implied to
be important for the transition from in-hive tasks to foraging. I have studied in detail
expression of all of these genes during the transition from nursing to foraging to understand how their expression patterns change during this important phase of life. My focus
lay on gene expression in the honeybee brain and fat body. I found an increase in the
AmOARα1 and the Amforα mRNA expression with the transition from in-hive tasks to
foraging and a decrease in expression of the other genes in both tissues. Interestingly,
I found the opposite pattern of the AmOARα1 and AmTAR1 mRNA expression in the
honeybee fat body during orientation flights. Furthermore, I closely observed juvenile
hormone titers and triglyceride levels during this crucial time. Juvenile hormone titers
increased with the transition from in-hive tasks to foraging and triglyceride levels decreased.
Furthermore, in-hive bees and foragers also differ on a behavioral and physiological level.
For example, foragers are more responsive towards light and sucrose. I proposed that
modulation via biogenic amines, especially via octopamine and tyramine, can increase
or decrease the responsiveness of honeybees. For that purpose, in-hive bees and foragers were injected with both biogenic amines and the receptor response was quantified
1
using electroretinography. In addition, I studied the behavioral response of the bees to
light using a phototaxis assay. Injecting octopamine increased the receptor response and
tyramine decreased it. Also, both groups of honeybees showed an increased phototactic
response when injected with octopamine and a decreased response when injected with
tyramine, independent of locomotion.
Additionally, nutrition has long been implied to be a driver for division of labor. Undernourished honeybees are known to speed up their transition to foragers, possibly to
cope with the missing resources. Furthermore, larval undernourishment has also been
implied to speed up the transition from in-hive bees to foragers, due to increasing levels
of juvenile hormone titers in adult honeybees after larval starvation. Therefore, I reared
honeybees in-vitro to compare the hatched adult bees of starved and overfed larvae to
bees reared under the standard in-vitro rearing diet. However, first I had to investigate
whether the in-vitro rearing method affects adult honeybees.
I showed effects of in-vitro rearing on behavior, with in-vitro reared honeybees foraging
earlier and for a shorter time than hive reared honeybees. Yet, nursing behavior was
unaffected.
Afterwards, I investigated the effects of different larval diets on adult honeybee workers.
I found no effects of malnourishment on behavioral or physiological factors besides a
difference in weight. Honeybee weight increased with increasing amounts of larval food,
but the effect seemed to vanish after a week.
These results show the complexity and adaptability of the honeybee division of labor.
They show the importance of the biogenic amines octopamine and tyramine and of the
corresponding receptors AmOARα1 and AmTAR1 in modulating the transition from inhive bees to foragers. Furthermore, they show that in-vitro rearing has no effects on
nursing behavior, but that it speeds up the transition from nursing to foraging, showing
strong similarities to effects of larval pollen undernourishment. However, larval malnourishment showed almost no effects on honeybee task allocation or physiology. It seems
that larval malnourishment can be easily compensated during the early lifetime of adult
honeybees.
The goal of the project VascuBone is to develop a tool box for bone regeneration, which on one hand fulfills basic requirements (e.g. biocompatibility, properties of the surface, strength of the biomaterials) and on the other hand is freely combinable with what is needed in the respective patient's situation. The tool box will include a variation of biocompatible biomaterials and cell types, FDA-approved growth factors, material modification technologies, simulation and analytical tools like molecular imaging-based in vivo diagnostics, which can be combined for the specific medical need. This tool box will be used to develop translational approaches for regenerative therapies of different types of bone defects. This project receives funding from the European Union's Seventh Framework Program (VascuBone 2010).
The present study is embedded into this EU project. The intention of this study is to assess the changes of the global gene expression patterns of endothelial progenitor cells (EPCs) and mesenchymal stem cells (MSCs) after direct cell-cell contact as well as the influence of conditioned medium gained from MSCs on EPCs and vice versa. EPCs play an important role in postnatal vasculogenesis. An intact blood vessel system is crucial for all tissues, including bone. Latest findings in the field of bone fracture healing and repair by the use of tissue engineering constructs seeded with MSCs raised the idea of combining MSCs and EPCs to enhance vascularization and therefore support survival of the newly built bone tissue. RNA samples from both experimental set ups were hybridized on Affymetrix GeneChips® HG-U133 Plus 2.0 and analyzed by microarray technology. Bioinformatic analysis was applied to the microarray data and verified by RT-PCR.
This study gives detailed information on how EPCs and MSCs communicate with each other and therefore gives insights into the signaling pathways of the musculoskeletal system. These insights will be the base for further functional studies on protein level for the purpose of tissue regeneration. A better understanding of the cell communication of MSCs and EPCs and subsequently the targeting of relevant factors opens a variety of new opportunities, especially in the field of tissue engineering.
The second part of the present work was to develop an ELISA (enzyme-linked immunosorbent assay) for a target protein from the lists of differentially expressed genes revealed by the microarray analysis. This project was in cooperation with Immundiagnostik AG, Bensheim, Germany. The development of the ELISA aimed to have an in vitro diagnostic tool to monitor e.g. the quality of cell seeded tissue engineering constructs. The target protein chosen from the lists was klotho. Klotho seemed to be a very promising candidate since it is described in the literature as anti-aging protein. Furthermore, studies with klotho knock-out mice showed that these animals suffered from several age-related diseases e.g. osteoporosis and atherosclerosis. As a co-receptor for FGF23, klotho plays an important role in bone metabolism. The present study will be the first one to show that klotho is up-regulated in EPCs after direct cell-cell contact with MSCs. The development of an assay with a high sensitivity on one hand and the capacity to differentiate between secreted and shedded klotho on the other hand will allow further functional studies of this protein and offers a new opportunity in medical diagnostics especially in the field of metabolic bone disease.
SPRED proteins are inhibitors of the Ras/ERK/MAPK signaling pathway, an evolutionary highly conserved and very widespread signaling cascade regulating cell proliferation, differentiation, and growth. To elucidate physiological consequences of SPRED2 deficiency, SPRED2 KO mice were generated by a gene trap approach. An initial phenotypical characterization of KO mice aged up to five months identified SPRED2 as a regulator of chondrocyte differentiation and bone growth. Here, the loss of SPRED2 leads to an augmented FGFR-dependent ERK activity, which in turn causes hypochondroplasia-like dwarfism. However, long term observations of older KO mice revealed a generally bad state of health and manifold further symptoms, including excessive grooming associated with severe self-inflicted wounds, an abnormally high water uptake, clear morphological signs of kidney deterioration, and a reduced survival due to sudden death. Based on these observations, the aim of this study was to discover an elicitor of this complex and versatile phenotype.
The observed kidney degeneration in our SPRED2 KO mice was ascribed to hydronephrosis characterized by severe kidney atrophy and apoptosis of renal tubular cells. Kidney damage prompted us to analyze drinking behavior and routine serum parameters. Despite polydipsia, which was characterized by a nearly doubled daily water uptake, the significantly elevated Na+ and Cl- levels and the resulting serum hyperosmolality could not be compensated in SPRED2 KOs. Since salt and water balance is primarily under hormonal control of aldosterone and AVP, we analyzed both hormone levels. While serum AVP was similar in WTs and KOs, even after experimental water deprivation and an extreme loss of body fluid, serum aldosterone was doubled in SPRED2 KO mice. Systematic investigation of contributing upstream hormone axes demonstrated that hyperaldosteronism developed independently of an overactivated Renin-Angiotensin system as indicated by halved serum Ang II levels in KO mice. However, aldosterone synthase expression in the adrenal gland was substantially augmented. Serum corticosterone, which is like aldosterone released from the adrenal cortex, was more than doubled in SPRED2 KOs, too. Similar to corticosterone, the production of aldosterone is at least in part under control of pituitary ACTH, which is further regulated by upstream hypothalamic CRH release. In fact, stress hormone secretion from this complete hypothalamic-pituitary-adrenal axis was upregulated because serum ACTH, the mid acting pituitary hormone, and hypothalamic CRH, the upstream hormonal inductor of HPA axis activity, were also elevated by 30% in SPRED2 KO mice. This was accompanied by an upregulated ERK activity in paraventricular nucleus-containing hypothalamic brain regions and by augmented hypothalamic CRH mRNA levels in our SPRED2 KO mice. In vitro studies using the hypothalamic cell line mHypoE-44 further demonstrated that both SPRED1 and SPRED2 were able to downregulate CRH promoter activity, CRH secretion, and Ets factor-dependent CRH transcription. This was in line with the presence of various Ets factor binding sites in the CRH promoter region, especially for Ets1.
Thus, this study shows for the first time that SPRED2-dependent inhibition of Ras/ERK/MAPK signaling by suppression of ERK activity leads to a downregulation of Ets1 factor-dependent transcription, which further results in inhibition of CRH promoter activity, CRH transcription, and CRH release from the hypothalamus. The consecutive hyperactivity of the complete HPA axis in our SPRED2 KO mice reflects an elevated endogenous stress response becoming manifest by excessive grooming behavior and self-inflicted skin lesions on the one hand; on the other hand, in combination with elevated aldosterone synthase expression, this upregulated HPA hormone release explains hyperaldosteronism and the associated salt and water imbalances. Both hyperaldosteronism and polydipsia very likely contribute further to the observed kidney damage.
Taken together, this study initially demonstrates that SPRED2 is essential for the appropriate regulation of HPA axis activity and of body homeostasis.
To further enlighten and compare consequences of SPRED2 deficiency in mice and particularly in humans, two follow-up studies investigating SPRED2 function especially in heart and brain, and a genetic screen to identify human SPRED2 loss-of-function mutations are already in progress.
Cuticular hydrocarbons (CHC) abound on the surface of arthropods. In spite of their simple structure (molecules of carbon and hydrogen atoms), they provide pivotal functions in insects: their hydrophobic properties confer the insects a means to regulate water balance and avoid desiccation, whereas their diversity has enhanced their use as signals and cues in a wide range of communication and recognition processes. Although the study of CHC in insects over the past two decades has provided great insight into the wide range of functions they play, there is still a gap in understanding how they diversify and evolve. In this thesis, I have used members of the family Chrysididae to explore patterns of diversification of CHC. Most of the species of cuckoo wasps in this study are specialized parasitoids or kleptoparasites of mainly solitary hymenopteran hosts. Other hosts of the family include butterflies or stick insects. Cuckoo wasps are a particular interesting model to study the evolution of cuticular hydrocarbons because of their chemical adaptations that allow them to remain unrecognized by their hosts. Chemical insignificance (the reduction of the total amount of CHC on the cuticle) and chemical mimicry (the de novo production of CHC profiles resembling those of their female host) have been described in some representatives of the family and unpublished evidence suggests chemical deception is widespread in Chrysididae (Chapter 2). Nonetheless, to trace the evolution of any trait of interest, a reliable phylogenetic reconstruction of the family is required. Therefore, the first study of this thesis constitutes the largest and to-date most reliable phylogenetic reconstruction of the family Chrysididae, which includes representatives of 186 species of cuckoo wasps. While the results of this phylogenetic reconstruction are consistent with previous ideas on the relationships of subfamilies and tribes, it shows the existence of several non-monophyletic genera (Chapter 3). CHC are involved in intraspecific recognition, often acting as contact sex pheromones. Nevertheless, it is not yet understood to what extent CHC profiles differ between the two sexes and whether some compound classes are more prevalent in one or the other sex. So far, no comparison of CHC profiles of males and females has been done for more than a dozen of related species. In Chapter 4, I describe and compare CHC profiles of females and males of 58 species of cuckoo wasps in order to evaluate whether and to what extent CHC profiles of these species differ between the sexes. I demonstrated that CHC profiles of cuckoo wasps are frequently (more than 90% of the species analyzed) and strongly dimorphic (both sexes of a given species tend to produce very different CHC compounds). Methyl-branched compounds tend to be more prevalent in males (especially dimethyl-branched compounds) and unsaturated compounds prevail in females. Moreover, a sex-specific pattern in the distribution of the double bond position of alkenes was evident: internal double bond positions (> 11) occur predominantly in males, whereas alkenes with the doublé bond at position 9 were more abundant and frequent in females (Chapter4). In Chapter5, I investigated how CHC profiles of cuckoo wasps differ across species. Are CHC profiles of cuckoo wasps species-specific, enabling their use as cues for species recognition? How do CHC profiles resemble phylogenetic relatedness? In Chapter 5, I try to answer these questions by comparing CHC profiles of 59 species of cuckoo wasps. CHC profiles of cuckoo wasps are shown to be species (and sex-) specific. I show that CHC profiles are useful as a complementary tool to help delimiting taxonomically difficult sibling species. Moreover, the evaluation of CHC profiles of five commonly occurring species within a genus, showed little or no geographical variation. However, CHC profiles of closely related species may differ strongly among each other, not being useful to track the evolutionary history of species (Chapter 5). Sexual selection is generally credited for generating striking sexual dimorphism by causing changes in male traits. Most often, sexual selection has a stronger effect on males, who compete for access to and may be selected by females, thus male traits may rapidly evolve. Nevertheless, in cuckoo wasps, it appears that it is the female sex the one evolving faster changes, with females of very closely related species showing extremely divergent profiles. One plausible reason for this disparity is that natural selection acting on female’s CHC profiles may be stronger than sexual selection on males (Chapter 6). Since females of cuckoo wasps are most probably engaged in an evolutionary arms race with their female hosts, CHC profiles of female cuckoo wasps are likely rapidly evolving, thus explaining part of the strong observed sexual dimorphism of CHC (Chapter 6). In fact, Chapter 7 shows evidence of a possible ongoing evolutionary arms race between five cuckoo wasps of the genus Hedychrum and their hosts. Hedychrum species parasitize either Coleoptera-hunting or Hymenoptera-hunting digger wasps. Since the coleopteran prey of the former digger wasps is naturally better protected against fungus infestation, these wasps do not embalm their prey with alkene-enriched secretions as do the Hymenoptera-hunting digger wasps. Thus, Coleoptera-hunting digger wasps can apparently diversify their profiles to escape chemical mimicry. Interestingly, only female cuckoo wasps of these hosts have started producing the same compound classes and even the same CHC compounds as those of their hosts. Male cuckoo wasps, however retain an alkene-enriched CHC profile that reflects the molecular phylogeny of the genus (Chapter 7). Whereas, a larger number of parasite-host comparisons may be needed to further conclude that an arms race between cuckoo wasps and their hosts is capable of generating sexual dimorphism of cuckoo wasps, this thesis constitutes the first effort towards this, providing a starting point for further studies. Finally, I provide some methodological tools that may help in speeding up the sometimes cumbersome process of analyzing and identifying CHC profiles. One of the most time-demanding steps in the processing of CHC data is the alignment of CHC chromatograms. This process is often done manually, because alignment programs are mostly designed for metabolomics or are just recently being developed. I analyzed CHC profiles using a combined approach with two freely available programs. I used AMDIS (Automated Mass Spectral Deconvolution and Identification System, http://chemdata.nist.gov/mass-spc/amdis/) to deconvolute and automatically identify all CHC of interest present in a chromatogram. I then developed a series of R scripts to correct for potential, unavoidable errors while processing CHC chromatograms with AMDIS. Chapter 8 explains this procedure. In the next chapter, I developed a program that helps in the identification of one commonly occurring class of hydrocarbons. The limited number of linear alkanes (only one per carbon atom) and their characteristic diagnostic ion allows a rapid and unambigous identification of these substances. In opposition, unsaturated and methyl-branched compounds are more difficult to identify, as a result of the much larger diversity of existing compounds. To identify unsaturated compounds a derivatization is necessary to determine the position of the double bond. Methyl-branched alkanes, however can be identified from the original chromatogram if their diagnostic ions are known. Nonetheless, polymethyl-branched alkanes (e.g., compounds with two or more methyl groups along the chain) are often difficult to identify, because they may appear in mixes (e.g., 3,7 diMeC27 and 3,9 diMeC27), and tables containing the diagnostic ions are not easily available. Therefore, I developed a program that creates a table with all possiblemethyl-branched compounds containing up to 4 methyl groups, and that provides their diagnostic ions and a calculated retention index. This may allow a much faster identification of the methyl-branched compound a researcher is dealing with, without having to lose time in the tedious calculations by hand. The program is able to correctly identify, or at least, greatly reduce the number of possible options for the identification of an unknown methyl-branched compound. Thus, using this tool, most methyl-branched compounds can be readily identified (Chapter 9). This thesis ends with a general discussion (Chapter 10). Overall, this work provides a comprehensive overview of the diversity of cuticular hydrocarbons of cuckoo wasps. The analyses presented here shed light on the emergence and evolution of interspecific diversity and intraspecific sexual dimorphism of CHC profiles. In addition, two technical methods have been developed that could greatly facilitate the CHC analysis of insects.