Behaviour genetics deals with complex phenotypes which respond flexibly to environments animals experience. Change of phenotype in response to environment is phenotypic plasticity. A central question is how genes influence plasticity. I study plasticity and gene by environment interactions (GEI) relating to behaviours, metabolic, and genomic phenotypes of adults of the fruit fly Drosophila melanogaster. Chapters 1-3 study cuticular hydrocarbon (CH) levels of male flies. Chapter 1 shows male CH levels respond to time of day and light. Methods are developed to reduce high variability of CH. I show variation in CH parallels activity of two classes of CH synthesis hormones. Analysis of rate of variation gives estimates of turnover rates of CH and the metabolic cost of signaling. Chapter 2 studies mixed groups of genetically different flies, “hosts” and “visitors”. GEI of CH are found with both abiotic factors and with social mix. Social mix results in GEI as strong as abiotic factors. Indirect Genetic Effects (IGE) theory is used to show frequency-dependent IGE interactions. Chapter 3 shows that males in mixed social environments reduce expression of clock and CH synthesis genes, resulting in different signals. Females mate more often with males in a mixed group than with single-genotype males. Plasticity in male gene expression in response to social environment leads to different signals, mating levels, and potentially different fitness. Chapter 4 deals with behaviour, metabolite, and genomic phenotypes in flies differing in foraging gene alleles, as the food environment is changed. Strong GEI is found, structured by food type, chemical class of metabolite, and gene metabolic roles. A concept called “relative nutrient sensitivity” suggests an interaction between foraging and the insulin signaling pathway. I demonstrate epistasis between for and insulin with quantitative genetic methods and bioinformatics. These results lead to the conclusion that GEI are common in many fly phenotypes in response to well studied environments such as food and less studied ones such as social group. Some implications of this for maintenance of genetic variance are discussed.

Behaviour genetics deals with complex phenotypes which respond flexibly to environments animals experience. Change of phenotype in response to environment is phenotypic plasticity. A central question is how genes influence plasticity. I study plasticity and gene by environment interactions (GEI) relating to behaviours, metabolic, and genomic phenotypes of adults of the fruit fly Drosophila melanogaster. Chapters 1-3 study cuticular hydrocarbon (CH) levels of male flies. Chapter 1 shows male CH levels respond to time of day and light. Methods are developed to reduce high variability of CH. I show variation in CH parallels activity of two classes of CH synthesis hormones. Analysis of rate of variation gives estimates of turnover rates of CH and the metabolic cost of signaling. Chapter 2 studies mixed groups of genetically different flies, “hosts” and “visitors”. GEI of CH are found with both abiotic factors and with social mix. Social mix results in GEI as strong as abiotic factors. Indirect Genetic Effects (IGE) theory is used to show frequency-dependent IGE interactions. Chapter 3 shows that males in mixed social environments reduce expression of clock and CH synthesis genes, resulting in different signals. Females mate more often with males in a mixed group than with single-genotype males. Plasticity in male gene expression in response to social environment leads to different signals, mating levels, and potentially different fitness. Chapter 4 deals with behaviour, metabolite, and genomic phenotypes in flies differing in foraging gene alleles, as the food environment is changed. Strong GEI is found, structured by food type, chemical class of metabolite, and gene metabolic roles. A concept called “relative nutrient sensitivity” suggests an interaction between foraging and the insulin signaling pathway. I demonstrate epistasis between for and insulin with quantitative genetic methods and bioinformatics. These results lead to the conclusion that GEI are common in many fly phenotypes in response to well studied environments such as food and less studied ones such as social group. Some implications of this for maintenance of genetic variance are discussed.

The aims of this thesis were to investigate whether characteristics associated with animal welfare are genetically and genomically determined by using quantitative and molecular genetic approaches and to develop strategies indicating how these traits could be used in breeding programmes. Two traits that are closely related to animal welfare and associated with high socio-economic values are piglet survival at birth and aggressive behaviour in pigs. Piglet survival traits were analysed based on quantitative Bayesian approaches using phenotypic and pedigree information only, while aggressive behaviour was analysed based on molecular genetic approaches such as genome-wide association studies and genomic selection using additionally a dense panel of single nucleotide polymorphisms (SNP). The latter approach was validated using behavioural traits related to welfare characteristics in a welldocumented mouse data set. Selection for piglet survival at birth is expected to be effective, because all lines and breeds in this thesis showed considerable variation for this trait and relatively high heritabilities, particularly in lines with low average birth weight. Maternal heritabilities of individual birth weight were mostly at moderate magnitude and thus of great interest for selection. The genetic correlations between piglet survival and birth weight indicated that selection for either individual or average birth weight or variation of birth weight within litter would indirectly increase survival. The genetic associations of piglet survival with economically important (re)production traits are of great importance for breeding organisations. Undesirable genetic correlations between piglet survival and (re)production traits were generally of low magnitude, so that simultaneous improvement of all traits could be achieved. A comparison of five breeds and lines showed that differences in genetic parameters between breeds and lines can be substantial and no single selection strategy would be optimal for all. A unique study of a sire and a dam line originating from one breed but selected for more than 25 years with different breeding goals demonstrated how selection pressure can alter the genetic parameters over years. Breeding organisations should therefore consider selection strategies per breed or line individually to achieve maximum overall improvement. This study gives new insight into the use of genomic selection for traits associated with animal welfare. It is one of the first to present estimates for linkage disequilibrium in the pig using a new 60K SNP panel and the first to evaluate the efficiency of genomic selection against aggressive behaviour in pigs. Genomic selection showed a high predictive ability in comparison to traditional polygenic selection. It was especially advantageous for traits with lower heritabilities. In particular in situations where little family information was available, the performance of polygenic selection was low and genomic selection increased the performance considerably. Reducing the number of SNPs did not significantly change the performance of genomic selection. The consistently high performance across models indicates that low-density SNP panels may be sufficient to ensure a high efficiency of genomic selection, thus reducing the high costs associated with genotyping in pig breeding with its short generation interval. To summarize, this thesis has shown how to optimise quantitative and genomic approaches to improve animal welfare related characteristics efficiently in pig breeding programmes.

Recent clinical studies suggest that white matter pathology rather than grey matter abnormality is the major neurobiological substrate of age- related cognitive decline during “healthy” aging. According to this hypothesis, cerebrovascular (e.g. chronic cerebral hypoperfusion) and molecular (e.g. APOE, epigenetics) factors might contribute to age-related white matter pathology and cognitive decline. To test this, I used a new mouse model of chronic cerebral hypoperfusion and examined the following predictions: 1) hypoperfusion- induced white matter pathology might be associated with cognitive deficits, 2) APOE deficiency might be associated with white matter anomalies under normal physiological conditions and more severe hypoperfusion- induced white matter pathology, 3) chronic cerebral hypoperfusion might impact on hydroxymethylation (a newly discovered epigenetic marker) in white matter, via perturbations in associated epigenetic pathways, namely methylation and/ or TETs. I. Effects of chronic cerebral hypoperfusion on white matter integrity and cognitive abilities in mice To test the hypothesis suggesting that hypoperfusion- induced white matter pathology is associated with working memory and executive function impairment in mice, behavioural performance and neuropathology were systematically examined in two separate cohorts of sham and hypoperfused C57Bl6J mice. Spatial working memory, memory flexibility, learning capacity, short and long term memory recall were taxed using radial arm maze and water maze paradigms one month after surgery. At the completion of the behavioural testing white and grey matter integrity, inflammation were evaluated using standard immunohistochemistry with antibodies recognizing neuronal axons (APP), myelin sheath (MAG) and microglia (Iba1) as well as H&E histological staining to examine neuronal morphology and ischemic injury. In agreement with previous reports, the behavioral data indicated spatial working memory impairment in the absence of spatial memory flexibility, learning, short- and long- term memory recall deficits in hypoperfused mice However, in contrast to previous reports, a spectrum of white and grey matter abnormalities accompanied by an increased inflammation were observed in hypoperfused mice Although there was a significant association between hypoperfusion- induced inflammation in white matter and performance on a working memory radial arm maze task (p<0.05), the present pathological findings suggest that white matter abnormalities, neuronal ischemia and increased inflammation might be at the basis of hypoperfusioninduced cognitive impairment in mice. Further, chronic cerebral hypoperfusion might have affected alternative, non- examined brain processes (e.g. cerebral metabolism, neurotransmission) which might have contributed to the observed cognitive deficits in hypoperfused mice. II. Effects of APOE on white matter integrity under normal physiological and chronically hypoperfused conditions in mice To test the hypothesis suggesting that mouse APOE deficiency might be associated with white matter anomalies under normal physiological conditions and the development of more severe white matter pathology following chronic cerebral hypoperfusion, white and grey matter integrity, inflammation were examined in APOE deficient mice on a C57Bl6J background (APOEKO) and C57Bl6J wild- type (WT) counterparts one month after chronic cerebral hypoperfusion or sham surgery. A combined neuroimaging (MRI- DTI)/ immunochemical approach was attempted in these mice as an additional step towards translation of this research to human subjects. The ex vivo MRI- DTI findings demonstrated APOE genotype effects on the development of white matter abnormalities following chronic cerebral hypoperfusion in mice. Significant reductions in MRI metrics (FA and MTR) of white matter integrity were observed in examined white matter areas of APOEKO hypoperfused mice compared with WT hypoperfused counterparts (p<0.05). However, the neuroimaigng findings were not supported by the pathological analysis where no significant APOE differences were observed in hypoperfusion- induced axonal (APP), myelin (MAG, dMBP) pathology and inflammation (Iba1) (p>0.05). No significant differences in MRI parameters and pathological grades of white matter integrity were evidenced between APOEKO and WT sham mice (p>0.05). An absence of grey matter abnormalities was evidenced on T2- weighted scans and corresponding H&E stained brain sections in all experimental animals. However, significant reductions in MTR values and dMBP immunoreactivity (myelin pathology) (p<0.05) were observed in grey matter (the hippocampus) following chronic cerebral hypoperfusion in the absence of significant APOE genotype effect (p>0.05) suggesting the existence of both white and grey matter abnormalities in this animal model. Overall, the present neuroimaging data, but not pathological analysis, partially validated the main study hypothesis suggesting that APOE deficiency might be associated with the development of more severe white matter abnormalities in hypoperfused mice. III. Characterization of methylation and hydroxymethylation in white matter under normal physiological and chronically hypoperfused conditions in mice Lastly, I sought to test the hypothesis that chronic cerebral hypoperfusion might alter oxygen dependent DNA hydroxymethylation (5hmC) in white matter regions via perturbations in methylation (5mC) and/ or Ten- eleven translocation proteins (e.g. TET2) in mice. DNA methylation (5mC), hydroxymethylation (5hmC) and TET2 were immunochemically studied in white and grey matter of sham and chronically hypoperfused C57Bl6J mice a month after surgery. The immunochemical results demonstrated significant increases (p<0.05) in 5hmC in the hypoperfused corpus callosum (CC) in the absence of significant hypoperfusion- induced alterations in the distribution of 5mC and TET2 (p>0.05) in white matter. Significant hypoperfusion- induced increases were evident for TET2 in the cerebral cortex (Cx) (p<0.05). These data partially validated the main study hypothesis suggesting hypoperfusion- induced alterations in 5hmC in white matter. However, in contrast to the study hypothesis, the observed hypoperfusion- induced alterations in 5hmC occurred in the absence of changes in 5mC and TET2 in white matter. A subsequent correlation analysis between hydroxymethylation and 5mC, TET2 in the CC failed to show significant associations (p>0.05). In search of the cellular determinants of 5hmC in the CC, hydroxymethylation was examined in relation to some of the cell types in white matter- mature oligodendrocytes, oligodendrolial progenitors (OPC) and microglia both in vivo and in vitro. Specifically, a separate parametric correlation analysis between the proportion of 5hmC positive cells and the respective proportions of mature oligodendrocytes, OPC and microglia in the CC demonstrated that hydroxymethylation correlated significantly only with microglia in vivo (p<0.05). Following this, 5hmC immunochemical distribution was studied in vitro in oligodendroglia cells at different stages of maturation, and interferon γ/ lypopolisaccharide activated and nonactivated microglia. The in vitro analysis demonstrated that 5hmC is high in OPC, activated and nonactivated microglia, but it is low in mature oligodendrocytes. Taken together the in vivo and in vitro cellular analyses suggest that the processes of hydroxymethylation in white matter might be immunoregulated. However, it is possible that in vivo in addition to microglia, other cell types (e.g. astrocytes, OPC) contributed to the presently observed 5hmC upregulation in the hypoperfused CC. Conclusion The experimental work presented in this thesis further developed and characterized a new mouse model of chronic cerebral hypoperfusion by confirming previous behavioural findings (e.g. working memory deficits) and revealing previously undetected spectrum of white and grey matter pathology in this animal model. The thesis demonstrated for the first time by using a newly developed ex vivo MRI procedure that APOE might modulate hypoperfusion- induced white matter pathology in mice. Additional immunochemical analysis revealed important hypoperfusion- induced epigenetic alterations in white (5hmC) and grey (TET2) matter in this animal model. Future experiments on chronically hypoperfused mice would allow to get a better insight into the neurobiological determinants (e.g. white vs. grey matter) underlying the observed cognitive deficits in this animal model, the involved cellular and molecular pathways as well as the functional significance of genetic (APOE) and epigenetic (5hmC, TETs) alterations in the hypoperfused brain. Future experimental work on this animal model would potentially reveal new biological targets for the pre- clinical development of therapies for age- related cognitive decline. Further development and optimization of the newly developed ex vivo MRI procedure would allow its broader application in preclinical settings and would facilitate the translation of experimental findings to clinics.

In this thesis I investigated the genetic basis of several behaviours to answer questions surrounding the evolution and mechanistic basis of behaviour. Firstly, I took a single-gene approach to investigate the influence of fruitless (fru) on the courtship behaviour of Drosophila. fru is an alternatively-spliced transcription factor that is necessary for the production of male sexual behaviours, and has also been implicated in producing species-specific differences in courtship song. I investigated the patterns of selection acting on fru at the sequence level and found that positive selection was restricted to the alternatively spliced exons of fru. From this I hypothesised that the positively selected changes in fru would contribute to species-specific differences in courtship song. To test this I examined how isoform-specific fru loss-of-function mutants influence courtship song, and generated “species-swapped” flies whereby regions of fru that showed evidence for positive selection were transferred from four species of Drosophila, into D. melanogaster. Contrary to prediction, I found flies that lacked isoforms containing positively selected regions did not show any differences in courtship song. Unfortunately “species-swapped” flies were not generated in time to examine phenotypes and neuroanatomy as intended. Next, I examined the genetic basis of cold acclimation in two species of Drosophila using a transcriptomic approach. I found that the genes differentially expressed in response to cold acclimation were largely different in each of the species; however, the biological processes they were involved in were broadly similar. Finally, I investigated the transcriptomic changes associated with parental care in the burying beetle Nicrophorus vespilloides to determine if males and females alter the genes they express when parenting alone versus with a partner. I found that males greatly reduced their transcriptional response when parenting with a partner, suggesting they reduce the care they provide when present with a female.