All living organisms consist of cells and the identity of a cell is defined by the genes it expresses. To assure proper function, a cell receives continuous information on which genes to turn on and off. This information is, to a large extent, provided by transcription factors. Krüppel-related zinc finger proteins probably constitute the largest family of transcription factors in mammals and many of these proteins carry a potent repressor domain called Krüppel-associated box (KRAB). The human genome alone encodes more than 200 KRAB zinc finger proteins but still very little is known about their biological functions. The Krüppel-related zinc finger genes appear to have been involved in a massive expansion throughout evolution. To unravel some of the secrets underlying this evolutionary success, we studied the molecular evolution of KRAB zinc finger genes. We show that the frequently occurring duplications of these genes are accompanied by a low sequence constraint in their zinc finger region. In addition, we show that the number of zinc finger motifs carried within these proteins is far from fixed. New zinc finger motifs are frequently added while others are inactivated or even discarded from the coding region. The structurally independent Krüppel zinc finger motif has, through these mechanisms, served as a highly adaptive building block for the generation of new transcriptional regulators. The mouse, rat and human genomes carry four different variants of the KRAB domain – KRAB(AB), KRAB(Ab), KRAB(AC) and KRAB(A). This thesis presents the identification of a novel KRAB domain, KRAB C, as well as a functional analysis of the different KRAB domains. We conclude that all different KRAB domains share a common co-repressor, TIFβ, and effectively repress transcription. These functions are mainly mediated by the KRAB A box but are clearly influenced by the presence of a KRAB B, b or C box. Furthermore, we show that all KRAB zinc finger gene subfamilies originate from the KRAB(AB) zinc finger genes. In addition, this thesis includes a structural and functional analysis of four novel mouse and human KRAB zinc finger genes; MZF6D, HKr18, HKr19 and HZF12. Whereas HKr18 and HZF12 seem to be ubiquitously expressed, MZF6D and HKr19 show a more restricted expression pattern. Northern blot and in situ hybridisation analyses of MZF6D showed that the expression of this gene is restricted to meiotic germ cells. MZF6D might thus be involved in the formation of male gametes. The expression of HKr19, on the other hand, seems to be restricted to lymphoid cells, indicating a possible role for this KRAB zinc finger gene in the regulation of lineage commitment.

Although the practice of releasing fish into the wild is common in the management and conservation of fish populations, the success of release programmes and the potential harmful genetic and ecological effects that may follow are rarely considered. This thesis focuses on genetic and ecological consequences of fish releases, exemplified by supportive breeding of brown trout (Salmo trutta) and translocation of European eel (Anguilla anguilla). Specific questions addressed include: What is the relative performance of hatchery produced fish released to support wild populations, and do released hatchery fish contribute to the natural productivity? What is the variation in reproductive success in the wild, and how does it affect the genetic consequences of a supportive breeding programme? Is there a spatial genetic structure in the European eel that must be considered in the management of this rapidly declining species? Experiments conducted under natural and near-natural conditions in the River Dalälven, Sweden, suggest that hatchery produced trout can reproduce in the wild. In fact, when the pronounced variation between individual breeders was accounted for, there were no detectable differences between hatchery produced and wild born trout in reproductive success or offspring survival. These results were supported by molecular genetic data suggesting a pronounced gene flow from hatchery to wild trout in the river. Hatchery reared trout were, however, found to exhibit reduced survival rates immediately following release into the wild, an effect that was most likely due to phenotypic responses to the hatchery environment during ontogeny and a lack of experience of the wild. In sharp contrast to recently published studies, the present genetic analyses of European eels sampled across the whole distribution range suggest no spatial genetic structure but a subtle temporal genetic heterogeneity within sampled locations. These results emphasise the need to consider temporal replication when assessing population structure of marine species. The results obtained have general implications for the management and conservation of fish populations. First, supportive breeding of threatened salmonid populations might be successful, not only for boosting the census size and thereby reducing the short-term probability of extinction, but also for reducing the risks of inbreeding depression and loss of adaptive potential in future generations. However, the results also highlight the need to restore the natural productivity of a population under supportive breeding to avoid a potential reduction in fitness due to hatchery selection. Further, the lack of a detectable spatial genetic structure in the European eel suggests that the management strategy of translocating juvenile eels from locations were they are overabundant to other suitable freshwater habitats does not necessarily have to include genetic considerations with respect to the geographical origin of the translocated eels.

This thesis examined neurotoxic effects of nicotine exposure during a defined critical period of neonatal brain development in mice. In our environment there are numerous hazardous contaminants that an individual can be exposed to during its entire lifetime. In many mammalian species the neonatal period is characterised by a rapid development of the brain. The present studies have identified a defined critical period during the neonatal brain development in mice, where exposure to low doses of nicotine causes permanent disturbances in the cholinergic nicotinic receptors and altered behaviour response to nicotine at adult age. This adult reaction to nicotine, a hypoactive response, was the opposite of that observed in control animals and animals exposed to nicotine before or after this period. Animals showing a hypoactive response to nicotine lacked nicotinic low affinity binding sites in the cerebral cortex. Furthermore, neonatal exposure to nicotine affected learning and memory in adult animals, an effect that was time-dependent. This thesis also showed that neonatal exposure to nicotine increased adult susceptibility to a repeated exposure of nicotine, manifested as an even more pronounced effect in spontaneous behaviour after challenging doses of nicotine. In these animals the nicotinic receptors in the cerebral cortex, assayed by a-bungarotoxin, was decreased. Neonatal exposure to nicotine was also shown to increase adult susceptibility to the organophosphate paraoxon, a known cholinergic agent, and to the brominated flame retardant 2,2´,4,4´,5-pentabromodiphenyl ether, a novel environmental agent, at adult age. This was seen at doses that did not affect behaviour in control animals, and was manifested as deranged spontaneous behaviour and reduced habituation, aberrations that also worsened with age. The results indicate that differences in adult susceptibility to environmental pollutants are not necessarily an inherited condition. Rather they may well be acquired by low dose exposure to toxic agents during early life.

A gill filament-based assay for the cytochrome P450 1A (CYP1A)-catalysed activity ethoxyresorufin O-deethylase (EROD) was developed in rainbow trout (Oncorhynchus mykiss) and applied to Atlantic salmon (Salmo salar), Arctic charr (Salvelinus alpinus), Atlantic cod (Gadus morhua), saithe (Pollachius virens), and spotted wolffish (Anarhichas minor). Exposure to waterborne β-naphthoflavone (βNF; 10-6 M) induced branchial EROD activity in all species but the spotted wolffish. In rainbow trout exposed to low concentrations of benzo[a]pyrene (BaP; 10-9 M) and the textile dye indigo (10-8 M) the gills responded more rapidly than the liver to BaP, and indigo induced branchial but not hepatic EROD activity. A CYP1A-dependent BaP adduct formation was shown in gills of fish exposed to waterborne 3H-BaP, i.e. the adduct formation was enhanced by βNF and blocked by ellipticine (CYP1A inhibitor). The predominant location for BaP adducts was the secondary lamellae (most exposed part of the gill filament), whereas the CYP1A enzyme was also present in the primary lamellae of the gill filament. Hence, in addition to the cell-specific expression of CYP1A an important determinant for the localisation of adducts seemed to be the bioavailability of BaP. This idea is supported by the fact that the CYP1A enzyme was induced only in secondary lamellae by BaP (10-7 M) and indigo (10-6 M), whereas it was induced in both primary and secondary lamellae by 3,3´,4,4´,5-pentachlorobiphenyl (10-8 M). Apparently, readily metabolised inducers (BaP and indigo) are biotransformed in the secondary lamellae. My results show that gill filament EROD activity is a sensitive biomarker of exposure to waterborne dioxin-like pollutants, and that the assay has potential for use in monitoring. Furthermore, the results suggest that readily metabolised dioxin-like compounds absorbed via the gills may undergo first-pass metabolism in the gill cells and therefore remain undetected by monitoring of EROD activity in the liver.

Speciation is the process in which reproductive barriers evolve between populations. In this thesis I examine how sexual signals contribute to the maintenance, reinforcement or breakdown of reproductive barriers. Male pied flycatchers (Ficedula hypoleuca) and collared flycatchers (F. albicollis) differ in song and plumage traits. However, where the two species coexist, several pied flycatchers sing a song resembling the collared flycatcher (mixed song). Mixed song is not caused by introgression from the collared flycatcher but is due to heterospecific copying. Mixed song provokes aggressive behaviour in collared flycatcher males and leads to heterospecific pairing and maladaptive hybridization. The species differences in song were found to be larger in an old than a young hybrid zone. This was due to a reduction in the frequency of mixed song in the pied flycatcher and a divergence in the song of the collared flycatcher. Apparently, mixed song causes maladaptive hybridization, which over time leads to reinforcement of reproductive barriers by a song divergence. Previous studies have shown that a character displacement in male plumage traits reinforces species barriers. Hence both plumage and song divergence reduce the incidence of hybridization. The evolution of male plumage traits has been so rapid, or selection has been so strong that rapidly evolving molecular markers are unable to trace it. Hybrid females mate with a male of the same species as their father. Previous studies have shown that females use male plumage traits controlled by genes linked to the sex chromosomes (the Z) in species recognition. An association between preference and a sex-linked trait through the paternal line may render reinforcement of reproductive barriers more likely. In conclusion, sexual signals are affected by species interactions that cause breakdown or reinforcement of reproductive barriers.

Estimating substitution frequencies at sites that influence (Ka) and do not influence (Ks) the amino acid sequence is important for understanding the dynamics of molecular sequence evolution and the selective pressures that have shaped genetic variation. The aim of this work was to gain a deeper understanding of the driving forces of substitution frequency variation in human pathogens. Rickettsia prowazekii, the causative agent of epidemic typhus and Helicobacter pylori, which has been implicated in gastric diseases were used as model systems. A specific focus was on the evolution of orphan genes in Rickettsia. Additionally, adaptive sequence evolution and factors influencing protein evolutionary rates in H. pylori were studied. The comparative sequence analyses of orphan genes using Typhus Group (TG) and Spotted Fever Group (SFG) Rickettsia, indicate that orphan genes in the SFG correspond to pseudogenes in the TG and that pseudogenes in the SFG correspond to extensively degraded gene remnants in the TG. The analysis also showed that ancestral gene sequences could be reconstructed from extant gene remnants of closely related species. The studies of split genes in R. conorii indicate that many of the small fragmented ORFs are probably pseudogenes. Analysis of the H. pylori carbamoyl phosphate synthetase provided an opportunity to understand natural selection acting on a protein undergoing adaptive evolution. Factors such as network properties, protein-protein interactions, gene essentiality and chromosomal position on protein evolutionary rates in H. pylori were studied, of which antigenicity and gene location were identified as the strongest factors. In conclusion, high Ka/Ks ratios in human pathogens may reflect either adaptive sequence evolution or gene deterioration. Distinguishing between the two is an important task in molecular evolution and also of great relevance for medical microbiology and functional genomics research.

A central goal of evolutionary biology is to understand the processes responsible for morphological, genetic and behavioural differentiation between sexes and among geographically distinct populations. Perhaps the most significant processes are genetic drift, natural selection, phenotypic plasticity and sexual selection. The main aim of this thesis was to investigate differentiation among individuals and populations of the sex-role reversed pipefish (Syngnathus typhle) and, consequently, determine which processes may be responsible for emerging patterns. This unique species is characterised by males predominately choosing amongst displaying females. In this thesis I revealed, on a microgeographic scale, morphological differentiation without genetic divergence among populations. Interestingly, females differed in size whereas the males did not. For females in this sex-role reversed species, the costs of expressing a plastic phenotype may be outweighed by the potential gains from greater survivorship, higher fecundity or increased mating success. Thus, females gain the ability to make themselves as conspicuous and attractive to males as possible in the specific environment they are living. Moreover, behavioural experiments, which focussed on describing “personalities”, reproductive investment strategies, and mate-sampling tactics, also indicated that males as well as females had the behavioural plasticity required to adjust to the environment in which they live. To this end, using video playbacks as experimental stimuli may be especially rewarding in this species. Overall, the studies in this thesis acknowledge the ability of species to fine-tune their phenotype to maximise fitness and, therefore, highlight the importance of considering patterns of differentiation in an environment-specific context.

Syftet med detta arbete är att studera utomhusundervisning inom biologiämnet i grundskolans senare år. Arbetet syftar även till att fastställa Ekobussens roll i utomhusundervisningen. Frågeställningarna lyder som följande: •Vilken inställning har lärare till utomhusundervisning inom biologi? •Vad finns det för fördelar och nackdelar med utomhusundervisning? •Vad får elever ut av utomhusundervisning? •Hur använder sig lärare av Ekobussen och vilken inställning har de till den? För att få svar på dessa frågor genomfördes en enkätundersökning med lärare och elever samt djupintervjuer av tre lärare. Resultatet i undersökningen visar att både lärare och elever anser att utomhusundervisning inom biologiämnet inte bedrivs i tillräcklig utsträckning. Orsaker till detta är bland annat osäkerhet hos lärare, brist på bra exkursionsplatser i närområdet samt att det är schematekniskt svårt att få tid att komma ut. Undersökningen visar att de stora fördelarna med utomhusundervisning inom biologi är att eleverna anser sig koppla teoretisk och praktisk kunskap samman samt att många sinnen stimuleras vilket underlättar inlärningen. / The purpose of this work is to study outdoor education in biology, in the later years of compulsory school, and to investigate the role of “Ekobussen” in outdoor education. The questions at issue runs: •What do teachers think about outdoor education in biology? •What benefits and disadvantages are there with outdoor education? •What do the pupils gain by outdoor education? •What are the attitudes of “Ekobussen” by teachers and how do they use it in their work? To answer these questions we handed out a questionnaire to both teachers and pupils, and we also interviewed three teachers. The result of the investigation shows that both teachers and pupils believe that they are not having enough outdoor education in biology. The causes of this are among other things; unsure teachers, no good outdoor areas to examine in the immediate surroundings of the school and lack of time. The investigation shows that the greatest advantages of outdoor education in biology are that the pupils feel that they can connect theoretical- and practical knowledge and that many senses are stimulated which facilitates learning.

The global carbon cycle is intimately linked with the earth’s climate system. Knowledge about carbon cycling in the biosphere is therefore crucial for predictions of climate change. This thesis investigates the carbon dioxide balance of Swedish boreal lakes, its regulation, significance to the carbon budget of the boreal landscape, and sensitivity to climate change. Swedish boreal lakes were almost exclusively supersaturated in CO2 with respect to the atmosphere, resulting in an emission of CO2 from lakes to the atmosphere. Lake pCO2 was closely related to the concentration of terrigenous dissolved organic carbon (DOC), indicating that the utilization of terrigenous DOC by lake bacteria is a major source of CO2. This conclusion is supported by independent field studies, showing that net plankton respiration accounts for most of the CO2 emitted from Swedish boreal lakes, while photochemical mineralization and sediment respiration were less important. Mineralization of terrigenous DOC and subsequent emission of CO2 from lakes to the atmosphere was a major carbon loss factor in 21 major Swedish boreal catchments, removing 30-80% of the organic carbon exported from terrestrial soils to surface waters. Lake CO2 emission is in the same order of magnitude as organic carbon accumulation in boreal forest soils, and should therefore be included in the carbon budget of the boreal landscape. In a set of nearly 5000 global lakes, DOC concentration was a much more important regulator of lake pCO2 than temperature. Climate change will therefore affect the carbon balance of lakes primarily via alterations in terrestrial DOC export, rather than via changes in temperature per se. Both current observations and models of future climate suggest an increasing export of terrigenous DOC from many Scandinavian catchments. Hence, there probably is a current trend towards higher CO2 emission from Swedish boreal lakes, which is likely to continue in the future.

One of the large lineages of parasitic wasps, the Cynipoidea, exhibits three distinctly different life modes. Slightly more than half of the about 3000 species are parasitoids in insect larvae, whereas the remaining species are associated with plants, either as gall inducers or as inquilines (guests feeding on plant tissue in galls). The main focus of this thesis has been to identify morphological changes associated with the shifts between life modes. Particular attention was paid to structures believed to be important in gall initiation. Comparative anatomical studies of the egg, larva and venom apparatus were performed, including representatives of parasitoids, gall inducers and inquilines. Examination of gross morphology and ultrastructure revealed that the eggs of the gall inducers are larger and surrounded by a thicker shell than the parasitoid eggs. These differences may be related to the fact that the gall inducer egg contains sufficient egg yolk for the embryo during the entire egg period, whereas the parasitoid egg often absorbs nutrients through the eggshell. Furthermore, the gall inducer egg is probably more exposed to desiccation and therefore a thicker and more resistant eggshell is crucial. Comparing the terminal-instar larvae of about 30 species of parasitoids, gall inducers and inquilines, extensive morphological variation was found, particularly in the head and mouthpart features. The variation was summarized in 33 morphological and one life-history character and parsimony analyses were performed. The resulting phylogenetic estimates were largely in accordance with previous analyses of adult morphology and molecular data. The larval data point to a single origin of the inquilines, in agreement with adult morphology but in conflict with molecular data. The venom apparatus was found to be quite uniform in structure among a sample of 25 species of cynipoid species. It consists of a very short venom duct, a reservoir and a single unbranched venom gland. With few exceptions, the venom apparatus is conspicuously larger relative to the female metasoma in the gall inhabiting species than in the parasitoids. We found little evidence of anatomical structures that could facilitate chemical communication between the gall-inducer embryo and the surrounding plant tissue through the thick eggshell. On the other hand, the enormous venom glands and reservoirs, which are apparently not used for defence, suggest that the adult female plays a significant role in gall induction by injecting secretions into the host plant when laying eggs.