<p>The importance of cryptic female choice, i.e. female post-copulatory influence over male reproductive success, in driving the evolution of male traits remains controversial. The main aim of this thesis was to understand the post-copulatory consequences of sexual interactions and the importance of cryptic female choice in two species of beetle.</p><p>Males of the red flour beetle Tribolium castaneum use their legs to rub the lateral edges of the female elytra during mating. When manipulating female perception of this behaviour, I found that females preferentially use the sperm of males with vigorous leg rubbing when they mate with more than one male. Leg rubbing also appeared to increase female rate of oviposition. Females do not seem to gain any indirect benefits by preferring males with an intense leg rubbing behaviour since this behaviour was found to have very low narrow sense heritability and did not appear to be condition dependent in its expression.</p><p>Males of the bruchid beetle Callosobruchus maculatus have spiny genitalia that harm their mates. Females kick males during copulation and when prevented from kicking, suffered reduced lifetime offspring production as a consequence of more extensive injuries. Males were not able to delay female remating, increase rate of oviposition or increase sperm precedence by inflicting relatively severe injuries to non-kicking females. Hence, the injuries appear to be side effects of male efforts to remain in copula. When copulation duration was manipulated, ejaculate size and female lifetime offspring production increased with the length of copulation. Females reduced their mating rate when they had access to water, suggesting that they obtain water from the large ejaculates and trade-off their need for additional water against the costs of mating. Males may then reduce the benefits of remating by providing their mates with a large amount of water. Females did not increase their remating propensity to avoid inbreeding when they had mated to brothers. Together, these studies reveal the complexity of sexual interactions and the importance of post-copulatory processes for the fitness of both males and females.</p>

Multiple mating by females is common in nature. Yet, the evolution and maintenance of polyandry remains a bit of an evolutionary puzzle. It was my aim in this thesis to reach a greater understanding of this phenomenon as well as to investigate the consequences of polyandry on the evolution of reproductive divergence in insects. In an extensive meta analysis addressing the direct effects of multiple mating on female fitness in insects, I found that insects gain from multiple matings in terms of increased lifetime offspring production. In species without nuptial feeding, increased mating rate leads to decreased female lifespan and my results strongly support the existence of an intermediate optimal female mating rate. However, results from an experimental study where I examined the relationship between female fitness and mating rate in the bean weevil (Callosobruchus maculatus) showed that female fitness was maximized at two alternative mating rates, indicating that some species may exhibit a more complex relationship between the costs and benefits of mating. In the meta analysis on species with nuptial feeding, I found only positive effects of increased mating rate and the puzzle is rather what constrains the actual mating rates of females in these groups. Sexual selection is a very potent driver of rapid evolutionary change in reproductive characters. Most research has focussed on precopulatory sexual selection, but in promiscuous species sexual selection continues after copulation and variance in male fertilization success gives rise to postcopulatory sexual selection. In this thesis I found that three allopatric populations of the red flour beetle (Tribolium castaneum) have diverged in traits related to reproduction. Male genotype affected all aspects of female reproduction, but more interestingly, males and females interacted in their effect on offspring production and reproductive rate, showing that the divergence was due to the evolution of both male and female reproductive traits. When studying postcopulatory sexual selection, sperm competition has been put forward as the main source of variance in fertilization success. The results from a set of double-mating experiments, using the same populations of flour beetles, provided strong evidence that cryptic female choice is also important in generating variance in male fertilization success. I found not only main effects of female genotype on male fertilization success but also male-female interactions which provide more unambiguous evidence for cryptic female choice. Finally, I attempted to uncover which male signals-female receptors are involved in the reproductive divergence observed in the Tribolium populations. In a double-mating experiment I manipulated female perception of two male reproductive signals, copulatory courtship and cuticular hydrocarbons, and the results indicate that, within populations, both signals are sexually selected. However, only male cuticular hydrocarbons seem to be involved in the reproductive divergence between the populations. In conclusion, multiple mating by female insects can be understood solely in terms of direct fitness benefits resulting from increased offspring production. I have shown that postcopulatory sexual selection can lead to rapid divergence in reproductive traits related to mating and that cryptic female choice plays an important role in this divergence.

The importance of cryptic female choice, i.e. female post-copulatory influence over male reproductive success, in driving the evolution of male traits remains controversial. The main aim of this thesis was to understand the post-copulatory consequences of sexual interactions and the importance of cryptic female choice in two species of beetle. Males of the red flour beetle Tribolium castaneum use their legs to rub the lateral edges of the female elytra during mating. When manipulating female perception of this behaviour, I found that females preferentially use the sperm of males with vigorous leg rubbing when they mate with more than one male. Leg rubbing also appeared to increase female rate of oviposition. Females do not seem to gain any indirect benefits by preferring males with an intense leg rubbing behaviour since this behaviour was found to have very low narrow sense heritability and did not appear to be condition dependent in its expression. Males of the bruchid beetle Callosobruchus maculatus have spiny genitalia that harm their mates. Females kick males during copulation and when prevented from kicking, suffered reduced lifetime offspring production as a consequence of more extensive injuries. Males were not able to delay female remating, increase rate of oviposition or increase sperm precedence by inflicting relatively severe injuries to non-kicking females. Hence, the injuries appear to be side effects of male efforts to remain in copula. When copulation duration was manipulated, ejaculate size and female lifetime offspring production increased with the length of copulation. Females reduced their mating rate when they had access to water, suggesting that they obtain water from the large ejaculates and trade-off their need for additional water against the costs of mating. Males may then reduce the benefits of remating by providing their mates with a large amount of water. Females did not increase their remating propensity to avoid inbreeding when they had mated to brothers. Together, these studies reveal the complexity of sexual interactions and the importance of post-copulatory processes for the fitness of both males and females.

Harmony and cooperation was for long believed to dominate sexual interactions. This view slowly started to change 25 years ago and is today replaced with a view where males and females act based on what is best from a costs-benefits perspective. When sex specific costs and benefits differ, concerning reproductive decision influenced by both sexes, sexual conflict will occur. The basis for discordant reproductive interests between the sexes is that males produce many small gametes, while females’ produce few and large gametes. One result of this difference is that the optimal mating rate differs between the sexes. Males, with their many small sperm, maximize their reproductive output by mating with many females, while females often do best by not mating more frequently than to fertilize their eggs, since mating often entails a cost. Sexual conflict over mating is thus an important factor shaping the interactions between the sexes. In this thesis I study this and related conflicts between the sexes, using mathematical models, fruit flies and comparative methods. Mathematical modelling was used to explore how males and females may coevolve under sexual conflict over mating. This model shows that sexual conflict over mating results in the evolution of costly female mate choice, in terms high resistance to matings, and costly exaggerated male sexual traits, aimed to manipulate females into mating. A key assumption in this model is that males which females find attractive also are more harmful to females. This assumption was tested by housing fruit fly females with either attractive or unattractive males. Females kept with attractive males were courted and mated more, and suffered a 16 percent reduction in lifetime offspring production. In another study I measured genetic variation in two antagonistic male traits used to compete over females; offence - a male’s ability to acquire new mates and supplant stored sperm, and defence - a male’s ability to induce fidelity in his mates and prevent sperm displacement when remating occurs. Independent additive genetic variation and positive selection gradients were found for both these traits, indicating an ongoing arms race between these male antagonistic traits. This arms race also had a negative impact on females, since high values of offence compromised female fitness. Genetic variation in female ability to withstand male harm was also tested for and found, indicating that females evolve counter adaptations to reduce the effect of harmful male traits. Finally, the proposed link between sexual conflict and speciation was tested. Theory suggests that perpetual sexual arms races will cause allopatric populations to evolve along different evolutionary trajectories, resulting in speciation. This theory was tested using comparative methods by contrasting the number of extant species in taxa with high and low opportunity for sexual conflict. The study showed that taxa with high opportunity for sexual conflict, on average, has four times as many species as those with low opportunity, supporting that sexual conflict is a key process in speciation.

Against the background of past, recent and future climate change, the present thesis addresses elevational shifts of alpine treelines in the Swedish Scandes. By definition, treeline refers to the elevation (m a.s.l.) at a specific site of the upper trees of a specific tree species, at least 2 m tall. Based on historical records, the first part of the thesis reports and analyzes the magnitude of treeline displacements for the main trees species (Betula pubescens ssp. czerepanovii, Picea abies and Pinus sylvestris) since the early 20th century. The study covered a large and heterogeneous region and more than 100 sites. Concurrent with temperature rise by c. 1.4 °C over the past century, maximum treeline advances of all species amount to about 200 m. That is virtually what should be predicted from the recorded temperature change over the same period of time. Thus, it appears that under ideal conditions, treelines respond in close equilibrium with air temperature evolution. However, over most parts of the landscape, conditions are not that ideal and treeline upshifts have therefore been much smaller. The main reason for that discrepancy was found to be topoclimatic constraints, i.e. the combined action of geomorphology, wind, snow distribution, soil depth, etc., which over large parts of the alpine landscape preclude treelines to reach their potential thermal limit. Recorded treeline advance by maximum 200 m or so over the past century emerges as a truly anomalous event in late Holocene vegetation history. The second part of the thesis is focused more on long-term changes of treelines and one specific and prevalent mechanism of treeline change. The first part of the thesis revealed that for Picea and Betula, treeline shift was accomplished largely by phenotypic transformation of old-established stunted and prostrate individuals (krummholz) growing high above the treeline. In obvious response to climate warming over the past century, such individuals have transformed into erect tree form, whereby the treeline (as defined here) has risen. As a means for deeper understanding of this mode of positional treeline change, extant clonal spruces, growing around the treeline, were radiocarbon dated from megafossil remains preserved in the soil underneath their canopies. It turned out that Picea abies in particular may attain almost eternal life due to its capability for vegetative reproduction and phenotypic plasticity. Some living clones were in fact inferred to have existed already 9500 years ago, and have thus persisted at the same spot throughout almost the entire Holocene. This contrasts with other tree species, which have left no living relicts from the early Holocene, when they actually grew equally high as the spruce. Thereafter they retracted by more than 300 m in elevation supporting that also on that temporal scale, treelines are highly responsive to climate change. The early appearance of Picea in the Scandes, suggests that Picea “hibernated” the last glacial phase much closer to Scandinavia than earlier thought. It has also immigrated to northern Sweden much earlier than the old-established wisdom. The experiences gained in this thesis should constitute essential components of any model striving to the project landscape ecological consequences of possible future climate shifts.

The bgl operon of Escherichia coli, involved in the uptake and utilization of aromatic β-glucosides salicin and arbutin, is maintained in a silent state in the wild type organism by the presence of structural elements in the regulatory region. This operon can be activated by mutations that disrupt these negative elements. The fact that the silent bgl operon is retained without accumulating deleterious mutations seems paradoxical from an evolutionary view point. Although this operon appears to be silent, specific physiological conditions might be able to induce its expression and/or the operon might be carrying out function(s) apart from the utilization of aromatic β-glucosides. The experiments described in this thesis were carried out to test these possibilities. In cultures exposed to prolonged stationary phase, majority of the bacterial population dies and a few mutants that have the ability to scavenge the nutrients released by the dying cell mass survive. Bgl+ mutants were found to be enriched in twenty-eight-day-old Luria Broth grown cultures of E. coli that are wild type for bgl but carry the rpoS819 allele. Out of the five Bgl+ mutants that were isolated, four carried a mutation in the hns locus while one of them, ZK819-97, had an activating mutation linked to the bgl operon. Further analysis of ZK819-97 by DNA sequencing revealed the existence of a single C to T transition at the CAP binding site in the regulatory region. ZK819-97 was chosen for further analysis. Competition assays were carried out in which Bgl+ strain, ZK819-97 (Strr), and the parental Bgl- strain, ZK820 (Nalr), were grown independently for twenty-four hours in Luria Broth and then mixed in 1:1,000 (v/v) ratio reciprocally, without addition of fresh nutrients. ZK819-97, when present in minority, was found to increase in number and take over the parental strain, ZK820, i.e. ZK819-97 showed a Growth Advantage in Stationary Phase phenotype. To determine whether the GASP phenotype of ZK819-97 is associated with the bgl locus, the bgl allele from this strain was transferred by P1 transduction to its parental strain, ZK819. The resulting strain, ZK819-97T (Bgl+, Tetr), when competed with the parental strain, ZK819 Tn5 (Bgl-, Kanr), also showed a GASP phenotype when present in minority in the mixed cultures. To reconfirm this further, the bgl locus was deleted from ZK819-97T. The resulting strain, ZK819-97Δbgl, showed a loss of the GASP phenotype. When the bglB locus was disrupted in ZK819-97T, the resulting strain, ZK819-97ΔB, also failed to show a GASP phenotype, indicating that the phospho-β-glucosidase B activity is essential for this phenotype. The strain, ZK819-IS1, carrying an activating IS1 insertion within the bgl regulatory region also showed a GASP phenotype, confirming that this phenotype of the Bgl+ strain is independent of the nature of the activating mutation. All the above mentioned strains used in the competition assays carry a mutant allele of rpoS, rpoS819. Introduction of the wild type rpoS allele in these strains resulted in the loss of the GASP phenotype of the Bgl+ strain, suggesting that the two mutations work in a concerted manner. The Bgl+ strain was found to show the GASP phenotype only when present in minority of 1:1,000 or 1:10,000 in the mixed cultures and showed a slight disadvantage at higher ratios, indicating that the GASP phenotype of the Bgl+ strain is a frequency dependent phenomenon. In competition assays carried out between 24-hour-old cultures of Bgl+ and Bgl- strains resuspended in five-day-old spent medium prepared from a wild type E. coli strain, Bgl+ strain did not show any extra or early GASP phenotype. In addition, a reporter strain, which has a lacZ transcriptional fusion with the activated bgl promoter, was resuspended in spent medium prepared from a five-day-old culture of wild type strain of E. coli and bgl promoter activity was measured by β-galactosidase assay. The bgl promoter did not show any induction in this medium. These experiments suggest the absence of any β-glucoside like molecules in the spent medium within the sensitivity of these assays. A reporter strain that has a lacZ transcriptional fusion to the wild type bgl promoter was used to measure the expression level of this promoter during exponential and stationary phase of growth in LB. Expression of the wild type as well as various activated promoters of bgl was found to be enhanced in stationary phase. To investigate a possible role of the rpoS encoded stationary phase specific sigma factor, RpoS (σs), and another stationary phase factor, Crl, known to be important for the regulation of many genes of the σs regulon, the bgl promoter activity measurements were carried out in the presence or the absence of RpoS and/or Crl. RpoS along with Crl was found to negatively regulate the expression of wild type as well as activated promoters of bgl, both in exponential and stationary phase. In the absence of the negative regulation by RpoS and Crl, the increase in the bgl promoter activity was more pronounced as compared to that in its presence. rpoS and crl mutations are common in nature and it has been suggested that crl deletion gives a growth advantage to the strain in stationary phase. To test this possibility crl deletion was created in wild type as well as in attenuated rpoS allele background. The strain carrying the crl deletion was found to have a growth advantage in stationary phase over the wild type strain in the presence of wild type rpoS allele, while it shows a slight disadvantage in combination with mutant rpoS. Over expression of LeuO or BglJ is known to activate the bgl operon. To study a possible role of these factors in the regulation of the bgl expression in stationary phase, the bgl promoter activity was measured in strains that were deleted for leuO and/or bglJ, in the absence or presence of crl. These studies indicated that BglJ had a moderate effect on the bgl promoter activity in stationary phase in the absence of Crl but not in its presence. LeuO did not have a significant effect on the bgl promoter activity in either condition. Thus under the conditions tested, the physiological increase in the levels of LeuO and BglJ in stationary phase was insufficient to regulate the bgl expression. Preliminary results show that the bgl operon might be involved in the regulation of oppA, an oligopeptide transporter subunit, in stationary phase. Implications of these findings are discussed. The studies reported in this thesis highlight the involvement of the bgl operon of E. coli in stationary phase. This could be mediated by genetic as well as physiological mechanisms. This study also underscores the importance of observing organisms closer to their natural context and the need to reconsider the concept of ‘cryptic genes’.

No description available.

Introduction: Les cadres de lectures alternatifs (CLA) sont utilisés par de multiples virus afin de générer plusieurs protéines à partir d'une seule séquence nucléotidique. Les épitopes dits « cryptiques », c’est-à-dire les épitopes dérivés de protéines codées dans des CLAs, ont étés dernièrement l’objet de différentes études portant sur la réponse immunitaire antivirale et les lymphocytes T cytotoxiques. Méthodologie: Afin de vérifier le potentiel immunogène d'épitopes encodés dans des CLAs programmés, trois cassettes ont été construites pour mener à l'expression de trois épitopes bien caractérisés (épitope GAG77–85 du virus de l'immunodéficience humaine de type 1; épitope NS31406-1415 du virus de l'hépatite C; épitope core18-27 du virus de l'hépatite B) à partir de trois cadres de lectures superposés. La première cassette permet une initiation alternative de la traduction, la deuxième comprend deux signaux bipartites en tandem permettant un frameshift ribosomique et la troisième est une cassette contrôle. Ces éléments ont été introduits dans des vecteurs adénoviraux. Les virions générés ont servi à immuniser des souris C57BL/6 transgéniques pour HLA-A*0201 et HLA-DR1. La réponse immunitaire induite une semaine post-immunisation a été mesurée par essai ELISpot IFN . Résultats: Dans le contexte de cassettes vaccinales, les peptides dérivés d'une initiation alternative de traduction et de changement de cadre de lecture ribosomique ribosomal peuvent être exprimés et détectés par le système immunitaire dans un modèle animal. Conclusion: Ces expériences suggèrent la possibilité de développer de nouvelles stratégies vaccinales dans le but de prévenir ou de guérir certaines maladies associées aux infections virales chroniques telles que celles causées par le virus de l’immunodéficience humaine et le virus de l’hépatite C. / Introduction: Alternative reading frames (ARFs) are used by multiple viruses in order to generate different proteins from a single nucleotide sequence. Cryptic epitopes, which comprise antigens derived from proteins encoded in ARFs, have recently been the focus of studies pertaining to antiviral immunity and cytotoxic T lymphocytes. Methodology: In order to verify the immunological potential of epitopes encoded in programmed ARFs, three cassettes were constructed to permit the expression of three welldescribed epitopes (GAG77–85 epitope of human immunodeficiency virus type 1; NS31406-1415 epitope of hepatitis C virus; core18-27 epitope of hepatitis B virus) from three overlapping reading frames. The first cassette permits alternative translation initiation, the second cassette includes signals inducing ribosomal frameshifting and the third cassette serves as a control. These elements were introduced into adenoviral vectors. Recombinant adenoviruses were used to immunize C57BL/6 transgenic mice expressing HLA-A*0201 and HLA-DR1. The immune response induced was measured one week following immunization using IFN ELISpot assays. Results: In the context of vaccine cassettes, peptides derived from alternative translation initiation and ribosomal frameshifting can be expressed and detected by the immune system in an animal model. Conclusion: These findings suggest the possibility of designing vaccination strategies in the hope of preventing or curing certain diseases associated with chronic viral infections, such as those caused by human immunodeficiency virus and hepatitis C virus.

Vernal pools are small, seasonally filling wetlands found throughout forests of north eastern North America. Vernal pools have been proposed as potential 'hot spots' of carbon cycling. A key component of the carbon cycle within vernal pools is the decomposition of leaf litter. I tested the hypothesis that leaf litter decomposition is more rapid within vernal pools than the adjacent upland. Leaf litter mass losses from litterbags incubated in situ within vernal pools and adjacent upland habitat were measured periodically over one year and then again after two years. The experiment was carried out at 24 separate vernal pools, over two replicate years. This is a novel degree of replication in studies of decomposition in temporary wetlands. Factors influencing decomposition, such as duration of flooding, water depth, pH, temperature, and dissolved oxygen were measured. Mass loss was greater within pools than adjacent upland after 6 months, equal after 12 months, and lower within pools than adjacent upland after 24 months. This evidence suggests that vernal pools of Central Ontario are 'hot spots' of decomposition up to 6 months, but not after 12 and 24 months. In the long term, vernal pools may reduce decomposition rates, compared to adjacent uplands.

Paropsis atomaria (Coleoptera; Chrysomelidae) is a eucalypt feeding leaf beetle endemic to southern and east coast Australia, and it is an emergent pest of the eucalypt hardwood industry. Paropsis atomaria was suspected to be a cryptic species complex based on apparent differences in life history characteristics between populations, its wide geographical distribution, and extensive host range within Eucalyptus. In this study genetic and ecological characters of P. atomaria were examined to determine the likelihood of a cryptic complex, and to identify the nature and causes of ecological variation within the taxon. Mitochondrial sequence variation of the gene COI was compared between populations from the east coast of Australia (South Australia to central Queensland) to assess genetic divergence between individuals from different localities and host plant of origin. Individuals from four collection localities used for the molecular analysis were then compared in a morphometric study to determine if observed genetic divergence was reflected by morphology, and common-garden trials using individuals from Lowmead (central Qld) and Canberra (ACT) were conducted to determine if morphological (body size) variation had a genetic component. Host plant utilisation (larval survival, development time, and pupal weight) by individuals from Lowmead and Canberra were then compared to determine whether differential host plant use had occurred between populations of P. atomaria; individuals from each population were reared on an allopatric and sympatric host eucalypt species (E. cloeziana and E. pilularis). Finally, developmental data from each population was compared and incorporated into a phenology modelling program (Dymex(tm)) using temperature as the principle factor explaining and predicting population phenology under field conditions. Molecular results demonstrated relatively low genetic divergence between populations of P. atomaria which is concomitant with the single species hypothesis, however, there is reduced gene flow between northern and southern populations, but no host plant related genetic structuring. Morphometric data revealed insufficient evidence to separate populations into different taxa; however a correlation between latitude and size of adults was discovered, with larger beetles found at lower latitudes (i.e., adhering to a converse Bergmann cline). Common garden experiments revealed body size to be driven by both genetic and environmental components. Host plant utilisation trials showed one host plant, E. cloeziana, to be superior for both northern and southern P. atomaria populations (increased larval survival and reduced larval development time). Eucalyptus pilularis had a negative effect on pupal weight for Lowmead (northern) individuals (to which it is allopatric), but not so for Canberra (southern) individuals. DYMEX(tm) modelling showed voltinism to be a highly plastic trait driven largely by temperature. Results from across all trials suggest that P. atomaria represents a single species with populations locally adapted to season length, with no evidence of differential host plant utilisation between populations. Further, voltinism is a seasonally plastic trait driven by temperature, but with secondary influential factors such as host plant quality. These data, taken combined, reveal phenotypic variability within P. atomaria as the product of multiple abiotic and biotic factors and representing a complex interplay between local adaptation, phenotypic plasticity, and seasonal plasticity. Implications for pest management include an understanding of population structure, nature of local adaptation and host use characteristics, and predictive models for development of seasonal control regimens.