Genomic imprinting, the parent-of-origin specific silencing of alleles, plays an important role in phenotypic plasticity and consequently evolution. The leading explanation for genomic imprinting is Haig's conflict theory, which suggests that alleles from each parent have evolved under different selectional pressures, resulting in the differential expression of patrigenes and matrigenes. Previous studies have mainly used mammals and flowering plants to test Haig’s theory. However, there is a lack of independent evidence to support the theory. My PhD thesis attempts to conduct an independent test of Haig’s conflict theory using buff tailed bumblebee Bombus terrestris. A methylation system to facilitate genomic imprinting has not been found in this species. Therefore the first aim of the study was to establish the presence of a functional methylation system in B. terrestris before testing Haig's conflict theory using worker reproduction in queen-less colonies. The initial finding is that a methylation system exists in B. terrestris. The next study, investigating the presence of methylated genes, revealed differential methylation patterns in caste and life stages. Finally, genes involved with worker reproduction in a range of social insects were identified, but distinguishing the matrigene and the patrigene for each gene was unsuccessful. Therefore the final study investigating the presence of imprinted genes in B. terrestris and whether they conform to the expression patterns hypothesised by Haig’s conflict theory could not be analysed. Although this study did not provide conclusive evidence to support Haig’s conflict theory, the presence of methylation in genes involved with worker reproduction in reproducing and non-reproducing B. terrestris workers suggests that further analysis is needed. With adequate evidence, proving Haig’s conflict theory will not only expand our knowledge of invertebrate methylation, but also our understanding of conflict within social insect societies and our knowledge of how genomic imprinting affects phenotypic plasticity.

Neonicotinoid exposure has been recognised as potentially impacting upon bee health, but whether realistic exposure scenarios are driving declines in bee health is not known. This thesis contributes new insights and perspectives to this research field investigating the use of molecular, physiological and behavioural endpoints as potential ecotoxicological markers for pesticide risk assessment. The thesis presents experimental data for dietary exposures of the European honey bee, Apis mellifera, and the buff-tailed bumble bee, Bombus terrestris, to one of two neonicotinoid pesticides, imidacloprid and thiamethoxam. The first part of this thesis explores impacts of chronic dietary exposures to neonicotinoid pesticides on bee immunocompetence- the ability to mount an immune response- using an artificial challenge to invoke an immune response in adult workers. Levels of phenoloxidase, an enzyme involved in melanisation and part of the bee’s defence system, were largely constitutive and resilient to exposure in honey bees and bumble bees. In honey bees, transient transcriptional changes in antimicrobial effector genes were observed following neonicotinoid exposure, but the physiological antimicrobial response was unaffected. In bumble bees, the induced antimicrobial response was impaired following neonicotinoid exposure, but only when exposed to concentrations likely higher than realistic environmental exposure scenarios. The next phase of this thesis investigates whether transcriptional, physiological and behavioural endpoints associated with the functioning of the honey bee hypopharyngeal gland were altered by imidacloprid exposure. Imidacloprid exposure led to transcriptional changes in foraging genes (associated with the control of temporal polyethism) and major royal jelly proteins (fed to developing larvae by nurse workers) and enzymatic changes in glucose oxidase (an enzyme involved in social immunity), which I hypothesise are linked with hypopharyngeal gland development. Despite these laboratory observations, no behavioural effects were observed in a field setting, monitored using Radio Frequency Identification transponders. Lastly, using RNA-Sequencing to investigate changes across the honey bee transcriptome, this thesis identified a suite of genes that were differentially expressed in adult workers in response to immune challenge and/or dietary neonicotinoid exposure. Wounding and bacterial-like infection led to upregulation of known immune genes, including a peptidoglycan recognition protein and antimicrobial effectors. Chronic exposure to thiamethoxam and imidacloprid led to downregulation of genes associated with several metabolic pathways, such as oxidative phosphorylation, pyruvate- and purine- metabolic pathways, as well as ribosomal activity. Some of these genes identified provide candidates for further study to elucidate functional effects mechanisms and better understand health outcomes, as well as potential new biomarkers for use in pesticide risk assessment. This thesis presents novel findings and offers opportunities for future research that will be of interest to a wide audience, including risk assessors and policy makers, as well as the broader biological community, including ecotoxicologists, insect physiologists and molecular biologists.

The Western Honeybee (Apis mellifera) is an important species, not only ecologically and economically, but as a source of recreation to many. The pollination services the species provides benefit a number of crops worldwide, and, as the honeybee is domesticated and kept in hives, can be directed commercially. Recently, although overall global stocks are growing, there have been reports of high colony losses worldwide. Due to the value of this species, this is a worrying trend. There are many stressors facing the honeybee, both natural and anthropogenic in origin. Two of the most prevalent, both in the popular media and in monitoring studies of colonies are insecticidal pesticides and the parasitic mite Varroa destructor. Due to the difficulties and expense of carrying out large-scale field studies required to properly investigate the multiple stressors and their interaction, the use of modelling to explore the problem and direct field work is a vital resource. In this thesis, I present research using the BEEHAVE model and a novel model to explore the exposure and potential impacts of pesticides and the varroa mite. The results show that the timing of a pesticide exposure in the year greatly changes the resultant impact on the colony. Pesticides can have many impacts on different stages of the honeybee, and I show that increased mortalities of different life stages of the honeybee (larvae, in-hive adults, foragers) and decreasing egg-laying rate, affect the development of the colony to different extents at different times of the year, with the colony being highly sensitive to losses of in-hive bees during the summer, and the over-wintering bees at the beginning and end of the year. A novel model is presented exploring the in-hive distribution of pesticide-containing nectar and the effect it has on the exposure of in-hive receiving bees and larvae. The results from this model show that, in-hive distribution is not important to consider for the adults, but may be for the larvae. The landscape, specifically the distance to pesticide-treated forage in relation to untreated forage also has an impact on the result of a pesticide exposure, and this is a potential avenue for the mitigation of pesticide impacts. I also present work towards the validation of BEEHAVE with regards to varroa mite infestation, finding that the model results are close to empirical data, both for datasets from the UK and USA, but the impact of varroa is underestimated. The results are discussed in the context of pesticide risk assessment, the mitigation of potential stressors and the modelling of the varroa mite. The BEEHAVE model is a vital tool for many applications, one being the risk assessment of pesticides. A review of the model by the European Food Security Agency (EFSA) highlighted extensions to the model required before it can be incorporated. This research begins to answer some questions asked in that review.

Sex allocation and sexual selection have been heavily studied, but rarely linked. In this thesis I investigated the interface between them in the gregarious parasitoid wasp Nasonia vitripennis, both directly and through their interactions with the mating system and sexual conflict. Chapter 2 investigated sexual selection and mating at the natal site: earlier eclosing males mated more females independently of body size. Nasonia follows Local Mate Competition, which describes how a female laying eggs alone on a patch of resources (a so-called single-foundress) should lay an extremely female-biased brood to minimise competition between her sons, yet ensure all her daughters are fertilised. Based on this I predicted that males with with fewer brothers would be better inseminators. Despite finding significant among-strain variation in (1) single-foundress sex ratio, (2) mate competitiveness when alone and (3) when in competition, (4) sperm resources, but not (5) sperm-depletion (Chapters 3 & 4), I did not find the predicted relationship. Conversely males from strains with more brothers had a higher mating success under competition (Chapter 3) leading to the question: does mating success select on sex ratio or vice versa? Either way it is a result of an interaction between sexual selection and sex allocation. Chapter 5 investigated the role of male post-copulatory courtship on female re-mating, and found that among- strain variation in female re-mating was not associated with variation in the duration of the post-copulatory courtship. Chapter 6 reviewed sexual conflict in the Hymenoptera: their haplodiploid genetics, newly sequenced genomes and varied life- histories provides a base for future research to build on. Finally I highlight the novel links between sexual selection, sex allocation, sexual conflict and the mating system found during my studies that will hopefully prompt future research on this topic.

Microsporidia are obligate intracellular eukaryotic parasites related to fungi, possessing greatly reduced genomic and cellular components. The microsporidian Nosema ceranae threatens honeybee (Apis mellifera) populations. Nosemosis has a complex epidemiology affected by host, pathogen and environmental factors. Although a draft of the N. ceranae genome has been published, the molecular basis underpinning pathogenicity is not known. The lack of established culturing techniques and a tractable genetic system necessitates use of model systems for both host and parasite such as Saccharomyces cerevisiae. I hypothesise effectors essential to disease progression exist amongst N. ceranae secretome genes. In this study I have started characterising these genes using a combination of established and novel techniques for studying microsporidia proteins including bioinformatics, heterologous expression in S. cerevisiae, and the genome-wide analysis platform of Synthetic Genetic Arrays. This effort has yielded new insights into N. ceranae secreted proteins which lack similarity to known sequences. I identified N. ceranae protein NcS77 as a candidate effector implicated in targeting host nuclear pores. NcS50 and NcS85 co-localise with ERG6 a marker for lipid droplets (an organelle known to be targeted by another obligate intracellular pathogen Chlamydia trachomatis) when expressed in S. cerevisiae. N. ceranae polar tube proteins (PTP) induce filament formation when expressed in S. cerevisiae and PTP2 co-localises with the cell wall. Interestingly this phenotype is replicated by another secreted protein which may infer a common function. Together these data contribute to knowledge on N. ceranae pathology bringing us closer to understanding the disease and ultimately lead the way to mitigation.

The decline of bumblebee (Bombus spp.) populations in the UK and worldwide has been well reported. It has been generally assumed that such declines result in the genetic impoverishment of some species, potentially leading to reduced fitness and increased extinction risk. This study tested the fundamental assumption linking population fragmentation with fitness, in a model system of two Bombus species native to the UK. Bombus monticola has declined significantly in range across the UK in recent years and occupies fragmented upland areas, while Bombus pratorum has remained abundant and widespread over many habitat types. The effects of genetic diversity on fitness have been addressed in wild Bombus species, but this is the first study to explicitly compare data from species of differing levels of population connectivity and hence test the assumptions of traditional population genetic theory. As genetic diversity has often been linked with immunocompetence, aspects of the innate immune response were quantified, together with parasite load. These empirical measures of fitness showed lower than expected variability between the two study species, and no evidence was found to support the theory of lower fitness in fragmented populations. However, the considerable variability between sample sites in both species for all parameters measured raised interesting questions as to the underlying evolutionary processes; it is postulated that B. monticola populations may maintain a higher than expected Ne, despite their fragmented distribution. This study also provided methodological developments. An alternative method for the quantification of wing wear as a proxy for age was proposed, which could be easily applied to other Bombus species and possibly adapted for use in other flying insects. In addition possible sources of error in AFLP analysis were highlighted which have not been adequately discussed in the current literature, namely the effects of sample storage. Given the utility of AFLPs for non-model species, this is an important avenue for future research, and would be applicable to studies in other systems. Overall, the data presented here emphasise the challenges of studying fitness in wild populations, and underline the requirement for research into the fundamental principles underlying many assumptions made by conservation genetic theory.

Over 80% of wild angiosperms are reliant upon animal pollination for fruit and seed set and bees and other insects provide a vital pollination service to around a third of the crops we produce. Habitat loss, climate change and disease spread all threaten pollinator populations, with local declines and range contractions in honeybees and bumblebees leading to concerns that crop production may suffer as a result of pollinator shortages. Whilst agriculture and wildlife are often presented as being at odds with one another, the relationship between farmers growing pollination dependent crops, and the bees and insects that service them could be mutualistic. Flowering crops planted by farmers can provide an important source of forage to wild bees, whilst in return wild bees can contribute to ensuring farmers achieve adequate yields of marketable crops. The potential of this mutualistic relationship can be maximised by farmers by adopting management practices that reduce harm to, and enhance the wellbeing of, the wild bees around their farm. A group of common pesticides (neonicotinoids) used by farmers have recently been linked to pollinator ill health. Sub-lethal effects resulting from exposure to the neonicotinoid imidacloprid have been reported in honeybees and bumblebees, with bumblebee reproductive success found to diminish as a result of exposure to field realistic doses of this agrochemical. Here, the mechanism behind the reduced queen production in bumblebee colonies is suggested, with bees exposed to imidacloprid showing reduced efficiency in foraging for pollen. Farmers dependent upon pollinating insects for crop production can opt to avoid the use of pesticides known to harm these insects, however future studies are needed to identify safer alternatives that can be use in their place. Farmers can choose to increase the number of bees at their farms by utilising domesticated honeybees and purchasing commercially reared bumblebees. The use of these pollinators can ensure a minimum number of bees in the vicinity of a crop, and facilitate the production of crops at times when wild bee numbers are low. Concerns have been raised, however, regarding the use of commercially reared bees, mostly in regard to pathogen and parasite transmission, but also in respect to the possibility of outcompeting native species. Here the frequency and severity of attacks on commercial Bombus terrestris colonies, by the wax moth, an understudied bumblebee pest, are examined. Wax moths were found to infest almost half of the bumblebee nests deployed at fruit farms, with around a third of infestations resulting in nest destruction. Farmers investing in commercial bees will want to reduce the impact of harmful pests that may result in a reduced pollination service being delivered. Wax moth infestation rates at the study farms using commercial bees were high and the potential of a ‘spill- back’ effect on wild bees was examined. No evidence was found to suggest that nests in close proximity to these farms were any more or less likely to suffer from an attack than nests situated further away. Nest size was found to be the most significant predictor of an infestation, with larger nests more prone to wax moth attacks. Whilst farmers can utilise domesticated and commercially reared bees, relying on one source of pollination is inherently risky, and the most robust service will likely be provided by a range of pollinators. As well as reducing the use of chemicals known to harm beneficial insects, farmers can improve the habitat around their farms to help encourage and sustain wild pollinator populations. Sowing flower strips has been found to increase the abundance and diversity of pollinating insects, however, studies linking the use of these strips to crop production are lacking. Here we demonstrate for the first time that sowing small flower strips, adjacent to strawberry crops serviced by both wild and managed bees, can increase the overall number of pollinators foraging on the crop. This thesis contributes to our understanding of the implications of farm management decisions on pollinator health. It provides experimentally based evidence to guide farmers in making informed decisions regarding the future of crop pollination services and highlights the need for an integrated approach to managing pollination services for sustainability.

Many bumblebee species are suffering from the effects of habitat fragmentation and population isolation. In some cases, populations have lost genetic diversity due to genetic drift and it is possible they are now at heightened risk of extinction. Inbreeding may be particularly costly to bumblebees because, as Hymenoptera, their complementary sex determination system can lead to the production of sterile or inviable diploid males. However, little is known about the effect that diploid male production has on bumblebee colony fitness. Here, the consequences of brother-sister mating in the bumblebee Bombus terrestris are investigated, and the production of diploid males was found to exert considerable costs at the colony level by reducing productivity and survival. Diploid males may therefore act as indicators of the genetic health of populations, and their detection could be used as an informative tool in hymenopteran conservation. Due to the costs associated with inbreeding, selection may have favoured the evolution of kin recognition systems in bumblebees. Data are presented that suggest that B. terrestris can discriminate between kin and non-kin as gynes were less willing to mate with siblings compared to non-relatives. Theory predicts that inbreeding may impose further costs on bumblebees through increased levels of parasitism, but empirical data are scarce. The relationship between population genetic diversity and parasite prevalence is assessed using Hebridean island populations of Bombus muscorum and Bombus jonellus. In the more outbred B. jonellus, there was no relationship between parasite prevalence and population heterozygosity. But prevalence of the gut parasite Crithidia bombi and the tracheal mite Locustacarus buchneri were found to be higher in populations of B. muscorum that had lower genetic diversity. In addition to assessing infection status, the activity of the immune system was assessed in each individual bee. However, there was no relationship between population heterozygosity and these immune parameters. This suggests that, in some Hymenopteran species, as populations lose genetic diversity the impact of parasitism will increase, potentially pushing threatened populations closer to extinction. Therefore, preventing population fragmentation by the creation of suitable habitats and by ensuring connectivity between habitat patches are important aspects of hymenopteran conservation. Finally, this thesis investigates the potential threat of pathogen spread from commercially reared bumblebees used for crop pollination to wild bumblebees. Although no direct evidence for parasite spillover is found, the prevalence of C. bombi was significantly higher in B. terrestris by the end of the season on farms that used commercial bumblebees compared to farms that did not. This high prevalence does suggest that pathogen spillover is a potential threat and it would be preferable to reduce the usage of commercial bumblebees where possible. For example, sowing wild flower mixes could boost natural pollinator populations, which in turn would benefit soft fruit pollination. Overall, this thesis contributes to our knowledge of the consequences of inbreeding in bumblebees and the relationship between genetic diversity and parasite prevalence. It provides a greater understanding of the factors that might be pushing threatened pollinators towards extinction and as a whole provides important information that may inform conservation practitioners, whose aim is to protect the future of our hymenopteran pollinators.

Bumblebees have shown both long and short-term declines throughout their range. These declines may be attributed to a range of factors including changes in land use, alterations in climatic conditions and species introductions. However, management strategies for bumblebee conservation often focus on provision of summer forage resources and other factors are frequently overlooked. Provision of spring forage and nesting sites for bumblebee queens are rarely considered, though colony foundation and early colony growth are two of the most sensitive stages in bumblebee life history. Here, the efficacy of certain agri-environment prescriptions for providing spring forage and nest sites for bumblebees is assessed, highlighting a need for specific schemes targeted towards the provision of these vital resources in the rural environment. The nesting ecology of bumblebees is poorly understood because wild colonies are difficult to locate. However, a greater knowledge of the colony-level effects of environmental change is crucial to understanding bumblebee declines. Attracting bumblebee queens to nest in artificial domiciles could provide a valuable tool for studying colony-level responses. However, domicile trials and the findings of a literature review presented here demonstrate that this approach may be largely impractical for use in the UK. Conversely, a nationwide public bumblebee nest survey produced numerous data regarding nest site preferences among bumblebee species and also demonstrated that citizen science may also provide a sensitive method for detecting declines in currently common bumblebee species. An understanding of the ecology of species interactions and coexistence can provide valuable insights into factors that may influence declines. Data presented here suggest that coexistence between some bumblebee species may be maintained by resource partitioning based on diel activity patterns that are linked to species-specific environmental tolerances. If this is the case, the potential role of climate change in bumblebee declines may be severely underestimated. There is also increasing evidence that genetic factors may play a role in bumblebee losses, accelerating declines of small, fragmented populations as a result of reduction in genetic diversity and inbreeding depression. Here, the feasibility of reintroducing British B. subterraneus (now extinct in the UK) from New Zealand into England is assessed using population genetic techniques. The findings suggest that the population history of B. subterraneus in New Zealand has resulted in a dramatic loss of genetic diversity and high genetic divergence from the original UK population, suggesting that it may not be a suitable for use in the reintroduction attempt. This work draws together some understudied aspects of bumblebee ecology with a particular focus on nest site requirements, availability of spring forage, mechanisms of avoidance of inter-specific competition and population genetic processes. The potential role of these in bumblebee declines is considered and new data relevant to the conservation of these important species is presented. It is hoped that this work will inform future management strategies for bumblebee conservation, highlight areas in need of further study and provide a sound starting point for future research in these areas.

Bumblebees are ecologically and economically important as pollinators, but some species are suffering severe declines and range contractions. In this thesis, three cryptic bumblebee species are studied to elucidate differences in their distribution, ecology and population genetics. As a result of their high morphological similarity, very little is known about the lucorum complex species: B. lucorum, B, cryptarum and B. magnus. In this study, their distributions across Great Britain were assessed using molecular methods, revealing that B. lucorum was the most abundant and most generalist of the three species, whereas B. magnus was the rarest and most specialised, occurring almost exclusively on heathland. Additionally, both B. magnus and B. cryptarum were more likely to be present at sites with cooler summer temperatures. Cryptic species represent interesting models to investigate the levels of niche differentiation required to avoid competitive exclusion. Characterising the niches of these species at a single site across the flight season revealed differences along three niche dimensions: temporal activity, weather sensitivity and forage-resource use. These species exhibited asymmetric niche overlap; a combination of ecological divergence and spatio-temporal heterogeneity may contribute to maintaining them in sympatry. Population genetic studies can be highly informative for understanding species ecology and for conservation management. The differences in habitat specialisation exhibited by these bumblebee species provide the opportunity to test conflicting hypotheses about links between dispersal and ecological specialisation: are habitat specialists selected to have low or high dispersal ability? Based on microsatellite analysis, the generalist B. lucorum had high levels of genetic diversity and little population structure across large spatial scales. The habitat specialist B. magnus had the lowest genetic diversity but similar levels of population differentiation to the moderate generalist, B. cryptarum. However, unlike B. cryptarum, B. magnus population differentiation was not affected by geographic distance, suggesting that this specialist species may maintain effective dispersal across large scales despite being restricted to a fragmented habitat. Bergmann’s rule is a well-known ecogeographic rule describing geographical patterns of body size variation, whereby larger endothermic species are found more commonly at higher latitudes. Ectotherms, including insects, have been suggested to follow converse Bergmann’s gradients, but the facultatively endothermic nature of bumblebees makes it unclear which pattern they should adhere to. This thesis reports caste-specific differences in body size between the three lucorum complex species in agreement with Bergmann’s rule: queens and males of B. cryptarum and B. magnus, which were found more commonly at higher latitudes and at sites with cooler temperatures, were larger than those of B. lucorum. Population genetic studies of invertebrates generally require the destruction of large numbers of individuals, which is often undesirable. Testing a variety of faecal collection and DNA extraction methods demonstrated that it is possible to obtain DNA of sufficient quality for genotyping from bumblebee faeces, without harming the individuals. This method would be valuable for studies of rare or declining bee species, for queens in reintroduction projects, and may be applicable to other arthropods. Overall this thesis contributes substantially to our knowledge of the ecology and population genetics of three important pollinator species. It provides data to inform species conservation, as well as understanding of ecosystem functioning and population dynamics. Furthermore, it successfully uses these cryptic species as a model to test several fundamental ecological theories.