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Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva. The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva-infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria-specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parvaspecific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETDFMK was shown to inhibit killing by CD8+ T cell clones.

The chytridiomycete Batrachochytrium dendrobatidis (Bd) is a parasite which has been blamed for amphibian declines across the world. This study was designed to investigate the effects of Bd on natterjack toads (Bufo calamita), following the discovery of Bd populations of this species in the UK. The effect of Bd on natterjack toads was assessed by fieldwork and experiments. Wild adult natterjacks were tagged and repeatedly tested for Bd during 2009-2011. Captive adults and juveniles from infected populations were also tested in response to changes in environmental conditions. Swabs were used to collect Bd DNA from the skin of study animals. Swabbing did not reliably diagnose infection but the quantity of Bd DNA in swabs (Bd score) provided an indication of infection activity. Immersion in water appeared to trigger Bd zoospore emergence from the skin, resulting in an increase in the likelihood of Bd detection and increases in Bd score. Bd dynamics in natterjack populations were also influenced by salinity. Natterjacks in the UK are found mainly in coastal habitat, where ponds are often inundated by high tides. Adults captured in brackish water were less likely to test positive than those caught in fresh water. Bd isolated from coastal natterjacks was killed in vitro by a salinity equivalent to 50% seawater. The isolate grew fastest at low salinities, suggesting that it may have become adapted to brackish conditions. Despite this adaptation, tidal inundation may be sufficient to disinfect ponds and limit Bd transmission. Capture-mark-recapture (CMR) data from adult natterjacks revealed a weak negative correlation between Bd score and survival in the wild. Males had higher Bd scores than females but survival did not differ between sexes and there was no correlation between Bd score and growth. An experiment showed BD could kill natterjacks if infection activity was boosted by wet conditions. However only 6% of wild adults recorded Bd scores in excess of a mortality threshold derived from this experiment. Many adults and juvenile natterjacks can tolerate Bd infection and act as reservoirs of this pathogen. Despite detection of Bd in at least 14 UK natterjack populations by 2011, no mass mortalities of adult natterjacks have been reported and spawning has continued at all sites. Bd does not appear to have a major effect on natterjacks but this situation may not persist and vigilance should be maintained. Bd isolated from natterjack toads in this study belomgs to a global panzootic lineage (GPL) of Bd which Farrer et al (2011) claim has achieved a global distribution as a result of human activities.

Comparative analysis of a recent isolate (J) and a laboratory-adapted strain (W) of the parasitic nematode Nippostrongylus brasiliensis found that the former had higher fecundity and gave rise to more persistent infections, although these traits were partially abolished after three years of laboratory passage, suggesting that infection dynamics can be modified by continuous high-dose propagation. Host immune responses to the two strains were similar in mode and magnitude. Proteins secreted by infective larvae (L3) and adult parasites showed some subtle differences between strains, although the activity of enzymes which might impact on persistence such as acetylcholinesterases and nucleotide metabolising enzymes were similar. Activation of N. brasiliensis L3 was not influenced by host serum, but a 37°C temperature cue was sufficient to induce feeding and protein secretion. Rat skin extracts induced chemotaxis of L3 and also induced the secretion of pre-synthesised proteins, although feeding and subsequent protein secretion were unaffected. Analysis of L3 secreted products by two-dimensional immunoblotting revealed differential immune recognition of specific proteins. Analysis of host resistin-like molecules showed that they had no effect on parasite chemotaxis and feeding activities, in contrast to published data. The venom-allergen homologue/ASP-like (VAL) proteins are important therapeutic targets found in all parasitic nematodes studied to date, and eight secreted variants of VALs have been discovered in N. brasiliensis. Although N. brasiliensis VALs (NbVALs) were found to be immunogenic during natural infection, immunisation with recombinant NbVAL-7 did not protect mice against challenge. Moreover, natural infection induced antigen-specific IgE and Type I hypersensitivity reactions to NbVALs, suggesting that this may be an intrinsic property of these proteins which limits their use in immunoprophylaxis of nematode infection.

Avian malaria (Plasmodium spp.) is a popular model system to study the ecology and evolution of parasite-host-vector interactions in the wild. These studies have historically focused mostly on the avian hosts and the malaria parasites. Knowledge regarding the role of vectors is essential to our understanding of these wild systems, but has only very recently started to accumulate. This thesis aimed to contribute to this field by assessing mosquito-malaria-host associations for British mosquitoes and the role of mosquito ecology in shaping these parasite systems in a British woodland study site, using molecular, field ecology and statistical modelling methodologies. From the 12 mosquito species or species groups found, I showed that the Cx.pipiens/torrentium mosquito group is likely to have a major role in avian malaria transmission in Great Britain, while Cs. annulata may be transmitting P. circumflexum. I also demonstrated a positive spatial association between mosquito density per host and avian malaria prevalence, in accordance with theoretical expectations for malaria transmission. Findings here provide evidence that avian malaria transmission in British woodlands is limited mainly to June-August, being preceded by relapse of previous infections or, alternatively, by maintenance of chronic blood parasitaemia through the colder months; this agrees with theoretical expectations and findings elsewhere for temperate climates. This thesis also described local-scale spatial heterogeneity and seasonal variation in adult mosquito abundance within a British woodland where avian malaria is endemic, with differing patterns found between species or species groups. Spatially, variation in adult mosquito abundance was associated with microclimatic and landscape variables such as distances to mosquito breeding sites, microclimate and canopy height; seasonally, variation in mosquito abundance was associated with temperature and rainfall, alongside calendar date. The heterogeneity in mosquito parameters and associations with environmental variables found at a site where avian malaria is endemic highlights the need to anticipate such complexity when trying to understand Plasmodium transmission. By doing so, we further extend the potential of these parasite systems to improve our knowledge regarding the ecology and evolution of parasite-host-vector associations.

The role of parasites as agents of selection on their hosts has been well established, but less is known about how parasites facilitate divergence among host populations. In this thesis, I used the three-spined stickleback, Gasterosteus aculeatus, as a model organism to explore spatial variation in host-parasite interactions and the consequences for divergence of host traits. First, I established the extent of spatial variation in natural infection in the study system, North Uist, Outer Hebrides, Scotland, by conducting a survey of macroparasite communities in twelve freshwater lochs over two years. I found substantial geographic variation in parasite communities that was remarkably stable in time. Assuming that differences in parasite community composition correspond to differences in parasite-mediated selection, it suggests that North Uist stickleback populations experience divergent parasite-mediated selection that is consistent in time. Next, I carried out a series of artificial infection experiments with lab-reared sticklebacks from five populations using three widespread macroparasite species (Gyrodactylus gasterostei, Diplostomum spathaceum and Schistocephalus solidus), to assess geographic variation in parasite resistance and a component of the innate immune system, the respiratory burst response. There was significant variation among populations in resistance to G. gasterostei and D. spathaceum, and the innate immune response. To some extent the variation was related to natural infection levels, suggesting that divergent parasite-mediated selection may drive investment in these traits. Lastly, I conducted a growth experiment with the five stickleback populations and showed that there was significant population-level variation in juvenile growth rate, an important life history trait. In spite of considerable variation in all traits, I found no evidence for genetic trade-offs across populations between juvenile growth rate, and macroparasite resistance or the innate immune response. This thesis adds to a growing body of work that emphasises the importance of space in shaping host-parasite interactions.

The purpose of this thesis is to investigate associations between parasite infection and host life-history variation in the wild Soay sheep population of the islands of St Kilda, NW Scotland. Studying host-parasite interactions in wild animal populations is of interest because of the importance of heterogeneity in resource availability, genetics, and environmental conditions in determining resistance to parasites, with implications for human populations and wildlife conservation and management. However, very few studies are able to investigate these associations in a longitudinal manner, which is essential in order to understand how infection is associated with life-history variation across ages and environmental conditions. In this thesis, I investigate associations between parasite resistance and ageing and the importance of maternal effects on offspring parasite resistance. I also establish the shape of natural selection on parasite resistance, and associations between measures of parasite burden and antibody responses. The principle findings of the analyses presented in this thesis are: i) Adult sheep of both sexes show a decline in parasite resistance in old age which is consistent with senescence. Furthermore, the rate of decline in parasite resistance with age is accelerated in individuals that have experienced more stressful environmental conditions over their lifespan. ii) Aspects of maternal phenotype and lamb early life performance are significantly associated with parasite resistance in lambs. Some of these effects persist into adult life and may even affect late-life changes in parasite resistance with age. iii) Analysis of ageing in five female reproductive traits shows that the contributions of individual senescence, terminal effects, and selective disappearance vary across traits, and that therefore multiple traits should be studied in order to understand ageing more fully. Most strikingly, there was no evidence for significant senescence in the probability of producing twins. iv) The first estimate of the strength of natural selection on parasite resistance in a longitudinally-monitored population provided evidence for positive selection on parasite resistance in lambs but not adults. Selection in lambs also varied across environmental conditions, being stronger in years of more favourable conditions. v) Analysis of associations between estimates of parasite burden and antibody responses showed that an estimate of parasite burden was not correlated with either a general or parasite-specific antibody response. However, antibody responses were positively correlated, and there was some evidence for a genetic correlation between the two in lambs but not adults.

Immune responses are presumed to contribute to host fitness, either by fighting off infections or via immunopathology. Research in this thesis sought to relate the magnitude of a putative immune response to infection and host and parasite fitness. The experiments and field studies presented here all focus on the interactions between the freshwater crustacean, Daphnia magna and its sterilizing bacterial endoparasite, Pasteuria ramosa, using the number of circulating haemocytes as a measure of host immune activity. I found substantial genetic variation in Daphnia’s cellular response to P. ramosa, and that Daphnia genotypes that mount the strongest cellular responses are the most likely to get infected and suffer sterilization. Thus, a strong cellular response is associated with low, as opposed to high host fitness potential. There were also some host genotypes that mounted a weaker cellular response and did not go on to suffer infection, and some that lacked a cellular response and also never suffered infection with P. ramosa. These findings led to a heuristic two-stage model for infection, where the parasite has to (1) pass from the host gut to haemolymph and then (2) successfully overcome haemolymph-based immune effectors to reproduce and achieve fitness. I also demonstrate that both the magnitude of host cellular response and likelihood of infection increases with initial parasite dose in susceptible host genotypes, and that host cellular response is associated with likely infection under both host and parasite genetic variation. Parasitised Daphnia also have substantially more circulating haemocytes than their healthy counterparts in both the laboratory and in the wild, where there is substantial genetic and environmental variation. This is one of the very few examples of how an immune response designates low host and high parasite fitness potential in a wild system. Finally, using a mixture of field study and common garden experiment, I demonstrate evolution in parasite infection traits over the course of an epidemic in a wild population, and that this evolution is associated with a decline in host abundance.

The African trypanosome Trypanosoma brucei evades the immune system of the mammalian host by periodically switching its surface coat which is made up of Variant Surface Glycoprotein (VSG). T. brucei shows monoallelic expression of one VSG out of a repertoire of ~1200 genes with the active VSG gene expressed from one of ~15 telomeric expression sites (ESs). The mechanism behind the monoallelic exclusion of ESs is unclear. NLP was identified as a novel and essential AT-hook protein binding transcriptionally silent simple sequence repeats in T. brucei. I depleted NLP using RNAi in various T. brucei reporter lines containing an eGFP in different transcriptionally silent areas of the genome and monitored derepression of eGFP using flow cytometry. After NLP knock-down, I observed 45-65 fold derepression of silent ESs, and up to 5 fold derepression of other transcriptionally silent areas. Using chromatin immunoprecipitation (ChIP) I found an enrichment of NLP in certain non-transcribed loci including the rDNA spacers. I also found that blocking NLP synthesis results in a rapid fall in levels of the active VSG transcript. Lastly, I discovered using tandem affinity purification (TAP) followed by mass spectrometry that NLP is a part of a novel TbISWI complex in T. brucei, which also includes two previously unidentified protein partners. The high mobility group B (HMGB) protein family constitutes a major abundant class of non-histone chromatin associated DNA-binding proteins which play a role in chromatin architecture in a wide range of eukaryotes. In T. brucei, the HMGB protein TDP1, which contains two HMG boxes and one DEK C terminal DNA-binding domain, was first identified as binding to VSG ES promoter oligomer sequences. I report that TDP1 is an essential nuclear protein enriched in the nucleolus and expression site body, and is involved in facilitating transcription. Blocking TDP1 synthesis using RNAi mediated knock-down results in approximately 40-90% reduction in transcription of RNA polymerase I transcribed genes. Using ChIP, I find that TDP1 is enriched in the rDNA and on the active VSG ES in bloodstream form T. brucei. Additionally, the relative proportion of TDP1 binding the procyclin promoter compared with the upstream spacer and downstream EP1 genes is greater in procyclic form compared with bloodstream form T. brucei. Lastly, I performed TAP experiments with TDP1 and found that TDP1 interacts with the core histones. These results indicate that TDP1 is an architectural chromatin protein important for transcription control in T. brucei.

Insects are often infected with facultative endosymbiotic bacteria, which can have a range of important ecological effects. The grain aphid, Sitobion avenae, harbours diverse facultative symbionts, which suggests their importance in grain aphid biology. This thesis attempts to explain the ecological roles of the facultative endosymbionts in S. avenae. It also examines the question of whether the horizontal transmission of symbionts between aphid clones and species can be important for shaping the ecology and evolution of multi-species aphid communities. Novel techniques developed for research with the grain aphid study system are presented. Grain aphid clones vary in their tolerance to low temperatures, but this trait is not affected by their facultative endosymbionts. Strains of a symbiont Hamiltonella defensa do not protect grain aphids from hymenopterous parasitoids, regardless of the host genotype. However, experienced parasitoid females preferentially oviposit in aphids which do not harbour symbionts. Comparison of the fitness consequences of infection with the same Hamiltonella strains in their original and in novel grain aphid host clones reveal no consistent differences. Symbiont strains establish easily following artificial transfer between clones of the grain aphid, but the symbionts transferred from other aphid species form less stable infections. Hamiltonella strains do not affect the fecundity of their grain aphid host clones regardless of their host species of origin, but also do not generally confer protection against parasitoids. There are no clear patterns in the distribution of parasitoid-resistant phenotypes across phylogenetic trees of Hamiltonella and its bacteriophage APSE. Strains of four unrelated species of endosymbionts, Rickettsia, Spiroplasma, Rickettsiella and Regiella, confer the same pathogen-resistant phenotype to a single pea aphid clone. The same symbiont strains can confer resistance to clones of two different aphid species. Some strains in multiple infections may compensate for the costs of infections with other symbionts. The importance of these results for understanding the ecological and evolutionary role of facultative endosymbionts in aphids and other insects are discussed, and directions for further research are proposed.

Epidemic disease outbreaks pose a significant risk to the stability and survival of many populations on earth. Current methods to understand how epidemic diseases transmit are often confounded by heterogeneity in infection rates amongst host populations. Endemic parasites, which are often less severe compared to epidemic diseases, may contribute to that variation by impacting on host biology and therefore altering the transmission of epidemic diseases. The effects of an endemic, gastrointestinal infection on host fitness and host interactions with an epidemic parasite were explored in a novel invertebrate system developed for this study. The chosen host was the German cockroach, Blattella germanica, which was infected with an endemic protozoan parasite, Gregarina blattarum alone or in co-infection with an entomopathogenic nematode, Steinernema carpocapsae, which causes epidemic outbreaks in host populations. There was evidence of density dependent regulation by the endemic parasite. Reductions in both host survival and fecundity during endemic infection both contributed to this regulation. The endemic parasite also had fitness costs for offspring from infected parents, who took longer to reach adulthood and were less likely to survive. Protozoan infected host populations had lower densities and showed less variance in population fluctuation compared to parasite free populations and the endemic parasite was generally found at high prevalence within the infected groups. When hosts infected with G. blattarum were exposed to S. carpocapsae, the resulting co-infection led to reduced host survival but also reduced emergence of nematode transmission stages. Hosts infected with G. blattarum also had differential immune responses to macro and microparasites which could alter host susceptibility to different types of infections. Infection with an endemic parasite caused substantial changes in the biology of the host, which may have important effects on host population ecology. Endemic parasites can also have considerable consequences for the transmission potential of more a virulent pathogen. These findings demonstrate the important of endemic infections which should be given greater consideration in future host-parasite studies.

The main parasite affecting the Scottish Atlantic salmon farming industry is Lepeophtheirus salmonis costing approximately £29 million annually through lost production and treatments. As such, the primary aim of this study was to investigate the infection pressure, fecundity and infection dynamics of L. salmonis in a west coast sea loch containing salmonid aquaculture. Atlantic salmon held in small sentinel cages were used to monitor sea lice infection pressure in Loch Shieldaig. Investigation of the factors affecting infection pressure within the loch found no evidence of a direct link between monthly gravid lice counts on local farmed salmon and lice burdens on sentinel cage fish. Copepodid L. salmonis were present throughout the year despite there being low numbers of wild salmonids in the system during much of the year and as such the source of this infection was unclear. In subsequent laboratory trials, L. salmonis from wild salmon were found to be more fecund than those from farmed salmon and similarly, fecundity and survival were also higher in summer compared to winter populations of L. salmonis. These findings raise the possibility that L. salmonis infecting wild salmonids may “seed” previously fallowed systems. Alternatively, trials with atypical hosts found that L. salmonis of farmed origin did re-infect Atlantic cod and produce viable egg strings which moulted through to the infective copepodid stage. Infection challenges demonstrated that L. salmonis copepodids were observed to settle on atypical hosts such as saithe and Atlantic cod in low numbers although failed to develop to the chalimus stages and complete its lifecycle. Challenges utilising the mobile pre-adult stage were similarily unsuccesful in allowing L. salmonis to moult to the adult stage on these atypical hosts. Previous work conducted on Pacific three-spined sticklebacks demonstrated the species could support large burdens. In infection trials with three-spined sticklebacks from Scotland’s west coast, settlement was found to be low, which suggests they play no role in the Scottish inshore infection dynamics of L. salmonis. These series of studies provide novel and timely data on the biology and infection dynamics of L. salmonis on typical and atypical hosts in Scottish marine aquaculture areas. The data will form part of the knowledge used to make informed pest management and policy decisions assisting the future development of the aquaculture industry in Scotland.

Bats (Chiroptera) are one of the most successful and diverse of mammalian orders, with an estimated 1100 species worldwide. Due to protected species legislation, studies that focus upon bat endoparasites are limited. As such, many fundamental questions concerning bat-parasite relationships remain unanswered, including evolutionary aspects of such associations, hostparasite interactions and factors that may influence the composition of bat parasite communities. To further knowledge of bat parasitology, one hundred bats, that had either died of natural causes, or had been euthanized due to severity of injury, were acquired across Greater Manchester and Lancashire between September 2005 and September 2008. Molecular typing methods confirmed 93 specimens to be common pipistrelle (Pipistrellus pipistrellus), six to be soprano pipistrelle (P. pygmaeus) and one to be a whiskered bat (Myotis mystacinus). Development of PCR-based methodologies, coupled when possible with morphological analyses, confirmed the presence of the following microparasites (prevalence data in parenthesis): Babesia vesperuginis (23%), Trypanosoma spp (36%), Bartonella sp. (2%) and Eimeria sp. (20%), and the following macroparasites: Lecithodendrium linstowi (80.4%), Lecithodendrium spathulatum (19.6%), Prosthodendrium sp. (35.3%), Plagiorchis koreanus (29.4%) and Pycnoporus heteroporus (9.8%). Potential factors affecting the parasite community composition including host sex and age, season, year, geographic location and parasite co-infection are explored. The detection of Eimeria sp. would appear to be the first record of coccidia in British bats, and also the first global record of Eimeria sp. from the common pipistrelle. Phylogenetic analysis of bat-associated Bartonella sp. ITS region, clusters the isolate in a well supported clade with B. grahamii, B. elizabethae and B. queenslandensis, all known to infect rodents, in addition to B. grahamii and B. elizabethae being recognised human pathogens. The first molecular sequence data for L. spathulatum is presented, and sequence data for Prosthodendrium specimens is also novel; both are incorporated into a phylogenetic analysis of the Lecithodendriidae, which questions the current taxonomic status of Prosthodendrium. Lastly, in an attempt to assist the evolutionary study of haemosporidian parasites, ectoparasitic bat flies were collected in the field from Puerto Rican bats and additional samples were acquired from Germany. Dissection and cytochrome b-specific PCR analysis of DNA extracted from insect digestive tract tissue confirmed the presence of haemosporidian DNA within two of the German samples. Phylogenetic analysis of the order Haemosporidia, incorporating the novel cytochrome b gene sequence derived from these German samples, showed the latter to cluster strongly with samples isolated from Madagascan bats and illustrates likely host-switching between birds and mammals.

The Giant African Snail, Achatina (Lissachatina) fulica Bowdich, is a tropical crop pest species with a widespread distribution across East Africa, the Indian subcontinent, Southeast Asia, the Pacific and the Caribbean. It is also a known intermediate host of the rat lungworm, Angiostrongylus cantonensis, which can infect humans and cause eosinophilic meningitis. The phylogenetic position of A. fulica within the Achatinoidea and the Achatinidae was investigated using segments of the nuclear ribosomal (r) RNA cluster, actin and histone 3 genes and the mitochondrial CO1 and 16S rRNA genes. Results from molecular data support the monophyly of the Achatinidae based on the taxa surveyed as well as the morphological distinction of the Eastern Achatina (Lissachatina) from the Western and Central Achatina (Achatina); Lissachatina should therefore be elevated to genus status. The results also show non-monophyly of the Coeliaxidae, Ferussaciidae and Subulinidae; the taxonomy of these families must therefore be reassessed. The extent of genetic diversity in global A. fulica populations was also determined using an SSCP molecular marker developed from the 16S rRNA gene. Results reveal only one haplotype (C) emerged from East Africa and spread globally. The rat lungworm (Angiostrongylus cantonensis) has a parallel distribution with A. fulica, and the possible role of the snail in the spread of the parasite is investigated using a molecular marker derived from the small subunit (SSU) rRNA gene. A survey of the parasite within the route of dispersal of A. fulica detected A. cantonensis only in the Philippines and the French Polynesian territory of Tahiti, the latter of which being the first reported case of A. cantonensis infection for Achatina fulica in that territory. Due to the limited sampling of the snail and the patchy distribution of the parasite, there are insufficient data at this time to assess the role of Achatina fulica in the spread of Angiostrongylus cantonensis.

Teladorsagia circumcincta is a major financial burden on the UK sheep farming industry. Disease control is becoming increasingly difficult due to the rapid emergence of anthelmintic resistance. This has prompted the search for alternative, sustainable control measures, including vaccination. Vaccine design would be aided by a thorough knowledge of the mechanisms involved in immunity to T.circumcincta. Most research has focussed on humoral and cellular responses to infection with this nematode. This thesis focuses on the impact of infection with regards to the proteins found locally within the abomasum. Using a well established infection model, proteomic analysis of lymph draining the abomasum was carried out by means of 2-dimensional electrophoresis (2-DE). The identity of many of the proteins in gastric lymph was revealed by means of MALDI-TOF analysis. The relative quantities of the lymph proteins were monitored over time using gel analysis software in both primary infection and immune challenged infection models. This study revealed a number of proteins of interest, including the acute phase proteins serum amyloid A, alpha-1 acid glycoprotein and haptoglobin, as well as the actin depolymerising protein, gelsolin. The effect of infection and immunity to T.circumcincta on these proteins was investigated further by means of biochemical assays, western blotting and real-time PCR. The impact of infection on the permeability of the abomasal mucosa will affect the resultant gastric lymph proteome. This “leak lesion” phenomenon is well documented in T.circumcincta infection but the underlying cause is unknown. Tight junction proteins in the abomasum were studied, using immunofluorescence techniques, in an attempt to define the role of these proteins in this important immunological/pathological event. The aim of this thesis is to contribute to the knowledge of innate immune responses and local pathology occurring within the abomasum during T.circumcincta infection.

The hemiparasite Rhinanthus minor is a common component of many northern temperate grasslands. It can have major impacts on ecosystem processes, and is often present at very high densities, therefore constituting an important potential food source for invertebrate herbivores. Thus, the aim of this thesis is to investigate the interaction between this hemiparasite and its invertebrate herbivores, and to explore the various ecological factors which are likely to affect this interaction. In the first series of experiments the thesis explores how the density of the hemiparasite affects the composition of the vegetation, the performance of the hemiparasite and the levels of invertebrate herbivore damage it receives. The results of a field experiment and a greenhouse study demonstrated that hemiparasite density can adversely affect its own performance and survivorship and dramatically change the composition of the vegetation, but surprisingly appeared to have no impacts on the levels of herbivore damage the hemiparasite receives. The second series of experiments investigated the impacts if host identity on the performance of the hemiparasite and how this affects its invertebrate herbivores. The results demonstrated that host identity can have a major impact on the performance of the hemiparasite and its herbivores; however, the indirect effects on the invertebrates appear to be species specific. Thirdly, the thesis examines the effects of multiple host plants on the performance of R. minor and the knock-on effects for its invertebrate herbivores. Experiments demonstrated that multiple hosts are beneficial to R. minor, and that the antiherbivore defensive properties conferred to the hemiparasite by certain host plants are maintained in the presence of a second host species. Finally, the impact of nutrient addition and host plant damage on the performance of the hemiparasite and on the performance of its invertebrate herbivores was examined. The experiments showed that while certain host plants have highly contrasting effects on the performance of the hemiparasite‟s herbivores, the addition of nutrients and impact of host plant damage largely remove these differences, while neither factor appeared to affect the performance of the hemiparasite.

Exploitation of RNA interference (RNAi) has revolutionised work on Caenorhabditis elegans, and considerable success has been recently reported with plant-parasitic nematodes. It has proven difficult to transfer this technology to animal parasitic species, and previous attempts in this laboratory by feeding Nippostrongylus brasiliensis larvae with Escherichia coli expressing double-stranded RNA (dsRNA) gave no consistent reductions in levels of target transcripts. The aim of this study was to develop methods for RNAi in N. brasiliensis, a rodent strongylid nematode which is closely related to gastrointestinal nematodes of humans and livestock, in order to explore the biological functions of parasite secreted proteins. In order to promote uptake of exogenous macromolecules such as dsRNA and small interfering RNA (siRNA) by infective larvae, the process of activation, whereby larvae are induced to resume feeding and development, was studied in vitro. Activation could be induced solely by exposure of larvae to elevated temperature (37oC), whereas host serum or glutathione had no effect. Neither a membrane permeant analogue of cyclic GMP nor muscarinic receptor agonists promoted activation, suggesting that a cholinergic neural pathway is not involved in the process. Activation at 37oC could be blocked by inhibitors of phosphatidylinositol 3-kinase, Akt protein kinase & cytochrome P450. These data indicate that the early signalling events for larval activation in N. brasiliensis differ substantially from the hookworm Ancylostoma caninum, most probably acting through thermosensory rather than chemosensory neurons, but that they may converge downstream of a step dependent on phosphatidylinositol 3-kinase. Stimulation of protein secretion paralleled resumption of feeding, suggesting that these processes were tightly linked and regulated by similar pathways during activation. Temperature-activated larvae were exposed to dsRNA and siRNA via electroporation or soaking in the presence of a variety of transfection reagents, but RNAi was unsuccessful. Serotonin was demonstrated to increase the rate of uptake of macromolecules, yet larvae exposed to exogenous dsRNA in the presence of serotonin still failed to show RNAi-mediated gene silencing. In addition, RNAi was also observed to be irreproducible in adult N. brasiliensis using the same methods of delivery. The use of mRNA encoding firefly luciferase identified uptake into larvae or adult worms as a major impediment in the process. Venom Allergen Homologue/ASP-Like (VAL) molecules have been identified as major components of secreted proteins from many species of parasitic nematodes. In this study, eight N. brasiliensis VALs were identified and sequenced, and all were shown to be present in products secreted by adult parasites. NbVAL-7 and NbVAL-8 were also detected in secreted products of infective larvae. Structural similarities to other members of the Pathogenesis- Related protein superfamily are discussed, as are possible functions for these proteins in N. brasiliensis.

Theory predicts that hosts should evolve highly specific immune defences when there are significant fitness costs associated with parasitic infection. Historically, mechanistic studies have defined invertebrate immunity as innate and non-specific. However, recent evidence from ecological studies challenges this view by finding a high degree of specificity between host and parasite and evidence of immune priming, that are indicative of a more complex system. Critics of the ecological perspective assert that without mechanistic evidence, there is no sound reason to assume that these phenomena are generated by innate immunity. To begin bridging the gap between mechanistic and ecological fields of innate immunity, I have examined the molecular basis of specificity and priming in the model Bombus-Crithidia host-parasite system. My studies show that immune gene expression mirrors the interaction effect found with indirect, ecological measures of immunity, providing unequivocal evidence of innate immune specificity in invertebrates. A similar examination of immune priming suggested that the genes I analysed were not involved in this phenomenom and is most likely to be indicative of the relative importance of different arms of immunity in the primed immune response. Finally, I examined more general aspects of the immune response to Crithidia by characterising the temporal dynamics of immune gene expression throughout infection for the first time in this model host-parasite system. I also confirmed that the assumed link between virulence and intensity of Crithidia infection is valid. To conclude, my studies have shown that integration of molecular knowledge into natural host-parasite systems can only serve to enrich our understanding of the wider capabilities of invertebrate innate immunity.

Ixodes ricinus is the most important human-biting tick in Europe and the principal vector of Lyme borreliosis. In addition, this hard tick species transmits a large number of microbial pathogens that are of importance to animal and human health. Little is known about the diversity and genetic population structure of I. ricinus across Europe. Genetic diversity of these tick populations may have implications on disease transmission. I. ricinus primers were designed for a number of mitochondrial genes and a Multilocus Sequence Typing-like Scheme (MLST) was devised. This was termed mitochondrial MLST (mtMLST). MLST has so far mainly been used for typing microbes, and the development of a MLST scheme for an arthropod vector is novel. Understanding the geographic structure of I. ricinus populations, in combination with studies regarding the migration of tick-borne microbial infections, e.g. Lyme borreliosis, is likely to illuminate important processes in the evolution and spread of tick-borne diseases.

Lyme borreliosis (LB) is the most frequent vectorborne disease in the Northern Hemisphere. It is a complex bacterial zoonosis involving vertebrate hosts and hard ticks of the genus Ixodes. The causative agents, bacteria of the LB group of spirochaetes, form a species complex comprising 17 named species. As is the case for most microbial pathogens, epidemiological and ecological studies require appropriate genotyping. Although the use of single loci may provide rapid results, there are serious disadvantages, in particular when inferring evolutionary relationships or geographic population structure. A novel multilocus sequence analysis (MLSA) system of the LB group spirochaetes has been developed based on housekeeping genes to overcome these problems. Here, the system is optimized and tested using extracted spirochaetal DNA directly from ticks and then utilized to obtain insights into the migration and spread of individual species as well as to investigate the evolutionary origins of the species complex. Species belonging to the LB group of spirochetes display different patterns and levels of host specialisation which makes this an ideal system to study the impact of host associations on spread of zoonotic tickborne diseases. For example, Borrelia valaisiana and B. garinii are transmitted exclusively by birds while B. afzelii is transmitted by rodents. I demonstrate that the migration of the LB species is dependent on, and limited by, the migration of their respective hosts. I also show the presence of B. afzelii strains in England and, through the use of the MLSA scheme, demonstrate that the strains are highly structured. A close evolutionary relationship between B. afzelii and its rodent host species is shown. Furthermore, through phylogenetic analyses, some evidence of a coevolutionary relationship between the LB group species and their major group of vector species, the Ixodes persulcatus species complex, is presented and a Eurasian origin for the species group is suggested.

Trichuriasis, caused by the intestinal nematode Trichuris, is a disease that affects up to a billion people worldwide. To date, most of our knowledge of this disease comes from the mouse model, Trichuris muris, which has been successfully used to dissect the immune-mediated effector mechanisms that elicit the expulsion of the nematode. Numerous studies have shown a temporal association between intestinal nematode expulsion and goblet cell hyperplasia; however their precise role in response to nematode infection remains elusive. Goblet cells found at mucosal surfaces secrete many constituent components of the mucus barrier, including the gel-forming mucins (Muc2 in the intestine); mucins are large multifunctional glycoproteins that provide the structural framework of the barrier. The studies presented in this thesis demonstrate that the mucosal barrier and in particular its mucin components, changes in response to acute and chronic T.muris infection. In animals resistant to chronic T. muris infection, IL-13-mediated increase in Muc2 production and secretion was observed at the site of infection. Critically, expulsion of the nematode was significantly delayed in the absence of Muc2. Further investigation subsequently showed that Muc5ac, a mucin normally expressed in non-intestinal mucosa was, in fact, expressed in the intestine following nematode infection and was associated with nematode expulsion in the resistant mice. Moreover, mice deficient in Muc5ac were susceptible to chronic infection, despite a strong underlying TH2-type immune response which is essential to eliminate the nematodes, suggesting that Muc5ac acts as a critical effector molecule. Several qualitative changes in the mucins were also noted during resistance: mucins were more highly charged and more sulphated during nematode expulsion. Overall, the changes within the mucus barrier during resistance result in altering the rheological properties of the mucus layer making it less porous and mucins were shown to directly 'damage' the nematodes during nematode expulsion as reflected by a significant reduction in ATP levels. Chronic infection was accompanied by decreased levels of low charged and highly sialylated Muc2. Additionally, we demonstrated that the excretory secretory products released by the nematode consist of serine proteases capable of depolymerising the Muc2 mucin network, which may be part of the nematodes regime to improve its niche and/or aid movement through the mucus layer. Overall, this resulted in a porous mucus layer and a favourable environment for the parasite.Data is presented to show that the intestinal mucus barrier and its constituent mucins are an integral part of the co-ordinated expulsion mechanisms that occur in animals resistant to T.muris infection and we identify a mechanism whereby the nematode exerts its effects on the mucin environment to promote its survival within the host.

Larval schistosomes infect the human host by penetration of unbroken skin, before gaining access to a blood vessel and beginning their intravascular life. The work in this thesis focuses on the mammalian infection process, as it would be the ideal point to interrupt the life cycle. A whole organism approach was taken, and the life cycle stages before, during and after skin penetration were studied, namely the intra-molluscan germ ball (embryonic cercaria), infective cercaria, and the in vitro cultured day 3 schistosomulum (equivalent to skin stage larva). Confocal microscopy was used for a morphological survey of these life cycle stages, establishing the timeline of cercarial embryogenesis and documenting the impressive changes they undergo. The temporal and spatial gene expression patterns underlying the changes were investigated using the first genome-wide microarray for S. mansoni for transcriptional profiling of the three stages. The known repertoire of molecules likely to be secreted during host entry was greatly expanded, particularly the proteases and venom allergen-like proteins. Genes involved in energy production and conservation were up-regulated in the cercaria, as were several genes encoding proteins deployed immediately on arrival in the skin. Additionally, micro exon genes (MEGs) encoding variant secreted proteins were highly upregulated in the schistosomulum, emphasising their likely role after entry into the mammalian host. The transcription of many tegument and gut-associated genes was also increased in the schistosomulum; cathepsins were particularly notable, even in the cercaria, implying that the larval gut becomes active long before blood feeding begins. The first application of whole mount in situ hybridisation to germ balls confirmed localisation of invadolysin and VAL-10 to the nascent acetabular glands. However, SmKK7 and Sm16 were not expressed in these glands, questioning their putative roles in immunomodulation. Finally, three MEGs were revealed to be expressed in tissues at the host-parasite interface.

Anthelmintic resistance in parasitic nematodes of small ruminants is widespread and, in some parts of the world, threatens the sustainability of sheep production. The mechanisms whereby parasitic nematodes become resistant to anthelmintics, particularly ivermectin, remain to be determined. The majority of studies to date have investigated target site mutations; relatively little attention has been paid to the role of gene expression changes. The present study focused on Teladorsagia circumcincta; the predominant parasitic gastrointestinal nematode species in the UK and the predominant resistant species. The role of changes in gene expression were investigated in an ivermectin-susceptible isolate (CVL) and a multidrug resistant isolate (MOTRI), utilising a range of molecular biological techniques. In the first experiment, a panel of novel putative ivermectin resistance genes were identified from T. circumcincta, comprising 11 partial P-glycoprotein (Pgp) and 3 partial Cytochrome P450 (CYP) sequences. Both Pgps and CYPs have been implicated in the handling and metabolism of xenobiotics in other biological systems, but have not been investigated in T. circumcincta to date. Initial results, using semi-quantitative PCR identified changes in expression of this panel of genes between the CVL and MOTRI isolates. Constitutive differences in expression of the Pgps and CYPs between CVL and MOTRI were determined using the ΔΔCt TaqMan® real-time PCR method. A statistically significant increase in expression was observed for TeciPgp-9 NBD2 across all life-cycle stages but most notably in eggs (55-fold increase). A statistically significant reduction in expression of TeciPgp-2 NBD2 was observed in all but the adult stages of MOTRI compared to CVL. Analysis of a 208 base pair sequence of TeciPgp-9 NBD2 identified high levels of polymorphism, with at least four non-coding SNPs evident in the MOTRI isolate. These results merit further investigation. Inducible changes in the expression of the Pgps and CYPs were investigated in MOTRI before and after ivermectin treatment, using real-time PCR. Statistically significant fold changes in expression in most of the genes occurred in at least one life-cycle stage. Inducible expression of TeciPgp-2 NBD2 and TeciPgp-9 NBD2 was investigated further by comparing adult MOTRI parasites with those recovered three days after in vivo ivermectin exposure, and by exposing pools of MOTRI xL3 to ivermectin in the larval migration inhibition test. The survivors of ivermectin exposure exhibited a statistically significant reduced 13.68-fold expression of TeciPgp-2 NBD2 compared to MOTRI. Similarly, the MOTRI xL3 able to migrate in the presence of ivermectin in the LMIT had a 1.88-fold reduction in TeciPgp-2 NBD2 expression compared to MOTRI xL3 unexposed to ivermectin. These results indicate that inducible changes in TeciPgp-2 NBD2 and TeciPgp-9 NBD2 expression can occur, but the experimental design is critical to being able to identify the changes. In a more global approach, the transcriptomic response of MOTRI adults to in vitro ivermectin exposure was investigated using Roche 454 sequencing, generating 98,685 novel EST sequences, providing an important resource for a genome resource-poor organism. Objective bioinformatic analysis of the two datasets revealed statistically significant differences in the mean expression levels of the KEGG orthologous groups for ‘translation’, ‘amino acid metabolism’ ‘carbohydrate metabolism’ and ‘xenobiotic degradation and metabolism’. On combining the two datasets, and through application of a novel statistical method, 16 clusters of ESTs were identified as containing statistically significant differences in the mean proportion of exposed reads compared to unexposed reads under the conservative model, whilst a further 355 clusters were found to have statistically significant differences under the liberal model. One-way suppression subtractive hybridisation (SSH) was used to identify genes exhibiting increased expression in MOTRI adults compared to CVL adults. 28 contiguous sequences were identified from the SSH experiment; 6 contiguous sequences were selected for validation; 5 of these results were confirmed using semi-quantitative PCR. Each contig was BLAST searched against the Roche 454 dataset; contig SSH14 aligned most closely to one of the statistically significant clusters in the conservative model, SSHs 5, 6, 10 and 23 aligned most closely to statistically significant clusters in the liberal model. This suggests that changes in expression in these sequences occur both constitutively, between CVL and MOTRI isolates, and inducibly, following ivermectin exposure. This work has shown that changes in gene expression, particularly the constitutively reduced expression in TeciPgp-2 NBD2 and the constitutively increased expression in TeciPgp-9 NBD2 (coupled with the presence of SNPs) could play a role in allowing multidrug resistant T. circumcincta to survive ivermectin exposure. Roche 454 sequencing and SSH approaches identified gene expression changes associated with in vitro ivermectin exposure and ivermectin resistance. These could form the basis of a novel panel of candidate resistance genes whose altered expression profiles may allow multidrug resistant T. circumcincta to survive ivermectin exposure by some, as yet identified, mechanism. Finally, we have also shown that a multidrug T. circumcincta isolate is affected by ivermectin exposure and that changes in gene expression could have a role to play in the ivermectin resistance phenotype in T. circumcincta. The genetic changes underpinning these changes in gene expression remain to be elucidated, and need to be investigated in other isolates. These changes could form the basis of an ivermectin resistance molecular marker, to monitor the spread of resistance, and to evaluate management practices aimed at delaying its spread.

The processes through which parasites are able to survive in their susceptible host are complex and not fully understood. The platyhelminth parasite, ‘Schistosoma mansoni’, completes part of its life cycle in the snail ‘Biomphalaria glabrata’. During its intra-molluscan development, the parasite produces excretory-secretory products (ESPs), a cocktail of uncharacterised proteins and lipids. ‘Schistosoma mansoni’ ESPs may play a role in modulating snail-host immune responses. Two laboratory strains of ‘B. glabrata’ were used in this study; a schistosome-susceptible strain (NHM1742) and a schistosome-resistant strain (NHM 3017) refractory to infection. Snail haemocytes, the main type of circulating defence cells, which have similar characteristics to macrophages, were investigated. The extracellular signal-regulated kinase (ERK) signalling pathway is known to regulate defence reactions in cells. Activated ERK-like proteins were identified in ‘B. glabrata’ haemocytes using phosphor-specific anti-ERK antibodies. The phosphorylation (or activation) of ERK was reduced by 60% in susceptible snail haemocytes following ‘S. mansoni’ ESP exposure (20 [mu]g/ml for 1 h). In contrast, resistant snail haemocytes did not exhibit any changes in phosphorylated ERK levels following ESP-challenge. Nitric oxide (NO), a reactive molecule, which plays a role in host defence mechanisms, increased 3.3 fold in resistant snail haemocytes when challenged with ESPs. ESP-challenged susceptible snail haemocytes did not show any significant modulation in NO levels. The use of scanning electron microscopy also highlighted an increase in haemocyte spreading (on glass coverslips) in ESP-challenged haemocytes from resistant snails only. Heat shock protein 70 (HSP70), an evolutionarily conserved protein with immune-modulation properties was investigated using western blotting and scanning confocal microscope using anti-HSP70 antibodies. HSP70 was significantly reduced in susceptible (P[less than or equal to]0.01) and resistant (P[less than or equal to]0.05) snail haemocytes following 1 h exposure to 20 [mu]g/ml ESPs. Interestingly, the ERK cell signalling pathway was found to coordinate both NO and HSP70 levels, as the ERK inhibitor, U0126, significantly reduced levels NO output and HSP70 expression in ‘B. glabrata’ haemocytes. Finally, haemocyte gene expression was analysed after ESP-challenge using a ‘B. glabrata’ cDNA microarray. The microarray analysis highlighted differentially expressed genes (DEGs) between ESP exposed resistant and susceptible snail haemocytes. These DEGs transcribe proteins involved in protein degradation, protein transcription, cell-to-cell interaction and immune responses. This study has broadened our knowledge on the differences that exist between resistant and susceptible ‘B. glabrata’ haemocytes and their differential response to ‘S. mansoni’ ESPs; providing important insights into snail-schistosome interactions.

Lernaeocera branchialis (Linnaeus, 1767) is a parasitic copepod possessing a complex dual-host lifecycle. The “definitive” gadoid hosts, including Gadus morhua (Atlantic cod), Melanogrammus aeglefinus (haddock) and Merlangius merlangus (whiting), are infected by the fertilised female, which penetrates the host’s ventral aorta or bulbus arteriosus whilst undertaking extensive metamorphosis and a haematophagous lifestyle. The pathogenic effects of this activity upon the host have been well documented and mortality may occur, especially when multiple parasites are present. These negative impacts on cod, particularly juveniles, by L. branchialis have the potential to adversely affect cod aquaculture in the future, and already vulnerable wild cod stocks. This PhD project therefore, investigated the immune response of wild haddock and cultured-cod post-infection by L. branchialis, and the possible mechanisms by which the parasite modulates/evades the host’s immune response. The systemic immune response of both wild haddock and cultured-cod post-infection by L. branchialis depended on the maturation stage of the parasite, and in the former host species, upon the infection intensity. Wild haddock harbouring fully metamorphosed females showed an increase in circulating thrombocytes and a decrease in serum protein levels however; if multiple mature L. branchialis were present the haddock possessed reduced circulating monocytes, and increased circulating thrombocytes and serum anti-trypsin activity. Infection by L. branchialis was also associated with a suppressive effect on haddock serum spontaneous haemolytic activity. These responses were thought to be due to the host trying to counteract the increased damage caused by the massive increase in size and the feeding of the mature parasite, which is more pronounced when multiple parasites are present, resulting in the increase in some parameters and the ‘consumption’ of others. However, the effect of parasite-derived secretions and other pathogens due to observations on wild fish could not be discounted. The laboratory-infection of cultured-cod from two different sources was also performed in order to study the immune response over time. The two groups of cod showed differences in their immune response to L. branchialis. The first group showed suppressed respiratory burst activity of phagocytes, as the parasite reached the early penella sub-stage, whilst no suppression in phagocyte respiratory burst activity was found in the second group. The parasite was found to migrate along the afferent branchial artery of the cod where a thrombus formed and was present throughout its migration into the ventral aorta. At 14 d post-infection, leukocytes expressing Interleukin 8 mRNA were observed within the free-flowing blood at the periphery of the organising thrombus within the lumen of the ventral aorta. This was speculated to aid the recruitment and activation of leukocytes to the site, and the maturation and neovascularisation of granulation tissue. The infection of the second group subsided with the death of the parasite, and none of the parasites metamorphosed past the early penella sub-stage. The live parasites infecting the first group of cod did not possess IgM or complement component C3 binding on their cuticle, however, both IgM and C3 binding occurred on the dead parasites in the second infection trial. This may highlight the importance of these opsonins and the cytotoxic effect of phagocytes in the elimination of L. branchialis by some cod. However, the first infection was terminated as the parasite reached the early penella sub-stage due to a loss of stock cod prior to the study, so the long-term success of the infection can not be concluded. Therefore, the immune response to infection needs to be determined over the entire metamorphosis of L. branchialis to determine whether the infection was successful or not, and preferably in populations with varying susceptibility to L. branchialis. This will not be possible without further studies into the resistance of different stocks of cultured-cod. Many arthropod parasites, such as ticks and salmon lice, have been previously documented to produce pharmacologically active secretions, aiding host invasion and parasite feeding, preventing the host immune response from working effectively against the parasite, all aimed at improving survival of the parasite. Therefore, the effects of the secretory/excretory products (SEPs) produced during the initial infective stage and by the mature, fully metamorphosed female on the immune response of cultured-cod in vitro, and the location of exocrine glands associated with the oral region of the parasite were investigated. The SEPs from the infective stage of the parasite were found not to affect the intracellular hydrogen peroxide (H2O2) production of phagocytes. The practical difficulties in collecting large quantities of the SEPs from the infective stage meant that their effects could not be tested on the other host immune parameters studied. The SEPs from fully metamorphosed female L. branchialis, however, had a number of suppressive effects on the host immune response in vitro including: 1) suppression of the intracellular production of cytotoxic H2O2 during the respiratory burst of phagocytic leukocytes post-PMA stimulation, 2) suppression of the production of macrophage activating factor by leukocytes with a priming effect on naïve phagocyte function, and 3) suppression of the chemo-attraction ‘power’ of zymosan activated cod serum, i.e. anaphylatoxin activity, on head kidney-derived leukocytes. These effects were dose-dependent, and highlight the capacity of L. branchialis to suppress its host’s innate immune response at the local feeding area. Further work is required to establish the mechanisms by which the parasite-derived SEPs suppress these host immune parameters, and to identify which molecules produced by the parasite are responsible. The correlation between these in vitro results, and systemic immune parameters measured from laboratory-infected Atlantic cod and wild infected haddock are discussed. Host immuno-modulation by other arthropod parasites is mediated by pharmacologically active secretions produced by exocrine glands. Therefore, the exocrine glands of the infective and fully metamorphosed female L. branchialis were also investigated in order to identify those that might be responsible for the secretion of host-modifying products. Adult female exocrine glands were mapped using diaminobenzidine (DAB), most commonly known to stain peroxidases and catalases. These compounds are known to be involved in the neutralisation of harmful free radicals which are released during the respiratory burst and tissue damage. Such products may therefore be important protective secretory components at the site of feeding / infection. Exocrine glands were located in the infective stage associated with the oral region, one pair termed the anterior gland complex (AGC), and the other pair extending either side of the oral cone termed the circum-oral glands (CG). These were further investigated using light microscopy and transmission electron microcopy. The AGC and CGs possessed multi-component secretions and they possessed secretory vesicles, abundant and highly active rough endoplasmic reticulum and Golgi apparatus suggesting that protein is an important component of the secretory products. These glands were also observed in the fully metamorphosed females where they had increased in size within the cephalothorax post-metamorphosis. It is hoped that the identification of these glandular structures, which are thought to secrete within the local vicinity of the oral cone, will aid future studies regarding the identification and secretion kinetics of parasite-derived molecules during the infection and feeding process.

Virulence (the harm to the host during infection) is the outcome of continuous coevolution between hosts and parasites. This thesis adds to a growing body of work on host-parasite interactions, and describes experiments that study the effects of variation in the genetic and the environmental contexts of infection. All of them focus on interaction between the planktonic freshwater crustacean Daphnia magna and a naturally occurring parasite, the spore-forming bacterium Pasteuria ramosa. I show that elevated minimum temperatures that facilitate parasite growth drive natural epidemics of this parasite. I also demonstrate that the expression of infection traits in P. ramosa is temperature-dependent in a genotype-specific manner [genotype-by-environment (GxE) interactions]. These GxE interactions could maintain polymorphism through environment-dependent selection. Next, I test if GxG interactions for infectivity can be altered by environmental variation (GxGxE interactions), and find that this trait is quite robust to thermal variation. Infectivity is also more important in determining parasite fitness relative to the production of transmission stages, highlighting the importance of considering natural infection routes, an aspect sometimes overlooked in studies of host-parasite systems. Another experiment under different food and temperature regimes showed evidence for environment-dependent virulence-transmission relationships, a fundamental component of virulence evolution models. Lastly, I show that variation in temperature does not increase the cost to the host of resisting infection.

Almost 2 billion people world-wide are infected with parasitic helminths. These complex multicellular eukaryotic organisms are capable of establishing long-term infections even in the face of an intact immune response. Typically, in these settings regulatory components of the immune response, such as Foxp3+ T regulatory cells (Tregs), become dominant, limiting protective effector responses towards the parasite. Helminths are thought to have evolved mechanisms, including release of immunomodulatory molecules termed excretory-secretory products (ES), to sway the balance between the regulatory and effector arms of the immune response to favour their persistence. In this thesis both the development of a protective immune response toward, and the potential manipulation of the immune response by, the rodent gastrointestinal nematode Heligmosomoides polygyrus have been studied. Firstly, the effects of H. polygyrus ES (HES) on bone-marrow derived dendritic cells (DCs) were analysed. Although HES did not alter the phenotype of the DC it was found to be able to suppress the ability of the DC to respond to inflammatory stimuli. This activity was lost when HES was heat-inactivated (hiHES). After adoptive transfer, HES-pulsed DCs were able to induce a HESspecific T helper (Th)2-type response even if co-treated with an inflammatory stimulus. Th2-type responses are protective against H. polygyrus infection. Surprisingly, the ability of HES to generate a Th2-response in a co-treatment situation was not related to its anti-inflammatory properties; DCs co-treated with hiHES and an inflammatory stimulus were able to drive an equivalent Th2-response to HES in this situation. Next, making use of mouse strains with different susceptibility phenotypes to primary H. polygyrus infection, potential mechanisms of resistance were characterised. Development of granulomas in the gut wall were found to be associated with reduced worm burdens. Furthermore, in highly susceptible C57BL/6 mice, production of IL-23 was shown to be counter-regulatory to this process, as mice on the same background but deficient in this cytokine have increased numbers of granulomas and dramatically enhanced resistance. Susceptibility to H. polygyrus was also considered at the level of epigenetic regulation. A protein that binds specifically to methylated DNA, methyl-CpG binding domain protein (MBD)2, was found to affect the proportion of Foxp3+ Tregs within the CD4+ T cell population in vivo. Additionally, in vitro induction of Foxp3 in response to TGF-β was enhanced in MBD2-/- CD4+ T cells. MBD2-/- mice had a trend towards increased worm burdens when infected with H. polygyrus, suggesting that the difference in proportion of Tregs may limit generation of an effector response. Finally, the ability of HES to directly affect the regulatory arm of the immune response was focussed upon. It was found that HES was able to induce Foxp3 expression in naïve peripheral T cells, and that this was mediated by stimulation of the TGF-β pathway. The TGF-β mimic was of parasite origin as a pan-vertebrate TGF-β antibody was unable to block its effects but sera from H. polygyrus infected animals was competent to do this. Activity of this type was not limited to HES as ES from the ovine helminth Haemonchus contortus was found to have the same property. These data imply that some helminth parasites have evolved mechanisms to support generation of Foxp3+ Tregs, thus favouring the regulatory arm of the immune response and hence their own persistence.

A conserved acto-myosin motor complex is implicated in parasite motility and host invasion by a wide variety of Apicomplexan zoites. Until recently, only actin, myosin A (MyoA) and its putative light chain (MLC1 in Toxoplasma gondii) or myosin tail domain interacting protein (MTIP) in Plasmodium spp., had been identified as central to the function of this motor. Identification of two further components in T. gondii, the gliding-associated proteins (GAP45 and GAP50), has provided a valuable insight into how the motor may be anchored in the inner membrane complex (IMC) that lies below the plasma membrane. Results presented here demonstrate that Plasmodium falciparum (Pf)GAP45 and PfGAP50 are expressed and co-localise with PfMTIP at the periphery of merozoites. Both GAPs are found to be in complex with PfMyoA, and PfMTIP. Pulse-chase experiments indicate that the motor complex is assembled in two stages. PfGAP50 is incorporated after the formation of a ternary complex comprising PfGAP45, PfMyoA and PfMTIP. PfGAP45 is shown to be N-myristoylated and palmitoylated and may therefore function as a linker protein tethering the motor to the outer leaflet of IMC. Additionally, PfGAP45 is phosphorylated by calmodulin-dependent protein kinase 1 (CDPK1); a process that may be important in the regulation of the motor. Recombinant PfGAP50 is a well-ordered protein, whereas PfGAP45 has a low content of secondary structure. Potential interaction of GAPs with other motor components has been examined. Co-immunoprecipitation experiments, circular dichroism (CD) and fluorescence spectroscopic analyses have not provided any evidence of direct interaction with any other motor proteins.

The saproxylic habitat harbours a remarkable diversity of Coleóptera and associated parasitoids. Owing to the inaccessibility of this habitat and the often concealed nature of its associated organisms, we know very little about most saproxylic species. Many species are fundamental ecosystem components and have the potential of being economically important pests or biocontrol agents. Likewise, vast numbers of saproxylic Coleóptera and parasitoids are under threat. To date, the reasons and manner in which saproxylic organisms partition themselves within the saproxylic habitat is inadequately understood. Thus, the reasons behind saproxylic diversity and the reason why so many species are under threat remains unknown. Furthermore, our understanding of saproxylic pests and biocontrol agents is minimal. There is thus a pressing need for continued and thorough investigations of this unique habitat, in particular, the chemical ecology of saproxylic Coleóptera and their parasitoids is clearly a subject of considerable scientific and practical interest. This thesis investigates the role of fungi and volatiles in saproxylic systems, in particular, it questions their importance in the chemical ecology of saproxylic beetles and parasitoids. In the first study of its kind, Chapter 2 samples volatile organic compound (VOC) emissions from felled beech logs during their initial 30 months of decay. Volatile emissions were found to be in accordance with amounts detectable to insects suggesting that, in a natural decay situation, saproxylic volatiles are readily available as insect semiochemicals. Emissions of particular VOCs were found to differ significantly through time, indicating that saproxylic insects can potentially use semiochemical information to partition themselves according to decay stage. Potential sources of the detected compounds include wood, fungi and other saproxylic organisms. Fungal volatiles are speculated to have a particularly important involvement in these volatile emissions because fungal decay dynamics are shown to coincide with the release of particular volatiles. Chapter 3 expands upon concepts developed in Chapter 2 by investigating the role of fungi in the volatile emissions of decaying beech. Volatiles were sampled from beech logs inoculated with basidiomycetes (Stereum hirsutum, Coriolus versicolor, Phanerochaete velutina, Hypholoma fasiculare, Bjerkandera adusta and various combinations thereof). Fungal community structure was found to significantly affect the volatile profile of decaying wood, with specific VOCs associated with particular fungal treatments. The study concludes that volatiles of fungal origin are a strong potential source of semiochemicals for insects utilising and partitioning themselves within the saproxylic habitat and their natural enemies. Chapter 4 describes an insect attraction experiment and develops the findings of the previous studies in an entomological context. Wood-decaying fungi of the same species and combinations used in Chapter 3 (fungi known to have an effect on volatile emissions of decaying wood) were applied as bait in insect traps. Various beetles and parasitoids exhibited preferences for particular treatments. The dataset is notable for the absence of wood-dwelling insects suggesting that stimuli from fungi alone are insufficient in attracting such species. In light of the conclusions of Chapter 4, Chapter 5 explores the role of saproxylic fungi in a real wood-decay context. Beetles were reared from beech logs inoculated with the same fungal treatments as used in preceding chapters. Various beetle species exhibited significant associations with particular fungal treatments indicating that fungal community structure of decaying wood has marked effects on beetle species composition. Despite the fact that the same fungal species were used as Chapter 4, the dataset is notable for having a vastly different species composition to that of this preceding study (including the presence of numerous wood-living species) confirming that decaying wood is a rich source of stimuli and fungi alone tend to be an insufficient information source. This thesis concludes that saproxylic fungi have a marked influence on beetle communities and volatile emissions of coarse woody debris. Saproxylic fungi are also likely to be strong mediators of the ecology of parasitoids and other saproxylic insects groups.

Changes in wing development and development through the stadia of Aphis fabae are examined after parasitisation by Aphidius colemani along with the underlying mechanisms. Parasitoid oviposition into nymphs of winged gynoparae of A. fabae shows that wing development is inhibited and the earlier parasitism takes place, the stronger the effect. Consideration of the timing of events indicates that the active component(s) is most likely injected during oviposition and is not related to hatching and growing of the parasitoid larva. When extracts of venom glands are injected into late-second stadium aphids, many develop to fourth-stadium possessing rudimentary wingbuds, show developmental arrest and often die while moulting to the adult stage. Injections with extracts of female or male parasitoids into later stadia give similar results with regard to development to the adult but aphids injected in the late-fourth stadium develop normally. Experiments with long-day winged virginoparae reveal similar results in development to the adult but injections into long-day wingless aphids, which develop more rapidly, show this effect only when performed early in the third stadium. The earlier the injection before the final moult the greater the effect of the extract on preventing adult development. The results indicate that there is an active factor(s) in the female parasitoid’s venom that disrupts wing development and/or inhibits development to the adult; the loss of activity after treatment at 100 °C or with protease makes it likely that the factor(s) is a protein. Surprisingly, injections of extracts from male parasitoids have similar effects. The location and function of such a factor(s) in males are unknown. Fast Protein Liquid Chromatography is used to fractionate the parasitoid extracts and bioassays reveal activity in fractions 16-25. Aphidius ervi venom also inhibits development of A. fabae (a non-host) whereas A. colemani venom has no effect on the development of Megoura viciae (a non-host). Injection with lipopolysaccharides and infection with a fungal pathogen are used to test whether stimulation of the immune response affects wing development in the host and the possibility that the host is in control of inhibiting its own wing development in order to support its immune system is discussed.

Based on the published protein-protein interaction maps of five organisms and other public databases for domain-domain and protein-protein interactions, two new approaches are proposed to infer the protein-protein interaction network of the Anopheles gambiae (A. gambiae) mosquitoes. Our main contributions are: i) Adopted an orthologous protein pair transfer method that has so far not been seen in literature; ii) Proposed a new hybrid Bayes method; iii) Used voting machines at two levels of the combined classifier/predictor; iv) Used heterogeneous datasets as the training data; v) And finally, used the trained classifier to predict the protein interactions maps for A. gambiae, arguably one of a few least known organisms in terms of protein interaction mechanism. With the first method, the orthologous and in-paralogous protein clusters are extracted for both species. The relations between two peer-to-peer proteins in the two species are identified so that the interactions in the D. melanogaster protein interaction maps are transferred to pairs of interacting proteins in A. gambiae. The second strategy, namely the hybrid Bayes, is based on the domain composition of proteins, with which we utilize a probability model to build virtual domain-domain maps by integrating large-scale protein interaction data from five organisms, namely Saccharomyces cerevisiae, Caenorhabditis elegans, Escherichia coli, Mus musculus and Drosophila melanogaster. For the hybrid Bayes method, once the virtual domain-domain interaction maps are constructed, we propose two ways to predict the protein-protein interaction maps. These two methods are compared and then combined to form a voting machine to collectively decide a protein-pair's candidacy. The users could adjust the weights for different methods to flexibly control the output. Parameters are chosen through running different experiments on the training data set. While both the orthologous cluster and hybrid Bayes methods produce encouraging results the second one predicts more protein-protein interaction than the first. Yet these two data sets share a very small fraction of common interactions. We adopt a second voting machine and calibrate the parameters with the putative protein interaction data. Those parameters for the voting machine are used to predict the protein-protein interaction maps of the A. gambiae and produces reasonably good results.

Parasites can impose fitness effects on host populations: there may be seasonal and geographic variation in the interaction as well as over a longer time scale, host-parasite cospeciation. I investigate the Leucocytozoa parasites of birds as a model system to try to understand the role of diversity of infection, dispersal and scale on the nature of the coevolutionary process between the hosts and parasites. Leucocytozoa had effects on the breeding success of a population of the blue tit, Cyanistes caeruleus. Number of infections was important, and old birds infected with mixed lineage infections raised broods of significantly lower quality than those infected by a single lineage or uninfected birds. There was also evidence for individual lineage effects. The parasites showed seasonal dynamics, and overall prevalence followed a model previously proposed for the dynamics of avian malaria. Individual lineages, however, displayed different dynamics; this emphasises the importance of consideration of lineage identity. These results may have consequences for the evolutionary dynamics of this host-parasite interaction. The geographic context of the Leucocytozoa-bird interaction was investigated, and little phylogeographic structuring was found in parasite lineages across Europe. Contrary to predictions, the distribution of individual lineages followed the abundance-occupancy relationship, suggestive of widespread dispersal. At a larger phylogenetic scale I looked for evidence of host-parasite cospeciation in this system. There was a strong signal of coevolution due to cospeciation in 40% of the host-parasite associations. Migrant hosts harboured significantly fewer cospeciating parasites than residents and partial migrants. The implications and potential applications of this type of cospeciation analysis are discussed. I conclude by suggesting that the bird-Leucocytozoon system is an excellent model system for the study of host-parasite interactions, attaining high diversity and prevalence in some cases. Furthermore, the role of diversity, mixed lineage infections and dispersal are key to our understanding of the evolutionary dynamics of this system.

The Trichinella genus can be broadly divided into two clades that differ in respect to the formation of the host-parasite complex within the muscle cell.  Parasite excretory/secretory (ES) proteins may be responsible for the developmental modification of host cells, as well as the differences observed between the two clades.  Serine proteases are known to be involved in parasite tissue/cell degradation and invasion, and are abundant in the ES protein fraction of mature L1 larvae (ML) of Trichinella spiralis and Trichinella pseudospiralis (representative of the two clades).  Presented here is analysis of the T. spiralis secreted ML serine protease, TspSP-1 and its T. pseudospiralis homologue TppSP-1.  Using the T. spiralis draft genome assembly TspSP-1 was shown to be encoded by a multi-copy gene family, with three genes tandemly repeated.  The putative proteins share high levels of similarity at the N-terminus, which includes the catalytic domain and potentially all encode functional serine proteases.  Comparative analysis of TppSP-1 was carried out and at least one full length cDNA transcript was characterised, though initial results indicated that TppSP-1 may also be encoded by a multi-copy gene family.  Based on TspSP-1, the putative TppSP-1 protein also contained a catalytic domain, though it was lacking one of the catalytic triad residues, indicating a loss of serine protease function.  Several serine protease homologues that have lost catalytic residues have gained other functions, which may also be the case for TppSP-1.  Given the timing of expression is it possible that TspSP-1 and TppSP-1 may be involved in the invasion of the intestinal epithelial cells.  Whether or not these proteins interact with host or parasite proteins and function as typical serine proteases is yet to be determined.

Nippostrongylus brasiliensis is a rodent intestinal nematode with an important pulmonary migrating stage. Previous studies have observed a lack of TNF-α production and minimal recruitment of neutrophils, which led us to the belief that anti-inflammatory mechanisms could be active in the lung stage. In this study, lipopolysaccharide (LPS) stimulated alveolar macrophages (NR8383) or rat lungs were used as in vitro or in vivo inflammation models respectively. Both live N.brasiliensis larvae and NES significantly reduced the production of LPS-induced pro-inflammatory mediators, TNF-α and NO, but not IL-1β, in NR8383 cells. The inhibition of TNF-α production was related to the heatlabile and trypsin-sensitive fraction of NES concentrate. 1-D protein gel of NES concentrate revealed that the molecular weights of proteins are between 6kDa and 100kDa. Glycoproteins were found abundant in NES concentrate. The inflammatory processes, including NF-κB translocation and TNF-α gene transcription were significantly inhibited by NES and/or NES concentrate. In vivo, we observed a significant reduction of neutrophil recruitment (≈ 40%) by NES on a background of LPS (100ng/ml) induced inflammation. This reduction was associated with the significant inhibition in gene transcriptions of proinflammatory mediators TNF-α, IL-1β, iNOS, ICAM-1 and MIP-2 in bronchoalveolar lavage (BAL) cells. The down-regulation of pro-inflammatory mediators and inflammatory processes observed in this study suggests that N.brasiliensis larvae and/or NES are capable of modifying the normally potent LPS inflammatory response, both in vitro and in vivo. This study and planned future studies could be fundamental in developing anti-inflammatory agents with immune-active molecules in N.brasiliensis-derived products.

Neospora caninum and Toxoplasma gondii are closely related intracellular protozoan parasites associated with abortion in mammals. N. caninum was identified in 1984, described as a new species in 1988 and often previously misidentified as T. gondii. In this study molecular techniques were used to investigate the presence of N. caninum and T. gondii in naturally infected populations. To facilitate this a nested PCR technique, targeting the Nc5 region of N. caninum, was developed. Using this and an established T. gondii specific PCR the prevalence of N. caninum and co-infection with T. gondii were investigated in naturally infected mammals. The fox has been identified as a potential definitive host of N. caninum. PCR analysis of DNA extracted from tongue tissue determined a low prevalence of 1.4% (1/70) in British foxes. None of these foxes had detectable T. gondii DNA. Small mammals are a potential source of N. caninum infection. N. caninum was present in the brain tissues of all species investigated: Mus domesticus 3.1% (4/130), Rattus norvegicus 4.4% (2/45), Apodemus sylvaticus 3.4% (4/118) and Oryctolagus cuniculus 10.5% (6/57). T. gondii prevalences were significantly higher in all species tested. Co-infection was observed in all species except Rattus norvegicus. Investigation of vertical transmission of T. gondii in wild Mus domesticus, determined it was a common occurrence with 74.6% of foetuses from infected dams having detectable parasite DNA. A relatively high frequency of infection with N. caninum was detected in brain tissue of aborted lambs (18.9%). No significant relationship was found between N. caninum and T. gondii infection. Detection of N. caninum DNA was restricted to the brain in aborted lambs but was more widely distributed in rabbits. T. gondii DNA was detected in multiple tissues of both host species. These studies demonstrate that N. caninum is widely distributed in natural populations of mammals.

Gnathiid isopods (peracarida: Isopoda: Gnathiidae) are marine ectoparasites, in which the juvenile stages associate with fishes for several hours to days to feed on blood, plasma and tissue fluids, in order to complete their intricate life cycles. These cycles each include two juvenile moults and a final metamorphosis into non-feeding, adult stages. The blood meal ingested provides resources for adult survival, reproduction and embryological development. In the model gnathiid species Paragnathia formica, embryological development occurs in internal uterine sacs of the adult female, rather than in a brood pouch, as in most Peracarida. In this study, by means of traditional histology, nuclear staining and scanning electron microscopy, changes occurring throughout embryological development to the pre-hatching juvenile stages were examined, including gastrulation and three post-gastrulation stages. Loss of the egg shell/membrane as well as an intra-uterine moult were observed, and conversion of the yolk mass into larval tissues and organs was recorded. The internal morphology of the digestive tract was examined in third stage juveniles to track changes in cells lining the digestive glands with blood meal digestion. Dark, sub-cuticular cells were also detected prior to the metamorphosis into adult stages, which may function in the moulting process. The final moult accompanied dramatic tissue re-arrangement in the male cephalosome, during which the small head of praniza 3 juveniles adapted for feeding, transformed into an enlarged cephalon with prominent mandibles. The structural trait of the mandibles with other morphological male characteristics, form the basis of the taxonomic classification and identification of individuals within the Family Gnathiidae. The parasitic juvenile stages, if caught separately from males, are difficult to identify due to intra-specific polymorphisms. Thus attempts were made to link adult and juvenile stages of a variety of species including P.formica, using two approaches, namely telson morphometries and molecular typing. However, adult and juvenile telsons varied within a single species, and were often too conserved between species to be used as identification tools morphometric ally. On the other hand, molecular typing methods were more successful in identifying the host fishes on which juveniles had fed, rather than identifying the ectoparasite species themselves. This anomaly was possibly due to the long-lasting storage of nucleated fish red blood cells in the anterior hindgut of P.formica juveniles before being processed. Blood meal digestion was studied using in situ histochemistry and spectrophotometric proteinase assays on fed (praniza 3) juveniles, with unfed (zuphea 3) stages used as controls. It demonstrated predominantly protein breakdown by cathepsin-like cysteine proteases within the digestive glands during early, mid- and late digestion, with only limited activity in the anterior hindgut and rectal vesicle. Furthermore, serine protease inhibitors were partially purified, from first stage (zuphea 1) unfed P.formica juveniles using chromatographic techniques. These partially purified inhibitors were shown to display anticoagulant activities, possibly to facilitate feeding of the ectoparasite by preventing the host haemostatic reactions. This project succeeded in complementing information previously gathered on the developmental biology of Pi formica, with novel observations made of intra-uterinë moults during embryological development in females, of dark, sub-cuticular cells growing as digestion progressed in fed final stage juveniles, and of tissue re-arrangement occurring in the cephalosome during the final juvenile metamorphosis into males. Furthermore, a potentially novel species of Gnathia was discovered after microscopy observations of telsons, and discriminant function analysis was applied to gnathiid telson measurements for the first time to separate different species statistically. Finally, both biochemical investigations, namely on digestive proteinases and anticoagulant compounds in P.formica, were original, with new fmdings including protein digestion by cathepsin-like enzymes in the digestive glands offed fmal stage juveniles, and trypsin inhibitors displaying anticoagulant properties in unfed first stage juveniles.

I describe the isolation and characterisation of new sigma-viral isolates collected from Europe and North America. With these new isolates, I show that sigma virus has very low levels of viral genetic diversity across Europe and North America compared to other RNA viruses. I examine the susceptibility of Drosophila melanogaster to five of the viral isolates to investigate whether specificity exists in this system. The results suggest that there is little constraint on flies evolving resistance to all five viruses and that trade-offs between resistance against the five viruses are unlikely to explain why variation is susceptibility exists in wild populations of Drosophila melanogaster. Sigma-viruses increase the susceptibility of flies to the fungus Beauveria bassiana – that commonly infects insects in the wild. This could have profound effects in the wild where flies are constantly exposed to bacteria and fungus during feeding. One interpretation of the increased susceptibility of sigma-infected flies, is that the sigma virus is suppressing the Toll-pathway. However, I found no evidence for viral suppression of the Toll-pathway, nor did I find evidence that flies mount a Toll-dependent immune response against the sigma virus. This suggests that either Drosophila do not mount an immune response against the sigma virus, or that the immune response is controlled by other pathways. Finally, I describe the hypermutation of adenosines to guanosines in the genome of the sigma virus. The clustering of these mutations and the context in which thy occur indicates that they have been caused by ADAR–RNA editing enzymes that target double stranded RNA. This is the first evidence that ADARs target viruses outside of mammals, and it raises the possibility that ADARs could play a role in the antiviral defences of insects.

Currently, three species of the eukaryotic obligatory intracellular microsporidian parasites of the genus Nosema have been identified in bees, namely Nosema bambi infecting bumblebees (Bombus spp.) and Nosema apis and Nosema ceranae found in honeybees (Apis spp.). Given difficulties in identifying them Cj visually, I developed molecular genetic markers for these species and used them to understand their distribution and modes of transmission. I developed a PCR based method for the specific and sensitive diagnosis of N. bombi in bumblebees through amplification of partial ribosomal RNA gene fragments. A survey of N. bambi in European bumblebees allowed me to reveal that the parasite is common and widespread. Furthermore, partial rRNA sequences of microsporidians of North American bumblebees were very similar to European ones, suggesting that N. bambi has a Holarctic distribution. I conducted an , experiment to test for horizontal transmission of N. bambi via artificially contaminated flowers, and I analysed secondary hosts and wild flowers using molecular genetic ·tools for the presence of N. bombi. I was unable to demonstrate horizontal transmission, but suggest that the phoretic mite Parasitellus fucarum may act as a secondary host of N. bambi. In a further study, I found bumblebee ovaries to contain N. bombi, which hints at vertical transmission. Using in situ hybridisation of bumblebee ovaries, I was unable unequivocally to locate N. bambi. I then developed a method to differentiate between N. apis and N. ceranae based on PCR-RFLPs of partiat' small subunit rRNA and employed it to analyse microsporidian isolates of the Western honeybee Apis mellifera from across the world. From the data generated, I conclude that N. ceranae most likely jumped host from the Asian honeybee A. cerana to A. mellifera, probably within the last decade;N. ceranae is now found across most of the world. Using a temporal sequence of Nasema isolates from Finland from 1986-2006, I found that N. ceranae has been present in Europe since at least 1998.