Investigations have been made into the genetic control of immunity to the nematode Trichuris muris. Both background genes and genes within the mouse major histocompatibility complex (MHC), H-2, were shown to influence the expulsion of T. muris with the former having the stronger influence. At least two genes within the H-2 complex determined response phenotypes, the effects of "resistance" or "susceptibility" alleles at I-A being modulated by resistance or susceptibility alleles at aD end locus/loci. Differential responsiveness within slowly responding mouse strains suggested that parasite-dependent effects were also important. The primary antibody response to T. muris excretory/secretory (E/S) antigen, predominantly an IgG response, was also shown to be controlled by background and H-2-linked genes. In general, mouse strains less resistant to infection developed higher levels of IgG than- more resistant strains of mice. However strains of mice possessing the H-2q haplotype, irrespective of their genetic background, rapidly developed higher levels of IgG1 antibodies than strains of other haplotypes, H-2q haplotype mice tending to be more resistant to infection. Recognition of two high molecular weight (MW) E/S antigens by IgG as revealed by immunoprecipitation was also found to be almost exclusively H-2q restricted. This restriction may be partly quantitative but as such would operate in vivo due to the restriction on the ability to produce high levels of specific IgG. Both H-2q restricted phenomena may be part of, but not absolute requirements for, protective immunity. Parasite-induced effects on host immunity were also studied. Later larval and adult stages of T. muris were shown to be immunosuppressive, immunosuppression being long lasting and preventing the expulsion of subsequent infections. Vaccination with E/S antigen was shown to protect strains of mice which are slow to expel worms (poor-responder) or totally unable to expel worms (non-responder) from a primary infection with T. muris. However protection was slow to be expressed. Antigen recognition profiles of vaccinated strains of mice differed from their primary infection recognition profiles and included the recognition of the two high MW antigens shown to be H-2q restricted in a primary infection. Thus altering the mode or route of E/S antigen presentation may lead to shifts in responsiveness of H-2 genotypes to specific determinants and/or boost specific antibody levels sufficiently to reveal recognition of these antigens. Prior experience of a patent primary infection prevented vaccination protecting non-responder mice against subsequent infections. This inability was correlated with suppressed IgG1 antibody levels and failure to recognise three high MW antigens including the IL-2q restricted antigens. Using a panel of monoclonal antibodies raised against E/S antigen it was shown that E/S antigens, apparently including both immunogenic and immunosuppressive molecules, were localised to granules within the stichocyte cytoplasm of the adult T. muris stichosome.

The hoverflies (Diptera:Syrphidae) represent an apparently paradoxical visual Batesian mimicry complex, with what appear to be "poor" Mimics outnumbering their more accomplished counterparts. The purpose of this thesis is to determine how far conventional mimicry theory is capable of explaining the apparent paradoxes of mimicry in the hoverflies. It becomes obvious that determining the mimetic status of the supposedly poor Mimics is not a trivial task. Conventional experimental tests of mimicry, using captive predators, seem incapable of predicting the degree of protection enjoyed by a Mimic in the field. The research therefore concentrates on developing some novel empirical approaches to the study of mimicry. This includes developing a method of image analysis which yields an objective, single-value measure of the similarity between Model and Mimic patterns. This index of similarity is used to produce unique descriptions of the structure of mimetic communities in terms of Mimic frequency and similarity to the supposed Model. These profiles indicate that there is an objective basis to the perceived paradox, and suggest that there is not a simple relationship between the actual and perceived similarity of two patterns. The perceived similarity of Model and Mimic will be a key determinant of mimetic success. The index of similarity is also used as a basis for direct comparison of the supposedly mimetic hoverflies with a more established example of mimicry in the butterflies. This exercise demonstrates that an index of pattern similarity enables a unique comparative analysis of mimicry. It is proposed that an index of similarity also provides a unique opportunity to test our theoretical understanding of mimicry, if it is used in conjunction with a mathematical model that possesses some specific attributes. A suitable prototype model is developed and demonstrated. The thesis concludes with an indication that the novel empirical approaches developed here, have been adopted elsewhere. This latter work indicates that those hoverfly species which are apparently "poor" Mimics, may be exploiting some constraint in predator perceptual and cognitive systems to achieve mimetic protection, despite a relatively low degree of actual similarity to the Model species.

The development of the gastrointestinal nematode Heligmosomoides polygyrus (syn. Nematospiroides dubius) in the mouse was studied. The stage specific production of acetylcholinesterase was measured in both excretory/ secretory products and in worm homogenates and found to be maximal between days 4-6 post infection, corresponding to the fourth larval stage of the parasite's life cycle. Analysis of the proteolytic enzymes found in the same preparations of the parasite again revealed a stage specific release. Quantitative examination showed a maximum concentration of proteolytic enzymes in the early third larval stage, whilst qualitative analysis revealed a number of molecules at 96, 76, 42, 33, 18, 16, and 13 kDa in the early stages, which gradually disappeared as the parasite aged until only those at 76, 18, 16, 13 kDa remained by day 120. The molecules present on the surface of the various stages of the parasite were extracted using a number of procedures. Various stage specific surface molecules were identified as were two possible sex specific molecules at 76 and 145 kDa. The immune response to a primary infection of the parasite was characterised in three strains of mice with different degrees of susceptibility to infection (SJL, BALB/c and CBA). It was noted that the better the strain was at expelling the parasite, the greater and swifter was the response as assessed through the use of a number of criteria. These included white blood cell counts, differential cell counts, the Band T cellularity of the secondary lymphoid organs, the response of these cells to mitogens, the mucosal mast cell response, quantitative antibody response (Mancini and ELISA) and qualitative antibody response to parasite antigens (immunoblot). In each case SJL responded better than BALB/c which, in turn responded to higher degree than CBA. Functional host protective immunity was stimulated in the same three strains of mice using a challenge infection following a 9-day anthelmintic abbreviated infection. The same criteria were used to measure the immune response to the parasite as for the primary infection and, as for the primary infection, it was found that the high responder strains gave a more rapid and more intense reaction to the parasite than the low responder strain. Immunisation prevented the establishment of a proportion of the challenge infection and also resulted in the premature expulsion of parasites. The parasite surface molecules which were recognised by mice undergoing either a primary infection or an immunising infection were identified. It was revealed that molecules at 208, 145, 92, 76 and 62 kDa on adult parasites were recognised by mice which had expelled a primary infection. Mice which were immune to a challenge infection recognised molecules at 62 and 20-15 kDa on larval parasites. A molecule at 30.5 kDa was also recognised by immune mice and corresponded to the molecular weight of acetylcholinesterase in the ES.

Hoverflies (Diptera: Syrphidae) vary widely in their mimetic associations, comprising wasp-mimetic, bee-mimetic and non-mimetic species. Social wasp mimics are dominated by 'imperfect mimics' which outnumber their supposed models (Hymenoptera: Vespidae) by large factors. The purpose of this thesis is to determine to what degree Batesian mimicry can account for these paradoxes, and to test alternative hypotheses for the evolution of the yellow-and-black patterns. There is little evidence of an effect of wasp abundance on 'imperfect mimic' abundance across 23 years of trapping data, as predicted if mimics are protected from predators through their resemblance to wasps. The seasonal asynchrony and high abundance of 'imperfect mimics' relative to their models is also notable, as well as the possible significance of wasp predation on hoverflies. Predictions concerning the function of the colour patterns of 'imperfect mimics' are tested using the association between similarity to the model and flight agility (indirectly measured assuming a trade-off between reproductive potential and flight agility). There is no strong indication that mimetic protection is the primary function of the colour patterns, but the evidence concurs with an aposematic function, signalling to predators the unprofitability of attempting capture. These conclusions are tentatively supported by direct measures of flight agility, though the small differences among species are difficult to pick up. The data on reproductive morphology of hoverflies show considerable variation across species, especially in males. The existence of giant testes in some species suggests that methods of dealing with sperm competition in hoverflies are diverse and deserve further study. The high ratio of 'imperfect mimics' to both models and good wasp mimics is also partly explained by habitat disturbance; undisturbed habitats show significantly less 'imperfect mimics' as a proportion of the hoverfly population. Current relative abundance in the UK may therefore be very different to when the colour patterns evolved.

The seaweed fly, Coelopa frigida, lives in piles of rotting seaweed deposited on beaches by tides and winds. In all populations studied it is polymorphic for two gene arrangements on Chromosome I. A polymorphism at the alcohol dehydrogenase locus is strongly associated with this inversion and can be used to estimate karyotype frequencies. An extensive series of samples from natural populations has revealed a seasonal cycle in inversion frequencies but otherwise frequencies are remarkably constant both geographically and temporally. There is a consistent excess of heterokaryotypes in these samples. Three selective forces influencing inversion frequencies have been investigated. 1) An association between karyotype and development time, previously observed in the laboratory, has been demonstrated in conditions case to those in natural populations. 2) Viability differences between karyotypes have been examined. In natural populations there is some evidence that the excess of heterokaryotypes increases with larval density. In the laboratory heterokaryotypes are shown to have higher viability than either homokaryotype but the strong density dependence reported previously has not been observed. Viability differences are concentrated in the first two days of larval life and are probably related to the rate of supply of nutrients. 3) An association is demonstrated between karyotype and adult size - especially in males. Adult size is shown to correlate with longevity and fecundity of both sexes. Several experiments indicate that large males enjoy greater mating success than small males. The relationship between larval density, development time and adult size is described. The possibility that the effect of the inversion varies between populations or between alcohol dehydrogenase genotypes has also been investigated. A simulation has been used to study how these selective forces interact with one another, and with the changeable environment in which the flies live, and to examine the extent to which they can account for the observed karyotype frequencies.

Aggregation of the abundant neural protein α-synuclein contributes to cellular toxicity in Parkinson‘s disease. We have created transgenic nematodes carrying fusion constructs encoding human α-synuclein (S) tagged with YFP (V) and/or CFP (C) as a fluorescent marker. Using the unc-54 myosin promoter, a synuclein-YFP (unc-54::SV (NI)) fusion construct was abundantly expressed in the body wall muscles of Caenorhabditis elegans. Permanent integrated lines were successfully generated for unc-54::V (NI), unc-54::S+V (I), unc-54::SC+SV (I), unc-54::C+V (I), and unc-54::CV (I) using gamma irradiation. The outcrossed transgenic synuclein strains were radiation sensitive and have shorter life span and lower pharyngeal pumping compared to wild type N2 and unc-54::V (I) worms. Fluorescence Resonance Energy Transfer (FRET) was measured for all the transgenic strains. The unc-54::SC+SV (I) worms showed FRET signals intermediate between the negative (unc-54::C+V (I)) and positive (unc-54::CV (I)) control strains. Confocal images were taken to confirm the presence of FRET. FRET signals increase markedly during early adult life in unc-54::SC+SV (I) worms. RNA interference by feeding was performed in unc-54::SC+SV (I) worms to knock out the Hip-1 co-chaperone function, thereby increasing the FRET signal. unc-54::SC+SV (I) fusion worms were also exposed to pesticides such as chlorpyrifos and rotenone, and we observed an increase in the size and intensity of fluorescent aggregates thereby increasing the FRET signal. Finally we have quantified reactive oxygen species (ROS) for unc-54::SC+SV (I) fusion worms and NL5901 strains by using the H2DCF-DA assay, showing that ROS levels were increased by pesticide exposure.

The carrot fly (Psila rosae F.) is an important pest of the cultivated carrot (Daucus carota) and other crop species in the family Apiaceae, since the larvae burrow into and feed on the developing roots. Current P. rosae control relies heavily upon the use of chemical insecticides, but these are inadequate. The aims of this study were to investigate the chemical ecology of P. rosae, particularly with regard to long range attractant and repellent semiochemicals suitable for incorporation into integrated pest management strategies; the incorporation of attractant host plant extracts, or semiochemical attractants, into the monitoring programme; and the development of an autodissemination trap for release of the pathogenic fungus Entomophthora schizophorae in the field for biological control. A number of techniques for the extraction of volatile semiochemicals from a wide range of host and non-host plant species, and P. rosae adults themselves, were employed and compared. Samples were analysed by gas chromatography (GC), and the biologically active components in these complex natural product extracts were located by coupled GC-electrophysiological techniques and identified by coupled GC-mass spectrometry (GC-MS). Responses to the electrophysiologically active compounds were compared using electroantennographic (EAG) analysis: one unusually high EAG response was observed to a toxic component in hemlock (Conium maculatum) leaf extract (y-coniceine). Of the forty-two EAG active components identified from common crop species and C. maculatum, eight had not previously been reported. A range of bioassay techniques (including four-arm star olfactometers, V-tube olfactometers, and oviposition bioassays) were employed to determine behavioural activity of the samples and identified compounds, but only the oviposition bioassay showed significant behavioural discrimination to y-coniceine. Further studies of longer range behavioural responses to volatile semiochemicals were performed in the field. Significant responses were seen to a known field attractant (combined trans-asarone and hexanal) and, for the first time, to a microwave assisted solvent extract of celery (Apium graveolens) leaf. A prototype autodissemination trap for E. schizophorae was produced and evaluated.

This thesis examines the relationship between insect life history and behavioural decisions and underlying cellular biochemistry, with particular focus on bethylid parasitoid wasps in the genus Goniozus. This comprises the first major body of work attempting to draw links between the underlying metabolome of an organism and its behaviour. This thesis further optimised the first known example of a combined LC-MS and NMR metabolomic approach capable of analysing extremely low biomass samples (<1 mg), a vital requirement when studying the behaviour of individual organisms. Part 1 of this thesis details the optimisation and validation of this metabolomic approach, whilst also examining the effects of aging on the metabolome of adult Goniozus wasps. Part 2 applies this approach to examine the effects of diet, host species and host aging on Goniozus wasp behaviour and biochemistry. Comparisons of the metabolomes of starved and honey fed wasps indicate that G. legneri is capable of utilising a carbohydrate rich diet as an energy source. Aged honey fed wasps possessed higher levels of large storage lipids, such as tri- and diacylglycerides, than starved wasps of the same age. Metabolomic analysis also detected a legacy effect on the metabolome of G. legneri associated with differences in the species of host each wasp was reared on. A similar legacy effect was confirmed when examining the metabolomes of wasps reared on artificially aged hosts. Whilst Goniozus wasp oviposition behaviour was altered by the species of host presented, no links between changes in a wasp’s metabolome and its resulting contest behaviour were found. Part 3 of this thesis examines the morphological, behavioural and chemical mimicry of another wasp, the hyperparasitoid Gelis agilis. G. agilis demonstrated an enhanced predation avoidance rate compared with control species, similar to that of the black garden ant Lasius niger. Agitation of G. agilis also resulted in the chemical emission of a known ant alarm pheromone.

Background. Necator americanus is one of the major causes of human hookworm infection, affecting over 800 million people worldwide. Hookworm infections cause gastro-intestinal bleeding, anaemia and iron deficiency, and are associated with high rates of morbidity, especially in children. Although chemotherapy has proven effective, high rates of reinfection are reported in socioeconomically developing countries, possibly due to the short-term efficacy of anthelmintic drugs in addition to individual predisposition to these infections, raising interests in developing suitable alternatives to chemotherapy which are capable of providing complete, long-term protection against hookworms. Understanding of the molecular mechanisms used by Necator americanus larvae to penetrate the human skin and the vasculature would therefore aid the development of effective vaccines against this important pathogen. Methods. First, Necator americanus larval exsheathing fluid (EF) and excretory/secretory products (ES) were profiled using gel electrophoresis and enzyme assays. Protease inhibitors against the main protease classes were used to determine which proteases are present in larval products. Second, the interaction of larval EF and ES products with human skin and extracellular matrix (ECM) macromolecules including collagens I, III, IV and V, fibronectin and laminin was investigated using western blots and protein separation by gel electrophoresis. Third, the impact of Necator americanus larval EF and ES on the endothelial barrier was examined using human umbilical vein endothelial cells (HUVEC). Permeability, an essential endothelial barrier function, was assessed during treatment with larval products, using transendothelial electrical resistance (TEER), and post-treatment using albumin-tracer flux. Finally, at the cellular level, responses to treatment with larval products were assessed by investigating molecular changes at cell-cell vascular endothelial (VE)-cadherin junctions and actin filaments, and by determining levels of secreted inflammatory cytokines, IL-6 and IL-8, and vascular endothelial growth factor (VEGF) in the culture medium. Results. It would appear that a repertoire of larval proteases, including serine, cysteine, aspartyl and metalloprotinases, caused partial degradation of skin macromolecules, collagens I, III, IV and laminin while fibronectin was fully degraded. Proteolysis of skin- and ECM macromolecules was related to the characteristic presence of proteolytic enzymes in larval products. The presence of transglutaminase activity was confirmed in both EF and ES products. Larval proteases caused a dose related increase in endothelial permeability, characterised by a decrease in monolayer resistance (TEER) with increased permeation of albumin tracer, which was minimal in the presence of a cocktail of protease inhibitors. These barrier changes were associated with disruption of junctional VE-cadherin and F-actin, the formation of intercellular gaps and an increase in endothelial secretion of IL-6 and IL-8. Conclusions. Necator americanus larvae produce a repertoire of proteolytic enzymes which could play an important role in negotiating the skin and breaching the endothelium to gain access to the host’s blood circulation.

Despite major advances in the diagnosis of infections for many pathogenic organisms, there is still a problem obtaining an accurate immunodiagnosis of Echinococcus infection due to its serological cross-reactivity with other species of taeniid cestodes or at higher taxonomic levels. For this reason, there are ongoing efforts to develop a better method for diagnosis of echinococcosis, especially when the parasite has a crucial role in hypersensitivity reactions. Like other helminthic infections, one of the immunological hallmarks is an elevated serum concentration of parasite-specific IgE. Our aim was to assess the use of IgE reporter system as a possible new method for diagnosis Echinococcus spp infection, using RS-ATL8 NFAT Reporter System, which is a humanised rat basophilic leukaemia (RBL) cell line that can be used to detect the presence of specific human IgE directed against Echinococcus allergens and cross-link their receptors, depending on luciferase generation as an indication of presence of parasite-specific IgE, pointing to infection. Towards this goal, we first optimised the use of the humanised RS-ATL8 Reporter System. This was achieved by optimisation of experimental conditions, such as cell density, stimulation time, optimum conditions for sensitising factors and stimulant optimum concentration. Once a robust standard operating procedure had been elaborated, the second goal was to choose a few Echinococcus antigens for the investigation into their immunogenic properties and potential diagnostic value and to express them recombinantly for testing through the RS-ATL8 NFAT Reporter System. The chosen antigens were EF1-alpha, EgAg5, AgB2, Cyclophilin, Eg19, EgTeg, and EgTPx.