Parasitic platyhelminths are a major burden on humanity through their direct impacts on human health and the huge economic losses they cause, predominantly by undermining agricultural productivity and associated anti-parasite treatments. Previous studies on helminth parasites have highlighted the potential of the neuropeptide signalling system as a source of novel parasite control targets. The use of model organisms is preferable when the target organism is prohibitively difficult or unethical to study directly. The free living microturbellarian Macrostomum lignano is an ideal candidate for the study of parasitic flatworms due to its ease of culture, small size, availability of genomic and transcriptomic datasets and its basal phylogenetic position within the phylum Platyhelminthes. Firstly, this project mapped the distribution of four novel neuropeptides identified from the M. Iignano genome and uses RNA interference (RNAi) on one of those associated neuropeptide genes in an attempt to knockdown the associated transcript and delineate function. Subsequently, the expression of these neuropeptides was examined in selected parasitic flatworms. Despite being successful in the treatment of various parasitic flatworms of animals and humans, the mechanism of action of the anthelmintic praziquantel (PZQ) remains open to debate, hampering efforts to identify mechanisms of resistance and the design of novel drugs which could emulate its effects in parasitic flatworms. The mechanism of action of PZQ was investigated in M. lignano by first determining if the worm was PZQ sensitive. Next the genome was screened for the presence of voltage operated calcium channel (VOCC) β subunit genes (one of the proposed targets of PZQ) which was subsequently silenced using RNA interference to determine if it had a role in PZQ sensitivity in M. lignano. A role for these VOCCs in PZQ action in M. lignano was confirmed using RNAi.

Biological invasions continue to alter the structure and functioning of ecosystems worldwide. While the vectors and pathways of establishment of Invasive species are understood, predicting the ecological changes, or impacts, these species can exert on recipient ecosystems is lacking. Using invasive freshwater amphipods as models, this thesis aimed to investigate the role of intraspecific variation and interspecific interactions in understanding and predicting invasive species' impacts. This work first looks at the inherent variation in feeding behaviour within and between populations of an invasive amphipod, and discusses the implications of such variation for the use of functional responses, or the relationship between feeding rate and prey density. in invasion ecology. Secondly, behaviours associated to dispersal and impacts, as well as parasitic profiles, are compared between two UK populations of Dikerogammarus villosus. Differences between the populations show that monitoring population level differences, including behaviour and parasitism, can inform on invasive species' spread and impacts. Thirdly, interspecific interactions are used in order to elucidate the potential impac s of Dikerogammarus haemobaphes. Two aspects in particular are highlighted: predator-prey interactions, using the comparative functional response framework, and interference competition with D. villosus in terms of habitat use. Finally. cross-ecosystem interspecilic interactions are considered and their effects on a critical ecological process: leaf litter degradation. Using two invasive/native plant pairings, the individual leaf shredding abilities of an invasive and a native amphipod are compared in a laboratory setting. While both native plant species were consumed more readily, no difference was detected between the two amphipods. However, an in situ leaf litter bag experiment showed that the indirect effect of the invasive amphipod on macroinvertebrate community diversity may modify the degradation of certain plant species. This thesis therefore provides empirical evidence of the importance of intraspecific variation and interspecific interactions for the furthering predictive ecology

Molecules in the secretions of S. mansoni cercariae (CTF) aid the penetration of cercariae into host skin. Similarly, molecules in the saliva of 0. moubata (TSG) allow successful feeding. Examples of such molecules are anti-platelet factors, vasodilatory agents and anti-coagulants. These molecules are also exposed to the immune system of the host and induce potent immune responses which may be used for epidemiological analyses of exposure to these parasites. In one-dimensional SDS-PAGE, sera from rabbits immunized with crude homogenates of mouse platelets or human platelets have antibodies (anti-mph and anti-hph respectively) which cross-react strongly with a 46 kDa antigen and weakly with 20 kDa and 70 kDa antigens present in soluble extracts of different life-cycle stages of S. mansoni. Interestingly, the antibodies specific to the 46 kDa S. mansoni protein reacted to a similar-sized molecule in TSG from 0. moubata. Initial mass spectrometric analysis of the 46 kDa band in extracts of S. mansoni and 0. moubata, presumed to be this Platelet-like molecule (PLM46), indicated the molecule was actin. In order to determine whether other proteins of CTF and TSG are present at 46 kDa and to identify the 20 kDa and 70 kDa molecules, two-dimensional gel electrophoresis was performed. The cross-reactive antibodies, affinity purified by acid elution from the electro-blotted 46 kDa protein of both CTF and TSG and antibodies from anti-hph produced by acid elution from nitrocellulose membrane bound CTF and TSG significantly inhibited human and mouse platelet shape change and aggregation induced by agonists such as collagen, thrombin and ADP. When antibodies specific for actin were removed from the anti-hph using purified rabbit actin on .a n affinity column, the inhibitory effect of the cross-reactive antibodies on platelet aggregation still occurred. In order to determine the mechanism of platelet aggregation inhibition by these cross-reactive antibodies, P-selectin expression and fibrinogen binding to platelets were investigated.

No description available.

No description available.

No description available.

Leishmania parasites are the causative agents of a diverse spectrum of infectious diseases termed the leishmaniases. These digenetic parasites exist as intracellular, aflagellate amastigotes in a mammalian host and as extracellular flagellated promastigotes within phlebotomine sand fly vectors of the family Phlebotominae. Within the sand fly vector’s midgut, Leishmania has to undergo a complex differentiation process, termed metacyclogenesis, to transform from non-infective procyclic promastigotes into mammalian-infective metacyclics. Members of our research group have shown previously that parasites deleted for the L. (L.) major cDNA16 locus (a region of chromosome 23 that codes for the stage-regulated HASP and SHERP proteins) do not complete metacyclogenesis in the sand fly midgut, although metacyclic-like stages can be generated in in vitro culture (Sádlová et al. Cell. Micro.2010, 12, 1765-79). To determine the contribution of individual genes in the locus to this phenotype, I have generated a range of 17 mutants in which target HASP and SHERP genes are reintroduced either individually or in combination into their original genomic locations within the L. (L.) major cDNA16 double deletion mutant. All replacement strains have been characterized in vitro with respect to their gene copy number, correct gene integration and stage-regulated protein expression, prior to phenotypic analysis. HASPA1 was not detected in cultured promastigotes, but was expressed in mouse isolated amastigotes. Parasite mutant lines were passaged through susceptible BALB/c mice, during which HASPA2 gene containing mutant lines, in the absence of a HASPA1 gene, were shown not to develop lesions. Mouse-passaged parasites were used to infect the L. (L.) major specific sand fly vectors, Ph. (Ph.) papatasi and Ph. (Ph.) duboscqi. The progress of parasite metacyclogenesis was then monitored over twelve days, by midgut dissection and microscopy. Metacyclogenesis was not fully recovered in any of the replacement mutants tested. Surprisingly, HASPB protein expression could not be detected in the replacement mutants within the sand fly midgut, although HASPB protein was readily detected when the same parasite lines were cultured in vitro. The same was true for SHERP, although in situ expression was recovered in the presence of a HASPB gene, which itself did not expressed detectable HASPB protein levels. These observations suggest a requirement for one or multiple as-yet-unidentified regulatory component(s) for HASPB expression within the sand fly midgut and these are not required in culture. Quantitative PCR data suggested HASPB upregulation to be essential for metacyclogenesis completion, suggesting a sand fly specific function for HASPB.

Many helminth parasites are able to survive for long periods in immunocompetent hosts. It has been suggested that the successful establishment of helminths is in part due to the induction of regulatory T cells (Tregs) which suppress anti-parasite immune responses. A possible mechanism by which parasites induce Tregs would be production of TGF-β, which can induce immunosuppressive Treg cells and also directly suppress immune responses. TGF-β homologues have been identified in parasitic nematodes including Brugia malayi, Schistosoma mansoni and Ancylostoma caninum. The B. malayi TGF-β homologue, Bm-TGH-2, has been demonstrated to signal through the mammalian TGF-β receptor, which indicates the TGF-β homologues may have a function in the host. We hypothesised that Treg induction through parasite-derived TGF-β could be a potent method by which parasites could evade the host immune system. To address this, molecular techniques were first used to identify and characterise the transcription of novel TGF-β homologues from the intestinal parasites Haemonchus contortus, Heligmosomoides polygyrus, Nippostrongylus brasiliensis and Teladorsagia circumcincta, showing that TGF-β homologues are present in a wide range of parasites. TGF-β activity was then shown in products from both S. mansoni and H. polygyrus, and indeed the H. polygyrus products were found to induce Tregs through the TGF-β pathway. Antiserum against bacterially expressed H. polygyrus TGF-β homologue (Hp-TGH-2) was produced, and used to probe H. polygyrus excretory/secretory products, showing that Hp-TGH-2 is secreted. Attempts were also made to express recombinant Bm-TGH-2 (B. malayi TGF-β homologue) in insect cells, however the purified protein proved not to be functional. - v - In vivo mouse models of B. malayi infection were tested to examine the phenotype of T cell responses at the site of infection. An accumulation was found of CD4+Foxp3+CD25+CTLA-4hiCD103+ T cells, which resemble activated natural Tregs, and which were suppressive in vitro. This proportion of Tregs at the site of infection diminishes over time, however CD103 expression (which is associated with activated Tregs) is increased on Tregs present at all timepoints up to day 21 postinfection, indicating that although a growing effector response may outgrow Tregs over time, the Treg population remains activated. Using an adoptive transfer model, it was shown that the Treg induction could spread to other bystander responses. In IL-4R-deficient mice, Treg accumulation was unaffected, indicating alternatively activated macrophages (which are absent in these mice) are not required for Treg induction.

The study of social evolution and virulence in parasites is concerned with fitness consequences of trade-offs between parasite life history traits and interactions between parasite species and/or genotypes with their hosts. I develop our understanding of social evolution and virulence in parasites in several ways. (1) I review empirical evidence for the fundamental predictions of virulence-transmission trade-off theory and demonstrate that the fit between theory and data is primarily qualitative rather than quantitative; that parasites differ in their degree of host generalism, and this is likely to impact virulence in four ways. (2) I take a comparative approach to examine the underlying causes of an observed statistical variation in the size of parasite infectious doses across taxa, revealing that mechanisms used by parasites to infect hosts are able to explain variation in both infectious dose and virulence. (3) I formally compare data on human pathogens to explain variation in virulence across taxa, revealing that immune subversion and not growth rate, explains variation in virulence. This allows me to predict that immune subverters and not fast growing parasites are likely to cause the most virulent clinical infections. (4) Using bacteria and their naturally infecting viruses (bacteriophage), I take an experimental approach to investigate the consequences of coinfection for parasite life history traits, and find that viruses cultured under a mix of single infections and coinfections evolved plasticity; they killed hosts more rapidly when coinfecting, and this resulted in high fitness under both single infection and coinfection conditions. (5) I experimentally investigate how selection within and between hosts and patches of hosts affects the fitness and virulence of populations of these viruses. I find that limited host availability favours virulent, faster killing parasites with reduced transmission; suggesting high, rather than low, virulence may be common in spatially structured host-parasite communities.

Parasites are thought to have large effects on their host populations, driving genetic change, population density changes, speciation and be a major selective force maintaining sexual reproduction. Indirect signatures of parasite-mediated selection are common, but explicit examples of parasite-mediated selection in nature are lacking. In this thesis I examine parasite-mediated dynamics in a natural population of Daphnia magna that experiences an annual epidemic of the bacterial pathogen Pasteuria ramosa. I also test a novel hypothesis investigating the relationship between parasitism and the production of resting eggs. In chapter 2 a combined field study and laboratory infection experiment illustrates one of the best examples of parasite-mediated selection in a natural population, with Daphnia collected after a parasite epidemic having higher levels of parasite resistance than those collected before. This chapter also explored the relationship between parasitism and resting eggs, which are only produced during the sexual phase of reproduction. Daphnia that were reproducing sexually in the field prior to the parasite epidemic were more susceptible, supporting higher levels of parasite growth, than their asexual counterparts. This supports the idea that some genotypes invest in sex at the expense of parasite resistance. In chapter 3 I used molecular markers to investigate genotype frequency changes in the same population in relation to the parasite epidemic. The parasite epidemic was found to be associated with genetic change in the population, and a laboratory infection experiment revealed that the genotype most resistant to the parasite was also most common following the peak of the parasite epidemic. While chapter 2 explored a genetic relationship between susceptibility and resting eggs, chapter 4 explores whether crowding conditions, cues indicating parasite prevalence in the population, or direct exposure to parasite spores can induce resting egg production. I found that crowding conditions or parasite prevalence enhance levels of male and resting egg production, but patterns were entirely dependent on Daphnia genotypes. There was no indication that exposure to parasite spores affects levels of sexual reproduction.