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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.

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.

From an evolutionary perspective, natural selection is expected to maximize transmission to new hosts. If a live, mobile host often benefits parasite transmission, the question arises as to why malaria parasites are virulent? The favoured trade-off view of virulence evolution assumes that virulence arises as an unavoidable consequence of parasite resource exploitation within the host that is necessary to maximise parasite transmission. However, virulence is not always a simple function of parasite density and can arise as a result of immune-mediated virulence (immunopathology). This thesis explores how immunopathology contributes to virulence on the one hand, and parasite transmission on the other, in order to improve our understanding of parasite virulence evolution. In tackling this question, the role parasite genetic diversity plays in determining immunopathology induced during malaria infection was also addressed. Using the rodent malaria Plasmodium chabaudi chabaudi (P.c.c.) in C57BL/6 mice, I explored whether immune factors – in terms of specific host cytokines central to the protection-pathology balancing act of the immune response elicited against malaria parasites – help to determine the virulence induced during infection with genetically distinct parasites, and if so, what effect this may have on transmission-stage parasites. I showed that the cytokine milieu induced by P.c.c. parasites during primary infection varies with parasite genotype and that virulence can arise independent of parasite density, via immunopathology. Specifically, I showed propensity to induce the pro-inflammatory cytokine tumour necrosis factor [TNF]-a contributes to the virulence induced, regardless of P.c.c. clone. Importantly, I also showed that across P.c.c. genotype, TNF-a reduces the density of transmission-stage parasites. Thus, virulence is not always a simple function of parasite replication, having an immune-mediated component which acts to reduce transmission potential. The importance of parasite genotype in determining the degree of immunopathological virulence induced during malaria infection was revealed by studying the anti-inflammatory arm of the immune response. The extent to which the anti-inflammatory cytokines interleukin [IL]-10 or transforming growth factor [TGF]-b limited the immunopathology induced during P.c.c. infection depended on parasite clone. In addition, parasite genotype played a key role in determining how such anti-inflammatory manipulations affected the density of transmission-stage parasites; being detrimental, beneficial or incidental to parasite fitness, depending on P.c.c. clone. Although the general mechanisms of immune regulation are qualitatively unchanged across distinct P.c.c. clones, these data emphasize the importance of parasite genotype: distinct clones differ quantitatively in immune regulation, which contributes towards their distinct virulence and fitness schedules. Overall, I found that even within a parasite species – in this case P. chabaudi – the effect of immunopathology on the virulence-transmissibility relationship may be genetically variable and may not conform to that predicted by the trade-off hypothesis, having the potential to alter the costs and benefits of virulence, depending on parasite genotype. Thus, the host immune response may play a role shaping virulence evolution and defining the limit to malaria virulence in nature.