Saprolegnia parasitica is one of the most devastating animal pathogenic oomycetes causing massive economic losses every year worldwide. The disease – saprolegniosis, is characterised by the appearance of white cotton-like spots on the skin, fins and gills of freshwater fish, leading to its death by haemodilution. More efficient control methods will be derived from a better understanding of S. parasitica, but inefficient transformation protocols currently are limiting the basic molecular knowledge This thesis describes the first application of transient gene silencing in S. parasitica. A gene that codes for a putative tyrosinase from S. parasitica, SpTyr, was selected to investigate the suitability of the RNAi technique to functionally characterise genes. Transient gene silencing can be used to functionally characterise genes in S. parasitica. In order to infect a host organism S. parasitica needs to attach to it. We found that detachment of S. parasitica cysts requires strength 3 times higher when compared to other Saprolegnia spp. I also demonstrate through functional genomics that a gene, SPRG_018636, that encodes for a fibronectin protein is involved in spine formation. Aquacultures often use, as a method of sterilisation, UV systems. This method has proven to be efficient against bacteria, virus and protozoa. However, we studied the effect of UVB and UVC on the growth of several oomycetes including S. parasitica and Saprolegnia diclina and it was found that S. parasitica is UV resistant. Key players from the resistance mechanism were not identified but significant knowledge was acquired on recovery from UV damage. Small steps were taken towards the development of a stable transformation protocol with the work on this thesis. Nevertheless, further studies will be necessary to accomplish this goal. Functional characterisation of a homolog of PiNuk7 found in S. parasitica genome was performed. This protein unlike Phytophthora infestans homolog did not translocate into the host cell.

Some of the most devastating plant and animal pathogens belong to the oomycetes. Saprolegnia parasitica, a major pathogen of freshwater fish and the causal agent of Saprolegniosis, causes large economic losses in aquaculture due to the death of freshwater fish which contract Saprolegniosis. Losses due to Saprolegniosis are also incurred by natural fish populations. Saprolegniosis was previously controlled through the use of a biocide called malachite green, until the dye was found to have potentially carcinogenic effects. Following this discovery, the use of malachite green on fish destined to enter the food chain was banned worldwide and subsequently there has been an increase in the occurrence of Saprolegniosis. This work investigates various aspects of S. parasitica with the view to inform the development of new control strategies. Three cDNA libraries, including two interactive libraries, were analysed and bioinformatics provided valuable insight into the genes expressed not only by S. parasitica but also by the host. The annotation and expression profiles of several of these genes are described in this work and the potential of S. parasitica genes as vaccine targets is discussed. One protein, identified as a vaccine candidate, is annotated, cloned and overexpressed in order to evaluate its efficacy as a vaccine. It has been shown previously that the plant pathogenic oomycete Phytophthora infestans is able to utilise a protein localisation motif, the RxLR, in order to translocate effector proteins into the host. The question as to whether S. parasitica possesses a similar translocation system is addressed in this work. Localisation studies on an RxLR protein of S. parasitica origin are described and the microscopic interaction between S. parasitica and its host are described and attempts to optimise production of interaction structures are discussed.

Thesis (M.S.)--University of North Carolina at Wilmington, 2005. / Includes bibliographical references (leaves: [13]-17)