Parasitic isolates of Saprolegnia diclina Humphrey from Coho salmon (Oncorhynchus kitsutch Walbaum) were grown under controlled laboratory conditions to test the effect of environmental parameters on growth. This study points out the significance of medium selection and methods of determining growth in introducing variability into the results of experimental studies of parasitic Saprolegniaceae. The results indicate that there is a significant effect of temperature and pH on growth and morphological development while the effects of phosphate concentration, aeration and nitrogen sources appear to be as significant but vary with medium used or method in determining growth. / Science, Faculty of / Botany, Department of / Graduate

The success of eukaryotic oomycete pathogens depends largely on effector proteins, molecules that manipulate or interfere with host defence mechanisms in the extracellular space or inside their host cells. One economical important oomycete parasite is the fish pathogen Saprolegnia parasitica, which is the causal agent of saprolegniosis. S. parasitica is responsible for devastating losses in the aquaculture industry worldwide. In order to effectively fight S. parasitica it is crucial to understand its pathogenicity strategies. With the focus on cytoplasmic effector proteins, the genome of S. parasitica was screened in the present study for secreted proteins that possess Ricin-B-like lectin domains or potential N-terminal RxLR-motifs. A list of more than 30 potential effector candidates was compiled and gene expression analysis was performed with RNAseq. Possible functions of the candidate effectors are discussed. SPRG_21856.2 (Ricin-B-like lectin candidate) and SpHtp3 (RxLR candidate) were selected for further analysis. Although two different expression systems, Escherichia coli and Pichia pastoris, were used, recombinant protein production of SPRG_21856.2 was not successful. Possible reasons and alternative overexpression methods are discussed. For SpHtp3 recombinant protein production was successful. Functional characterisation revealed that SpHtp3 is an active nuclease capable of destroying both DNA and RNA molecules. Furthermore, SpHtp3 was found to specifically enter fish cells in an O-sulfate and pH-dependent manner. Interestingly, the potential N-terminal RxLR-motif of SpHtp3 was not required for translocation. In support of that finding also PsHtp3, a Phytophthora sojae homolog of SpHtp3 that lacks an RxLR-sequence, was capable of translocating into fish cells. After translocation SpHtp3 localised in small vesicles from which it was only released when the host cells were under direct attack by S. parasitica. SpHtp3 is the first protein described from an oomycete that exhibits both self-translocation and nuclease activities combined in one molecule. Potential functions and future applications are discussed.

Oomycetes are a class of organisms within which there are a range of devastating pathogens with a variety of different hosts including plants and animals. Phytophthora infestans, the causal agent of late blight in potato, is one of the most important plant pathogens in economical terms. Saprolegnia parasitica is a fish pathogenic oomycete capable of causing disease in freshwater fish species. This study uses both pathogens to work towards understanding the fundamental biology of oomycetes and the development of effective control strategies.

Saprolegnia parasitica is a pathogenic oomycete that cause saprolegniosis. Freshwater fish like salmon and trout species are particularly vulnerable to infection, which is characterized by cotton-like grayish mycelial growth on the surface of the fish. Currently, an effective treatment for this disease is not available. This pathogen has a great impact on freshwater fish species world-wide. An initial step to keep this devastating disease at bay is the ability to detect the responsible pathogen, so that appropriate actions could be taken before it becomes widespread. The development of molecular tools that will accurately and rapidly detect S. parasitica is the main goal of this project. This project is divided into two main sections. The first section describes initial marker design efforts that were focused on the internal transcribed spacer (ITS) regions. Efforts were also made for the collection of field samples and the generation of our own ITS data that includes a number of Saprolegnia spp. Compiled sequence data allowed the identification of unknown samples and the adoption of the clade taxonomic system that other researchers had established for species designations. The accumulated sequence data helped to clarify the taxonomy within the genus Saprolegnia and complemented previous studies. The design of broad specificity PCR primers also allowed a quick initial detection of Saprolegnia spp., which could then be identified to species either by determining ITS nucleotide sequence or by a subsequent step of RFLP marker. Isolates sequence data in the compiled sample collection could be used for validation purposes in further marker development. The second section of the project described the development of higher specificity molecular markers for the detection of S. parasitica. These were based on the study of three different gene loci as potential markers. These included the Pumilio RNA-binding protein (Puf), Glutathionylspermidine synthetase (Gsp) and the thiazole biosynthetic enzyme (Thi4). The nucleotide sequence of each locus was studied to develop suitable PCR primers that were then refined through testing against our isolate collection to improve their specificity for the target species. Saprolegnia parasitica-specific markers were developed for the Puf and Gsp loci and these were further evaluated using our field collected samples. / Graduate

Thesis (Ph.D.)--Aberdeen University, 2008. / Title from web page (viewed on March 9, 2009). Includes bibliographical references.

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

Saprolegnia is a fresh water fish parasite responsible for significant economic losses in the aquaculture industry worldwide. The disease caused by this organism is termed saprolegniosis, being characterized by a mycosis-like infection of gills and fish skin. Without sustainable treatments available for controlling or preventing saprolegniosis, it has become a significant problem especially for salmon farming, frequently affecting the fish after vaccination. Little is known regarding how the fish immune system responds to infection and such information is vital for developing future treatments and preventive measures to saprolegniosis. To study the immune response of Atlantic salmon to Saprolegnia, a detailed immune profile of experimentally infected presmolts was performed by analyzing the expression levels of several immunity-related genes. Infected fish exhibit a very strong inflammatory response while the majority of genes associate with the adaptive immunity were found to be down regulated. The mechanisms behind this response were then investigated: It was discovered that the cell wall of Saprolegnia can be recognized by fish immune cells, triggering an inflammatory response. A protease secreted by the parasite that has the ability to degrade fish antibodies was identified and, for the first time in an oomycete, the production of prostaglandin E2 was characterized, a molecule that was later shown to potentiate inflammatory responses while suppressing host adaptive immunity genes. Lastly two metabolic pathways of the oomycete were explored as novel targets for control, prostaglandin and sterol metabolism by the use of cyclooxygenase and CYP51 inhibitors, respectively.

No description available.

I examined two amphibian communities to assess factors that may impact amphibian biodiversity. The results suggest that the potential factors which influence the maintenance of amphibian biodiversity are multi-faceted and thus, attempts to understand these factors must reflect these complexities. I investigated factors that influenced the susceptibility of western toad (Bufo boreas), Cascades frog (Rana cascadae), and Pacific treefrog (Hyla regilla) embryos to infection with the fungal pathogen Saprolegnia ferax. I found that there were considerable interspecific differences in susceptibility of anuran embryos to infection with Saprolegnia. Interspecific differences can be attributed to differences in egg-laying behavior and sensitivity to ambient levels of ultraviolet radiation. I studied the effect of Saprolegnia on competitive interactions between larval R. cascadae and H. regilla. The presence of Saprolegnia differentially affected larval recruitment of the two species and mediated competitive interactions. These results suggest that pathogens may have strong effects on species interactions and thus, when present may have strong influences on community composition. I examined population differences in response of native red-legged frogs (R. aurora) to introduced bullfrogs (R. catesbeiana). Syntopic R. aurora tadpoles reduced their activity and increased their refuge-use when presented with the chemical cues of R. catesbeiana, whereas allotopic R. aurora did not. Predation by R. catesbeiana was lower for syntopic R. aurora compared with animals from allotopic populations. Individuals that are unfamiliar with novel, introduced organisms may not possess adaptations that would prevent a negative encounter. In field experiments I demonstrated that introduced R. catesbeiana, and smallmouth bass Micropterus dolomieui, influenced the microhabitat use, growth, and survival of larval and metamorphic R. aurora. These results illustrate the potential complexities of interactions between native and exotic species. These results also stress the importance of understanding the mechanisms of interactions between native and exotic species to allow for the persistence of native biodiversity. / Graduation date: 1998

Graduation date: 1991