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abstract: Emerging infectious diseases (EIDs) in vulnerable populations are a proposed cause of reduced global biodiversity due to local and regional extinctions. Chytridiomycosis, a fungal disease caused by Batrachochytrium dendrobatidis (Bd), is affecting amphibian populations worldwide. Chapter 1 of this thesis reports using lab-raised larval tiger salamanders (Ambystoma tigrinum nebulosum), collected as eggs, to test if Bd infects them. Bd infects metamorphosed tiger salamanders; however, it is currently unknown if larvae can be infected by Bd. Adult frogs tend to host Bd on ventral surfaces and hind legs while tadpoles host Bd in keratinized mouthparts. No research has considered differences in infection between life stages of salamanders. It was hypothesized that Bd can colonize larvae in the same manner as metamorphosed animals. Larval salamanders were inoculated to test if Bd concentrations differ among body regions in larvae compared to metamorphosed salamanders. Larvae can carry Bd with the concentration of Bd varying between body region. Chapter 2 report using native tiger salamanders (Ambystoma tigrinum nebulosum), from northern Arizona and Bd as a study system to test if Bd is native or introduced to Arizona. It was hypothesized that Bd is not endemic to Arizona, but is introduced. There are multiple hypotheses regarding potential routes Bd may have traveled through Arizona and into Mexico. These hypotheses were tested using the Kaibab Plateau in Coconino County, Arizona, as a study site. The plateau is isolated from surrounding areas by the Grand Canyon to the south and the Vermillion Cliffs to the north serving as major biogeographical barriers. It is hypothesized that tiger salamanders are not dispersing into or out of the Kaibab Plateau due to geological restrictions. Bd, therefore, should not be present on salamanders on the Kaibab Plateau due to geological restriction. Tiger salamanders in stock tanks located on the Kaibab as well as preserved museum specimens housed in the Arizona State University Natural History Collection were sampled. The results indicate that Bd occurs at low levels on Kaibab Plateau tiger salamanders. / Dissertation/Thesis / Masters Thesis Biology 2019

A mouse model was used to study the pathogenesis of systemic infection by the opportunistic pathogen <i>Candida albicans</i>. Using this model it was demonstrated that <i>C. albicans</i> yeast cells were more pathogenic for mice than hyphal forms. Polymorphonuclear leukocytes were shown to be important in resistance to systemic infection by <i>C. albicans</i>. Studies on conditions which promote germination of <i>C. albicans</i> yeasts showed that maximum numbers of yeast cells produced germ tubes when incubated in tissue culture media at 37°C, by 2 hours. A comparative ultrastructural examination of yeasts, germ tubes and hyphal forms demonstrated marked differences in the thickness and organisation of the cell walls between these forms. Furthermore, germination of <i>C. albicans</i> yeasts was shown to be accompanied by significant release of cell wall antigens. <i>In vitro</i> interactions between mouse polymorphonuclear leukocytes and <i>C. albicans</i> yeasts, germ tubes and hyphae in the absence of serum were examined. Mouse neutrophils were found to adhere readily to the surface of germ tubes and hyphae but not yeasts. This adherence resulted in damage of the fungus. Studies on the degradation of killed <i>C. albicans</i> yeasts following phagocytosis by murine macrophages <i>in vitro</i>, showed that progressive removal of yeast cell wall layers occurred. This was followed by dissolution of the cytoplasmic contents. During this process, cell wall and cytoplasmic antigens were released into the surrounding medium. An enzyme linked immunosorbent assay was developed to measure IgM, IgA and IgG class antibodies to <i>C. albicans</i> mannan and cytoplasmic antigens in patient's sera, and was shown to have diagnostic potential for candida infection. In particular, use of this assay to monitor the kinetics of antibody levels to these antigens was found to be of diagnostic value for immunocompromised patients at risk of candida infection. Finally a number of monoclonal antibodies were produced to <i>C. albicans</i> cytoplasmic proteins and have been partially characterised.

The overall aim of the research project was to evaluate the potential of the fungal pathogen <I>Ascochyta caulina</I> as a biological control agent against <I>Chenopodium album</I>, a major weed in arable crops. A number of isolates of <I>Ascochyta caulina</I> were evaluated but isolate W 90-1 from Holland proved to be the most promising candidate because of its high virulence. It was therefore selected for more detailed greenhouse and field studies into the environmental parameters required for infection and disease development. Results of these studies showed that in order to achieve the maximum infection, a temperature of between 20-30°C, a relative humidity of >95% for 24 h and a spore density of approximately of 1-2 x 10⁶ spores per ml spore suspension were required. Mortality and plant necrosis levels after application of <I>A. caulina</I> decreased with plant age and treatment of <I>C. album</I> shortly after emergence or to juvenile plants (before 4-leaf growth stage) was most effective. The requirement for long periods of high relative humidity and the inability of <I>A. caulina </I>to cause satisfactory disease after the 4 leaf growth stage are the most important limiting factors for the development of <I>A. caulina</I> as a bioherbicide for <I>C. album. </I>A range of spore formulations was studied with the aim of reducing the requirement for long periods of high relative humidity for disease development. Studies indicated that disease development could be increased by incorporation of surfactants (Tween 80 or Sylgard) and nutrients (Czapek-Dox Broth and Yeast Extract) into inoculum suspension. Results of field trials indicated that if application were properly timed and optimum environmental conditions can be achieved the pathogen can give satisfactory control of the weed.

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<p dir="ltr">The Cdc14 phosphatase family is highly conserved in fungi. In Saccharomyces cerevisiae, Cdc14 is essential for down-regulation of cyclin-dependent kinase activity at mitotic exit. However, this essential function is not broadly conserved and requires only a small fraction of normal Cdc14 activity. In general, few conserved functions of Cdc14 phosphatase have been defined. Here, I present mechanistic biochemical and phenotypic characterization of Cdc14 phosphatases in fungi. I have demonstrated that fungal Cdc14 phosphatases possess an invariant motif in the disordered C-terminal tail that is required for full enzymatic activity. This motif, termed substrate-like catalytic enhancer (SLiCE), functions during the rate-limiting step of Cdc14-directed catalysis, by binding to the active site and supporting phospho-enzyme hydrolysis. Adjacent to the SLiCE motif exists a conserved minimal Cdk consensus motif that likely serves a regulatory function as phosphorylation of this site inhibits Cdc14 activity in vitro. Vertebrate Cdc14 enzymes also possess a distinct, but mechanistically similar SLiCE motif, which may be the first described biochemical difference between Cdc14 enzymes. Moreover, the vertebrate SLiCE motif lacks an adjacent Cdk consensus motif, which may point to differences in how Cdc14 activity is regulated in higher eukaryotes.</p><p dir="ltr">Mutation of this motif in vivo served as a tool to discover biological processes that require high Cdc14 activity. In S. cerevisiae strains expressing this hypomorphic mutant allele (cdc14hm), I discovered a novel sensitivity to cell wall stresses, including chitin-binding compounds and echinocandin antifungal drugs. This sensitivity was also observed in the distantly related fungi Schizosaccharomyces pombe deletion strain and the human fungal pathogen Candida albicans hypomorphic and deletion strains, suggesting that this phenotype reflects a conserved function of Cdc14 orthologs in mediating fungal cell wall integrity. I also revealed that high Cdc14 activity is required for C. albicans ability to develop hyphae, which is an important virulence trait. This led to our determination that high Cdc14 activity is critical for virulence in two animal models of invasive candidiasis. Together, these results argue that Cdc14 would be an excellent antifungal drug target for the treatment of invasive Candida infections and sensitization to existing antifungal drugs.</p><p dir="ltr">Lastly, I implemented the auxin-inducible degradation system in C. albicans. Using this system, we were able to deplete Cdc14 and other target protein levels to >95% within minutes. Depletion of Cdc14 was robust enough to phenocopy gene deletions, confirming previous results and demonstrating the utility of rapid target protein inactivation. This system will serve as a powerful tool for future functional characterization of Cdc14 in C. albicans and other pathogenic fungal species.</p>