<p>Sexual development enables microbial pathogens to purge deleterious mutations from the genome and drives genetic diversity in the population. <italic>Cryptococcus neoformans</italic> is a human fungal pathogen with a defined sexual cycle. Nutrient-limiting conditions and pheromones induce a dimorphic transition from unicellular yeast to multicellular hyphae and the production of infectious spores. <italic>C. neoformans</italic> has a defined <bold>a</bold>&ndash;&alpha; opposite sexual cycle (bisexual reproduction); however, >99% of clinical and environmental isolates are of the &alpha; mating type. Interestingly, &alpha; cells can undergo &alpha;&ndash;&alpha; unisexual reproduction, even involving genotypically identical cells. A central question is why would cells mate with themselves given that sex is costly and typically serves to admix pre-existing genetic diversity from genetically divergent parents? Sexual reproduction generates abundant spores that following inhalation, they penetrate deep into the alveoli of the lung, germinate, and establish a pulmonary infection growing as budding yeast. Therefore sex has been linked with virulence; however, hyphal development has been previously associated with reduced virulence and thus the roles of morphogenesis in virulence have not been extensively analyzed. To further understand the role of unisexual reproduction in <italic>C. neoformans</italic> we will investigate the evolutionary implications of &alpha;&ndash;&alpha; mating, explore its role in pathogenesis, and we will dissect the signaling pathway that regulates sexual development.</p><p>We isolated &alpha;&ndash;&alpha; unisexual reproduction progeny from the hyperfilamentous strain XL280 and subjected to a variety of phenotypic and genotypic assays (including whole genome sequencing and CGH). We found that unisexual and bisexual reproduction frequently generates phenotypic and genotypic diversity de novo, including aneuploidy. Aneuploidy was responsible for the observed phenotypic changes, as chromosome loss restoring euploidy results in a wild-type phenotype. Other genetic changes, including diploidization, chromosome length polymorphisms, SNPs, and indels, were also generated. Our study suggests that the ability to undergo unisexual reproduction may be an evolutionary strategy for eukaryotic microbial pathogens, enabling de novo genotypic and phenotypic plasticity and facilitating rapid adaptation to novel environments, such as the mammalian host.</p><p>Interestingly aneuploidy strains that were fluconazole resistant were as virulent as the WT parental strain XL280. Although XL280 belongs to the serotype D lineage that exhibits limited pathogenicity, in further studies we found that is hypervirulent in the murine model. It can grow inside the lung of the host, establishing a pulmonary infection, and then disseminates to the brain to cause cryptococcal meningoencephalitis. Surprisingly, this hyperfilamentous strain triggers an immune response polarized towards Th2-type immunity, which is characterized by less protective immunity and is usually observed in the highly virulent sibling species <italic>C. gattii</italic>, responsible for the Pacific Northwest outbreak. These studies: 1) provide a technological advance that will facilitate analysis of virulence genes and attributes in <italic>C. neoformans</italic> var. <italic>neoformans</italic> (serotype D), and 2) reveal the virulence potential of serotype D that is broader and more dynamic than previously appreciated.</p><p>Bisexual and unisexual reproduction are governed by shared components of the conserved pheromone-sensing Cpk1 MAPK signal transduction cascade and by Mat2, the major transcriptional regulator of the pathway. However, the downstream targets of the pathway are largely unknown, and homology-based approaches have failed to yield downstream transcriptional regulators or other targets. To address this question we applied an insertional mutagenesis via <italic>Agrobacterium tumefaciens</italic> transkingdom DNA delivery to identify mutants with unisexual reproduction defects. In addition to elements known to be involved in sexual development (Crg1, Ste7, Mat2, and Znf2), three key regulators of sexual development were identified by our screen: Znf3, Spo11, and Ubc5. Spo11 and Ubc5 promote sporulation during both bisexual and unisexual reproduction. Genetic and phenotypic analyses provide further evidence implicating both genes in the regulation of meiosis. Phenotypic analysis of sexual development showed that Znf3 is required for hyphal development during unisexual reproduction and also plays a central role during bisexual reproduction. Znf3 governs cell fusion and pheromone production through a pathway parallel to and independent of the pheromone signaling cascade. Surprisingly, Znf3 participates in transposon silencing during unisexual reproduction and may serve as a link between RNAi silencing and sexual development. In further studies we found that Znf3 is required for sex- and mitotic-induced (SIS and MIS). SIS is less efficient in <italic>znf3</italic> unilateral matings and is abolished in <italic>znf3</italic> x <italic>znf3</italic> bilateral matings, similar to the phenotypes of <italic>rdp1</italic> mutants (the RNA-dependent RNA-polymerase of RNAi pathway). Znf3 is also required for transgene-induced mitotic silencing; <italic>znf3</italic> mutations abrogate silencing of repetitive transgenes during vegetative growth. Znf3 tagged with mCherry is localized in the cytoplasm in bright, distinct foci. Co-localization of Znf3 with the P-body marker Dcp1-GFP further supports the hypothesis that Znf3 is a novel element of the RNAi pathway and operates to defend the genome during sexual development and vegetative growth. In concussion our studies provide further understanding of unisexual reproduction as an evolutionary successful strategy.</p> / Dissertation

This study reports the results of different staining techniques on the chromosomes of two Poecilia formosa lineages, providing evidence of two additional nucleolus organizer region (NOR) chromosomal clones in this gynogenetic fish. A comparative analysis of chromosomal clones detected in the Amazon molly, along with their frequency and distribution in different collecting sites, is also presented, and clonal heterogeneity resulting from chromosome changes is discussed.

No description available.

Mole salamanders (Ambystoma) and woodfrogs (Lithobates sylvaticus) are abundant in New England and depend on ephemeral wetlands for breeding. Their aquatic habitats have been well studied and are protected by several local and regional regulations. State endangered species laws also protect mabled salamanders (A. opacum), Jefferson salamanders (A. jeffersonianum), and blue-spotted salamanders (A. laterale). However, these amphbibians spend most of their adult lives in terrestrial habitats that remain poorly protected and elusive to researchers. In chapter 1, I developed a novel technique using passive integrated transponders for tracking small animals. I used this technique to track marbled salamanders walking up to 200 m from their breeding pond during post-breeding migrations. In Chapter 2, I examined the importance of multiple habitat variables for predicting the distributions of woodfrogs and spotted salamanders at 455 ponds in western Massachusetts. Based on a variable-comparison technique I developed, the best predictor for either species of amphibian was the amount of forest in the surrounding vii landscape. Both species were found more frequently in upland forests where the ponds are least protected by state and federal wetland regulations. In chapter 3, I used my data from chapter 2 and three other similar data sets to conduct an analysis of spatial scale and to parameterize a recently published resistant kernel model. The complex model parameterized by an expert panel did significantly worse than the null model. The distributions of both amphibians were best predicted by measuring the landscape at very large scales (over 1000 m). The most effective scales for conservation may be largest for organisms of intermediate dispersal capability. In chapter 4, I explored the evolution and genetics of the Jefferson/blue-spotted/unisexual salamander complex. I framed research into the fascinating unisexual reproductive system with a model that relates nuclear genome replacement, positive selection on hybrids, and biogeography of the species complex. I parameterized this model using genetic data taken from salamanders spanning Massachusetts and an individual-based breeding simulation. If paternal genomes are transmitted to offspring with the frequencies reported from laboratory experiments, then my model suggests that there must be strong selection favoring unisexuals with hybrid nuclei.

There are four members involved in the breeding complexes of poeciliid fishes found in the freshwaters of northeastern Mexico: males and females of a bisexual species, and diploid and triploid unisexuals. Both unisexuals reproduce by gynogenesis, i.e., an asexual type of reproduction where the sperm triggers egg development but the male genome is excluded to produce clonal offspring. The three types of females are closely related, which suggests that they are potential competitors since all three require the service of the same males. The potential for competition is compounded by a highly skewed sex ratio in favor of females. On the average the unisexuals comprise about 30% of the Poecilia females. This high frequency coupled with a close genetic relatedness to their bisexual hosts, raises the question of how the unisexuals are maintained in nature. Other investigators who work with bisexual/unisexual complexes in the related genus, Poeciliopsis, have postulated that male dominance hierarchies are responsible for restricting the access of subordinate males to their conspecific females. Consequently, these subordinate males mate with unisexual females. The current report tests whether or not this hypothesis applies to bisexual/unisexual complexes of Poecilia. We have found that linear dominance hierarchies appear to function in the defense of home ranges and do not restrict access of males to females. Dominant males exhibit less mating activity than subordinate males towards females. Previous reports showed that males are reproductively competent throughout the year, whereas females show striking asynchrony in their reproductive readiness. Such asynchrony limits the proportion of receptive females at any one time. Consequently, there are more males ready to mate than there are females receptive to their mating attempts. This may lead to mating frenzies. We postulate that these indiscriminate matings maintain the fertility of both unisexuals. When the relative reproductive outputs of adult females are compared, both unisexuals appear as fit as their bisexual congeners.

Several species‐specific proteins have been identified by polyacrylamide gel electrophoresis (PAGE) of skeletal muscle extracts from the diploid gynogen, Poecilia formosa, its related triploid unisexuals, and their sympatric, bisexual species, P. mexicana and P. latipinna. These water‐soluble, low molecular weight proteins (7,000‐13,000) comigrate with a fraction of purified rabbit parvalbumin on nondenaturing gels and show staining properties similar to rabbit parvalbumins. The electrophoretic patterns of these muscle proteins provide a set of distinctive phenotypic markers for each of the host species involved in naturally occurring breeding complexes with P. mexicana × P. latipinna show no evidence of sexual dimorphisms. Furthermore, the hybrid phenotypes are those that would be predicted from appropriate combinations of parental alleles at three gene loci. The patterns found by PAGE for several generations of pedigreed stocks of P. formosa show strictly matroclinous inheritance of a characteristic muscle protein phenotype and coupled with the electrophoretic patterns of several enzymic proteins reflect the probable hybrid origin of this diploid unisexual. Finally, paternal contributions by P. mexicana to the hybrid genome of triploid unisexuals are clearly demonstrated by comparative analyses of muscle protein phenotypes for P. formosa and its contemporary host species. Our identification of distinctive phenotypic markers in the muscle proteins of several poeciliid species involved in unisexual‐bisexual breeding complexes provides an important new tool for further studies on the adaptive significance of unisexuality, hybridization, and fixed heterozygosity in the evolutionary biology of these fishes.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal.

Thèse numérisée par la Division de la gestion de documents et des archives de l'Université de Montréal