Genetic mutation is the ultimate source of new phenotypic variation in populations. The importance of mutation cannot be understated, and constitutes a significant evolutionary force. Although single mutations may have little to no impact on organismal performance or fitness, when multiplied across the total number of potential sites within the genome, mutation can have a large impact. Accurate measurement of the rates, molecular mechanisms, and distributions of effects of mutations are critical for many applications of evolutionary theory. Despite the importance of both deleterious and beneficial mutations, their genome-wide patterns and phenotypic consequences are poorly understood when considering the mitochondrial genome. Mitochondria are organelles that are essential for eukaryotic life. They contain their own genome and generate bioenergy (ATP) necessary to sustain life via the electron transport chain (ETC). Because of their role in eukaryotic physiology, understanding how mitochondrial genetic and phenotypic variation can impact populations and evolutionary outcomes is essential. Past studies have implicated DNA-damaging oxidative stress as a source of mutations within somatic tissue, but there is a gap in knowledge regarding its role in heritable damage within the germ line. In this thesis, I aimed to test this possibility by characterizing the phenotypic and mutational consequences of high intracellular ROS levels caused by mitochondrial ETC genetic damage. I performed experiments using Caenorhabditis elegans ETC mutant, gas-1, and mutation-accumulation (MA) lines generated from this ancestral genotype. I quantified organismal fitness (fecundity and longevity), reactive oxygen species (ROS) levels, mitochondrial membrane potential (delta psi m), and ATP levels in these lines, and compared the results to those from a set of wildtype control lines. I begin with a general introduction to the hypothesis and the C. elegans system in Chapter I. In Chapter II, I report the findings from this work. In short, I found that while gas-1 MA lines began the experiment with low lifetime fecundity in comparison to the wildtype strain, their fecundity showed no further decline as expected, and even exhibited higher fecundity levels on days 3-5 of reproduction relative to the gas-1 progenitor. The gas-1 progenitor exhibited higher rates of ROS compared to wildtype, whereas the MA lines reverted back to wildtype levels; a similar pattern was observed for delta psi m, while ATP levels were low in the gas-1 progenitor and remained low in the MA lines. I interpret these findings in light of high-throughput sequencing results from these lines showing that, while nuclear and mitochondrial DNA mutation rates were equal to wildtype in these lines, the genomic pattern of mutation was highly nonrandom and indicative of selection for beneficial or compensatory sequence changes. Because ROS levels declined to wildtype in the evolved (MA), this study was unable to address whether ROS is a major contributor to heritable mutation in this system. I hypothesize that, in addition to their putatively compensatory genetic changes, gas-1 lineages experienced physiological compensation allowing them to survive, and that this was associated with a "slow living" phenotype. In Chapter III, I summarize general conclusions and implications of this study and end by providing suggestions for further study.

Evolutionary life history theory predicts that tradeoffs among fitness-related phenotypes will occur as a result of resource limitations and/or physiological constraints. Such tradeoffs are defined as the cost(s) incurred on one component of fitness (e.g., reproduction) by the increased expression of another fitness-related trait (e.g., longevity). Only recently have researchers begun to investigate the mechanistic bases of life history tradeoffs. A recent proposal is that reactive oxygen species (ROS) have a central role in shaping life history traits and tradeoffs. Research on disparate animal taxa has highlighted strong correlations between oxidative stress resistance and fitness-related life history traits, for example. Here, I use the model organism Caenorhabditis elegans to test several hypotheses concerning the effects of ROS on life history traits and the manifestation of life history tradeoffs. Additionally, I use heat stress and an alternate food source to explore the responses of life history traits to other forms of physiological stress. Relative fitness and other traits related to reproduction were found to be affected in mostly negative ways by increasing oxidative insult. Lifespan was surprisingly unaffected by oxidative stress, but was modified by temperature. In vivo ROS levels as measured by fluorescent microscopy reveal a tradeoff between antioxidant production and reproduction in this species.

Indiana University-Purdue University Indianapolis (IUPUI) / Methylmercury (MeHg) exposure from occupational, environmental and food sources is a significant threat to public health. MeHg poisonings in adults may result in severe psychological and neurological deficits, and in utero exposures can confer significant damage to the developing brain and impair neurobehavioral and intellectual development. Recent epidemiological and vertebrate studies suggest that MeHg exposure may contribute to dopamine (DA) neuron vulnerability and the propensity to develop Parkinson’s disease (PD). I have developed a novel Caenorhabditis elegans (C. elegans) model of MeHg toxicity and have shown that low, chronic exposure confers embryonic defects, developmental delays, reduction in brood size, decreased animal viability and DA neuron degeneration. Toxicant exposure results in an increase in reactive oxygen species (ROS) and the robust induction of several glutathione-S-transferases (GSTs) that are largely dependent on the PD-associated phase II antioxidant transcription factor SKN-1/Nrf2. I have also shown that SKN-1 is expressed in the DA neurons, and a reduction in SKN-1 gene expression increases MeHg-induced animal vulnerability and DA neuron degeneration. Furthermore, I incorporated a novel genome wide reverse genetic screen that identified 92 genes involved in inhibiting MeHg-induced animal death. The putative multidrug resistance protein MRP-7 was identified in the screen. I have shown that this transporter is likely expressed in DA neurons, and reduced gene expression increases cellular Hg accumulation and MeHg-associated DA neurodegeneration. My studies indicate that C. elegans is a useful genetic model to explore the molecular basis of MeHg-associated DA neurodegeneration, and may identify novel therapeutic targets to address this highly relevant health issue.