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Sperm senescence, a decline in sperm quality caused by male ageing and by sperm ageing before or after copulation, may have fitness costs manifested as infertility or lowered genetic quality of offspring. This thesis tested the distinct evolutionary roles of sperm senescence using a laboratory-adapted population of Drosophila melanogaster. We developed a practical approach to avoid confounding male age with sperm age by standardizing pre-copulatory sperm age and mating history in young and old male age groups. Applying this approach, we documented sperm senescence in D. melanogaster and discussed its potential evolutionary importance. First, ageing males declined in fitness as evidenced by the reduction in fertilization potential of their ejaculates but not by decreased offspring fitness (the ability that a fly can survive to adulthood, successfully mate and produce viable offspring). This suggests a decline in the quality or quantity of seminal fluid or spermatozoa, with no decline in the genetic quality of sperm that actually fertilized ova. Second, post-copulatory sperm senescence has significant negative impacts on offspring fitness, indicating degraded genetic integrity of the spermatozoa stored in females. In both cases, male ageing and sperm ageing had similar fitness impact on male and female offspring, different from what has been suggested by previous work. In addition, We demonstrated that female fecundity, fertility, and length of the fertile period after a single mating were positively associated with the concentration of yeast in their food, and were negatively associated with the duration of yeast restriction in their diet, which suggested that sperm storage is affected by the nutritional status of the females. By revealing the significance of sperm senescence on male and female fertilization success and the fitness of the next generation, this thesis sheds light on a number of evolutionary and applied issues, and provokes new questions for future research on sperm senescence. / Thesis (Ph.D, Biology) -- Queen's University, 2014-03-07 10:38:12.879

Genetic perturbations and foreign chemicals can result in a multitude of changes across a wide range of biochemical processes in a biological system. These perturbations may affect the metabolome, the small molecule metabolites in an organism. Recently, liquid-chromatography coupled to mass spectrometry (LC-MS) technology has been used to quantify large proportions of the metabolome, however standardized protocols are not yet available for use with Drosophila melanogaster. Here, I developed an ion-pairing LC-MS protocol for the metabolomic characterization of D. melanogaster and demonstrated its implementation in establishing the metabolomic profile of flies under oxidative stress and in the metabolic profiles of four different Drosophila species. I demonstrated that this new method allows for the detection of otherwise difficult metabolites and that it is repeatable and sensitive with acceptable levels of ionsuppression, matrix effects, limits of detection and quantification. I then used this method to determine and quantify the metabolomic fingerprints of loss of Superoxide dismutase activity and paraquat-induced stress. Comparing and contrasting the effects of these two sources of oxidative stress, I document both similarities and stressor-specific effects.

Bicaudal-C (Bic-C) encodes a KH-type RNA binding protein required maternally for anterior patterning of the Drosophila oocyte and correct migration of the centripetal follicle cells. In Drosophila, premature translation of the germ-plasm determinant Oskar in Bic-C mutant oocytes suggests a function for Bic-C in post-transcriptional gene regulation. / Purification and microarray analysis of Bic-C containing ribonucleoprotein complexes revealed that Bic-C associates with multiple transcripts encoding functionally-related components of the Wnt/Frizzled/Dishevelled signaling pathway that regulate actin dynamics, in addition to its own mRNA. Using transgenic reporter constructs, Bic-C was demonstrated to destabilize its own mRNA via cis-acting 5' UTR elements. When auto-regulation was bypassed and Bic-C was over-expressed in the female germline, premature cytoplasmic streaming was induced, disrupting axial patterning through displacement of both Gurken (Grk) and oskar. These phenotypes can also be induced by disruption of the actin cytoskeleton with pharmacological agents and are similar to those described for hypomorphic mutant alleles of orb, which encodes a CPEB-like protein that promotes polyadenylation of target mRNAs. The Bic-C overexpression phenotypes require its RNA binding activity, are substantially enhanced by mutations affecting orb and poly(A) polymerase, and are suppressed by mutations affecting the deadenylase CCR4 and its accessory protein NOT3. Co-immunoprecipitation experiments demonstrate that Bic-C associates with components of the deadenylase complex and with components of an ER-associated RNP complex that includes Me31B, PABP and Trailer-hitch. The latter complex is involved in Grk exocytosis. Accordingly, Grk secretion is defective in Bic-C mutants. / Taken together, these results support a model whereby Bic-C antagonizes Orb function by negatively regulating the expression of Orb target mRNAs, through recruitment of the deadenylase machinery, that are involved in coordinating cytoplasmic movements. Furthermore, this work identifies a novel function of Bic-C in dorsal/ventral patterning by promoting Grk secretion.

A series of recent studies using Drosophila melanogaster suggest that while males may benefit from having access to many partners, female fitness is reduced by extended cohabitation and sexual interaction with males. Yet, even if repeated sexual interactions are harmful to females, limited male exposure will ultimately be detrimental due to sperm-depletion and infertility. Females are therefore expected to balance mating opportunities and sperm storage capacity to maximize lifetime reproductive success. I introduced extended mating deprivation as a selective pressure to experimentally evolve lines of D. melanogaster for characters related to mating and postcopulatory sexual selection. Evolution of the mate-deprived lines over several dozen generations demonstrated that restricted sexual access was indeed a potent selective pressure. I consistently found that when males were removed for an extended time period, female fitness declined substantially, suggesting that mate-deprivation over nine days was harmful. Under these conditions, selected-line males responded to mate-reduced conditions and demonstrated a 13% increase in reproductive success compared to controls. Experimental females had a 15% increase in fertility compared to controls. I investigated a series of developmental characteristics that may have been altered by the selection regime, and while there was some evidence of evolved change, these results were not consistent. Although the data at hand do not substantiate a detailed characterization, both sexes in the experimental populations demonstrated increased fitness after extended mate-deprivation, thus evolutionary change appears to have occurred via selection on one or both relevant male ejaculate characteristics: sperm number and survival, and factors affecting female late-life fertility. / Thesis (Master, Biology) -- Queen's University, 2008-04-28 23:05:42.835

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I have investigated the mechanisms by which developmental asymmetry arises, using oocyte determination in Drosophila melanogaster as a model system. The Bicaudal-D (Bic-D) gene is required early in oogenesis for the asymmetric localization of specific mRNAs and proteins and for the differentiation of an oocyte from one of a cluster of 16 interconnected germarial cells. To better understand how Bic-D functions in creating this asymmetry, I took two approaches. First, I examined the role of Bic-D in the asymmetric localization of mRNA and other cellular components during later oogenesis. Second, I molecularly and genetically characterized a gene that interacts with Bic-D in oocyte determination. To determine the role of Bic-D in later oogenesis, I used an inducible source of Bic-D activity to selectively rescue the block at oocyte determination in Bic-D null mutants. Using this system, I find that Bic-D is indeed required in the later stages of oogenesis for the localization of specific mRNAs at both the anterior and posterior of the oocyte. Bic-D is also required for oocyte growth and nuclear positioning, processes which also depend on microtubules. / In the second part of this thesis, I describe the characterization of a Bic-D interacting gene which I have identified as the Drosophila homologue of the human Lissencephaly-1 (Lis-1) gene, DLis-1. Human Lis-1 is the causative gene for Miller-Dieker Syndrome and is required for neuronal migration in the developing brain, while fungal homologues have been implicated in dynein dependent nuclear migration. Like Bic-D , DLis-1 is essential for oocyte determination and for intracellular localization throughout oogenesis. DLis-1 is required for correct positioning of the oocyte nucleus, and appears to function upstream of dynein in this process. Immunolocalization studies suggest that DLis-1 functions as part of a cortical anchor that links microtubules and the oocyte nucleus, via dynein and microtubules, to the cell cortex. DLis-1 and Bic-D are also required for nuclear positioning during neural development in Drosophila, supporting a model in which the neuronal migration defects in Miller-Dieker Syndrome are due to a disruption of dynein/Lis-1 dependent nuclear migration.