Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at El Paso, 2008. / Title from title screen. Vita. CD-ROM. Includes bibliographical references. Also available online.

Sexual reproduction is an evolutionary innovation that arose 1.2 billion years ago, and in that time, has allowed a rapid diversification of species outpacing that of asexually reproducing organisms. Successful sexual reproduction in animals requires the incredible coordination of complex genetic and behavioral factors; from the most fundamental levels of ensuring correct chromosome segregation and ploidy to the most complex of behavioral mating rituals, any failure can result in a complete loss of evolutionary fitness. In this thesis, I have explored the developmental programs that function to ensure somatic sex determination, sexual differentiation, and mating behaviors in C. elegans. C. elegans is an androdiecious nematode species that has been extensively characterized in regard to the sexual dimorphism of its development, nervous system, and behavioral outputs. Sex determination pathways are widely diverged across phyla, and C. elegans has coopted a Gli family transcription factor to serve as a cell autonomous global regulator of somatic sex determination. I investigated the expression of this transcription factor, tra-1, with cellular, subcellular, sex-specific, and temporal resolution in both sexes of C. elegans and found that it is dynamically regulated to control sex determination. In contrast to the upstream sex determination pathway, genes that control downstream sexual differentiation in animals display much higher functional conservation, and many of the regulators of sexual differentiation belong to a family of transcription factors known as the DMRT family. Downstream of the tra-1 global regulator, I found that the highly conserved DMRT family gene dmd-4 acts much more specifically in adult hermaphrodites to generate sexual dimorphism at the level of the phasmid sensory neurons PHA and PHB. Furthermore, the sexual dimorphism of DMD-4 is regulated post-translationally by a ubiquitin-binding domain that I also found to be functionally conserved in the human ortholog, Dmrt3. Although these transcription factors both demonstrate the high degree of genetic control that contributes to sex determination and sexual differentiation, I also described male-specific effects of early life stress on sexual dimorphic synaptic connectivity and behavior generated by the phasmid sensory neurons, indicating that sexual differentiation is also plastic to environmental cues encountered during the life of an organism. This thesis provides insight into how genetic pathways function at multiple levels to give rise to extensive sexual dimorphism in the soma of an animal, both globally and in regard to the development on individual cells, in addition to the ways in which these genetic pathways can be modified by environmental factors and organismal life history.

The genetics of switchback evolution is largely unknown. While it is assumed that latent gene regulatory networks become reactivated to recreate an ancestral tissue, the details regarding this reactivation has yet to be elucidated. How has a network been maintained over the evolutionary history of this group? Are certain genes within these pathways more susceptible to suppression than other genes? In this study, I examined Euphilomedes carcharodonta, a member of the Sarsielloidea superfamily of ostracods, a clade which has demonstrated the loss and regain of the lateral eye multiple times over its evolutionary history. In particular, I looked at the genetic mechanisms for the development of the sexually dimorphic lateral eye, in which males have large, multifaceted image-forming eyes typical to those of other pancrustaceans (Hexapoda+Crustacea) while females do not. This sexual dimorphism is of particular interest because it allows me to study the genetic underpinnings of a regained trait using individuals of near-identical genetic backgrounds, as these organisms have a singlechromosome sex determination system (XXIXO). Examination of developmental eye genes ec-Dachshund, ec-Daughtless, ecChaoptic, ec-Shaven, and ec-Epidermal growth factor receptor showed differential gene expression patterns in which juvenile male eyes expressed these genes at a higher level than did juvenile female eyes. These genes are thus candidate genetic components of 6 switchback evolution, as this data is a first step towards comparative transcriptomics and gene expression studies comparing multiple species.

The placenta plays a vital role in a healthy pregnancy by supporting the intricacies of fetal development. Over 10% of pregnancies experience impaired placental function, resulting in the loss of critical neuroactive steroids the fetal brain cannot yet make, thus leaving them vulnerable to perinatal brain injury and abnormal neurodevelopment. However, this vulnerability is not always equal. Many neurodevelopmental disorders exhibit a sex bias in incidence and severity. I hypothesize that loss of placental support during pregnancy results in sex differences in both behavioral presentation as well as on the cellular and transcriptomic levels. Utilizing the akr1c14cyp19aKO (plKO) mouse model, which features placenta-specific allopregnanolone (ALLO) knockdown, I investigated the sex specific impact of placental hormones on cerebellar development. Here I show that placental ALLO is essential for cerebellar white matter development and inflammatory regulation via microglial function. Male mice without placental ALLO exhibit signs of placental inflammation, accelerated postnatal myelination, and defects in microglial phagocytosis of excess myelin. Alternatively, females seem to be more resilient with a progressive anti-inflammatory profile across development and reduced myelination. Additionally male plKO show autism-like behaviors such as deficits in social behavior and increased stereotyped behavior. The females do not exhibit this phenotype. My main goals were threefold; to investigate how male and female inflammatory profiles differ and where this difference originates, to investigate how this inflammation impacts microglia and thereby oligodendrocytes, and how I can alter microglial function in a way to improve plKO outcomes. Mechanistically, these changes appear to be in part due to baseline sex differences in response to inflammatory stimuli which prime microglia to differentially support the surrounding white matter. Together, this work supports a novel link between placental ALLO loss, microglial function, and sex specific presentation of neurodevelopmental disorders.

This study is primarily concerned with assessing the influence of the sex determination genes, transformer (tra), doublesex (dsx) and fruitless (fru) on three sexually dimorphic traits within Drosophila; pheromone blend, courtship song and sex comb tooth number. The sex determination loci have all been implicated as possible candidate genes affecting these important traits that contribute to sexual isolation, which is a major cause of speciation. Quantitative Trait Loci (QTL) analysis is used to assess the effects of these known candidate genes on the naturally occurring variation of mean interpulse interval (IPI) of courtship song and the differing pheromone blend profiles between Drosophila simulans and D. sechellia. The QTL analysis for both song and pheromone blend variation incorporated Multiple Interval Mapping (MIM), which enables the detection for epistasis. The desaturase loci desat1, desat2 and desatF were also included in the assessment on pheromone blends (cuticular hydrocarbon compounds), since they facilitate ecological adaptation and are also candidate genes, which are likely to exert a large affect on this particular trait. The sex determination genes were not significantly influential on the interspecific variation of the cuticular hydrocarbon compounds between these two sibling species. However significant effects were detected from two of the desaturase loci. desat1 was associated with a strong effect on the interspecific variation of a saturated hydrocarbon chain compound (unbranched-23). Additionally the candidate gene desatF potentially exerts an influence on the variation of 7,11-heptacosadiene, through a large epistatic effect with unidentified loci, situated between the markers pros and Mtn. The candidate gene eloF is situated in this region, and is known to affect the elongation of unsaturated hydrocarbon chains. The QTL associated with the marker desatF influenced the variation of both diene compounds (7,11-heptacosadiene and 7,11-pentacosadiene). Intriguingly epistasis was only detected for the variation of these two diene compounds. The MIM analysis assessing the affects of the sex determination genes on interspecific variation of mean IPI detected the candidate gene fru as the closest marker associated with a significant QTL on the third chromosome. The MIM also found a significant QTL associated with the marker Dgα situated on the second chromosome. Moreover significant epistatic interactions were detected between a further QTL situated nearest the marker forked on the X-chromosome with both of the other significant QTL situated on the third and second chromosomes. The analysis of a number of Recombinant Inbred (RI) lines was also carried out to test for the affects of the sex determination genes on both mean IPI and sex comb tooth number. The fru locus was associated with a significant increase in mean IPI, whereas the opposite was true for the dsx locus. In the analysis of sex comb tooth variation, it appears that all RI lines homozygous for D. sechellia alleles at the sex determination loci had significantly higher numbers of sex comb teeth. The final data chapter involves the sequence analysis of the fruitless locus, including all 13 fru proteins between ten recently sequenced Drosophilid genomes. The PAML program was used to detect the possible influence of natural selection on sequence divergence. There was no significant positive selection detected at the BTB functional domain and the sequences encoding for this domain were extremely conserved. Positive selection was found to be acting on the exon encoding for the Zinc-finger C domain. This domain is present in two protein isoforms including the male sex-specific isoform FRUMC, and the common non-sex-specific isoform FRUComC. Interestingly positive selection was also found at the non sex-specific Zinc-finger D domain.

Growth rates between individually tagged Atlantic halibut, from a single batch of farm produced eggs, on-grown in sea cages and pump ashore tanks for three years were significantly different. The tank reared fish 405g - 5992g showed a 29% premium in growth (final weight) compared to the cage reared fish 444g -4640g. Females in both systems reached a greater size (7352g tanks, 5836g cages) than males. Males that matured early (3819g tanks, 2877g cages) had a lower mean end weight than males maturing a year later (4326g tanks, 3086g cages). Early maturing males had the largest initial size. Seasonal variations in growth were observed for all groups. Major divergences in growth between males and females only became apparent when the males first matured at around 1.5 - 2 kg. No female maturation was observed during the trial. Halibut growth was determined to be positively allometric with growth of males being more linear then females. Condition factor increased with time whilst there was a decrease in Specific Growth Rate (SGR) from approximately 0.5%day⁻¹ to 0.1%day⁻¹ throughout the trial. Concentrations of plasma cortisol, osmolality, chloride and glucose measured through the trial provided no evidence of chronic stress at either site. Acute confinement stress (2, 12 and 30 minutes) was shown to elicit both primary and secondary stress responses in accordance with other marine teleosts. Increases in plasma cortisol, osmolality, CI⁻, Na⁺ and glucose were observed, reaching maximum concentrations within 80 minutes, although there was no effect on plasma K⁺. The duration of the confinement appeared to have no effect on the magnitude of the response. Following repeat confinements (4 days later) there was no evidence of either habituation or a cumulative effect in terms of cortisol or glucose whereas the effects on osmoregulatory function (Na⁺, CI⁻ and osmolality) appeared to be longer lasting. The results provided the first information regarding the stress response of the Atlantic halibut and enabled a better interpretation of the vales measured in the fish reared in tanks and cages (chapter 1). In vitro cortisol production (% above basal secretion), measured by radioimmunoassay, from perifused interrenal tissue of the Atlantic halibut was significantly stimulated by porcine adrenocorticotrophic hormone (ACTH) (0.01-1.0 μM) and [Asn¹, Val⁵] angiotensin II (All) (0.1-lO μm). No significant increase in cortisol production resulted from physiological levels of potassium (K⁺) although non-physiological levels (lOmMKl) did elicit a mild response in comparison to the effects of ACTH and All. Maximum steroid production was in response to 0.01μM ACTH (1351% above basal secretion) and 1.0μM All (397% above basal secretion). With increased concentrations above these levels of both ACTH and All there was a reduction in the degree of cortisol stimulation. The results show that the interrenal tissue of the Atlantic halibut responds in accordance to that of other teleosts to classical steroidogenic peptides.

Osteoarthritis is a degenerative joint disease characterized by progressive erosion of the articular cartilage. Epidemiological studies have established a relationship between osteoarthritis and menopause suggesting that estrogen may be important in the development of cartilage regeneration therapies. The overall goal of this research project was to advance the field of cartilage tissue regeneration by investigating the role of 17 ß -estradiol (E2), an active estrogen metabolite, on the chondrocyte phenotype. The central hypothesis was that E2 plays an important and sex-specific role in regulating chondrogenesis. Specific Aim-1 focused on establishing and characterizing a primary human articular chondrocyte (HAC) cell source, and then examining the response of the cells in culture to E2. It was demonstrated that the response of HACs to E2 treatment was sex-specific despite both male and females cells expressing estrogen receptors. Female HACs showed changes in proliferation, matrix production, and differentiation while male cells did not. In addition, the female response was regulated through a rapid membrane signaling pathway mediated by protein kinase C. Specific Aim-2 involved establishing an ovariectomized animal model to investigate the effects of E2 on orthopaedic tissue implants. Human demineralized bone matrix (DBM) was implanted intramuscularly into female nude mice and rats. Ovariectomy was shown to reduce the ability of DBM to induce the formation of cartilage and bone tissue. Moreover, the inductive properties of DBM were reestablished with subcutaneous E2 supplementation. Specific Aim-3 entailed developing and characterizing a microencapsulation method for in vitro culture and in vivo delivery of chondrocytes to study the effects of E2 on chondrogenesis. Rat growth plate chondrocytes and HACs were microencapsulated in alginate using an extrusion method in conjunction with high electrostatic potential. Chondrocytes maintained their phenotype in alginate suspension but were unable to form cartilage tissue when implanted into our animal model. Further optimization of the system is required before the role of E2 on chondrogenesis of tissue engineered constructs can be determined. In summary, our results suggest that the successful production of tissue engineered therapies will likely depend on understanding and manipulating the actions of sex hormones in both the in vitro and in vivo environment.

The European earwig exhibits a remarkable male-dimorphism in forceps morphology that is associated with alternative reproductive tactics under the control of a conditional evolutionarily stable strategy. Populations on the small, rocky islands of the Farnes off the Northumberland coast are known to sustain populations with dramatically higher morph ratios than observed on the UK mainland. A survey conducted of island and mainland sites around the UK showed that the dimorphic populations of the Farnes are similar to other islands and that mainland populations generally exhibit low morph ratios. Additionally, a correlation between morph ratio and population density was found lending support to the hypothesis that the ESS thresholds that define the morph ratios have diverged through local adaptation. A set of seven microsatellite markers are presented that were developed from a Farne island population of F. auricularia with one additional, previously published locus. These eight markers exhibit genetic variability within and between populations and as such can potentially be applied at a range of scales, from broad-scale phylogeography to within population parentage studies. A phylogeographic study of the UK populations using these markers suggests a single postglacial colonisation from mainland Europe and give further support to the local adaptation hypothesis of ESS threshold evolution. A study of ejaculate size in F. auricularia showed that the males transfer free sperm at a steady rate and that the morphs do not differ in the number of sperm per ejaculate. Measurements of change in body-mass were found to be ineffective measures of ejaculate size, but that macrolabic males lost more weight during copula than brachylabic males. This may be the result of differential investment in accessory ejaculate components between the morphs, as a result of the differing risk of sperm competition.

Many organisms are sexually dimorphic for ecologically and socially important traits. One of the major foci of biology is to understand the evolution of such sexually dimorphic traits. Here I present my work on the evolution of a dimorphic trait, female-limited Batesian mimicry, in Papilio swallowtail butterflies. I begin by developing a character state path network to study the diversity of mimicry types and directionality of trait change during the evolution of female-limited mimicry. My phylogenetic analysis showed that female-limited mimicry has evolved independently in several groups of swallowtails, mainly via single-step character changes from monomorphic non-mimetic ancestors to female-limited mimetic descendents. Mimetic polymorphism has evolved in tandem with female-limited mimicry, the two being tightly correlated among mimetic species. Most traditional explanations of female-limited mimicry and mimetic polymorphism invoke sexual selection. In reviewing these hypotheses, I show that their key assumptions and predictions remain untested, and that sexual selection cannot maintain female polymorphism under some conditions. Sexual selection hypotheses are also unable to explain community ecological aspects of mimicry rings. Hence, I developed a novel model of female-limited mimicry based on sex-specific, frequency- and density-dependent advantages of mimicry. This model shows that both-sex mimicry, female-limited mimicry and mimetic polymorphism are favored along a gradient of relative mimic frequency. My ecological data from south Indian mimicry rings support a key prediction of this model. Finally, I employ the patterns of female-limited mimicry among swallowtail butterflies to highlight the contrast between Darwin’s sexual selection model and Wallace’s natural selection model of sexual dimorphism. I show that most of the sexual dimorphism in swallowtail wing color patterns is a product of natural selection for protective female coloration, predominantly in the form of female-limited mimicry. Thus, swallowtails support Wallace’s model of sexual dimorphism, underlining the importance of natural selection. / text