Thesis (MSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: PELOMEDUSA SUBRUFA is a freshwater turtle widely distributed throughout Africa and Madagascar, and is described as a Tropical to Sub-tropical species. 1 examined the female and male reproductive cycles of P. subrufa, over a 20-month period to determine whether they display a typical Tropical to Sub-tropical type reproductive cycle (pre-nuptial) or a typical Temperate Zone type reproductive cycle (post-nuptial). Blood and tissue samples were collected from wild specimens captured in the Western Cape, South Africa and these samples were supplemented by tissue samples obtained from museum specimens. In female P. subrufa seasonal variation in related circulating reproductive hormones in the plasma (estradiol, progesterone, and testosterone) were analyzed using validated ELISA kits. Plasma vitellogenin (yolk precursor produced in liver) was measured using a newly developed universal vitellogenin ELISA for vertebrates (UNIVTG). Ovarian follicles were measured (± 0.1 mm) and female ovaries were staged macroscopically (non-active, pre-vitellogenic, vitellogenic, gravid), and results were confirmed via histological sectioning of ovaries and oviducts. Females exhibited a cyclic reproductive pattern, with distinct phases of follicular enlargement (vitellogenesis), ovulation and a gravid period. Seasonal timing of the reproductive cycle coincided with those of other temperate zone freshwater turtles. Vitellogenic recrudescence began in summer (late December), and continued unabated through winter with ovulation occurring in the following spring (September-October). My data suggested that P. subrufa females mostly lay a single clutch of eggs during the late-spnng summer period (September through January). Clutch size varied between 7 -3 7 eggs, with the number of eggs being significantly correlated with maternal body size (r = 0.82, P < 0 001). Plasma estradiol and plasma vitellogenin concentrations peaked once during the ovarian cycle, typically coinciding with the period of early- to mid-vitellogenesis in late summer. Plasma testosterone varied throughout the year, but significant increases were measured during the ovulation and mating period in spring. Plasma progesterone concentrations were significantly elevated during the gestation period prior to ovi-position in mid-summer (December). In male P. subrufa spermatogenesis in mature specimens was distinctly seasonal and timing of the reproductive cycle coincided with those of other temperate zone freshwater turtles. Spermatogemc recrudescence began in summer, following emergence from a winter hibernation period (brumation) and spring mating. Peak testicular volume and maximum spermiogemc activity occurred in late summer and early autumn. Testicular regression commenced in autumn through winter. Spermatozoa were abundant in the ducti epididymi throughout the year. Plasma testosterone concentrations peaked once during the testicular cycle, typically coinciding with spermio genes is in late summer, early autumn. Ducti epididymi diameter showed significant variation throughout the year, whereas the epithelial cell height showed no significant seasonal variation. Peak secretory activity coincided with spermiogemc activity and high circulating testosterone concentrations in late summer, early autumn. Testicular recrudescence was correlated with increasing ambient air temperatures, photopenod and summer rainfall, whereas testicular regression, during late autumn, corresponded conversely with decreasing ambient air temperatures, photopenod and rainfall. Female and male reproductive cycles were asynchronous in that the peak spermatogenic activity occurred in autumn at the time when most females were depositing yolk in growing ovarian follicles. Therefore, adult females displayed a typical postnuptial vitellogemc cycle and adult males displayed a typical post-nuptial spermatogenic cycle. Differences between sexes in body size are common in many animals, and the African helmeted turtle is no exception. Sexual size dimorphism (SSD) in P. subrufa was pronounced, and using principal component analysis, it was clear that adult male P. subrufa was significantly larger than adult females. Using carapace length as the measure of body size (covariate), adult males, adult females, and juveniles differed significantly in absolute size of the carapace width, carapace depth, plastron length, plastron width, and head depth. However, there was no significant difference between adult males, adult females and juveniles in head width and head length. Therefore, adult males were larger than adult females in the seven traits measured, except in carapace depth where the females were significantly larger In the occurrence of ontogenetic growth patterns, the adults grow at a slower rate than juveniles in plastron length. There was no significant difference between adults and juveniles in shell width, however in depth, the adults grow at a faster rate when compared to the juveniles. Adults significantly grow at a faster rate than juveniles in absolute head size as well. However, when these traits were used as a whole data set (eight traits measured), there was no difference in growth rate between adults of either sex. Similarly, there was no significant difference in adults compared to juveniles in shell size, however, adults grow at a faster rate than juveniles in absolute body size and head size. Differences in body size, and in the size of traits such as shell measurements and head measurements relative to absolute body size, were assessed to clarify SSD of P. subrufa in South Africa. / AFRIKAANSE OPSOMMING: PELOMEDUSA SUBRUFA is ‘n varswaterskilpad wat wyd verspreid oor Afrika en Madagascar voorkom en word beskryf as ‘n Tropiese tot Sub-tropiese spesies. Die manlike en vroulike voortplantingspatroon van P. subrufa is oor ‘n tydperk van 20 maande bestudeer om vas te stel of hul voortplanting ooreenstem met ‘n tipiese tropiesie tot sub-tropiese voortplantingspatroon of ‘n tipiese gematigde-sone voortplantingspatroon. Waterskilpaaie is uit damme in die Wes-Kaap, Suid-Afnka gevang en bloed- en weefselmonsters is versamel. Materiaal en data is aangevul deur weefselmonsters van waterskilpaaie wat in museumversamelings gehuisves word. Ovarium follikels in P. subrufa wyfies is gemeet en die wyfies se ovanums is makroskopies gegradeer (onaktief, pre-vitellogenies, vitellogemes, dragtig) en resultate is deur histologiese snitte van die ovaria en ovidukte bevestig. Wyfies vertoon ‘n sikliese voortplantingspatroon, met duidelike fases van follikulere groei (vetllogenese), ovulasie en dragtigheid. Sirkulerende voortplantingshormone in die bloedplasma (estradiol, progesteroon en testosteroon) is ook geanaliseer met behulp van gevalideerde hormoonspesifieke ELISA bepalings. Plasma vitellogeen (‘n dooiervoorloper wat in die lewer vervaardig word) konsentrasies is ook bepaal met ‘n nuut ontwikkelde, universele (spesifiek vir werweldiere) vitellogeen ELISA (UNIVTG). Seisoenale tydsberekemng van die voortplantingsiklus het ooreengestem met die van ander varswaterskilpaaie vanuit die Gemagtigde-sone. Vitellogenese het in die somer begin en duur voort deur die grootste gedeelte van die somer, herfs en winter gevolg deur ovulasie in die daaropvolgende lente (September - Oktober). Die data ingewin stel voor d a t/5. subrufa wyfies meestal een broeisel eiers tydens laat lente-somer le (September tot Januane). Broeiselgrootte het gewissel tussen 7-37 eiers, met die hoeveelheid eiers wat beduidend met moederlike liggaamsgrootte gekorreleer was (r = 0.82, P < 0.001). Plasma estradiol en vitellogeen konsentrasies het een keer tydens die ovariumsiklus gepiek, en gewoonlik saamgeval met vroee tot middel vitellogenese in die laat somer. Plasma testosteroon het dwarsdeur die jaar gevarieer, maar beduidende toenames is gemeet tydens ovulasie en die paartydperk in die lente. Plasma progesteroon konsentrasies was beduidend hoer tydens dragtigheid kort voor eierlegging in die middel van die somer (Desember). In volwasse P. subrufa mannetjies was spermatogenese sterk seisoenaal en het die voortplantingsiklus ooreengestem met die van ander varswaterskilpadspesies wat in die gematigde streke voorkom. Na ‘n oorwinteringsperiode (brumasie), volg die panngstydperk gedurende die lente. ‘n Nuwe spermatogemese siklus het in die somer begin. Maksimale spermatogeniese aktiwiteit en testis-volume word in die laat somer en vroee herfs bereik. Testikulere regressie neem in aanvang in die herfs en duur voort tot na paringstyd in die lente. Tydens testikulere regressie word spermatosoe in die ducti epididymi gestoor. Plasma testosteroon konsentrasies het in die laat somer en vroee herfs gedurende die testikulere siklus, spermiogenese (sperm produksie fase), gepiek. Die grootte (omtrek) van die ducti epididymi het beduidende vanasie dwarsdeur die jaar getoon, terwyl epiteel selhoogtes geen beduidende seisoenale vanasie getoon het nie. Piek sekretonese aktiwiteit het saamgeval met spermiogeniese aktiwiteit en hoe vlakke van sirkulerende testosteroon tydens laat somer en vroee herfs. Testikulere groei het goed gekorreleer met toenemende omgewingstemperatuur, fotopenode en reenval, terwyl testikulere regressie in herfs met ‘n daling in omgewingstemperature, fotopenode en reenval gekorrespondeer het. Die vroulike en manlike voortplantingspatrone was nie goed ge-sinkroniseerd nie, deurdat piek spermatogeniese aktiwiteit tydens herfs voorgekom het, gedurende die tyd waann meeste wyfies besig was om dooier in groeiende ovarium follikels neer te le. Daarteenoor vertoon die mannetjies testikulere regressie tydens die pre-ovulatoriese fase en ovulasie penode van die wyfies. Dus toon volwasse wyfies ‘n tipiese gematigde sone vitellogeniese patron en volwasse mannetjies ‘n tipiese gematigde sone spermatogeniese patroon. ‘n Verskil in liggaamsgrootte tussen die geslagte is 'n algemene verskynsel by baie diere en P. subrufa is geen uitsondering me. Daar was wesenlike geslagtelike grootteverskille (SSD) in P. subrufa en ‘n hoofkomponent analise (PC A) het getoon dat daar beduidende morfometnese verskille tussen volwasse mannetjies en wyfies was. Deur karapakslengte as 'n maatstaf vir liggaamsgrootte te gebruik (mede-veranderlike), het volwasse mannetjies, volwasse wyfies en onvolwassenes beduidend verskil ten opsigte van absolute grootte van hul karapaksbreedte, karapaksdiepte, plastronlengte, plastronbreedte en kopdiepte. Geen beduidende verskil in kopbreedte en koplengte in volwasse mannetjies, volwasse wyfies en onvolwassenes is gevind nie. Derhalwe was volwasse mannetjies groter as volwasse wyfies in sewe van die liggaamseienskappe wat gemeet is, buiten vir karapaksdiepte waar die wyfies beduidend groter was. In terme van die voorkoms van ontogenetiese groeipatrone het volwassenes teen ‘n stadiger tempo as onvolwassenes in plastronlengte toegeneem. Daar was geen beduidende verskil in die groeitempo van dopbreedte tussen volwassenes en onvolwassenes nie, alhoewel dopdiepte van volwassenes teen ‘n vinniger tempo gegroei het as die van onvolwassenes. Absolute kopgrootte van volwassenes het ook teen ‘n vinniger tempo gegroei as in onvolwassenes. Wanneer hierdie eienskappe as ‘n volledige datastel gebruik word (al agt gemete eienskappe), wil dit voorkom asof daar geen verskil in groeitempo van volwassenes van die onderskeidelike geslagte is me. Daar was geen beduidende verskil tussen volwassenes en onvolwassenes, ten opsigte van dopgrootte nie, alhoewel volwassenes klaarblyklik teen ‘n vinniger tempo in liggaamsgrootte en kopgrootte toeneem. Verskille in liggaamsgrootte en grootte van ander veranderlikes, soos byvoorbeeld dop- en kop-eienskappe, relatief tot absolute liggaamsgrootte, word aangebied om geslagsdimorfisme in P. subrufa vir die eerste keer te beskryf.

The advent of sexual reproduction in early evolutionary history had profound effects on the evolution of animals. In most sexually reproducing species, males and females have distinct morphological and behavioral differences that are shaped by the evolutionary imperatives of each sex. Underlying the behavioral differences between males and females are distinct and measurable dimorphisms in the nervous system. These dimorphisms can arise in the form of connectivity, neurotransmitter usage, gene expression or combinations of all three. The androdioecious nematode Caenorhabditis elegans, with its stereotyped development and simple nervous system, offers a remarkably powerful system for studying the conserved mechanisms of sex determination that shape neural development. In this thesis, I present my work on the characterization of several genes that regulate the development of sexual dimorphisms in the nervous system. The first part of the thesis concerns the characterization of the gene ham-3, which codes for a subunit of the C. elegans ortholog of the SWI/SNF chromatin remodeling complex. ham-3 is required for the proper terminal differentiation of the HSN, a serotonergic neuron of the sex-specific nervous system, which it manages by regulating the expression of transcription factors required for crucial steps of migration, axon guidance and serotonergic fate adoption. The second part of the thesis concerns the investigation of sexually dimorphic pruning mechanisms. I show that unc-6/Netrin is subject to direct transcriptional repression in hermaphrodites by tra-1, the master transcriptional regulator of sexual fate determination in C. elegans. This regulation is required for the proper timing of the sexually dimorphic pruning of synapses in the tail region in hermaprhodites. In males, where unc-6 is not repressed by tra-1, unc-6 expression perdures into adulthood and the synapse is maintained. Together, these data provide insight into the ways in which conserved genetic and developmental mechanisms manage the generation differentiation, connectivity, and maintenance of sexually dimorphic nervous systems.

Thesis submitted to the Faculty of Science, University of the Witwatersrand, Johannesburg in fulfilment of the requirements for the degree of Master of Science. / Breeding was observed in the foam nest frog, Chiromantis xerampelina, over three breeding seasons The mating pattern was characterized by an extended breeding season with a male-biased operational sex ratio and asynchronous and unpredictable female arrival. At more than 90% of nests, from one to seven unpaired males ('peripheral males') gathered around the amplexing pair during nest construction. Those peripheral males closest to the pair competed 'With each other, and with the amplexing male, to position their cloacae against the female's cloaca during oviposition bouts. In a detailed study of a single population, over 80% of males were observed as peripheral males, and 57% of males were observed both in amplexus and as peripheral males. Male mating success and participation at nests was unrelated to size or weight. Chorus participation was the best predictor of male, mating success and participation at nests. The most plausible explanation for the presence of peripheral males was a sperm competition hypothesis; namely that peripheral males compete with the amplexing male for fertilizations by shedding sperm into the nest. I collected data on body mass and testis mass for 13 African anurans. Using additional published data on 19 Japanese anurans, an allometric relationship between body mass and testis mass was calculated. using 16 genera as independent data points. This revealed that C. xerampelina have testes fourteen times heavier than predicted on the basis of body mass. This is consistent with a trend seen in several taxa where testis size is related to the intensity of sperm competition. An additional experiment, in which the arnplexing male was prevented from shedding sperm into the nest, showed that peripheral males are capable of fertilizing eggs. I conclude that peripheral males are engaged in an opportunistic alternative mating tactic involving sperm cosnpetition. More than half the observed females bred polyandrously, some mating with up to three males, This was the result of amplexing males dismounting between nesting sessions, and males displacing one another from amplexus. / Andrew Chakane 2018

Thesis (Ph. D.)--University of Chicago, Faculty of the Division of the Biological Sciences and the Pritzker School of Medicine, Department of Ecology and Evolution, June 2001. / Includes bibliographical references. Also available on the Internet.

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.