Thesis (Ph. D.)--University of Sydney, 2005. / Title from title screen (viewed 20 May 2008). Submitted in fulfilment of the requirements for the degree of Doctor of Philosophy to the School of Biological Sciences, Faculty of Science. Degree awarded 2005; thesis submitted 2004. Includes bibliographical references. Also available in print form.

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

Evolution can occur rapidly, along timescales that are traditionally regarded as 'ecological'. Despite growing acceptance among biologists of rapid evolution, a strong paradigm of contemporary evolution is still absent in many sub-disciplines. Here I apply a contemporary evolution viewpoint to conservation biology. Specifically, I examine the impact of cane toads (Bufo marinus) on Australian snakes. Toads were introduced into Australia in 1935, have spread rapidly and represent a novel, extremely toxic prey item to na�ve Australian predators (including snakes). Based on dietary preferences and geographic distributions I find that 49 species of Australian snake are potentially at risk from the invasion of the toad. Furthermore, examination of physiological resistance to toad toxin in 10 of these �at risk� species strongly suggests that most species of Australian snake are poorly equipped to deal with a likely dose of toad toxin. Even species that are highly resistant to toad toxin (such as the keelback, Tropidonophis mairii) face indirect fitness costs associated with consuming toads. Within a population of snakes however, the impact of toads is unlikely to be random. For example, the examination of several component allometries describing the interaction between snakes and toads revealed that, within a species, smaller snakes are more likely to ingest a fatal dose of toad toxin than are larger snakes. Further consideration of the interaction between snakes and toads suggests that toads will not only be exerting differential impact on snakes based upon morphology, but also exert non-random selection on prey preference and resistance to toad toxin in snake populations. To examine the possibility of a morphological response by snakes to toads, I examined changes in the body size and relative head size of four species of snake as a consequence of time since exposure to toads. Two of the species (green treesnakes and red-bellied blacksnakes) are predicted to face strong impacts from toads. These two species showed an increase in mean body size and a decrease in relative head size as a consequence of time since exposure to toads; both changes in an adaptive direction. In contrast, the other two species (keelbacks and swampsnakes) are predicted to face much lower impact from toads, and these two species showed little or no evidence of morphological change associated with time since exposure to toads. These results indicate an adaptive change in morphology at a rate that is proportional to the predicted level of impact for each species, strongly suggesting an evolved response. Red-bellied blacksnakes (a toad-vulnerable species) were further assessed for evolved responses in prey preference and toxin resistance. Comparisons between toad-exposed and toad-na�ve populations of blacksnakes revealed that snakes from toad-exposed populations exhibited slightly higher resistance to toad toxin and a much-reduced tendency to eat toads, when compared with toad-na�ve snakes. Na�ve snakes exhibited no tendency to learn avoidance of toxic prey, nor were they able to acquire resistance to toxin as a result of several sub-lethal doses, suggesting that the observed differences between populations is evolved rather than acquired. Together, these results strongly suggest that blacksnakes are exhibiting an evolved shift in prey preference and toxin resistance as a consequence of exposure to toads. Thus, it appears that snakes are exhibiting adaptation at multiple traits in response to exposure to toads. Given the high likelihood that these adaptive shifts have an evolved basis, it appears that the impact of toads will decrease with time in many snake populations. But what about toads? Because the outcome of the interaction between a toad and a snake is also mediated by the body size and relative toxicity of toads, it is important to understand how these traits vary in space and time. Exploratory analysis revealed that toads exhibit a decrease in body size and a decrease in relative toxicity as a consequence of time since colonisation, indicating that their impact on native predators decreases with time. Additionally, there appears to be meaningful spatial variation in toad relative toxicity, indicating that some populations of native predators are facing higher impact from toads than others. Overall, these results clearly indicate the importance of assessing the potential for rapid evolutionary response in impacted systems. Doing so may provide evidence that some species are in less trouble than originally thought. Additionally, and as more data accumulate, it may be possible to characterise certain categories of environmental impact by their potential for eliciting adaptive response from �impacted� species. This approach has strong implications for the way conservation priorities are set and the way in which conservation dependent populations are managed.

Thesis (Ph.D.)--Ohio University, November, 2009. / Release of full electronic text on OhioLINK has been delayed until December 1, 2014. Title from PDF t.p. Includes bibliographical references.

Evolution can occur rapidly, along timescales that are traditionally regarded as 'ecological'. Despite growing acceptance among biologists of rapid evolution, a strong paradigm of contemporary evolution is still absent in many sub-disciplines. Here I apply a contemporary evolution viewpoint to conservation biology. Specifically, I examine the impact of cane toads (Bufo marinus) on Australian snakes. Toads were introduced into Australia in 1935, have spread rapidly and represent a novel, extremely toxic prey item to na�ve Australian predators (including snakes). Based on dietary preferences and geographic distributions I find that 49 species of Australian snake are potentially at risk from the invasion of the toad. Furthermore, examination of physiological resistance to toad toxin in 10 of these �at risk� species strongly suggests that most species of Australian snake are poorly equipped to deal with a likely dose of toad toxin. Even species that are highly resistant to toad toxin (such as the keelback, Tropidonophis mairii) face indirect fitness costs associated with consuming toads. Within a population of snakes however, the impact of toads is unlikely to be random. For example, the examination of several component allometries describing the interaction between snakes and toads revealed that, within a species, smaller snakes are more likely to ingest a fatal dose of toad toxin than are larger snakes. Further consideration of the interaction between snakes and toads suggests that toads will not only be exerting differential impact on snakes based upon morphology, but also exert non-random selection on prey preference and resistance to toad toxin in snake populations. To examine the possibility of a morphological response by snakes to toads, I examined changes in the body size and relative head size of four species of snake as a consequence of time since exposure to toads. Two of the species (green treesnakes and red-bellied blacksnakes) are predicted to face strong impacts from toads. These two species showed an increase in mean body size and a decrease in relative head size as a consequence of time since exposure to toads; both changes in an adaptive direction. In contrast, the other two species (keelbacks and swampsnakes) are predicted to face much lower impact from toads, and these two species showed little or no evidence of morphological change associated with time since exposure to toads. These results indicate an adaptive change in morphology at a rate that is proportional to the predicted level of impact for each species, strongly suggesting an evolved response. Red-bellied blacksnakes (a toad-vulnerable species) were further assessed for evolved responses in prey preference and toxin resistance. Comparisons between toad-exposed and toad-na�ve populations of blacksnakes revealed that snakes from toad-exposed populations exhibited slightly higher resistance to toad toxin and a much-reduced tendency to eat toads, when compared with toad-na�ve snakes. Na�ve snakes exhibited no tendency to learn avoidance of toxic prey, nor were they able to acquire resistance to toxin as a result of several sub-lethal doses, suggesting that the observed differences between populations is evolved rather than acquired. Together, these results strongly suggest that blacksnakes are exhibiting an evolved shift in prey preference and toxin resistance as a consequence of exposure to toads. Thus, it appears that snakes are exhibiting adaptation at multiple traits in response to exposure to toads. Given the high likelihood that these adaptive shifts have an evolved basis, it appears that the impact of toads will decrease with time in many snake populations. But what about toads? Because the outcome of the interaction between a toad and a snake is also mediated by the body size and relative toxicity of toads, it is important to understand how these traits vary in space and time. Exploratory analysis revealed that toads exhibit a decrease in body size and a decrease in relative toxicity as a consequence of time since colonisation, indicating that their impact on native predators decreases with time. Additionally, there appears to be meaningful spatial variation in toad relative toxicity, indicating that some populations of native predators are facing higher impact from toads than others. Overall, these results clearly indicate the importance of assessing the potential for rapid evolutionary response in impacted systems. Doing so may provide evidence that some species are in less trouble than originally thought. Additionally, and as more data accumulate, it may be possible to characterise certain categories of environmental impact by their potential for eliciting adaptive response from �impacted� species. This approach has strong implications for the way conservation priorities are set and the way in which conservation dependent populations are managed.

Thesis (Ph.D.)--University of Western Australia, 2003.

Currently published data on the role of the lymphatic system in amphibians are inadequate and contradictory. Estimates of the rate of formation of lymph and the role of the lymph hearts in returning this fluid to the circulation are not based on actual volume determinations but rather estimates derived from changes in hematocrit using published values of plasma and blood volume. The lymph hearts are known to be vital to the maintenance of normal fluid compartment physiology and to increase their rate of activity during episodes of hypovolemic stress. Yet, significant redistribution of body fluids following hemorrage appears to occur in animals without lymph hearts. In this study, plasma and blood volumes were determined by the dye dilution technique using injected Evan's blue dye to label the plasma. Eight intact and 6 animals with their lymph hearts destroyed were hemorrhaged to 78% and 75% of their initial blood volumes, respectively. Changes in blood volume were measured following the hemorrhage by analysis of Evan's blue washout and hemodilution.

No description available.

To date, the majority of studies into sex determination and sexual development have focused on the mammalian system due to the mouse model being an excellent tool for developmental biology as well as obvious implications to human health and development. However, the focus on the mammalian system has caused a large fraction of other vertebrate groups to be overlooked. The last decade has seen an array of studies into various non-mammalian organisms like fishes, birds, reptiles, and amphibians. These studies have unveiled a remarkably conserved molecular background utilized for sexual differentiation amongst all vertebrates, ranging from the alligator which employs temperature dependent sex determination to the mouse, which employs genotypic sex determination In this project, I implemented molecular methods traditionally used to study model organisms, to investigate an amphibian species, the cane toad (Bufo marinus). The cane toad was chosen due to its invasive status in Australia, as well as being a representative of one of the most successful and specious families of vertebrates, Bufonidae. Since, its introduction, this species has rapidly spread across the continent, adversely effecting native species throughout its introduced range. Recent studies have identified the disruption of the sex-determination pathway as a realistic goal in an otherwise, seemingly futile, effort to curtail their expansion. We decided to approach the study of cane toad sex determination and development on three levels, in order to have a broader understanding of the similarities between toads and other groups, and additionally, to better assess the feasibility of utilizing molecular means to control cane toads in non-native habitats. Firstly, I used a candidate gene approach to clone and characterize five genes which were previously known to be involved in both mammalian as well as amphibian sex determination: Sox9, Dmrt1, p450arom, Sf1, and Dax1. I chose Sox9 and Dmrt1 due to their known involvement in the male-specific pathway of mammals. Dmrt1 was also known to be male specific in other groups, including amphibians. Conversely, I also decided to investigate p450arom and Dax1, due to their affiliation with the female pathway, while Sf1 has a role in both sexes. All of these genes were expressed in the gonads of both sexes of cane toads. However, Sox9 exhibited strong transcriptional up-regulation in testes at the time of sexual differentiation, similar to mammals. Thus, using the candidate gene approach, I was successful in identifying a sex-specific marker which could be utilized in the manipulation of the sex determination pathway (e.g. female to male sex reversal). VIIThe second approach involved the study of sex determination on a cytogenetic level. Traditionally, Bufonids have been assumed to utilize a ZZ/ZW (male homogametic/female heterogametic) sex chromosome system, yet only one recent study has been successful in the identification of sex chromosomes in a toad species. Moreover, the authors identified the sex chromosomes in only one population (of 18 studied), highlighting the scarcity of discernible sex chromosomes in toads. When we began to investigate the karyotype of cane toads, we were able to identify a female-specific length polymorphism in the nucleolus organizer region (NOR) of chromosome 7, making this chromosome pair a strong candidates for the Z and W sex chromosomes. In order to verify our hypothesis, we also performed chromomycin A3 staining to reveal a differential signal between the chromosome pair, indicative of heterochromatin accumulation on the brighter NOR. This study lent strong support to the observed difference between the chromosomes, yet we required further investigation in order to identify the W and the Z chromosomes, specifically. By utilizing comparative genomic hybridization (CGH), I was able to identify a female specific region on the chromosome with the larger NOR, identifying it as the W chromosome. This study was successful in identifying the sex chromosomes in the cane toad. Moreover, it allows us to make a more accurate prediction of the possible sex determination method utilized: either a dosage dependent male determination system, or a female-specific gene in a female determination system. The third approach focused on the Bidder’s organ and its role in sexual development of cane toads. The Bidder’s organ is a gonadal structure comprised of ovary-like tissue, found in both males and females, with no known function. Since our study was focused on female to male sex-reversal of cane toads, we decided to investigate the potential of the Bidder’s organ to interrupt this process. By utilizing the candidate genes, which I had previously cloned, I was able to identify the Bidder’s organ as having an entirely distinct transcriptional pattern in comparison to the gonads of either sex. Additionally, the Bidder’s organ showed significantly higher levels of p450arom expression than the gonads, identifying it as a possible key player in the production of aromatase enzyme for oestrogen production. Moreover, gene expression patterns in the Bidder’s organ strongly correlated with the significant developmental time points in the sexual development of the toads, implying a possible function in the development process of toads. In conclusion, I have performed the first study of the molecular, cytogenetic and anatomical aspects of sexual development in a toad. I was able to verify that cane toads utilize a ZZ/ZW chromosome system and furthermore, likely utilize the mammalian male-linked gene, Sox9, in male development. I was also able to show that the Bidder’s organ is transcriptionally active at key time points, likely indicative of a functional role during development.

Thesis (Ph. D.) -- University of Tennessee, Knoxville, 2005. / Title from title page screen (viewed on Sept. 6, 2005). Thesis advisor: Arthur C. Echternacht. Thesis advisior: Christine R. B. Boake. Document formatted into pages (xvi, 151 p. : ill. (some col.)). Vita. Includes bibliographical references.