Amphibians contain a rich chemical arsenal in their skin glands, vital for their defence against predators, both large and small. The peptides secreted by the frogs have a range of biological activities. These include both antibacterial and anticancer activity, others are neuropeptides, while some exhibit antifungal and antimalarial activity. Peptides are usually sequenced using positive ion mass spectrometry ( MS ). However, negative ion MS can also provide valuable sequencing information. Under negative ion MS conditions, the presence of a Cys residue is immediately identified by the facile side chain loss of H [subscript 2] S. The position of the Cys residue is determined by the formation of a side chain induced backbone cleavage ion. When a Cys residue is in the C - terminal position of a peptide, the spectrum is dominated by the loss of both H [subscript 2] S and CO [subscript 2]. Negative ion MS can also be used to identify and sequence intramolecular disulfide bridged peptides. The disulfide bridge is immediately identified by the facile loss of H [subscript 2] S [subscript 2] from the parent anion. Once the disulfide bridge is cleaved, further amide cleavages provide much of the sequence of the peptide, including the residues originally within the disulfide link. When one of the disulfide bridged Cys residues is in the C - terminal position, the major fragmentation is the loss of H [subscript 2] S [subscript 2] and CO [subscipt 2] from the parent ion. The negative ion mass spectra of citropin 1.1 and synthetically modified analogues show an unusual loss of an internal Val residue from the ( M - H ) - parent ion. This rearrangement requires the decomposing anion to have an α - helical structure. The skin secretions of Litoria peronii or Peron ' s Tree Frog contain five novel peptides which have been named peroneins. Four pro - peroneins are present in the summer secretions only. The biologically active peptides caerulein 1.1, caerin 1.1 and caerin 2.1 are also present in the glandular secretions. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.

Venoms, toxins and host-defence systems constitute rich sources of biologically active molecules, many of which have enormous therapeutic and biotechnological potential. In particular, peptides are often a significant component of these chemical arsenals, and are fundamentally important as biological effector molecules. The research presented in this thesis is centred on the isolation and investigation of peptides from both frogs and spiders, and endeavours to probe the important structural and mechanistic features of these bioactive compounds. The skin peptide profiles of interspecific hybrids between the green tree frog Litoria caerulea and the magnificent tree frog Litoria splendida have been investigated in a ninemonth survey. Fourteen peptides were characterised primarily using mass spectrometry, of which three had not been identified previously in the skin secretions of either parent. A number of these peptides are antibacterial agents, while others effectively inhibit the formation of nitric oxide by neuronal nitric oxide synthase. Implications for the genetics and expression of amphibian dermal peptides are also discussed. The majority of frogs of the genus Litoria contain at least one peptide in their glandular secretion capable of inhibiting the formation of nitric oxide by the enzyme neuronal nitric oxide synthase. This was proposed to occur by preventing the association of the regulatory cofactor, Ca²⁺ -calmodulin, with its binding site on the enzyme. Non-covalent binding of the amphibian peptides to calmodulin in the presence of Ca²⁺ has been confirmed using electrospray ionisation mass spectrometry, by the observation of complexes in the gas phase with a 1 : 1 : 4 calmodulin / peptide / Ca²⁺ stoichiometry. In addition, the structure and binding interactions of caerin 1.8, a potent nitric oxide synthase inhibitor, have been further probed using mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Recently a number of small, disulfide - containing neuropeptides of the signiferin and riparin families have been characterised from the skin secretion of frogs of the Crinia genus. Of these, signiferin 1 and riparin 1.1 are both ten residue peptides with similar primary sequences, however appear to have a significantly different spectrum of bioactivity. Although both act at cholecystokinin-2 receptors, signiferin 1 is smooth muscle active while riparin 1.1 is not, and instead causes proliferation of lymphocytes. The three-dimensional structures of these peptides were determined using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Both signiferin 1 and riparin 1.1 adopt β - turn type conformations, however differences in these structures may be responsible for the variation in biological activity noted for these peptides. The dermal secretions of most Australian frogs contain at least one broad-spectrum peptide antibiotic, and often a series of peptides with differing activity to afford greater protection against microbial pathogens. Solid state nuclear magnetic resonance spectroscopy studies were carried out to investigate the interaction of a number of these antibacterial peptides with anionic model membranes, and the results are compared with work previously reported using neutral lipids. It appears the peptides may have a different mode of interaction with the membranes depending upon the charge of the lipid head group. The cupiennin 1 peptides have been identified in the venom of the neotropical wandering spider, Cupiennius salei, and demonstrate potent wide-spectrum antibacterial activity. Primary sequence analysis of these peptides suggests a unique amphipathic structure distinctly different from that of other potentially helical cationic antimicrobial peptides isolated thus far. Using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations, cupiennin 1a was found to adopt an α- helical structure with a flexible central hinge region in membrane mimicking solvents. Following this, nuclear magnetic resonance spectroscopy methods were used to further probe the antibacterial and the newly identified neuronal nitric oxide synthase inhibitory activity of this peptide. / Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, Discipline of Chemistry, 2006

The skin extracts from amphibians have been investigated for over fifty years and have been found to contain numerous components with therapeutic and medicinal uses. Host - defence compounds are secreted onto the dorsal surface of the animal from specialised granular glands in response to a variety of stimuli, such as stress induced by a predator. Isolated peptides can exhibit either pharmacological properties or antibiotic activity. Previous studies isolated a potent hypotensive neuropeptide, crinia angiotensin II, within skin secretions of the Australian frog Crinia georgiana. This prompted further investigations into the isolation and sequence determination of host - defence compounds from other species in this genus - C. signifera, C. riparia and C. deserticola. Fifteen novel peptides were identified. The major peptide components were potent disulfide containing neuropeptides of a type not observed in other Australian anurans that have been previously investigated. The remaining peptides demonstrate either antibiotic activity or inhibit the enzyme neuronal nitric oxide synthase. The skin components from anurans of the Litoria genus have been extensively studied, with a number of peptides exhibiting both antibacterial and pharmacological activity. The skin secretion of Litoria dentata has been investigated, with five novel peptides identified. The neuropeptide tryptophyllin L 1.3 was previously isolated from the related frog L. rubella. Other components that are unique in structure have not yet been tested for biological activity. The parasitic disease malaria is responsible for over one million deaths per year. The increase in resistance of current antimalarial compounds has led to the development of new treatments from various animal - derived peptide antimicrobials. A number of amphibian peptides and their derivatives were investigated as potential antiplasmodial agents against the malaria parasite Plasmodium falciparum. Results indicate that these compounds inhibit parasite growth with minimal haemolytic activity, making them promising tools for malaria research.The defence chemistry of amphibian neuropeptides has been extensively studied and is important in understanding both the ecology and physiology of the vertebrate. Neuropeptides are classified into groups with similar structural characteristics. Biological activity occurs via interaction with a G protein - coupled receptor. The most studied of all amphibian neuropeptides is caerulein, which has a similar spectrum of activity to the mammalian peptide cholecystokinin. This includes smooth muscle contraction that occurs via interaction with cholecystokinin receptors. The pharmacological activity of Australian anuran neuropeptides from various genera was investigated. Two biological assays were conducted - a smooth muscle contraction test and a lymphocyte proliferation assay. A range of neuropeptides contracted smooth muscle at nanomolar concentrations, while others only proliferated lymphocytes. Some peptides were inactive in both assays. Young marsupials are born at an immature stage of development and rely on immune protection provided by the mother. Eugenin is a host - defence compound isolated from pouch secretions of the Tammar wallaby. The immunomodulator activates CCK2 receptors, resulting in lymphocyte proliferation. Therefore, eugenin stimulates immune cells in the pouch providing vital immune protection for pouch young. / Thesis (Ph.D.)--School of Chemistry and Physics, 2006.

Venoms, toxins and host-defence systems constitute rich sources of biologically active molecules, many of which have enormous therapeutic and biotechnological potential. In particular, peptides are often a significant component of these chemical arsenals, and are fundamentally important as biological effector molecules. The research presented in this thesis is centred on the isolation and investigation of peptides from both frogs and spiders, and endeavours to probe the important structural and mechanistic features of these bioactive compounds. The skin peptide profiles of interspecific hybrids between the green tree frog Litoria caerulea and the magnificent tree frog Litoria splendida have been investigated in a ninemonth survey. Fourteen peptides were characterised primarily using mass spectrometry, of which three had not been identified previously in the skin secretions of either parent. A number of these peptides are antibacterial agents, while others effectively inhibit the formation of nitric oxide by neuronal nitric oxide synthase. Implications for the genetics and expression of amphibian dermal peptides are also discussed. The majority of frogs of the genus Litoria contain at least one peptide in their glandular secretion capable of inhibiting the formation of nitric oxide by the enzyme neuronal nitric oxide synthase. This was proposed to occur by preventing the association of the regulatory cofactor, Ca²⁺ -calmodulin, with its binding site on the enzyme. Non-covalent binding of the amphibian peptides to calmodulin in the presence of Ca²⁺ has been confirmed using electrospray ionisation mass spectrometry, by the observation of complexes in the gas phase with a 1 : 1 : 4 calmodulin / peptide / Ca²⁺ stoichiometry. In addition, the structure and binding interactions of caerin 1.8, a potent nitric oxide synthase inhibitor, have been further probed using mass spectrometry and nuclear magnetic resonance spectroscopy techniques. Recently a number of small, disulfide - containing neuropeptides of the signiferin and riparin families have been characterised from the skin secretion of frogs of the Crinia genus. Of these, signiferin 1 and riparin 1.1 are both ten residue peptides with similar primary sequences, however appear to have a significantly different spectrum of bioactivity. Although both act at cholecystokinin-2 receptors, signiferin 1 is smooth muscle active while riparin 1.1 is not, and instead causes proliferation of lymphocytes. The three-dimensional structures of these peptides were determined using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations. Both signiferin 1 and riparin 1.1 adopt β - turn type conformations, however differences in these structures may be responsible for the variation in biological activity noted for these peptides. The dermal secretions of most Australian frogs contain at least one broad-spectrum peptide antibiotic, and often a series of peptides with differing activity to afford greater protection against microbial pathogens. Solid state nuclear magnetic resonance spectroscopy studies were carried out to investigate the interaction of a number of these antibacterial peptides with anionic model membranes, and the results are compared with work previously reported using neutral lipids. It appears the peptides may have a different mode of interaction with the membranes depending upon the charge of the lipid head group. The cupiennin 1 peptides have been identified in the venom of the neotropical wandering spider, Cupiennius salei, and demonstrate potent wide-spectrum antibacterial activity. Primary sequence analysis of these peptides suggests a unique amphipathic structure distinctly different from that of other potentially helical cationic antimicrobial peptides isolated thus far. Using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations, cupiennin 1a was found to adopt an α- helical structure with a flexible central hinge region in membrane mimicking solvents. Following this, nuclear magnetic resonance spectroscopy methods were used to further probe the antibacterial and the newly identified neuronal nitric oxide synthase inhibitory activity of this peptide. / Thesis (Ph.D.) -- University of Adelaide, School of Chemistry and Physics, Discipline of Chemistry, 2006

<p>Understanding the diversity of life is one of the main aims of evolutionary biology, and requires knowledge of the occurrence and causes of adaptive genetic differentiation among geographically distinct populations. Environmental stress caused by acidity may cause strong directional selection in natural populations, but is little explored from an evolutionary perspective. In this thesis, a series of laboratory experiments and field data was used to study evolutionary and ecological responses of amphibians to environmental acidity. </p><p>Local adaptation to acid stress was studied in the moor frog (<i>Rana arvalis)</i>.The results show that acid origin populations have higher acid stress tolerance during the embryonic stages than neutral origin populations, and that acid and neutral origin populations have diverged in embryonic and larval life-histories. The mechanisms underlying adaptive differentiation are partially mediated by maternal effects related to extra-embryonic membranes and egg size. Acid origin females invest in larger eggs and have a stronger egg size-fecundity trade-off than females from neutral areas, likely reflecting adaptive differentiation in maternal investment patterns. </p><p>Potential carry-over effects of low pH, and the effects of UV-b/pH interaction were investigated in the common frog (<i>R. temporaria</i>). The results suggest that amphibian larvae are able to compensate for the negative effects of acidity experienced early in life, if conditions later turn beneficial. <i>R. temporaria</i> populations differed in their sensitivity to synergistic effects of low pH/UV-B, indicating variation in population responses to environmental stress.</p><p>In conclusion, these results suggest rapid evolution in response to human induced environmental change, much of which may be mediated via adaptive maternal effects. Acidification may be a powerful selective force shaping life-history evolution.</p>

<p>This thesis is an attempt to describe how different environmental factors influence life history traits in different populations, sexes and developmental stages in the moor frog, <i>Rana arvalis. </i>The studied populations are located along 1100 km latitudinal gradient, with pH varying between 4.0 and 8.5. I have used data from both natural populations and common garden experiments. </p><p>Reproducing moor frogs were larger and older at high latitudes, indicating a selective advantage of large size at high latitudes and/or earlier reproduction at low latitudes. When controlling for age I found that frogs were older and smaller at low pH, which may be a result of a reduced growth rate due to acid stress. The both sexes respond differently to different environments, with the lowest sexual dimorphism in body size found in the acid environments. This is possibly caused by a trade-off between growth and reproduction. Being large is considered to be advantageous, in females due to increased fecundity, and in males due to higher ability to compete for mates, while the cost of high growth is a reduced possibility to survive until the next mating season. Moor frog embryos originating from an acid population survived better under acid stress than embryos from a neutral population. Using quantitative genetic techniques I found strong maternal effects and small additive genetic variation for the traits in acid and non acid populations. The variation in acid stress tolerance owed largely to non-genetic effects. Females from acid localities lay larger eggs, which probably improves the performance of tadpoles under acid conditions. The trade-off between egg size and fecundity was stronger in acid populations indicating that females in acid populations reduced fecundity to increase offspring size. Finally, frogs from acidified environments were more asymmetric in skeletal traits further indicating the developmental stress created by acidification.</p>

World-wide amphibian declines sparked concern and encouraged investigation into potential causes beginning in the 1980’s. Infectious disease has been identified as one of the major potential contributors to amphibian declines. For example, Ranavirus has caused amphibian die-offs throughout the United States. Investigators isolated Ranavirus from dead or moribund amphibians during large-scale die-offs of amphibians in the Cades Cove area of Great Smoky Mountains National Park in 1999-2001. In 2009, after nearly a decade without follow-up monitoring, I undertook an investigation to determine if the virus persisted in the area, and if so, to assess spatial, temporal, and taxonomic patterns in prevalence. Three amphibian breeding ponds, including Gourley Pond, the site of these earlier mortality events, were monitored for Ranavirus during the 2009 amphibian breeding season. A peak in prevalence occurred at Gourley Pond corresponding to a massive amphibian die-off. Prevalence varied among three different taxonomic groups during this mortality event with the highest prevalence, 84%, detected in larval Ambystomatids, 44.4% prevalence in adult Newts, and no virus detected in adult Plethodontids. I did not detect virus at either of the other two breeding ponds despite equivalent sampling effort, similar community composition, and close proximity to Gourley Pond. These results suggest that the severity and spatial extent of Ranavirus in Cades Cove remains unchanged since its initial detection a decade ago. Also, despite the observed massive die-offs there is no evidence of local amphibian extinction at Gourley Pond.

Understanding the diversity of life is one of the main aims of evolutionary biology, and requires knowledge of the occurrence and causes of adaptive genetic differentiation among geographically distinct populations. Environmental stress caused by acidity may cause strong directional selection in natural populations, but is little explored from an evolutionary perspective. In this thesis, a series of laboratory experiments and field data was used to study evolutionary and ecological responses of amphibians to environmental acidity. Local adaptation to acid stress was studied in the moor frog (Rana arvalis).The results show that acid origin populations have higher acid stress tolerance during the embryonic stages than neutral origin populations, and that acid and neutral origin populations have diverged in embryonic and larval life-histories. The mechanisms underlying adaptive differentiation are partially mediated by maternal effects related to extra-embryonic membranes and egg size. Acid origin females invest in larger eggs and have a stronger egg size-fecundity trade-off than females from neutral areas, likely reflecting adaptive differentiation in maternal investment patterns. Potential carry-over effects of low pH, and the effects of UV-b/pH interaction were investigated in the common frog (R. temporaria). The results suggest that amphibian larvae are able to compensate for the negative effects of acidity experienced early in life, if conditions later turn beneficial. R. temporaria populations differed in their sensitivity to synergistic effects of low pH/UV-B, indicating variation in population responses to environmental stress. In conclusion, these results suggest rapid evolution in response to human induced environmental change, much of which may be mediated via adaptive maternal effects. Acidification may be a powerful selective force shaping life-history evolution.

This thesis is an attempt to describe how different environmental factors influence life history traits in different populations, sexes and developmental stages in the moor frog, Rana arvalis. The studied populations are located along 1100 km latitudinal gradient, with pH varying between 4.0 and 8.5. I have used data from both natural populations and common garden experiments. Reproducing moor frogs were larger and older at high latitudes, indicating a selective advantage of large size at high latitudes and/or earlier reproduction at low latitudes. When controlling for age I found that frogs were older and smaller at low pH, which may be a result of a reduced growth rate due to acid stress. The both sexes respond differently to different environments, with the lowest sexual dimorphism in body size found in the acid environments. This is possibly caused by a trade-off between growth and reproduction. Being large is considered to be advantageous, in females due to increased fecundity, and in males due to higher ability to compete for mates, while the cost of high growth is a reduced possibility to survive until the next mating season. Moor frog embryos originating from an acid population survived better under acid stress than embryos from a neutral population. Using quantitative genetic techniques I found strong maternal effects and small additive genetic variation for the traits in acid and non acid populations. The variation in acid stress tolerance owed largely to non-genetic effects. Females from acid localities lay larger eggs, which probably improves the performance of tadpoles under acid conditions. The trade-off between egg size and fecundity was stronger in acid populations indicating that females in acid populations reduced fecundity to increase offspring size. Finally, frogs from acidified environments were more asymmetric in skeletal traits further indicating the developmental stress created by acidification.

Common carp (Cyprinus carpio) have been hypothesized to contribute to declines in aquatic macrophytes, waterfowl, and water clarity in Delta Marsh, an 18,500 ha freshwater coastal wetland on Lake Manitoba, Canada. Ten ponds (1-13 ha) were chosen for a two-year experimental manipulation study. Following a year of baseline monitoring, manipulations were conducted in 2002. To facilitate access by carp into isolated ponds, channels were blasted from the main marsh into two ponds. Meanwhile, to restrict or exclude carp access into ponds, channels were either screened or diked to four ponds. Two connected and two isolated ponds functioned as controls. Although common carp were the original subject of the study, it became apparent that hydrological connection to the surrounding marsh had a paramount importance on the abundance and diversity of the fish, amphibian and water-column invertebrate communities. Connectivity, or lack of connectivity, played an important role in the distribution of the fish community, and subsequently the composition and abundance of water-column invertebrates and amphibians. Ponds with direct connection had diverse, mixed-species fish assemblages, with fewer invertebrates and amphibians. Ponds with restricted connections had fish communities composed of tolerant small-sized species and increased abundance of invertebrates and amphibians. Ponds that lacked connection could freeze and lose all fish, and had higher numbers of invertebrates and amphibians. An absence of adult common carp may have been responsible for increased amphibian numbers in the screened ponds, however more study is needed. Confounding impacts of fluctuating water levels made it impossible to implicate common carp for most changes observed within ponds in Delta Marsh. / May 2006