Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

Australia's marine environment covers extended areas, from the warm northern tropical, to the sub tropical central water, the cool temperate water of the south and the cold sub-Antarctic and Antarctic water. Australia has rich area of coral reefs. The marine biodiversity in Australia is enormous. Despite incredible biodiversity, Australian research in the marine anticancer drug discoveries is low in comparison with other countries. In this research we investigated a collection of marine sponges as a source for new anticancer leads. This thesis comprises six chapters. Chapter 1 covers the importance of natural products as a source of new drugs, and an introduction to cancer as a disease, chemotherapy in cancer treatments, and the natural products as a source for anticancer drugs. Also, the basic anticancer drug development process is highlighted. Finally, a thorough review of anticancer alkaloids isolated from marine sponges is presented. Chapter 2 presents the chemical investigation into a southern Australian marine sponge Stelletta sp., which led to the isolation and structure elucidation of bistellettazines A-C the first reported examples of terpenyl-pyrrolizidines conjugate, and bistellettazole A, a unique cyclic terpenyl-imidazole conjugate. Bistellettazines A-C and bistellettazole A feature unprecedented carbon skeletons that are proposed to share a common convergent biosynthetic origin, arising via the biogenic equivalent of a Diels-Alder addition between two hypothetical polyenyl norsesquiterpene precursors. The cytotoxic activity (in vitro) for these new alkaloids is also discussed. Chapter 3 discusses the isolation and structure elucidation of four new discorhabdins analogues namely, dihydrodiscorhabdin A, debromodiscorhabdin A, discorhabdin X and dihydrodiscorhabdin L. In addition, the known compounds discorhabdin A and discorhabdin D, were isolated from two southern Australian marine sponge specimens of the genera Higginsia and Spongosorites. The cytotoxic activity (in vitro) for these new alkaloids was also discussed. Chapter 4 discloses chemical investigation into two southern Australian marine sponge specimens of the genera Clathria and Ptilocaulis. Four new mirabilin analogues (mirabilins H-K) were isolated and characterized along with known mirabilin C, F (for the first time as TFA salt) and mirabilin G. The cytotoxic activity (in vitro) for these new alkaloids was also discussed. Chapter 5 presents the 1H NMR data for the known compounds isolated during this study, and Chapter 6 is covering the experimental part.

Abstract Ascoviruses (AVs) are members of the family Ascoviridae that are transmitted by female endoparasitic wasps and cause lethal infection in lepidopteran insects. AVs possess large double stranded DNA genomes ranging from 116-186 kbp. Recently, genomes of four AV species have been completely sequenced and have revealed important genes potentially needed for virus DNA replication and infection. Phylogenetic analyses of several of these genes indicate that AVs are closely related to iridoviruses and likely evolved from them. Two unique features, mode of transmission and cytopathology which involves cleavage of cells into virus-containing vesicles, make AVs different from other insect pathogenic viruses. During this decade, tremendous advancements in the study of RNA silencing mechanisms have openned a new dimension in virology. It is now evident that viruses reshape the cellular environment by reprogramming host RNA silencing machinery. The process of RNA silencing involves small non-coding RNAs, which with the help of nuclease-containing regulatory proteins bind to complementary messenger RNA (mRNA) targets, resulting in inhibition of gene expression. This sophisticated style of gene regulation has attained a fundamental status in living organisms, since RNA silencing has been revealed to be ubiquitous from viruses to prokaryotes to eukaryotes. Two main categories of small RNAs, short interfering RNA (siRNA) and microRNA (miRNA), have been defined as major players in RNA silencing. Interestingly, viral genomes like that of their hosts, encode miRNAs that can be used during virus invasion to manipulate host genes as well as miRNA biogenesis. Here, we report on the identification of the first insect virus miRNA (HvAVmiR- 1) derived from the major capsid protein (MCP) gene of Heliothis virescens ascovirus 7 (HvAV3e). HvAV-miR-1 expression was found to be strictly regulated and specifically detected from 96 h post-infection. HvAV-miR-1 expression coincides with a marked reduction of the expression of HvAV3e DNA polymerase I, which is a predicted target. Ectopic expression of the full-length and truncated versions of MCP retaining the miRNA sequence significantly reduced DNA polymerase I transcript levels and inhibited viral replication. Our results indicate that HvAV-miR-1 directs degradation of DNA polymerase I transcripts and regulates replication of HvAV3e. Further, we investigated changes in the expression levels of host miRNAs upon HvAV3e infection in an insect cell line derived from Helicoverpa zea fat body and investigated the role of a host miRNA, Hz-miR24, in the hostvirus system. It was found that Hz-miR24 is differentially expressed following virus infection, with an increase in its expression levels late in infection. Functional analyses demonstrated that Hz-miR24 targets viral DNA-dependent RNA polymerase and its β subunit mRNAs. This was confirmed using ectopic expression of Hz-miR24 and a green fluorescent protein-based reporter system. Expression of the target gene was substantially enhanced in cells transfected with a synthesized inhibitor of Hz-miR24. These findings suggest that ascoviruses encode their own miRNA(s) and concurrently manipulate host miRNAs that in turn regulate the expression of their genes at specific time points after infection. In connection to RNA silencing, we characterized a ribonuclease III (RNase III) protein encoded by HvAV3e. We found that RNase III protein was functional in vivo as well as in vitro and catalyzed long and short double stranded RNAs. Expression analyses during virus infection revealed autoregulation of this protein by degradation of its RNA transcripts. Moreover, RNase III protein was found to be involved in suppression of RNA silencing and essential for virus DNA replication and infection. Finally, we studied another ascoviral 8 protein, a putative inhibitor of apoptosis (IAP), which was found to be essential for virus DNA replication and pathology. Further, despite inhibition of apoptosis by HvAV3e, the IAP-like protein was found dispensable for the inhibition of replication. In conclusion, for successful invasion and attenuation of host antiviral responses, ascoviruses seem to utilize viral encoded proteins as well as miRNAs. Since the genomes of these viruses have only recently been sequenced, the role of many of the encoded genes essential for pathogenesis and manipulation of antiviral defence mechanisms remains to be eluciated.

Landscape modification resulting in habitat loss, fragmentation and intensification of land use is a serious threat to the earth’s biological diversity and the primary cause of the current extinction crisis. Recent research suggests the human-modified area of the landscape (that is not ‘traditional’ habitat for native species but potentially once was), or the ‘matrix’ (as it is hereafter called) has a major influence on wildlife persistence in modified landscapes. However, the matrix is a poorly studied and inadequately understood element of a modified landscape. There are at least two reasons for this. Firstly, the predominance of island biogeography and metapopulation theories in ecological thinking ensured that the matrix has historically been ignored in ecological research. Secondly, it is difficult to analyse matrix effects without confounding effects of habitat loss, habitat fragmentation and other aspects of landscape modification. As landscapes are modified attributes of the matrix co-vary with attributes of patches and landscapes, entangling their relative impacts on wildlife. Thus to investigate the independent influence of the matrix on mammals I selected 19 study landscapes by rigorous criteria to control for all potentially confounding patch and landscape attributes such as remnant forest patch size, shape, vegetation type, condition and position in the landscape, presence of any large (potential ‘source’) native forest patches within the landscape unit, as well as matrix land use composition and history. A study landscape was defined as the area within a 500 m radius of a remnant forest patch edge. Landscapes were selected along a gradient of rural-suburban residential development spanning the broadest achievable range of what I call ‘matrix development intensity’. This gradient was quantified by a novel weighted road-length metric that considered multiple road attributes to give significance to the ecological impact of different roads. Mammals and their habitat were sampled in three landscape elements within each landscape: remnant patch core, remnant patch edge and matrix to allow a landscape level inference. Mammals were sampled by a combination of Elliott traps, wire cage traps, hair funnels, scats and direct sightings. Thirty environmental variables were measured including habitat structure and disturbance and a full floristic survey was conducted. The matrix intensity gradient was characterised by increased anthropogenic disturbance such as increased housing density, closer proximity of sample sites to houses and higher human disturbance across the landscape mosaic, including in the core of remnant patches. However, matrix intensity was not the greatest source of overall variation in structural and floristic habitat attributes. Therefore the confounding of matrix effects with effects of remnant forest patch habitat attributes were successfully ruled out. Management actions of individual landholders can shape habitat attributes essential to mammals across the landscape mosaic. Mammal response to matrix intensity was species specific. Several native species declined in abundance, others were more resilient to moderate levels of matrix intensity, one species increased in abundance, and at least one species appeared unaffected by matrix intensity. Native species richness peaked at moderate levels, while exotic species richness and feral predators increased with matrix intensity and were negatively correlated with native species. Species response to matrix intensity appeared related to their use of edge or matrix habitat. However, an ability to use the matrix per se may not translate into an ability to persist in a landscape where development substantially reduces the habitat or movement value of the matrix. Seven a priori models of various remnant patch habitat, landscape and matrix influences on terrestrial mammal species richness were tested. Matrix attributes were the most important determinants of species richness. Matrix development intensity had a strong negative effect while matrix vegetation structural complexity had a strong positive effect on mammal species richness. Distance to the nearest remnant forest habitat was relatively unimportant. I hypothesised that thresholds of matrix intensity would exist where native species decline in abundance and exotic and native synanthropic species increase to dominate the mammal community. Thresholds were found for abundance of all native terrestrial species combined, macropod abundance and exotic rodent abundance. However, threshold models were only better than linear or cubic models for exotic rodent abundance. Matrix development intensity has a pervasive impact across the whole landscape mosaic that results in a complex range of environmental changes that individually and collectively impact the mammal community. Drawing on all results, I present a conceptual model of the overall impact of matrix development intensity on mammal community integrity. I conclude that a structurally complex matrix within a human-modified landscape can provide supplementary habitat resources and increase the probability of successful species movement across the landscape. Research needs to incorporate empirical data of specific matrix effects into models and theory of species distribution and abundance in human-modified landscapes. This can help guide application of management actions and landscape planning principles across different landscapes. Planning authorities and land managers need to explicitly acknowledge the importance of the matrix and the numerous factors that could be manipulated, such as retention or restoration of a structural vegetation layer that can assist mammal movement across the matrix, for greater landscape-wide conservation outcomes.

Landscape modification resulting in habitat loss, fragmentation and intensification of land use is a serious threat to the earth’s biological diversity and the primary cause of the current extinction crisis. Recent research suggests the human-modified area of the landscape (that is not ‘traditional’ habitat for native species but potentially once was), or the ‘matrix’ (as it is hereafter called) has a major influence on wildlife persistence in modified landscapes. However, the matrix is a poorly studied and inadequately understood element of a modified landscape. There are at least two reasons for this. Firstly, the predominance of island biogeography and metapopulation theories in ecological thinking ensured that the matrix has historically been ignored in ecological research. Secondly, it is difficult to analyse matrix effects without confounding effects of habitat loss, habitat fragmentation and other aspects of landscape modification. As landscapes are modified attributes of the matrix co-vary with attributes of patches and landscapes, entangling their relative impacts on wildlife. Thus to investigate the independent influence of the matrix on mammals I selected 19 study landscapes by rigorous criteria to control for all potentially confounding patch and landscape attributes such as remnant forest patch size, shape, vegetation type, condition and position in the landscape, presence of any large (potential ‘source’) native forest patches within the landscape unit, as well as matrix land use composition and history. A study landscape was defined as the area within a 500 m radius of a remnant forest patch edge. Landscapes were selected along a gradient of rural-suburban residential development spanning the broadest achievable range of what I call ‘matrix development intensity’. This gradient was quantified by a novel weighted road-length metric that considered multiple road attributes to give significance to the ecological impact of different roads. Mammals and their habitat were sampled in three landscape elements within each landscape: remnant patch core, remnant patch edge and matrix to allow a landscape level inference. Mammals were sampled by a combination of Elliott traps, wire cage traps, hair funnels, scats and direct sightings. Thirty environmental variables were measured including habitat structure and disturbance and a full floristic survey was conducted. The matrix intensity gradient was characterised by increased anthropogenic disturbance such as increased housing density, closer proximity of sample sites to houses and higher human disturbance across the landscape mosaic, including in the core of remnant patches. However, matrix intensity was not the greatest source of overall variation in structural and floristic habitat attributes. Therefore the confounding of matrix effects with effects of remnant forest patch habitat attributes were successfully ruled out. Management actions of individual landholders can shape habitat attributes essential to mammals across the landscape mosaic. Mammal response to matrix intensity was species specific. Several native species declined in abundance, others were more resilient to moderate levels of matrix intensity, one species increased in abundance, and at least one species appeared unaffected by matrix intensity. Native species richness peaked at moderate levels, while exotic species richness and feral predators increased with matrix intensity and were negatively correlated with native species. Species response to matrix intensity appeared related to their use of edge or matrix habitat. However, an ability to use the matrix per se may not translate into an ability to persist in a landscape where development substantially reduces the habitat or movement value of the matrix. Seven a priori models of various remnant patch habitat, landscape and matrix influences on terrestrial mammal species richness were tested. Matrix attributes were the most important determinants of species richness. Matrix development intensity had a strong negative effect while matrix vegetation structural complexity had a strong positive effect on mammal species richness. Distance to the nearest remnant forest habitat was relatively unimportant. I hypothesised that thresholds of matrix intensity would exist where native species decline in abundance and exotic and native synanthropic species increase to dominate the mammal community. Thresholds were found for abundance of all native terrestrial species combined, macropod abundance and exotic rodent abundance. However, threshold models were only better than linear or cubic models for exotic rodent abundance. Matrix development intensity has a pervasive impact across the whole landscape mosaic that results in a complex range of environmental changes that individually and collectively impact the mammal community. Drawing on all results, I present a conceptual model of the overall impact of matrix development intensity on mammal community integrity. I conclude that a structurally complex matrix within a human-modified landscape can provide supplementary habitat resources and increase the probability of successful species movement across the landscape. Research needs to incorporate empirical data of specific matrix effects into models and theory of species distribution and abundance in human-modified landscapes. This can help guide application of management actions and landscape planning principles across different landscapes. Planning authorities and land managers need to explicitly acknowledge the importance of the matrix and the numerous factors that could be manipulated, such as retention or restoration of a structural vegetation layer that can assist mammal movement across the matrix, for greater landscape-wide conservation outcomes.

Australia's marine environment covers extended areas, from the warm northern tropical, to the sub tropical central water, the cool temperate water of the south and the cold sub-Antarctic and Antarctic water. Australia has rich area of coral reefs. The marine biodiversity in Australia is enormous. Despite incredible biodiversity, Australian research in the marine anticancer drug discoveries is low in comparison with other countries. In this research we investigated a collection of marine sponges as a source for new anticancer leads. This thesis comprises six chapters. Chapter 1 covers the importance of natural products as a source of new drugs, and an introduction to cancer as a disease, chemotherapy in cancer treatments, and the natural products as a source for anticancer drugs. Also, the basic anticancer drug development process is highlighted. Finally, a thorough review of anticancer alkaloids isolated from marine sponges is presented. Chapter 2 presents the chemical investigation into a southern Australian marine sponge Stelletta sp., which led to the isolation and structure elucidation of bistellettazines A-C the first reported examples of terpenyl-pyrrolizidines conjugate, and bistellettazole A, a unique cyclic terpenyl-imidazole conjugate. Bistellettazines A-C and bistellettazole A feature unprecedented carbon skeletons that are proposed to share a common convergent biosynthetic origin, arising via the biogenic equivalent of a Diels-Alder addition between two hypothetical polyenyl norsesquiterpene precursors. The cytotoxic activity (in vitro) for these new alkaloids is also discussed. Chapter 3 discusses the isolation and structure elucidation of four new discorhabdins analogues namely, dihydrodiscorhabdin A, debromodiscorhabdin A, discorhabdin X and dihydrodiscorhabdin L. In addition, the known compounds discorhabdin A and discorhabdin D, were isolated from two southern Australian marine sponge specimens of the genera Higginsia and Spongosorites. The cytotoxic activity (in vitro) for these new alkaloids was also discussed. Chapter 4 discloses chemical investigation into two southern Australian marine sponge specimens of the genera Clathria and Ptilocaulis. Four new mirabilin analogues (mirabilins H-K) were isolated and characterized along with known mirabilin C, F (for the first time as TFA salt) and mirabilin G. The cytotoxic activity (in vitro) for these new alkaloids was also discussed. Chapter 5 presents the 1H NMR data for the known compounds isolated during this study, and Chapter 6 is covering the experimental part.

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.

This thesis investigates aspects of the ecology, morphology, bioacoustics, genetic relatedness, biogeography and taxonomy of cicadas in the Pauropsalta annulata Goding and Froggatt species complex, across their entire eastern Australian geographical distribution. These cicadas seem to emerge at the same sites annually, although the exact duration of their life cycle is not known. They are wary and mobile insects, with individuals generally taking flight at the first sign of disturbance, which makes them challenging to record, capture and observe. The males produce distinctive calling songs that have a consistent rhythmic structure, which is critically important for attracting females. In most cases, the songs are biphasic, with a penetrating introductory or "buzzing" component that apparently functions in mate attraction and a strongly structured cueing or "lilting" component for mate localisation. Initially Pauropsalta annulata was thought to comprise a single species with some degree of geographically structured variation in its calling song. Consequently, various "song types" had been recognised, but their precise relationship to one another had never been investigated. Therefore the structure of their calling songs were compared statistically across individuals of three song types, and this revealed four discrete clusters that were demonstrated to be independent of one another and consistent in their calling song structure. Plotting the geographical distributions of these cicadas revealed that each of the P. annulata song types is independently distributed geographically, with areas of overlap that are relatively small. Calling song structure is consistent for each song type across extensive geographical space and this structure holds even into areas of sympatry. One song type showed consistent differences from the others in male genitalia structure, and female ovipositor length differs significantly among three of the other song types. Each song type was found to be strongly associated with a small number of tree species and these associations are maintained into areas of sympatry. The spatial ecological and morphological comparisons in song structure, plant associations and morphology made in this study demonstrate that the P. annulata song types actually represent a complex of cryptic species. Two of the song types appear to intergrade in areas of sympatric overlap in terms of calling songs, plant associations and morphology, and thus represent subspecies as defined in population genetics terms. The components of the male calling song were then investigated to determine their specific functions and thus how they could delimit species boundaries. Significant differences were found in dominant song frequency between three of the four species, and may contribute to differential mate attraction. Both components of the song of each species have the same dominant frequency. Consistent differences were also evident, among species and subspecies, in the rhythmic structure of the "lilting" component of the male calling song. This component contains repeated phrases and each one of these provides a cue to which the conspecific female may respond. Her response is timed for the brief silent interval between the phrases. At this point the calling song becomes a duet, which enables the male to locate the female, as he actively searches for her on the surrounding branches at this stage. The calling song is discussed in terms of random mating within gene pools of these cicadas and in terms of its role as part of their broader fertilisation mechanism. A molecular analysis of the P. annulata species complex was performed to examine the phylogenetic relationships across 12 species and four subspecies defined in this study, and estimate divergence times within the group. Individual specimens were sampled widely across the geographical distributions of the species and subspecies where possible to account for genetic variation across space. DNA sequences from two loci were amplified: mitochondrial CO1 ("barcoding region") and a large intron from the dynamin nuclear gene. Separate phylogenies were reconstructed for each locus using maximum parsimony procedures and Bayesian posterior sampling with implementation of a relaxed molecular clock. The phylogenies from both genes provided strong support for the monophyly of the P. annulata species complex, and nine of the species were monophyletic based on the CO1 gene. The remaining three emerged non-monophyletic. Based on a clock calibration of 0.0165s/s/myr, the monophyletic clades represented by extant P. annulata species diverged about 4.5-8.0 million years ago. Those species that emerged non-monophyletic had shallower divergences, with the exception of one species, which exhibited haplotype diversity that conferred up to 13.2% sequence divergence between allopatric populations in CO1. Dynamin produced a broadly similar phylogenetic pattern to that of CO1, but the relationships among individuals across the species and subspecies that emerged non-monophyletic differed substantially. This lack of congruence between the two genomes, in combination with the dominance of internal haplotypes in both loci, indicates an overall pattern of deep coalescence rather than interspecific hybridization. Therefore the molecular data do not provide an alternative definition of species limits in the P. annulata species complex, despite some emerging non-monophyletic in this analysis. Sound interpretation of the phylogenetic pattern discovered here would not have been possible without the acoustic, ecological and geographical investigations on species limits that preceded this work. To determine what biological and climatic factors influence the present day distributions of these cicadas, the distributions of two of the most closely related species in the P. annulata species complex were compared with the distributions of the tree species with which each is mainly associated. Because a large part of their life cycle is subterranean, soil texture, pH, electrical conductivity and force required for surface penetration were compared across sites where each of the cicadas occur in sympatry and allopatry. Finally, the influence of temperature and rainfall variables were investigated by testing 'predicted distribution' models (formed using positive distribution records) against negative records for both sets of variables, individually and in combination. The results show that the extent of the distribution of the cicada species is substantially less than that of the associated tree species. The geographical distributions of one of the species may be influenced more by rainfall, or a combination of temperature and rainfall, whereas the other species appears to be more influenced by temperature alone. Both species tolerate soils with a wide range of pH levels, electrical conductivity and forces required for surface penetration. They both showed a strong association with soils that had a silt loam texture, with only few records from sandy soils. However, none of the soils sampled where the cicadas occurred were heavy clays, which suggests that the physical properties of such soils may provide an unsuitable environment for the nymphal stages of the life cycle of these particular cicadas. The resolution of species limits within the P. annulata species complex allowed the redescription of Pauropsalta annulata Goding and Froggatt sensu stricto and the description of 11 new species belonging to the P. annulata species group, all from eastern Australia. Two of these species comprise two subspecies each, also all new. New distribution records and calling song data are documented for the allied species, P. ayrensis Ewart, which is redescribed to include the characters newly recognised in the present study as significant taxonomically with respect to Pauropsalta cicadas. The treatment includes comprehensive descriptions of the morphology and calling songs of the species and subspecies, and separate descriptive keys are provided for both sets of characters. The new taxa comprise P. artatus sp. nov., P. corymbiae sp. nov., P. decorus sp. nov., P. graniticus sp. nov., P. inversus inversus subsp. nov., P. i. laboris subsp. nov., P. notialis notialis subsp. nov., P. notialis incitatus subsp. nov., P. simplex sp. nov., P. subtropicus sp. nov, P. torrensis sp. nov. and P. tremulus sp. nov. Areas of hybridization between P. n. notialis subsp. nov. and P. n. incitatus subsp. nov. are also documented, together with their calling songs and morphology, which justifies their subspecific status. The P. inversus subspecies are allopatric, but consistently differ in the duration between phrases of the calling song. Finally, the results and conclusions are amalgamated into a critical reassessment of what defines species limits and the most appropriate approaches to investigating species limits in sexual organisms. Some historical discussions are revisited, such as the question of the reality of species and how species are perceived under the premises of neoDarwinism. The realism of species demands that species limits are most realistically defined in terms of their fertilisation mechanism, for this delimits the gene pool and thus the distribution of adaptations (the calling song of cicadas, for example).

This thesis investigates aspects of the ecology, morphology, bioacoustics, genetic relatedness, biogeography and taxonomy of cicadas in the Pauropsalta annulata Goding and Froggatt species complex, across their entire eastern Australian geographical distribution. These cicadas seem to emerge at the same sites annually, although the exact duration of their life cycle is not known. They are wary and mobile insects, with individuals generally taking flight at the first sign of disturbance, which makes them challenging to record, capture and observe. The males produce distinctive calling songs that have a consistent rhythmic structure, which is critically important for attracting females. In most cases, the songs are biphasic, with a penetrating introductory or "buzzing" component that apparently functions in mate attraction and a strongly structured cueing or "lilting" component for mate localisation. Initially Pauropsalta annulata was thought to comprise a single species with some degree of geographically structured variation in its calling song. Consequently, various "song types" had been recognised, but their precise relationship to one another had never been investigated. Therefore the structure of their calling songs were compared statistically across individuals of three song types, and this revealed four discrete clusters that were demonstrated to be independent of one another and consistent in their calling song structure. Plotting the geographical distributions of these cicadas revealed that each of the P. annulata song types is independently distributed geographically, with areas of overlap that are relatively small. Calling song structure is consistent for each song type across extensive geographical space and this structure holds even into areas of sympatry. One song type showed consistent differences from the others in male genitalia structure, and female ovipositor length differs significantly among three of the other song types. Each song type was found to be strongly associated with a small number of tree species and these associations are maintained into areas of sympatry. The spatial ecological and morphological comparisons in song structure, plant associations and morphology made in this study demonstrate that the P. annulata song types actually represent a complex of cryptic species. Two of the song types appear to intergrade in areas of sympatric overlap in terms of calling songs, plant associations and morphology, and thus represent subspecies as defined in population genetics terms. The components of the male calling song were then investigated to determine their specific functions and thus how they could delimit species boundaries. Significant differences were found in dominant song frequency between three of the four species, and may contribute to differential mate attraction. Both components of the song of each species have the same dominant frequency. Consistent differences were also evident, among species and subspecies, in the rhythmic structure of the "lilting" component of the male calling song. This component contains repeated phrases and each one of these provides a cue to which the conspecific female may respond. Her response is timed for the brief silent interval between the phrases. At this point the calling song becomes a duet, which enables the male to locate the female, as he actively searches for her on the surrounding branches at this stage. The calling song is discussed in terms of random mating within gene pools of these cicadas and in terms of its role as part of their broader fertilisation mechanism. A molecular analysis of the P. annulata species complex was performed to examine the phylogenetic relationships across 12 species and four subspecies defined in this study, and estimate divergence times within the group. Individual specimens were sampled widely across the geographical distributions of the species and subspecies where possible to account for genetic variation across space. DNA sequences from two loci were amplified: mitochondrial CO1 ("barcoding region") and a large intron from the dynamin nuclear gene. Separate phylogenies were reconstructed for each locus using maximum parsimony procedures and Bayesian posterior sampling with implementation of a relaxed molecular clock. The phylogenies from both genes provided strong support for the monophyly of the P. annulata species complex, and nine of the species were monophyletic based on the CO1 gene. The remaining three emerged non-monophyletic. Based on a clock calibration of 0.0165s/s/myr, the monophyletic clades represented by extant P. annulata species diverged about 4.5-8.0 million years ago. Those species that emerged non-monophyletic had shallower divergences, with the exception of one species, which exhibited haplotype diversity that conferred up to 13.2% sequence divergence between allopatric populations in CO1. Dynamin produced a broadly similar phylogenetic pattern to that of CO1, but the relationships among individuals across the species and subspecies that emerged non-monophyletic differed substantially. This lack of congruence between the two genomes, in combination with the dominance of internal haplotypes in both loci, indicates an overall pattern of deep coalescence rather than interspecific hybridization. Therefore the molecular data do not provide an alternative definition of species limits in the P. annulata species complex, despite some emerging non-monophyletic in this analysis. Sound interpretation of the phylogenetic pattern discovered here would not have been possible without the acoustic, ecological and geographical investigations on species limits that preceded this work. To determine what biological and climatic factors influence the present day distributions of these cicadas, the distributions of two of the most closely related species in the P. annulata species complex were compared with the distributions of the tree species with which each is mainly associated. Because a large part of their life cycle is subterranean, soil texture, pH, electrical conductivity and force required for surface penetration were compared across sites where each of the cicadas occur in sympatry and allopatry. Finally, the influence of temperature and rainfall variables were investigated by testing 'predicted distribution' models (formed using positive distribution records) against negative records for both sets of variables, individually and in combination. The results show that the extent of the distribution of the cicada species is substantially less than that of the associated tree species. The geographical distributions of one of the species may be influenced more by rainfall, or a combination of temperature and rainfall, whereas the other species appears to be more influenced by temperature alone. Both species tolerate soils with a wide range of pH levels, electrical conductivity and forces required for surface penetration. They both showed a strong association with soils that had a silt loam texture, with only few records from sandy soils. However, none of the soils sampled where the cicadas occurred were heavy clays, which suggests that the physical properties of such soils may provide an unsuitable environment for the nymphal stages of the life cycle of these particular cicadas. The resolution of species limits within the P. annulata species complex allowed the redescription of Pauropsalta annulata Goding and Froggatt sensu stricto and the description of 11 new species belonging to the P. annulata species group, all from eastern Australia. Two of these species comprise two subspecies each, also all new. New distribution records and calling song data are documented for the allied species, P. ayrensis Ewart, which is redescribed to include the characters newly recognised in the present study as significant taxonomically with respect to Pauropsalta cicadas. The treatment includes comprehensive descriptions of the morphology and calling songs of the species and subspecies, and separate descriptive keys are provided for both sets of characters. The new taxa comprise P. artatus sp. nov., P. corymbiae sp. nov., P. decorus sp. nov., P. graniticus sp. nov., P. inversus inversus subsp. nov., P. i. laboris subsp. nov., P. notialis notialis subsp. nov., P. notialis incitatus subsp. nov., P. simplex sp. nov., P. subtropicus sp. nov, P. torrensis sp. nov. and P. tremulus sp. nov. Areas of hybridization between P. n. notialis subsp. nov. and P. n. incitatus subsp. nov. are also documented, together with their calling songs and morphology, which justifies their subspecific status. The P. inversus subspecies are allopatric, but consistently differ in the duration between phrases of the calling song. Finally, the results and conclusions are amalgamated into a critical reassessment of what defines species limits and the most appropriate approaches to investigating species limits in sexual organisms. Some historical discussions are revisited, such as the question of the reality of species and how species are perceived under the premises of neoDarwinism. The realism of species demands that species limits are most realistically defined in terms of their fertilisation mechanism, for this delimits the gene pool and thus the distribution of adaptations (the calling song of cicadas, for example).

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.