Zoology - Theses
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The function of female and male ornaments in the lovely fairy-wren
Ornaments like plumage colours or complex song are generally regarded as male traits that are shaped by sexual selection. By contrast, the factors that shape female elaborate traits have often been overlooked, though they are expressed in females across many taxa. Understanding how trade-offs and selective pressures shape female ornamentation is crucial for advancing our understanding of trait evolution. In this thesis, I investigate the form and function of female and male plumage colour and song in the Lovely fairy-wren (Malurus amabilis), a tropical species in which females and males are both highly colourful and vocal. This was investigated over three consecutive years and field seasons in Far North Queensland, Australia. My thesis research employed field observations, behavioural experiments, and genetic analysis, to test the adaptive function(s) and mechanisms for the evolution of female and male ornamental traits. I explicitly contrast females and males so that we can address, in the light of the abundant work done on males, how females may or may not differ from males. To provide context for the ornamental traits that are exhibited by this species, I first provide a comprehensive overview of the ecology and breeding biology of the Lovely fairy-wren, since a detailed description on the species natural history prior to this work was lacking. To understand the function of plumage colouration, I studied whether plumage colour in females and males is a signal and experimentally tested if it functions in a competitive context. Additionally, I assessed whether plumage colour is sexually selected, by examining its signalling content, costs (survival), and its relationship with reproductive and paternity success. Lastly, I investigated the song function, by describing female and male song structure and examining sex-specific variation in song rate across different contexts. I also used experimental data to examine female and male responses to simulated territorial intrusion. Overall this thesis provides insight into the form and function of both female and male plumage colours and song. First, it shows that visual and acoustic ornaments are important signalling components in different contexts, suggesting that female ornaments are not just a correlated genetic by-product of traits in males, and that selection favours female (and male) expression of traits. Second, the information conveyed by plumage colouration seems to be context-dependent in relation to the sex of the bearer: in males, it may follow the classical pattern of sexual selection, functioning in mate choice and male-male competition, while in females, plumage colours do not seem to be influenced by male choice, but function in same-sex competitive contexts. Third, it highlights that song has convergent functions in both sexes, as females and males have similar song structure and used song year-round in identical contexts for within-pair communication and joint territorial defence. The fact that females and males sing and have bright colours year-round in parallel with their territorial and breeding behaviour, suggests that individuals use their traits to maintain (sexual and non-sexual) resources. This work highlights the importance of studying and considering the fundamental differences in females and males, a necessary step for a realistic understanding of ornament expression, and contributes to the ongoing discussion on the evolution of elaborate female signal traits.
Predicting ectotherm life cycles under a variable climate: physiological diversity of matchstick grasshopper eggs and their ecological and evolutionary implications
Understanding the processes underlying the phenology and distribution of species is a key problem in ecology. These relationships are important for predicting the responses to species to environmental change. Phenology and distribution are closely linked to climate and weather through the thermal dependence of life cycles. However, for many biodiverse taxa, like insects, we have a poor understanding of the mechanistic links between adaptive traits and how life cycles are adapted to seasonal and variable temperature patterns. Insect life cycles are synchronised with suitable climatic conditions at critical life stages, such as the egg stage. Variation in thermal sensitivity of development and dormancy are two mechanisms by which insects can generate adaptive life cycle phenotypes. Eggs, therefore, present a unique opportunity to link adaptive variation in traits with corresponding variation in life cycles and thermal environments to examine how life cycles are adapted to variable climates. To understand the adaptation of insect life cycles to variable climates, we require a mechanistic understanding of the interactions between adaptive developmental traits of eggs and variation in the thermal environment on adaptations. Our ability to test thermal adaptation in ectotherms is also limited by our ability to efficiently characterise thermal responses. In this thesis, I described how thermocyclers are an efficient means of characterising the thermal response of small ectotherms with enough precision and sample size. I then used the widely distributed, endemic and flightless Australian matchstick grasshopper genera Warramaba (Orthoptera: Morabidae) as a model system to examine the significance of variation in thermal responses at the egg stage for life cycles under a variable climate. I used a mechanistic modelling framework to tease apart developmental and environmental sources of variation in life cycles at the egg stage and simulate their consequences for phenology and distribution in the field. Matchstick grasshoppers showed remarkable diversity in developmental responses to temperature at the egg stage, primarily in the expression of dormancy. I found that diverse Warramaba life cycles are shaped by the interactions between such developmental variation and local environmental temperatures. I demonstrated that we can achieve a mechanistic understanding of life cycle adaptation by considering the evolution of temperature-dependent traits and the evolution of life history within the context of seasonal temperature cycles. Mechanistic models are powerful tools to investigate the sources of life cycle variation and their consequences for insect distribution and phenology. Such frameworks are directly transferrable to other socio-economically important or threatened species to understand how insects are adapted to local climatic conditions and predict responses to a changing climate.
Testing the decline of the threatened New Holland Mouse (Pseudomys novaehollandiae)
Delineating the distribution of a threatened species is critical for identifying threats and guiding conservation management. The process is challenging, however, especially when a species is rapidly declining, and so changing its distribution. In this context, species distribution modelling (SDM) often lacks the precision needed to inform fine-scale management decisions, but on-ground surveys to test species’ distributions are time and resource intensive. The dilemma can be mitigated to some extent by careful examination of historical data, and optimal monitoring. The New Holland Mouse (NHM; Pseudomys novaehollandiae) is one of many Australian rodent species to have undergone drastic distributional declines since European invasion. Initially recorded in Victoria in 1970, by 2015 NHMs were thought to occur in only 3 of 12 historically occupied regions. I tested this decline with statistical rigour, using extensive Elliott and camera trapping surveys at >500 sites across Victoria. Combining my survey data with 48 years of others’ efforts, I evaluated the utility of standard Elliott trapping surveys and the efficacy of camera trapping for NHMs. I tested whether NHMs were where we would expect based on state-government threatened fauna SDMs, and whether the species’ purported early-successional fire association explained occurrence or abundance. I confirmed the species’ persistence in 5 of 12 historical regions – including regions where NHMs had not been detected in 5-21 years – and expanded the species’ known distribution in two regions. However, these finds can be attributed to a paucity of prior survey effort and were partnered with greater declines elsewhere. Elliott trapping surveys were often inadequate to provide statistical confidence in the species’ absence; camera trap surveys provide a viable alternative for distribution assessments. Standard state-government SDMs provided limited guidance as to the true distribution of NHMs and SDMs for declining species should be interpreted with caution. Time-since-fire did not explain the species’ occurrence and poorly explains abundance, though in certain locations inappropriate fire regimes are a threatening process. Predator control, habitat management, and careful reintroductions are key priorities for conservation of NHMs in Victoria.
Biochemical mechanisms of biomineralization and elemental incorporation in otoliths: implications for fish and fisheries research
All vertebrates have small bioinorganic “earstones” in their inner ear labyrinth that are essential for hearing and balance. While otoliths play a vital anatomical role in fish, their true value to science is as biochronometers, largely due to their unique pattern of growth. Otoliths first form in embryo and continue to grow throughout the life of an individual, with a double-banded increment composed of a calcium carbonate-rich region and a protein-rich region being deposited daily. In addition to this, they are considered to be metabolically inert, and do not undergo remodelling or resorption. Consequently, otoliths are employed in a variety of ways in fish ecology. Firstly, an individual fish’s age and growth rate can be estimated through counting increments and measuring their widths. Secondly, analysis of increment trace element:calcium ratios, such as by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS), can allow for the reconstruction of environmental histories, aiding in the determination of natal origin, movement, habitat use, diet and the impacts of climate change. The utility of specific trace elements as indicators of environmental change, however, is unclear as there is considerable uncertainty as to whether a given trace element is interacting with the mineral or protein components of an increment. This uncertainty is a consequence of otolith research having been largely focussed upon either microstructure or inorganic chemistry, with very few studies on the protein-rich regions of the otolith. As a result, very little is understood about the biochemical mechanisms of biomineralization or trace element incorporation. This is important, as the mechanisms that govern otolith formation and growth underpin the assumptions made in traditional increment analyses. In this thesis, I initially undertook a systematic review of all the literature pertaining to otolith biochemistry, revealing the significant gaps that exist in otolith biochemistry as a discipline. Importantly, I determined that fewer than a score of otolith proteins had been identified – a stark contrast to the hundreds or thousands of proteins that have been identified in comparable biomineral systems such as enamel or bone. Working on black bream (Acanthopagrus butcheri), an extensively studied species endemic to southern Australia, I used size exclusion chromatography coupled with ICP-MS to determine the trace element:protein interactions in endolymph, the inner ear fluid that otoliths are submerged in, and the source of all of its constituents. In this study, I assayed 22 elements, and determined that 12 were solely present in a protein-bound form, 6 were present as free ions, and 4 were present in both forms. This allowed me to make recommendations as to their utility in environmental reconstructions. In my next study, I created a unique, multi-disciplinary workflow that combined transcriptomics with proteomics. In this study, I sequenced the transcriptome of the black bream inner ear and used this to identify proteins from the separated organic phase of otoliths and endolymph from wild caught adult black bream. This resulted in the discovery of hundreds of previously unknown proteins, providing new insights into the likely biochemical mechanisms involved in otolith formation and growth. In my final study, I tested the utility of trace element ratios in environmental reconstructions. Specifically, I compared the ability of different cluster analysis approaches to resolve spatial and temporal differences in the likely spawning and larval nursery habitats of juvenile black bream in the Gippsland Lakes, Australia. The results from my thesis have allowed me to make recommendations as to the utility of trace elements in environmental reconstructions and have revealed exciting new avenues of research that fuse ecology and biochemistry.
Novel gene therapy for the treatment of diabetes-induced heart failure
People with diabetes are at risk of developing myocardial abnormalities known as diabetic cardiomyopathy. This is characterised by diabetes-induced left-ventricular (LV) impairment, which develops independent of hypertension, coronary artery or valvular heart disease, leading to an increased risk of heart failure. To date, there is still no effective or specific treatment for diabetic cardiomyopathy. Hence, the overall aim of my thesis was to investigate the therapeutic potential of targeting two distinct novel pathways, the phosphoinositide 3-kinase (PI3K)p110α axis and the hexosamine biosynthesis pathway (HBP)/O-GlcNAcylation, in the setting of diabetic cardiomyopathy. PI3K(p110α) is a lipid kinase that regulates several physiological functions, including membrane trafficking, adhesion, actin rearrangement, cell growth, and survival. Recent findings from our laboratory and others have highlighted that PI3K(p110α) is cardioprotective in a range of different cardiac pathologies. In Chapter 3, I investigated whether cardiac-directed PI3K(p110α) gene therapy ameliorates diabetic cardiomyopathy in a mouse model of type-1 diabetes (T1D) in vivo. I revealed that administration of recombinant adeno-associated virus-6 constitutively-active PI3K(p110α) (rAAV6-caPI3K) attenuated diabetic cardiomyopathy, even when administered after the initial manifestation of LV diastolic dysfunction. I then proceeded to investigate the cardioprotective effects of rAAV6-caPI3K in the more clinically-prevalent type-2 diabetes (T2D) setting. In Chapter 4, I elucidated that restoration of cardiac PI3K(p110α) activity, through the administration of rAAV6-caPI3K gene therapy, attenuates T2D-induced cardiomyopathy, together with limiting ROS generation. In comparison to the cardioprotective nature of PI3K(p110α), the HBP and subsequent protein O-GlcNAcylation have been implicated in the development of diabetic cardiomyopathy. The generation of β-N-acetylglucosamine (O-GlcNAc) from HBP is a substrate for the post-translational protein modification (PTM) called O-GlcNAcylation. Two specific enzymes regulate the addition and removal of O-GlcNAc modification; O-GlcNAc transferase (OGT) catalyses the addition of GlcNAc to proteins and O-GlcNAc-ase (OGA) facilitates its removal. In Chapter 5, I aimed to elucidate the effect of cardiac manipulation of O-GlcNAcylation in the setting of diabetes-induced cardiac dysfunction in vivo. I demonstrated here that a cardiac-selective increase in OGT (via rAAV6-OGT gene delivery), the enzyme responsible for O-GlcNAcylation, is sufficient to drive cardiac dysfunction and remodelling, resembling that seen in diabetic cardiomyopathy. In contrast, increasing cardiac OGA (via rAAV6-OGA gene delivery), the enzyme responsible for the removal of the O-GlcNAc moiety, attenuates several characteristics of diabetic cardiomyopathy, likely at least in part through the improvement of mitochondrial function. Finally, in Chapter 6 I investigated the impact of O-GlcNAcylation on the PI3K(p110α), pathway and the effect of PI3K(p110α) gene therapy on HBP signalling. I elucidated that PI3K(p110α) can negatively regulate consequences of the HBP and O-GlcNAcylation as part of its cardioprotective actions in diabetic cardiomyopathy, while HBP/O-GlcNAcylation inhibits PI3K(p110α)-mediated signalling, likely contributing to the ability for this pathway to exert cardiac impairments. In conclusion, data from this thesis reveal that gene therapies targeting PI3K(p110α) and HBP/O-GlcNAcylation are viable therapeutic targets for diabetic cardiomyopathy. These results hence provide a basis for pursuing gene therapy for the treatment of diabetes-induced heart failure.
Taxonomy, ecology and conservation genomics of North-Eastern Australian Earless Dragons (Agamidae: Tympanocryptis spp.)
Land clearing and modification of natural habitats is threatening biodiversity globally. In Australia, most native grassland habitats have been heavily modified for agriculture, including cropping and grazing. Grassland specialist species, including earless dragon lizards (Tympanocryptis spp.) in north-eastern Australia, are of conservation concern due to this continued habitat loss and fragmentation. However, the north-eastern Australian group of earless dragons (including the recently described T. condaminensis, T. wilsoni and T. pentalineata) are at significant risk, due to the presence of multiple undescribed cryptic Tympanocryptis lineages within this region. It is imperative that the taxonomy is resolved for these cryptic lineages of conservation concern, so conservation of these species may occur. One of the major challenges for taxonomists in recent times has been the species delimitation of morphologically cryptic taxa. The detection of distinct molecular lineages within cryptic genera has increased exponentially over the past decades with advances in genetic techniques. However, there are discrepancies in the rate and success of detection of cryptic taxa between studies using genetic methods and those using classic external morphology analyses. Therefore, novel integrative methods for species delimitation of cryptic taxa provide an avenue to incorporate multiple lines of evidence, including the application of osteological variation assessment where external morphological assessment fails to distinguish species. I develop a new pipeline integrating genomic data using single nucleotide polymorphisms (SNPs) and osteological geometric morphometric evidence from micro X-ray computed tomography (CT) imagery to assess variation between cryptic lineages for confident species delimitation. Here, I use this novel integrative pipeline to delimit cryptic lineages of earless dragons in north-eastern Australia. Prior to this study, there was evidence of three undescribed species of Tympanocryptis in this region. Using single mitochondrial and nuclear genes along with >8500 SNPs, I assess the evolutionary independence of the three target lineages and several closely related species. I then integrate these phylogenomic data with osteological cranial variation from CT imagery between lineages. I find that the very high levels of genomic differentiation between the three target lineages is also supported by significant osteological differences between lineages. By incorporating multiple lines of evidence for species delimitation, I provide strong support that the three cryptic lineages of Tympanocryptis in north-eastern Australia warrant taxonomic review. Earless dragons are found in most environments across the Australian continent, including a variety of ecological niches, from stony desert to tropical woodland or cracking clay savannah, although each species is often restricted to s certain habitat-type. I investigate the phylogenetic relationships among currently described earless dragons and newly delimited putative species with an assessment of broad biogeographic divisions, focussing on the north-eastern Australian Tympanocryptis group. I found significant structure across the north-eastern Australian lineages, with deep divergence between lineages occurring in the inland Great Artesian Basin region and more coastal Great Dividing Range. Regional diversification is estimated to have occurred in the late Miocene with subsequent Plio-Pleistocene speciations, and divergence and distributions of these species may therefore be reflective of the climate induced grassland-rainforest oscillations during this time. Based on these phylogenetic geographic relationships and the species delimitation from the integrative taxonomy approach, I describe three new species of Tympanocryptis from the cracking clay grasslands of the Darling Riverine Basin (T. darlingensis sp. nov.) and Queensland Central Highlands (T. hobsoni sp. nov.), and the stony open eucalypt woodlands on the Einasleigh Uplands (T. einasleighensis sp. nov.). The revision of these species provides further taxonomic clarity within the Tympanocryptis genus, and is an imperative step in the conservation of the north-eastern Australian earless dragons. These three putative Tympanocryptis species and the other three recently described earless dragons in north-eastern Australia inhabit restricted niches and areas with varying levels of habitat fragmentation and modification, and are therefore of significant conservation concern. However, little is known about these six north-eastern Australian earless dragon species. I utilise genomic methods to investigate population connectivity and genetic structure to determine management units. I then use species distribution modelling (SDM) to assess habitat suitability and fragmentation of each species. I integrate results of these analyses to form conclusions on the distribution and population structure of these earless dragons. I then discuss the major threatening processes and potential conservation strategies. This thesis uses several integrative approaches in resolving the taxonomy and forming conclusions on the conservation management of the north-eastern Australian Tympanocryptis species. This study successfully delimits cryptic lineages, explores the phylogenetic and geographic relationships between species, and provides baseline population genomics and ecological data to be used for conservation assessments and management decisions of earless dragons in north-eastern Australia.
Spiders of wheat agroecosystems of southern Victoria, Australia
Spiders are important predators of insects in all terrestrial ecosystems. Their great diversity, broad diet and varied foraging strategies make their role in food webs complex and dynamic and are therefore not an easy group to utilise in biological pest control. Spiders have been extensively studied in wheat systems, revealing great diversity in the guild structure of spiders in this environment. This study is the first direct investigation into spider populations in Australian wheat systems, and the third from the southern hemisphere. As no prior information was available for spiders in this cereal crop, the primary aim was to collate a foundation of ecological information about the community. The composition of the spider community in Australian winter wheat fields differed from those of Europe and New Zealand as there were very few members of the Linyphiidae family detected. Instead, the Australian fauna was more comparable to Canada and Hungary, where the Lycosidae family dominates. In this study, spiders comprised a substantial proportion of the invertebrate community and were the most numerous predatory group. Three families comprised the majority of mature individuals across the region – Lycosidae, Miturgidae and Gnaphosidae. Five species made up more than 80% of mature spiders in each year. Although they were also found in surrounding non-crop habitat, many of the common species showed an affinity for the crop habitat, particularly late in the crop cycle. The limited utilisation of adjacent non-crop habitat was further evidenced by the lack of any aggregations of the common spider species near the edges of fields. Spiders were present in the crop area throughout the year, although the phenology and activity patterns of resident species differed. Notably, the most commonly collected species, the lycosid Venatrix pseudospeciosa, began reproductive activity several months earlier than less numerous species. The spider community differed dramatically between two consecutive years, a heavy reduction in the density and diversity of spiders, which was most likely due to extremely low rainfall in the second year. The central similarity between the two crop seasons was the dominance of the ground hunting guild. In accordance with their relative abundance, this guild may have the greatest impact on prey populations. However, abundance alone does not determine the predatory role of spiders. Features such as prey preference, consumption rate, temporal and spatial synchronicity with pests and response to increasing prey densities together influence their impact in the trophic food web. Laboratory feeding trials on V. pseudospeciosa suggested that the species may be capable of foraging on a wide array of prey types but may be less likely to feed on co-existing natural enemy taxa. This commonly occurring species also appeared to have a high capacity to kill large numbers of prey, although this was not explored in the field. Providing management recommendations on how to maximise the use of spiders in Australian broadacre cropping was beyond the scope of this project. Instead, the intended goal was to inform future work into the use of spiders in biological pest management. This project attracted both industry funding and grower interest, reflecting an appreciation that this knowledge will contribute to establishing ways in which ecosystem services provided by naturally occurring enemies of pests can best be harnessed. As chemical based pest control reveals escalating inadequacies, an understanding of the organisms that live in agricultural habitats becomes increasingly crucial in the quest to sustainably feed the human population.
Breeding biology of the platypus (Ornithorhynchus anatinus)
This thesis examines the different behavioural stages of the reproductive cycle in the platypus, Ornithorhynchus anatinus, the time and energy investment of the female in breeding, and use of burrows by wild juveniles during the period after they first emerge. Many aspects of platypus reproduction are poorly understood due to their cryptic, nocturnal, semi-fossorial and semi-aquatic behaviour, which makes studies in the wild difficult. I studied a group of captive platypuses at Healesville Sanctuary and newly emerged juveniles from the wild population within Badger Creek, Victoria. My aims were to examine prey selection and seasonal energy intake, quantify and describe courtship, mating, nesting behaviour and maternal care given to nestlings, and describe how juvenile platypuses use the habitat in their natal home range. In captivity, platypuses consumed the fewest kilojoules during the breeding season and most kilojoules during the post-breeding breeding season. They showed a preference for less-mobile prey (mealworms, earthworms and fly pupae). Crayfish formed the largest quantity of food in the diet and was highly nutritious for energy (kJ), vitamins and minerals. The platypus diet was influenced by nutritional content, the stage of the breeding season and the behaviour of the prey species. Female platypuses controlled breeding encounters with males via three strategies; avoidance, by having lower activity levels and changing their activity pattern to partially diurnal; flight, by leaving the area immediately upon encountering the male; and resistance, terminating breeding encounters with the male and using a non-contact courtship behaviour prior to contact courtship behaviours. These strategies are likely to protect females from injury and coercion. After mating, females invested 8 ± 1.5 hours over 3 nights collecting wet vegetation for their nesting burrow. The morphology of burrows varied each year, but contained the same structural features: narrow tunnels, dead ends, ‘pugs’ of backfilled earth and multiple entrances that lead to a nesting chamber containing a spherical vegetation nest. The female’s energy intake increased to twice that of a non-lactating female in the final month of lactation, indicating the high cost of milk production. The length of lactation dependence for platypus nestlings was 128 ± 1 days. Females spent less time in the nest with twins compared with a single nestling. I developed an infra-red camera technique which allowed platypus nestling behaviour, growth and development to be observed in their burrow. Weaning occurred as an instantaneous event when the nestlings emerged into the water. Newly emerged wild juvenile platypuses each used multiple burrows for single or multiple nights within their natal home range. There was no significant correlation with vegetation communities along the bank at burrow sites, indicating burrow site selection was not driven by vegetation structure. No juveniles dispersed, suggesting they persist in the natal home range until the sub-adult stage which may assist their survival as they develop their skills and complete their growth in high quality habitat. My study demonstrates that female platypuses invest a high amount of time and energy in breeding, from avoiding the male platypus, through courtship and mating, creating the nesting burrow, maternal care during lactation, and while juveniles persist in her home range after weaning. I have provided captive management recommendations based on my research to advance the animal welfare and captive breeding success of the platypus.
The role of web-based chemical communication in the mating and foraging strategies of the orb web spider Argiope keyserlingi
Chemical communication is critical in shaping the interactions of individual animals, both within and between species. Pheromones (species-specific chemical signals) occur in all species and arguably contribute to more animal interactions than any other mode of communication. Nevertheless, our understanding of the role, nature and expression of chemical signals are mostly informed by studies of insects. This thesis investigates the role and expression of web-based chemical components in the orb web spider Argiope keyserlingi. Given female sexual cannibalism, Argiope males are under strong selection to exhibit mate choice and previous studies suggest that males in this genus fine tune their mating decision using contact pheromones (species-specific chemical signals that benefit both the signaller and the receiver) present on the female’s web. Previous studies suggest that males use these web-based contact pheromones to avoid previously mated females. I predict that these web-based pheromones may also provide males with reliable information about the quality of the female as a reproductive partner. Further, as a sit-and-wait predator, I predict that selection will favour strategies that increase encounters between web and prey, including the use of prey-attracting allomones (inter-specific chemical signals that benefit the signaller but are harmful to the receiver). The expression of chemical signals is ultimately determined by a combination of environmental and genetic effects. There is considerable evidence that diet is the most significant factor shaping pheromone expression. This can lead to considerable intraspecific variation in the signal for opportunistic foragers such as spiders, whose diet is influenced by their choice of web placement. This variation presents specific challenges to the receiver who must recognise and respond appropriately to a range of signals. In Chapter 1, I provide a comprehensive review of the literature and argue that diet-mediated pheromones (and signature mixtures) can enforce signal reliability by providing receivers with reliable information about the identity or quality of the signaller. In the context of mate choice in Argiope spiders, this should allow the male to choose a partner that will maximise his reproductive fitness (for example, by providing the receiver with offspring that have inherited good foraging genes). In Chapter 2, I use chemical analyses (gas-chromatograph flame ionisation detection) of silk extracts to demonstrate that there is considerable variation in the web-based chemical components of female A. keyserlingi. My results suggests that this variation is most likely due to changes in the availability of dietary nitrogen following the introduction of the laboratory diet. The maintenance of mate choice requires variation in the signal, so that the choosy sex can distinguish between signallers. Further, the variation in the signal should reflect reliable information. I suggest that variation in the nitrogen-containing components of the silk provides reliable information to searching males about the quality of the female as a reproductive partner. In Chapter 3 I examine how these web-based components vary on a broader scale between geographically distinct populations, as well as their underlying genetic and environmental influences. I found that nitrogen containing web-based chemicals are the most variable between populations, consistent with the view that arthropod predators are nitrogen limited. Furthermore, I suggest that variation in the nitrogen containing components of silk may provide males with reliable information about the nutritional health and foraging ability of the female. Interestingly, I also found that a single unidentified nitrogen containing silk component (unknown amide 3) and the total amount of all nitrogen containing silk components are strongly correlated with female condition, and may therefore provide males with reliable information about the female as a reproductive partner. In Chapters 2 and 3, I characterise a number of interesting web-based chemical components whose functional roles are also worthy of future studies. Of these components, I was particularly interested in putrescine, which is a nitrogen-containing compound and thus an expensive investment for a nitrogen-limited species. Importantly, putrescine also has a distinctive smell (of rotting matter) and is attractive to certain species of Diptera. In Chapter 4, I use field experiments that manipulated the amount of putrescine on female silk to demonstrate that web-based putrescine increases prey encounters for females and therefore acts as a prey-attracting allomone. Additionally, I found differences in prey capture rates between females collected from different populations, suggesting that there is population-level variation in the web-based foraging strategy of these spiders. Given the cost of putrescine and its role in foraging, I investigate whether web-based putrescine may provide males with a reliable signal of the nutritional health and foraging ability of potential mates in Chapter 5. As mating preference in this species changes with the reproductive experience of the male, I test the preference of males with 1 – 2 or 1 remaining mating opportunity to webs built by females given diets differing in protein content and also where I artificially manipulate the putrescine content of silk. My findings suggest that males with just 1 remaining mating opportunity but not males with 1 – 2 remaining mating opportunities use web-based putrescine to make mating decisions. Surprisingly, female diet did not influence male preference for web-based putrescine. In Chapter 6 (appendix), I discuss how anthropogenic changes to the environment (including but not limited to the nutritional environment) can shape the expression of pheromones in insects, and the consequences this may have for sexual selection. On a broader scale, diet-mediated signals employed in either inter- or intra-specific communication are vulnerable to anthropogenic influences, as changes to the environment can affect nutrient availability. Together, my results suggest that variation in the web-based chemical components of Argiope keyserlingi are driven by the availability of dietary nitrogen, and I argue that this variation will allow males to make accurate mating decisions about the nutritional health and foraging ability of the female. In particular, my research suggests an important role for putrescine in both the foraging and mating strategy of this species. Diet-mediated chemical signals such as these can provide reliable information about the signaller, but are vulnerable to environmental shifts (anthropogenic or otherwise) in nutrient availability.
Defining the master regulator of urethral closure in mouse
Hypospadias is the ectopic placement of the urethral opening on the underside of the penis and is one of the most common developmental abnormalities in humans, occurring in approximately 1 in every 125 live male births. In addition, we have observed a doubling in the incidence of hypospadias over the past several decades suggesting an environmental component likely in the form of estrogen mimicking chemicals generally referred to as environmental endocrine disruptors (EEDs). Current models fail to explain these observations. The goal of this thesis is to produce a theory that describes the development and genetic regulation of urethral closure, and use it to explain the aetiology, spectrum, and rise in incidence of hypospadias observed in humans. The work presented in this thesis was performed using a novel mouse model (OVE442) with isolated hypospadias. This model was used to define the role of the urorectal septum (URS) during urethral closure. The process of urethral closure is generally thought to occur by tissue fusion. However, we provide immunohistological evidence that suggests the urethra is internalized by growth of the URS, which contributes tissue to the ventral aspect of the penis during embryonic development. The OVE442 model was next used to define a key regulator of the URS during urethral closure. Initial characterization of a genomic mutation in OVE442 model led us to discover a long non-coding RNA, designated Leat1, which was deleted near EfnB2. Loss of signalling through the EPHRINB2 protein was previously shown to cause severe hypospadias in mouse, however little is known about EfnB2 gene regulation during urethral closure. Leat1 was characterized, functionally examined, and shown to regulate EfnB2 expression through direct interaction with the EPHRINB2 protein. We further showed that Leat1 expression is differentially regulated in males and females, and that it is supressed by estrogen. These results showed that EfnB2 drives growth of the URS during urethral closure and provided the first experimental evidence revealing the genetic mechanism that causes male and female urethral anatomy to diverge. These observations were used together with our anatomical descriptions to produce a developmental theory that explains urethral formation in mouse. We extended our understanding further by using comparative time series RNA-Seq to describe global transcription and ChIP-Seq to identify genes actively regulated by estrogen and androgen during urethral. From these data, we identify potential urethral closure genes downstream of Leat1 and EfnB2 including genes that are likely responsive to sex hormones. This work has provided fundamental insights on the anatomy and genetic regulation of urethral closure. I have shown that male urethral closure is driven by growth of the URS and that this growth is regulated by the long non-coding RNA Leat1 in mouse. Furthermore, I have produced a list of potential EED targets that may lead to better understanding the causes of hypospadias. Through this work I have produced a theory that explains the spectrum of urethral malformations observed in human, defects associated with hypospadias such as chordee, and the genetic mechanism that is likely disrupted by EEDs. These findings fundamentally change the way we consider urethral development and may help to find ways to reduce the incidence or prevent hypospadias in humans.
Triple jeopardy in the tropics: assessing extinction risk in Australia's freshwater biodiversity hotspot
Freshwaters are the most degraded and imperiled ecosystem globally. Despite this high vulnerability, conservation efforts in freshwaters often lag behind those in terrestrial and marine ecosystems. In Australia this is particularly evident; despite high levels of river degradation, few freshwater fishes have had their conservation status assessed and only 14% of fishes are listed. Most listed species are restricted to southern Australia where rivers are particularly degraded. Northern Australia’s rivers are very diverse with many highly range restricted fishes. Yet almost no species are listed, despite potential vulnerability and an increasing number of threats across the north. Nowhere is this more evident than the Kimberley region in the north-west, where 49% of species are restricted to three or fewer rivers, and 10% are restricted to an area of <20 km2. Very little is known about the ecology of the region’s endemic fishes, so their vulnerability cannot be assessed. In my thesis I assess extinction risk in the freshwater fishes of the Kimberley using the triple jeopardy framework, that is whether they have small geographic ranges, low abundances and/or narrow ecological niches. Specifically I aim to (1) determine the relationships between range size, body size and abundance in all Australian freshwater fishes and (2) whether these relationships can be used to identify species at risk of extinction. I then determine whether (3) small ranged Kimberley endemics have narrow habitat, dietary or thermal niches compared to closely related widespread species and (4) synthesize these results to identify the fishes most at risk of extinction in the Kimberley. First, I test for a relationship between geographic range size and body size in all Australian freshwater fishes. I then investigate how this relationship varies with conservation status. I identify currently unlisted freshwater fishes that share traits with listed species and map their distribution, along with freshwater fish research effort, across Australia. I found a positive relationship between range size and body size. For a given body size, conservation listed species have a range less than one tenth the size of unlisted species. Based on this relationship, I identified 55 additional species that may be vulnerable to extinction. Most of these species are restricted to northern Australia where freshwater fishes are poorly known due to low research effort. Second, I test for abundance-geographic range size and abundance-body size relationships in Australian freshwater fishes and investigate how these relationships vary with conservation status. I identify and map currently unlisted freshwater fishes that are numerically rare, and combined with the results outlined above, map species with a double jeopardy risk of extinction. I found a negative body size-abundance relationship and no correlation between range size and abundance. Although relative abundance was a poor predictor of current conservation listing, I identified 59 consistently rare species. Twenty of these species (34%) currently suffer a double jeopardy risk of extinction and all were restricted to northern Australia. Third, using closely related widespread and endemic congeneric pairings of Kimberley freshwater fishes, I investigate whether endemic species have narrow dietary niches at any stage during their development. Using qualitative measures of habitat and presence/absence data, I also assess habitat specialization. Most range-restricted species have narrower ecological niches making them more vulnerable to extinction. Fourth I test the thermal performance of two pairs of congeneric species that are sympatric in the Drysdale River, with one widely distributed species and one range restricted species in each pair. In the Syncomistes pair, resting metabolic rate (RMR) was similar between species at low temperature but at higher temperatures the RMR of the widespread species was lower due to the onset of anaerobiosis. The range-restricted Syncomistes also has a higher critical thermal limit (CTL). In the Melanotaenia pair, the results were the opposite, with the widespread species having a higher CTL and RMR. The thermal performance of each species was related to their distribution within the catchment rather than their geographic range size, with the thermally sensitive species dominating the cooler, perennial downstream reaches, and the hardier species being more abundant in the hotter, more ephemeral upper catchment. Finally, I use the above information to assess the triple jeopardy extinction risk in the fishes of the Kimberley. Seventy-nine per cent of Kimberley endemic fishes are vulnerable on one or more axis, and two species had a triple jeopardy risk of extinction. The majority of vulnerable species are found in the remote rivers of the north-western Kimberley, but the most imperiled species (Hypseleotris kimberleyensis) is restricted to the heavily degraded Fitzroy River. My thesis shows that, despite fundamentally different environments, life histories and dispersal capacity, Australian freshwater fishes exhibit range size, body size and abundance relationships largely similar to terrestrial fauna. By identifying northern Australia as a hotspot of unrecognized vulnerable species, I provide an important context for guiding targeted research and informing future conservation management of Australia’s freshwater fishes. Combined with their small ranges and/or low abundance, the narrower niches of most Kimberley endemic species makes the region’s fishes particularly extinction prone. By identifying which endemic species are most vulnerable, my study provides specific information for targeting conservation efforts in the region. As the Kimberley and northern Australia more broadly are earmarked for major development, substantial effort is needed to effectively manage fish populations, design and manage developments with the environment as a major stakeholder and preserve remote rivers with high endemism and extinction risk. However, as northern Australia’s rivers are in good condition, with planning and research there is an excellent opportunity for proactive, properly informed freshwater conservation across the region.
Cognitive bias as an indicator of emotional state and welfare in captive zebrafish
Concerns regarding animal welfare are ultimately predicated on the assumption that animals can experience subjective feelings, which are the prerequisite to experiencing suffering, pain and pleasure. Assessing the subjective feelings of animals directly, however, is difficult and perhaps impossible due to their personal nature. Existing approaches in animal welfare research primarily focus on the physiological and behavioural components of emotional (affective) states of animals, as indices of the subjective feelings of animals, however these have several limitations. For example, many of these measures do not give information about emotional valence (whether an emotion is positive or negative), but rather emotional intensity, and in many cases confounding explanations make it difficult to interpret the data unambiguously. Recently, new methods of assessing animal welfare, based on the cognitive aspects of emotional states, have provided ways of overcoming the disadvantages of existing techniques. In humans, it is well known that cognition and emotion are closely linked and reciprocally related. By assessing the cognitive aspects of individuals, such as decision-making, attention and memory, we can gain valuable insight into their emotional state. These phenomena, known as cognitive biases, are increasingly being explored in mammals and birds, but no systematic research has yet been conducted on fish. Since very large numbers of fish are used in scientific research, as well as the commercial pet trade and fisheries industry, an understanding of their cognition can provide useful information for addressing welfare issues for this animal group. In this thesis, I investigated three forms of cognitive bias – judgment bias, attention bias, and sensitivity to reward shifts – using zebrafish (Danio rerio) as a model species. I aimed to determine if these cognitive biases can be detected in zebrafish, and if so, whether tests can be designed to make use of these cognitive biases to inform zebrafish welfare. Designing appropriate experimental set ups, while taking into account the cognitive abilities of zebrafish proved to be challenging. Judgment bias tasks generally require extensive pre-conditioning, and I was unable to condition fish to an appropriate level for subsequent judgment bias testing. Future experiments would have to refine the conditioning process to improve conditioning success. Attention bias, however, required no pre-conditioning, and I found that zebrafish to which I had applied a stressor would position themselves further away from a potentially threatening stimulus. I believe that this approach could potentially be used as a basis for assessing affective states in fish. In the sensitivity to reward shift task, I found evidence that the nature of conditioning to the task was mediated by habitual behaviour rather than goal-directed behaviour, thus affect did not seem to play a major role during that process. The empirical studies described in this thesis were, for the most part, preliminary attempts at investigating cognitive bias in a previously unstudied taxonomic group. I recommend that considerable further work be conducted on understudied taxa, especially for those where large numbers of individuals are subjected to experimental research that has the potential to negatively impact their welfare. I am optimistic that cognitive biases could eventually become a routine aspect of welfare monitoring for a range of species in a variety of animal husbandry settings, given the opportunity of funding and further research.