Zoology - Theses
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ItemThe role of web-based chemical communication in the mating and foraging strategies of the orb web spider Argiope keyserlingi( 2017)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.
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ItemDefining the master regulator of urethral closure in mouse( 2017)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.
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ItemTriple jeopardy in the tropics: assessing extinction risk in Australia's freshwater biodiversity hotspot( 2017)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.
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ItemCognitive bias as an indicator of emotional state and welfare in captive zebrafish( 2017)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.
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ItemDefining the role of endogenous estrogen in penile urethral development( 2017)Hypospadias, the ectopic placement of the urethral meatus along the ventral aspect of the penis, is one of the most common birth defects in boys. The prevalence of hypospadias has increased in recent decades, a phenomenon that has been attributed to growing exposure to endocrine disrupting chemicals(EDCs)in the environment that disrupt androgen and estrogen signaling. Mice have been used to model hypospadias using knockout and transgenic lines and hormonal manipulations. While mice present a tractable model to study penile development, several structures differ between mice and humans and there is a lack of consensus in the literature on their annotation and developmental origins. Furthermore, extensive postnatal development occurs in mice, complicating comparisons to human penis development. Using, section histology, gross morphology, and magnetic resonance imaging, this thesis first provides an extensive examination of the adult mouse penis and addresses differences in the mouse and human penis to facilitate comparisons between human and murine hypospadias phenotypes. Secondly, this thesis presents a hypospadias phenotype in mice with reduced estrogen signaling. Mice lacking estrogen receptor α(αERKO)have a mild hypospadias phenotype, which corresponds to the most common phenotype seen in humans. The third chapter of this thesis describes this phenotype through section histology and micro computed tomography and outlines the delamination events that occur postnatally to develop the mature mouse penis. This study offers the first direct evidence of a role for endogenous estrogen in distal penile urethral development. To identify how estrogen may be acting alongside androgen to drive penis development, this thesis next identifies direct targets of estrogen and androgen in the developing penis of human and mice using chromatin immunoprecipitation followed by deep sequencing (ChIP-Seq). Select target genes identified from ChIP-Seq were validated as being hormonally responsive using mouse genital tubercle cultures and quantitative polymerase chain reaction (qPCR). The result is a list of potential targets of androgen and estrogen in the development of the penile urethra. This data represents a key step in determining which genes are potential targets of endocrine disrupting chemicals and which may be affected by EDCs to cause hypospadias. Taken together, this thesis proposes a precise balance between estrogens and androgens is critical in driving normal urethral closure. Determining which EDCs can induce hypospadias will require examination of not only anti-androgenic and estrogen-mimicking chemicals, but those that reduce estrogen signaling as well. This thesis provides a comprehensive guide to postnatal mouse penis anatomy as well as a list of estrogen targets in the developing penis, which together represent a useful resource for the field of hypospadias.
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ItemEffects of fungicides on Australian amphipods and organic matter breakdown in aquatic environments( 2017)Fungicides are used widely in agriculture to control fungal diseases and increase crop yield. After application, fungicides may be transported off site via air, soil and water to ground and surface waters therefore have the potential to contaminate freshwater and marine/estuarine environments. However, relatively little is known about their potential effects on aquatic ecosystems. Amphipods are important in ecosystem service as they help with nutrient recycling through the decomposition of organic matter. The aim of this thesis is to investigate the effects of common fungicides on biological responses in two Australian amphipod species, Allorchestes compressa and Austrochiltonia subtenuis, through a combination of single and mixture laboratory experiments. In addition a field experiment investigated the effects of fungicides on organic matter breakdown. In laboratory studies, juveniles of the marine amphipod A. compressa and the freshwater amphipod A.subtenuis were chronically exposed to two commonly used fungicides, Filan® (active ingredient boscalid) and Systhane™ (active ingredient myclobutanil) at environmentally relevant concentrations. A wide range of endpoints that encompass different levels of biological organization were measured including survival, growth, reproduction, and energy reserves (lipid, glycogen, and protein content). Long term interaction effects of fungicides Filan® and Systhane™ on mature amphipod A. subtenuis was also investigated to evaluate how the results of mixture studies vary between endpoints and to determine suitable endpoints for mixture toxicity studies. In the field study, leaves and cotton strips were deployed at 26 sites, 24 study and 2 reference sites, in an intensive agricultural region in south-eastern Australia to investigate the effects of fungicides and other anthropogenic stressors on organic matter breakdown. Leaves and cotton strips were deployed at the sites for a three week period and repeated twice in winter and spring. Breakdown rates of leaf and cotton at studied sites were compared to that of the reference site two which has similar altitude to the study sites to determine the effects of fungicides and other stressors on functional stream health. Pesticide concentration and physico-chemical parameters of sites were monitored during the study. The relationship between organic matter breakdown rates and environmental variables was investigated. Laboratory results demonstrated that Filan® and Systhane™ caused significantly adverse effects on survival, growth, reproduction, and energy reserves of both species of amphipod at environmentally realistic concentrations. Female amphipods were more sensitive to fungicides than males in terms of growth. Reproduction was the most sensitive endpoint and most affected by fungicide exposure. The effects of fungicide mixtures on A.subtenuis were endpoint-dependent and antagonistic effects were observed only on reproduction. Field data showed that organic matter breakdown rate was significantly correlated with pesticide concentrations and nutrients but leaf breakdown was also strongly impacted by temperature. Leaf and cotton degraded differently but both indicated the same results on functional stream health for majority of the sites. This thesis provides the first evidence of the effects of common fungicides on survival, growth, reproduction, and energy reserves of two Australian amphipods at environmentally relevant concentrations. The results suggest that fungicide pollution could affect the viability of amphipod populations in the natural environments that consequently could cause cascading effects on the ecosystem. This is also the first study to investigate individual relationships between different pesticide groups with organic matter breakdown in a field environment. The results of this study emphasize the importance of considering the long-term effects of fungicides in risk assessments for aquatic ecosystems and contribute to the literature of fungicide toxicity on aquatic environments.