Dissertations / Theses on the topic 'Cane toads'

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

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

Cane toads, Rhinella marina, were brought to Australia in 1935. The toads spread rapidly, and currently are found over more than 1.2 million km2 of tropical and sub-tropical Australia. Cane toads have had devastating impacts on Australian wildlife, largely due to the vulnerability of predators to the toads’ toxins. As toads continue to spread, we urgently need to develop management strategies to reduce the impact of toads on native fauna. Two new aquatic-based methods of cane toad control show great promise. First, traps that exploit the chemical attraction of toad tadpoles to conspecific eggs (a cannibalistic response) can be used to remove toad tadpoles from waterbodies. Secondly, a ‘suppression cue’ involves the suppression of toad eggs and hatchlings by older conspecific tadpoles. Although both strategies work well under lab conditions, unanswered questions include how the mechanisms function under different environmental conditions, how chemical cues interact, and the feasibility of using these methodologies in the field. My thesis addresses these questions. I ran extensive lab-based tadpole trapping trials to investigate how the effectiveness of trapping is altered by factors such as temperature, tadpole developmental stage, and toxin and tadpole geographic origin. I investigated whether exposure to one chemical cue affects the response of tadpoles to other cues, and I ran mesocosm trials to determine whether removing some (but not all) of the cane toad tadpoles from a waterbody increases the viability of survivors. To determine whether ‘suppression’ can successfully be transferred to the field, I ran trials in field ponds. Finally, I investigated the way in which cane toads are able to invade areas that are theoretically considered ‘too cold’ for the species to persist. My thesis documents the ways in which the success of aquatic-based cane toad control methodologies can be maximised, and how lab-based trials might translate into effective field practices.

The near-extirpation of varanid lizards by Cane Toads has severely impacted ecosystem processes. Given the critical roles of apex predators in regulating ecosystem function, and the massively negative impacts of toads on populations of large goannas, we need to know the time-course of such effects. Limited data suggest that goanna populations eventually recover (e.g., in northeastern Queensland, where toads have been present for several decades) – but we know almost nothing about the nature or rate of such recovery. To develop robust priorities for conservation management, we need to understand how monitors adjust to Cane Toads over time.

One of Australia’s infamous pest, cane toads, were found to be infected with potentially pathogenic protozoan parasite within the genus of Entamoeba. It was reported that a number of cane toads in Northern Territory population was presented with gastrointestinal disease and therefore is the first agent of disease to be described in cane toads, causing morbidity and mortality. With its invasion dynamic, ecological relationship, and impact of cane toads on Australian native fauna still under evaluation, first report of entamoebiasis in cane toads is an emerging wildlife disease. This thesis is a part of investigation into the significance and impact of novel Entamoeba sp. CT1 and Entamoeba ranarum found in Australian native fauna and cane toad populations. This thesis focuses on two aspects: one is on the technical aspect of diagnostic of Entamoeba species of cane toads and native fauna, and the other is on the molecular analysis of the SSU-rRNA gene for identification and taxonomy of Entamoeba. This thesis evaluates the application of various methods of diagnostic of Entamoeba in faecal and colon of wild cane toads. Methods evaluated include direct wet mount, histology, conventional PCR and real-time PCR, where recommendations on future clinical diagnosis and epidemiological studies were made based on this evaluation. In order to investigate the distribution of Entamoeba in wild native fauna, non-invasive faecal detection of Entamoeba was implemented onto native snake populations that share prey and predatorial relationship with cane toads. Steps taken to obtain and analyse complete sequences (SSU-rRNA gene) of Entamoeba species was described and used to infer evolutionary relationship of the Entamoeba species of interest. Comparative data on performance of each diagnostic method for detection of Entamoeba (colon and faecal) in cane toads were obtained. For the purpose of non-invasive method of diagnosis (faecal detection), this thesis provided evidence for the use of molecular-based assays (real-time PCR and conventional PCR) as the recommended approach. By implementing this approach, this study was able to provide firsthand evidence for the presence of Entamoeba ranarum in faecal samples of wild keelback snakes. Molecular analyses of 18S rRNA gene of Entamoeba species in cane toads and snakes placed the two species well into the amphibian/reptilian clade and further expand the knowledge on Entamoeba genetic diversity.

Invasive species threaten biodiversity on a global scale, stimulating extensive research not only on the impacts of these species, but also on the factors that constrain invader distributions. To answer that latter question, we need to understand the developmental plasticity of a given invasive species in response to novel abiotic and biotic challenges. The Cane toad (Rhinella [Bufo] marina) is a large, toxic anuran, native to South and Central America that was introduced to Australia in a futile attempt to control agricultural pests. Since their introduction in 1935, toads have spread west from north Queensland across tropical Australia and south into New South Wales. Extensive research shows that toads have rapidly evolved an increased rate of dispersal across the Australian tropics, and exhibit physiological and behavioural adaptations to arid regions. However, the southern front of the toad invasion into New South Wales has attracted less study. Toads in this area face novel challenges, both biotic and abiotic. Models of the potential distribution of toads in New South Wales have ignored the potential for toads to adapt to lower temperatures, or to other abiotic challenges such as salinity and acidity. Such plasticity could influence the eventual geographic distribution of toads - and hence, their impact on native species. I focused on the egg and larval stages of the life history (rather than later terrestrial stages, the focus of most previous research). Thus, I conducted field surveys to measure abiotic conditions in potential spawning ponds near the current invasion front in northern New South Wales. I found wide variation in pH (3.9 to 9.8) and salinity (0 to 1200 ppm [3.5% the salinity of seawater]) among natural waterbodies. Temperatures in available ponds averaged around 20°C during the breeding period, 10°C lower than in many areas of the toads’ native range, or in the Australian tropics. I then conducted laboratory studies to evaluate the toads’ response to these conditions. Broadly, I found that early life stages of Cane Toads are resilient and can deal with relatively extreme abiotic conditions. Hatching success of eggs was high at low pH (down to pH 4.0), whereas rates of tadpole survival, growth, and development were enhanced by higher pH. Eggs and tadpoles tolerated the most saline conditions I found in potential spawning ponds during surveys. Indeed, high-salinity treatments increased tadpole body sizes, accelerated metamorphosis, and improved locomotor ability of metamorphs (but did not affect metamorph morphology). At very low salinity (40 ppm [0.1% seawater]), eggs hatched but larvae did not develop past Gosner stage 37. Paradoxically, lower water temperatures may enhance rather than reduce recruitment of cane toads, at least in areas where pond temperatures reach or exceed 20°C. Cane toad eggs and larvae died at 16°C, but hatching success and larval survival rates were higher at 20°C than in warmer conditions. Lower temperatures slowed growth rates, increasing the duration of tadpole life; but also increased metamorph body mass (and influenced metamorph body shape and locomotor performance). However, comparisons of cane toads from two geographic regions (tropical and temperate) revealed no divergence in cold-tolerance, despite the major difference in mean water temperatures between tropical versus temperate regions. Overall, my data reveal that embryonic and larval life stages of R. marina can successfully tolerate the wide range of acidity, salinity and thermal conditions that they are encountering in this new part of their range in northern New South Wales. I also examined one novel biotic challenge in this area. Cane Toads are encountering an invasive predatory fish (Mosquito Fish, Gambusia holbrooki) that, like the toad, has an American origin. I tested the responses to toad tadpoles of Mosquito Fish from populations that were sympatric versus allopatric with cane toads. Toad-sympatric fish generally ignored toad tadpoles, whereas toad-allopatric fish initially consumed a few tadpoles, but rapidly developed an aversion to these toxic prey items. The laboratory-reared progeny of these fishes differed in their propensity to approach toad tadpoles as potential meals, but not in the rate at which they learned toad-avoidance. Thus, Mosquito Fish show an innate aversion to cane toad tadpoles (presumably reflecting coevolution), as well as an ability to rapidly learn taste-aversion. As a result, any impact of Mosquito Fish on Cane Toads (and vice versa) is likely to be transitory. Overall, my study shows that the egg and larval life-stages of cane toads can tolerate wide variation in the biotic and abiotic conditions of natal ponds, and that their responses to the conditions found in northern New South Wales are likely to facilitate rather than constrain continued southwards expansion of the toad invasion front in eastern Australia. I also conducted a separate experiment, to test a more general idea. My finding that low pH enhances hatching rates of toad eggs suggested a novel hypothesis: that the benefit of spawning after heavy rain (as seen in many anurans) may reflect impacts of transitory changes in pond water pH. To test this idea, I measured feeding rates of two predators of toad eggs (Mosquito Fish and conspecific toad tadpoles) at a range of pH conditions. Predation rates on eggs likely are lower following heavy rain that reduces pond pH, and the effect may last long enough to allow eggs to hatch into mobile larva more capable of evading predators. Thus, anurans may benefit by spawning as soon as heavy rain when reduces the threat that predators pose to newly-laid eggs.

Invasive species must adapt to a suite of novel selective pressures as they colonise new territory. For example, theory predicts that selective forces at an invasion front will favour traits that enhance rates of dispersal and population growth, whereas selective forces in the range-core will favour traits that enhance competitive ability. To explore this idea, I conducted competition trials on three life stages of the invasive cane toad (Rhinella marina) from invasion-front and range-core populations in tropical Australia. For larval and early terrestrial-stage animals, I conducted experiments whereby the offspring of invasion-front and range-core adults were placed into mesocosms to compete for limited resources. To enable such experiments, I developed a simple method to mark each cohort of tadpoles, with minimal observed effects on growth or survival. Range-core larvae proved to be better competitors than invasion-front larvae, as predicted by evolutionary theory. Contrary to expectations, invasion-front metamorphs outcompeted range-core metamorphs, growing larger and winning more food. For adults, I focused on the trade-off between rapid dispersal versus success in male-male rivalry. Longer arms reduced the force required to displace an amplecting male from a female. When competing for a female, males with longer arms had lower likelihoods of retaining amplexus. I also explored how visual and acoustic cues influence the behaviour of sexually active males. Male toads used the presence of a chorus to increase their activity levels and as a cue to initiate amplexus. Males also used movement to identify potential mates, with increased rates of amplexus when a target was moving. My thesis presents empirical evidence that the evolutionary forces unleashed by a biological invasion can modify traits important in intraspecific competition at each life stage. My results are relevant to evolutionary theory and are directly translatable into proposed management strategies.

Amphibians are storehouse of bioactive compounds. Among them, the skin of toad species is rich in biologically active compounds such as peptides, proteins, steroids, alkaloids and opioids. Some of these compounds have found significant therapeutic applications, for example as antibacterials, antifungals, antiprotozoals, antidiabetics, antineoplastics, analgesics and sleep inducing agents. In Traditional Chinese Medicine (TCM) aqueous extracts of Chinese toad skins have been used for centuries to treat pain, swelling, heart failure and several types of cancer with minimal to no side effects, generating a 10 billion USD market in China. Numerous compounds have been identified from these Chinese toad skin extracts, which have reported therapeutic activities in various disease conditions, either as a single compound or as a group of compounds. In Traditional Korean Medicine, toad extracts have also been reported to show potential activity against anxiety and depression. In Australia and America cane toad skins have a history of recreational use for euphoric purposes. This information coupled with knowledge of its use in China and Korea enabled us to hypothesise that Queensland cane toad skin extracts would likely contain similar ‘biologically-active’ compounds in selective extracts. This thesis reports on research work carried out to identify such ‘biologically-active’ extracts and/or compounds, then functionally characterise these in cultured cells for investigation of their therapeutic relevance to neuropsychiatric disorders, especially in obsessive compulsive disorder (OCD) - potentially identifying potent therapeutics for future development.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

The skin of the toads is known to be rich in bufadienolide compounds (a group of cardiac glycosides) that exhibit antitumor activity. For example, Huachansu (Cinobufacini), the aqueous extracts from the dried toad skin of Bufo bufo gargarizans Cantor or Bufo melanostictus Schneider, has been widely used in clinical therapy for various cancers in China. Clinical data have indicated that Cinobufacini may have significant anticancer activity with low toxicity and few side effects. Data to date suggest that treatment with Cinobufacini may also enhance the quality of life for patients with cancer. Huachansu contains several groups of compounds including peptides, bufadienolides/cardiac glycosides, cholesterols, indole alkaloids, bufogargarizanines, organic acid, and others. Bufadienolides, such as bufalin, cinobufagin, resibufogenin, and telocinobufagin, are responsible for the anti-cancer properties of Huachansu through disruption of the cell cycle and consequent inhibition of cell proliferation, induction of apoptosis, suppression of the NF-B pathway, immunomodulation and reversal of multi-drug resistance. The Australian cane toad (Bufo marinus) is also known as a source of bufadienolides, therefore is also considered as a new source of candidate lead compounds for drug development. Previous studies have shown that cane toad skin aqueous extracts (CTSAE) exhibited a stronger cardiac glycosides-like activity than the extracts of other organic solvents and have a suppression effect on Na+, K+‐ATPase in experimental models. However, no assay was performed to clarify the chemical constituents and pharmaceutical effects of CTSAE on cancer cells in previous studies.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Medical Science
Griffith Health
Full Text

Cane toads are native to warm humid landscapes of the Americas, but since its introduction to Australia, the rapid geographic spread has exposed toads to climatic conditions well outside those of the native range, with many Australian sites hotter and seasonally drier than those in which the species evolved. I investigated the impacts of temperature and hydration on aspects of dispersal behaviour and physiological traits of toads, to clarify the ways in which toads may have changed as a result of encountering novel environments within their invaded range, by comparing populations within the native range (Brazil) to ones introduced to Hawai’i and Australia. When stimulated to run at high or low temperatures, or when dehydrated, Australian toads exhibited greater performance endurance than either Brazilian or Hawai’ian toads. When raised under the same conditions, toads whose parents were collected from the climatic challenging area of Western Australia exhibited better performance than the offspring of Queensland toads. Rates of evaporative water loss, skin resistance and rehydration showed that toads from eastern Australia had higher skin resistance to water loss whereas Brazilian toads had faster rehydration rates, suggesting a shift in water balance strategy after toads were introduced to Australia. Histological examination of the skin structure showed that Australian toads have thinner skin than do native-range conspecifics. Laboratory trials revealed that toads from Townsville heated faster than toads from northwestern Australian populations, especially when deprived of water. When exposed to extreme heat or desiccation, Brazilian toads exhibited higher levels of corticosterone and of heat-shock protein 70 than Australian toads, suggesting a more reactive stress response. My studies clarify some of the mechanisms that have allowed cane toads to colonize diverse areas that impose climatic challenges not present within the species’ native range.

Phenotypic evolution in the invasive cane toad (Rhinella marina): adaptations for dispersal Abstract Invasive species provide a rare opportunity to study how organisms adapt when colonising novel environments. Despite the negative ecological impacts caused by the introduction of alien species, biological invasions act as natural experiments that we can exploit for ecological and evolutionary research. This is particularly true for introductions with precise geographic and historical records, such as the cane toad (Rhinella marina) introduction to Australia. Arguably the most successful invasive anuran worldwide, the cane toad was first introduced to the Hawai’ian Islands in 1932, and subsequently to northeastern Queensland in 1935. Over the past 82 years, R. marina has spread rapidly and at an accelerating pace throughout northern and eastern Australia, causing massive ecological disturbances in its wake. This acceleration of dispersal capability is a well-documented phenomenon in invasive organisms that can occur via a combination of natural selection, and spatial sorting. Although the increasing rate of cane toad dispersal has been extensively researched, my thesis aims to address questions about the evolutionary changes that have taken place throughout the process. Namely, what morphological, behavioural, or physiological shifts in cane toad phenotypes are associated with increased dispersal ability? I began by taking gross morphological measurements from toads across a transect through their northern Australian range that included long-colonised and invasion-front populations. Additional populations from a prior invasion (Hawai’i), and the native range (French Guiana) were later added to compare the morphology of individuals from the source populations. I focused on the components of each limb (hand, radioulna, humerus [forelimb]; femur, tibiofibula, foot [hindlimb]), as limb morphology is strongly linked to locomotor ability, but also collected data on mass, snout-vent length, head width, and parotoid gland shape. A subset of Australian individuals were collected from the edges of the range, representing the oldest and newest toad populations to be used in a common-garden breeding experiment. These individuals, and their resulting offspring were held in captivity and subjected to a series of performance trials. A separate group of individuals from invasion-front and range-core populations was used for Computerised X-ray Tomography (CT) scanning for precise geometric morphometric comparison of skeletal structure. Captive breeding of cane toads from the invasion-front (in Western Australia) and long-colonised areas (in Queensland) allowed me to control for the influence of rearing environment on common-garden F1 individuals. This also enabled analyses on heritability of morphology and performance traits by comparing offspring to their parents, and siblings to each other. Common-garden F1 offspring were raised over a period of 27 months, and measured repeatedly throughout ontogeny. Performance trials consisted of climbing trials (where toads would have to escape from a mesh tube by climbing vertically) and anti-predator raceway trials (where toads were encouraged to hop down a raceway by prodding). These trials were conducted on wild and captive toads. Within this thesis I document reproductive differences between invasion-front and long-colonised populations, significant geographic variation in locomotor performance, regional changes to skeletal structure, shifts in sexual dimorphism with time since colonisation, and heritability of behavioural and morphological traits; and I demonstrate that these changes have arisen via the rapid evolution of a high-dispersal phenotype during the invasion process.

Magister Scientiae (Biodiversity and Conservation Biology) - MSc (Biodiv and Cons Biol)
The guttural toad, Amietophrynus gutturalis, Power 1927, is a common toad with a broad geographic range through much of temperate, sub-tropical and tropical southern and central Africa. Introduced to the islands of Mauritius and Reunion in the 1960’s, and subsequently to Cape Town in the 1990’s, the species has become invasive in its extra-limital ranges. Determining the invasion history of a species provides valuable information for conservation biologists and managers and it is fundamentally important for improving our understanding of the underlying processes of biological invasions. This study aimed to determine the source populations of the extra-limital populations from Mauritius and Cape Town. Furthermore, studies investigating genetic diversity and demographics of African Bufonidae are largely absent from the literature. Understanding the evolutionary history of the species may also assist with determining their invasive ability and identifying similar features in other bufonids such as Amietophrynus regularis and A. xeros. Using mtDNA sequence data from the 16S and ND2 markers four geographically distinct clades were identified through Bayesian phylogenies and haplotype networks. However, a spatial analysis of molecular variance (SAMOVA) indicated a grouping structure of three clades. A total of 16 haplotypes were identified from 53 samples for the 16S marker and 22 haplotypes were identified from 43 samples for the ND2 marker. Both the Mauritius and Cape Town invasive populations were found to have originated from the eastern clade. However, they matched the common haplotype from this region which was found across a vast area that spans the KwaZulu-Natal province and into the Mpumulanga and Limpopo provinces. This did not allow for identifying a more precise region for the origin of the founder populations. The presence of haplotypes unique to the Cape Town invasive population, which group with the eastern clade, indicates that there has potentially been more than one introduction event. Demographic analysis revealed a recent population expansion in both the northern (Fs = -2.92) and the eastern clades (Fs = -5.03). Significant genetic variation was found among groups (93.92%), with low variation among populations and among populations within groups. Population pairwise differences were found to be significantly different between all clades except between the central and the southern clade. There was a negligible difference in the genetic diversity of the invasive populations when compared to the eastern clade. The eastern clades’ genetic diversity was low compared to the two other clades and demographic analysis revealed that this region has undergone the most recent population expansion. The negligible difference between the eastern clades’ genetic diversity and both invasive populations indicate that founder effects and genetic bottlenecking should have no impact on the invasive populations.
National Research Foundation (NRF)

Sexual selection theory predicts that the sex with the lower parental investment, usually the male, will be the more competitive and more highly adorned sex. Species in which males have a high investment In the form of parental care or nuptial gifts are instructive for testing the predictions of sexual selection theories. In the Mallorcan Midwife toad (Alytes muletensis), the male performs the parental care by carrying the eggs wrapped in a string around his legs. In laboratory experiments, both sexes were active in courtship and females were observed to compete more frequently than males. Both males and females incurred growth costs as a result of reproduction; males were also susceptible to physical injuries including the loss of limbs during brooding. Because the costs of reproduction are high for both sexes, either sex might be expected to exercise choice when finding a mate. Large males were not significantly better at caring for the eggs than were small males, and there was no correlation between female size and clutch size. Both sexes could benefit by choosing large mates, however, because hatchling size was significantly associated with both male size and female size, and large hatchlings are likely to enjoy fitness benefits. No evidence for female mate choice was evident in two-speaker arena experiments, but it is possible that mate choice in this species is mediated by non-acoustic cues. Double clutching, which enables brooding males to reduce the mating cost of providing parental care, is less common in A. muletensis than in the other species of midwife toads. Experiments and models indicated that the low frequency of double clutching is probably due to a time limit which constrains males to obtaining the second clutch within three days of the first. The potential reproductive rate of females was higher than that of males, suggesting that a female-biased OSR is responsible for the observed competition between females, but the reproductive rates of both sexes are expected to vary with the seasons. The resulting shifts in the OSR should be accompanied by seasonal shifts in the competitive sex

Worldwide, one of the most catastrophic impacts of invasive species is to imperil populations of apex predators, thereby inducing trophic cascades that can substantially modify ecosystem function. Management strategies generally focus on controlling the invader; but we can, instead, manipulate the behavioural response of vulnerable native taxa to render them more resilient to the invader’s arrival. In Australia, large varanid lizards (“goannas”) are fatally poisoned when they ingest invasive cane toads (Rhinella marina). As a result, the spread of cane toads has caused catastrophic population declines in these important predators. My PhD investigated the potential for an adaptive learning mechanism, Conditioned Taste Aversion (CTA), to conserve populations of tropical Australia’s largest lizard, the Yellow-spotted Monitor (Varanus panoptes). Ahead of the cane toad invasion, I exposed free-ranging goannas to small, live, non-lethal toads to assess the influence of training on lizard survival post-invasion. After cane toads arrived, trained goannas had significantly higher survival than their untrained (control) counterparts. I investigated mechanisms underpinning CTA to clarify how best to apply it in the field; for example, Yellow-spotted Monitors develop stronger aversions to live prey than sausages laced with chemicals, and an individual’s behavioural phenotype influences the effectiveness of CTA. I documented a behavioural syndrome that correlates with aspects of an individual’s ecology, life history and microevolutionary fitness. Furthermore, some individuals are inherently toad-averse; therefore some goannas (or populations) may not need CTA training. This behavioural diversity demonstrates how inter-individual variation can shape a population’s response to a novel pressure. Identifying and exploiting pre-existing behavioural mechanisms to deal with new challenges holds great promise for amplifying the tools available for conservation efforts globally.

The Major Histocompatibility Complex (MHC) is a large gene complex vital to the vertebrate immune response. The antigen-presenting molecules of the MHC class I and class II are involved in the immune surveillance of intracellular and extracellular pathogens, respectively. The regions of the MHC involved in peptide binding and presentation are often highly polymorphic and have high allelic variation within populations. High MHC diversity is theorised to provide immunogenetic competence to a population, and these genes have become a popular adaptive genetic marker in population studies, often with a conservation context. This thesis presents the characterisation of MHC diversity in two anuran species undergoing very different population histories: the cane toad (Rhinella marina), undergoing range expansions over the course of the Australian invasion; and the New Zealand endemic Hochstetter’s frog (Leiopelma hochstetteri), where populations are highly fragmented. In both cases, I investigate MHC diversity alongside neutral genetic diversity to infer the relative influences of neutral genetic forces, predominately drift, and selection in shaping allelic variation. This involved characterisation of the class I and class II in the cane toad, prior to completing a diversity study utilising genetic markers from both classes. I also characterised a class II beta gene in the Hochstetter’s frog to characterise diversity across 5 populations. The characterisation of the cane toad class I revealed a single classical locus, and an expansion of non-classical loci. This is similar to the class I organisation found in the model anuran, Xenopus laevis and across its subfamily Xenopodinae. The cane toad and X. laevis diverged around 230MYA. The characterisation of the cane toad class II revealed four class II alpha loci and three class II beta loci. It also revealed the expression of MHC class II splice isoforms at several alpha and beta genes. This is the first observation of alternative splicing in the MHC of any anuran species. The diversity of the classical class I (UA) and class II beta gene (DAB) was studied in the source of the Australian introduction (Hawaii), in a long-colonised site (Cairns) and a site on the invasion front (Timber Creek). I found that little diversity was lost as a result of the introduction and that Cairns was highly genetically representative of Hawaii. I found genetic drift acting at the invasion front, decaying genetic diversity at microsatellite markers and the UA locus in the dispersing cane toads. DAB diversity was maintained, however. Selection has retained all three DAB alleles across the toad invasion, likely mediated by pathogenic bacteria and parasites infecting the population. I found very high MHC class II DAB polymorphism in the Hochstetter’s frog across 5 sampled populations. Populations showed extreme differentiation; only two DAB alleles were shared by more than one population. Populations generally had high DAB diversity, except Otawa. The Otawa population had only two DAB alleles present. Combined with low microsatellite diversity, Otawa has likely undergone a recent decline, which has impacted genetic diversity. Low immunogenetic diversity in Otawa may predispose this population to a greater risk of extinction from emergent disease.

Includes abstract.
Includes bibliographical references.
Declines and losses of amphibian populations are a global problem involving a complexity of interacting causes. Regardless of the fact that amphibians in Africa are among those predicted to be hit the hardest by anthropogenic global change, many species remain poorly studied. Capensibufo rosei, Rose's Mountain Toad, is a restricted range species that survives in a few small, isolated montane populations in the extreme south-western Cape of South Africa. A recent study of the genus revealed that C. rosei may in fact comprise several cryptic species, with a distinctive lineage potentially being confined to the Cape Peninsula. I test the hypothesis that breeding sites on the Peninsula form a single genetic lineage, but are distinct at a population level due to limited dispersal abilities and little if any gene flow.

Invasive species are notorious for their negative impacts on the environment and economy, but also provide useful systems for studying evolution in wild populations. Here, I utilize next-generation sequencing (NGS) technologies to approach mechanistic questions about the rapid adaptation to novel environments exhibited by invaders. Bioinformatics analyses of NGS data currently lack standardized parameter choices and there is growing concern that these choices may affect the evaluation of population structure. To address this issue, I analyze simulated GBS datasets and empirical RAD datasets of varying structure using multiple sets of filtering parameter choices. I demonstrate that such choices indeed affect the results, particularly in datasets with low structure; I also identify some common pitfalls to avoid when analyzing and publishing NGS datasets. Bearing these in mind, I approach questions about population structure during invasion by re-analyzing a RADSeq dataset on invasive Australian cane toads (Rhinella marina) in conjunction with our RNA-Seq dataset on this species. Since their introduction, cane toads have dispersed across northern Australia, displaying adaptive variation that allows them to surmount climatic challenges. RNA-Seq data provide transcriptome-wide coverage of single nucleotide polymorphisms (SNPs), allowing us to test whether cane toads represent a genetic paradox of invasion, and to identify loci putatively under selection by climate. Immune function may also change during invasion; to test the assumptions of the enemy release hypothesis, I use differential gene expression analysis, which provides a snapshot of immune pathways found in spleen transcriptomes. I separately test specific responses to immune activation using field assays. Together, this body of work informs methodology, demonstrates the importance of selection in shaping invasion, and furthers our understanding of immune regulation in invaders.

The proliferation of artificial water points (AWP) to enhance livestock production has fundamentally changed the ecology of dryland ecosystems. By providing a constant source of surface water, AWP can affect the density, distribution and activity of water-dependent native and invasive species. Thus, AWP have the potential to facilitate trophic and competitive interactions, thereby amplifying their impact on the structure and composition of resident animal and plant communities. Recent studies suggest that a strategic manipulation of surface water availability in dryland biomes could be a useful management approach to reduce the negative impacts of native herbivores on vegetation and to mitigate the spread and impact of water-dependent invasive species. However, there is only limited empirical evidence that water exclusion could provide benefits for the ecology and community structure of dryland ecosystems by returning them to their natural water-limited state. Especially for invasive species where the effectiveness of a large scale water exclusion strategy has not been sufficiently evaluated. This is particularly important for high-impact invasive species such as the cane toad Rhinella marina, an anuran that is currently invading northern and arid regions of Australia. Due to their potent chemical defences cane toads put a wide range of native predators at risk of toxic ingestion. During periods of prolonged hot and dry conditions which typically prevail in arid regions, cane toads need regular access to water to survive. In rangeland areas of the Tanami Desert in Australia’s Northern Territory, the existence of AWP fitted with two types of reservoirs provided an opportunity to examine whether exclusion from access to surface water at AWP could limit the abundance and ecological impacts of cane toads. Cane toads could readily access surface water at AWP fitted with earthen dams as reservoirs but cane toads could not access surface water at AWP fitted with water tanks. This study has implications for the management of cane toads in Australia’s arid and semi-arid rangelands. The findings demonstrate that excluding water-dependent species from access to surface water at AWP by changing the water reservoir system from traditionally used earthen dams to water tanks results in a reduction of both their population densities and ecological impacts.

The helminth fauna of native Australian amphibians and the introduced toad, Bufo marinus was studied. Species composition and ecological relationships of the helminths were considered in detail. In addition, the relationship of one helminth species, Rhabdias sp., to the health of the toad was considered. A total of 27 helminth species (14 Nematoda, 8 Digenea, 2 Cestoda, 2 Acanthocephala, 1 Monogenea) was collected from both the toad and native amphibians in this study. Six helminth species were found to only infect toads in this study: Dolichosaccus juvenilis, Zeylanurotrema spearei, Cosmocerca sp. 2, Cosmocerca sp. 3, Austraplectana sp., adult acanthocephalans. Two of these species (D. juvenilis and adult acanthocephalans) had been reported from native fauna in previous studies. Three species were found to infect only native amphibians in this study: Parapolystoma sp., Seuratascaris numidica, and Onchocercidae gen. sp. All of the helminth species collected from B. marinus in this study, with the possible exception of Rhabdias sp. and Mesocoelium sp. for taxonomic reasons, can be determined as having an Australian origin. The majority were acquired by the toad from native amphibians. Some species, however, were thought to have transferred to the toad from native reptiles. At least 70% of toads and native frogs were infected with at least one helminth species. Maximum number of helminth species for an individual toad was 6, whereas for native frogs it was 4. Bufo marinus had a more diverse helminth community than native frogs at both a host individual and host population level. The use of diversity indices in helminth community ecology and the concept of core and satellite species, particularly in relation to amphibian helminth communities, is discussed. Comparison of the helminth fauna of B. marinus and a native frog, Litoria inermis, was undertaken in detail. Relationships of total helminth intensity and species richness to various factors, including host sex and snout-vent length and month of collection were calculated for both host species. Reasons for the possible disparity between helminth infection levels for B. marinus and Lit. inermis are discussed. Only one helminth species, Rhabdias sp., was thought to have potential as a biological control agent for the toad in Australia. Detailed studies of the life cycle of Rhabdias sp., natural infection levels within a population of B. marinus and its relationship with the health of the toad were undertaken. Rhabdias sp. infected over 80% of toads collected from QDPI, with a mean intensity of 16 nematodes per infected toad. Intensity of infection had a significant relationship with length of toad for subadult toads only. Average length of Rhabdias sp. within an infrapopulation had a significant relationship to host length for subadult and middle size class toads. Distribution of Rhabdias sp. within the toad population was aggregated, with degree of aggregation increasing with toad size class. Sex of toad had a significant relationship with average length of Rhabdias sp. only in Class II toads, where male toads had larger nematodes. Rainfall was an important environmental factor influencing infection of toads with Rhabdias sp. The majority of Rhabdias sp. recruitment into the toad population occurred during the late wet season, although small amounts of recruitment occurred throughout the year. Development of Rhabdias sp. from embryonated egg to infective third stage larva, in the laboratory, took 4 days at 24°C. Development was only observed via a free-living sexual cycle, with only one larva produced per free-living female. Experimental infections were hampered by a high death rate among the metamorph B. marinus and Limnodynastes ornatus used. Over 50% of metamorphs exposed to infective larvae of, Rhabdias sp. became infected. Number of larvae penetrating the metamorph was significantly related to the success of infection. Lower infection dosages produced proportionately higher levels of infection. Haematological data for B. marinus in Australia is presented for the first time. Presence of a Rhabdias sp. infection significantly decreased levels of red blood cells, packed cell volume and haemoglobin concentration. Level of Rhabdias sp. infection also significantly decreased these levels, but not to the same extent as presence of Rhabdias sp. alone.

Invasive species are of major concern to ecologists, because of their impacts on native fauna, communities, and ecosystems. Invasive species may alter the evolutionary pathways of native species by competitive exclusion, niche displacement, hybridisation, introgression, and predation, at times ultimately causing extinction. Further, the economic cost associated with invasive species, through losses in agriculture, forestry, and tourism, as well as the costs of preventing and controlling these species, are of major concern to land managers and governments. Specifically, the management of vertebrate invasive species is a crucial component of biosecurity, ecology, and land management. There are a range of control methods for invasive vertebrates, including hand-capture, trapping, baiting, shooting, and biological and genetic control methods. These control strategies vary in efficacy, depending on the life history and behaviour of the target species, the area over which removal occurs, and the method of delivery of the control. Understanding these factors assists with designing targeted control strategies, in which the chance of removal of each individual, or the impact of each capture, or both, is increased. The success of control methods for some invasive vertebrates has improved considerably over the last several decades, due to the ever-increasing body of research about the behaviour and life history of certain invasive species, and the refinement of control regimes in relation to new information. The invasive capabilities and impacts of amphibians generally receive less attention than other invasive vertebrates; as such, control methods for invasive amphibians are rare. Some invasive amphibians are generalist feeders, have high reproductive rates, and attain large population sizes; however, specific behavioural and life history traits are varied, and are often unknown. Further, abiotic factors, such as atmospheric temperature and moisture, effect the behaviour and activity of many amphibians. Current control strategies for invasive amphibians (e.g., hand-capture, exclusion fencing, and habitat modification) are often non-targeted, under-researched, and ineffective. Cane toads (Rhinella marina) are highly invasive anurans, native to south and central America. Their invaded range extends through many tropical areas worldwide, including Australia. Cane toad paratoid (shoulder) glands secrete powerful bufotoxins that are lethal to some native predators, and domestic pets. The impact pathways of cane toads on native species include poisoning after ingestion (both at larval and adult stages), and competition with other anurans. Further, the presence of cane toad tadpoles may affect growth rates of native tadpoles, while the presence of adults may affect calling behaviour of some native anurans. Potential control strategies for cane toads within their invaded range include hand-capture, tadpole traps, and biological and genetic control methods. These strategies are often non-targeted (e.g., tadpole traps, biological and genetic control methods), have been ineffective at suppressing toads for long periods, on a large scale, and in some cases require extremely high effort (e.g., hand-capture events). Trapping adult individuals using a solar-powered light and acoustic lure that automatically plays a cane toad call to attract toads into traps may be a viable control method that is easily refined to increase captures by exploiting behavioural characteristics of the cane toad. The success of any control method is dependent on the ability to refine it by targeting specific demographics of the invasive population, and increasing the number of captures per unit effort spatially, and temporally. Many control methods for cane toads are ineffective because they do not consider the activity patterns of toads in response to abiotic factors; however, understanding and exploiting these patterns could allay wasted effort. For example, land managers could augment captures by understanding the environmental conditions that drive activity, at different times of year, and focusing trapping effort on periods when toads are most active. I examined cane toad activity (numbers of captures) in response to several environmental variables (humidity, temperature, rainfall, wind speed, and moon luminosity) over eleven months of trapping. Captures were highest (i.e., toads were most active) in the wet season (Dec – Feb), and lowest in the dry season (Jun – Aug). In the wet season, wind speed and minimum temperature effected activity (toads were most active on warm, still nights), while rainfall was the strongest predictor of activity in the dry season. I suggest that land managers could allay wasted trapping effort by focussing on nights with conditions conducive to toad activity (e.g., wet nights during the dry season). It is important to determine the area over which toads are attracted to the call used as a lure in traps (the active space of the call), to aid in trap placement and the design of large scale trapping regimes. A vocalisation's active space is the area within which a receiver responds to it, while its maximum extent occurs when a receiver stops responding. I mapped behavioural responses of male and female cane toads to advertisement calls by conducting experimental playbacks to quantify the active space of calls for both sexes, separately. Both sexes displayed positive phonotaxis 20 – 70 m from calls. Males also displayed positive phonotaxis 70 – 120 m from calls, whereas females' movement preferences were random >70 m from a call. Differences between male and female responses were likely driven by differences in their use of information provided by calls. I suggest that traps should be placed m apart, such that a female toad can never be more than 70 from a trap, but effort is not wasted by 'over-trapping' in the target area. Targeting reproductively active females is the best strategy for reducing recruitment into the next generation, and is a common control technique for vertebrate pests with high reproductive rates. Female cane toads can lay over 10 000 eggs per clutch, and should be targeted, however current control regimes do not focus on the removal of females. The lures used in adult cane toad traps play an advertisement call used by male toads to attract females. In many anurans, females select mates based on the structural parameters of advertisement calls (e.g., dominant frequency and pulse rate), therefore modifying the parameters of calls used as lures in cane toad traps, to create especially attractive calls, may augment gravid female captures. I altered the frequency and pulse rate of artificial calls used as lures, and conducted several trapping regimes in and around the Townsville region in northern Australia, to determine which calls were most attractive to gravid females. Overall, gravid females preferred a 'combination' call with a low dominant frequency, and high pulse rate (relative to the population median for these parameters). Approximately 91% of the females trapped using a low frequency and high pulse rate combination call were gravid, whereas in traps using a call with population median parameters only approximately 75% of captured females were gravid. Calls that indicated large-bodied males (low frequency) with high energy reserves (high pulse rate) are often attractive to female anurans, and were effective lures for gravid female toads in my study. Often, advertisement calls differ among populations. In this case, the attractive 'combination' call I identified in the Townsville cane toad population may be less attractive to gravid females in other populations. I sampled calls from 4 cane toad populations across Australia (south east Queensland, north Queensland, Western Australia, and the Northern Territory), and constructed artificial vocalisations based on the median parameters of the sampled calls. I conducted trapping at each population, using calls tailored to each population, to determine which call was most attractive to gravid females in those populations. I created 'median' calls based on median call parameters of each population. I also manipulated the frequency and pulse rate of tailored calls from the population median by the same percentages as the altered parameters of the Townsville combination call (an artificially manipulated call with a low frequency and a high pulse rate, relative to the median values of these parameters in Townsville) to create a unique combination call for each population. Median calls, from Townsville or the local population, were always less attractive to females than combination calls. In south east Queensland, Western Australia, and the Northern Territory, there was no significant difference in mean nightly female captures between traps producing the Townsville combination call, and traps producing tailored combination calls for each population. In north Queensland, traps producing the Townsville combination call caught significantly fewer females than traps producing the tailored combination call for that region. I suggest that calls used as lures in traps should have tailored parameters derived from vocalisations in the area in which trapping occurs, to maximise gravid female captures. Cane toad management strategies should increase the chance of removal of every individual, by exploiting behavioural characteristics, and by increasing the period over which removal occurs. The lures in cane toad traps start and stop automatically, and operate all night, thus managers need only be on-site to remove trapped toads. Conversely, 'toad-busting' hand-capture events require participants to be on-site to find and remove toads, and may therefore be less efficient, in terms of captures per person-hour, than trapping. I used capture-mark-recapture analysis to compare the efficacy of trapping, and hand capturing cane toads, over 10 weeks, in Townsville, Australia. I trapped 7.1% - 22.4% of the estimated population per week, and hand-captured 1.7% - 6% of the estimated population per week. Trapping was more efficient than hand-capture in my regime; overall, more toads were caught per trapping person-hour than per hand-capture hour. Traps attract toads and maximise the period over which removal occurs, thus the probability of removal for each toad was higher than by hand-capture. Also, many toads caught in traps were not encountered during active searches, and vice versa, so the use of both methods, together, may be beneficial. I conclude the thesis by placing my research into an applied context, and exploring future directions for cane toad management.

The eastern spadefoot (Scaphiopus holbrookii) is an ephemeral wetland breeding amphibian that ranges from southern Florida north and westward to southeastern Missouri and northward along the Atlantic coastal plain to Massachusetts. This species is listed as either "threatened" or "endangered" in the four states (Connecticut, Massachusetts, New York, and Rhode Island) in the northeastern United States where it is known to exist. Population declines and extirpations throughout the region over the past century have been documented and are largely believed to be the result of habitat loss and/or alteration. Very limited empirical results exist on many life history attributes of S. holbrookii anywhere in its range, including movement patterns, upland habitat selection, and breeding habitat preferences. These are critical information gaps that must be filled in order to effectively conserve and manage for this rare species in the northeastern U.S. While S. holbrookii is extremely rare throughout most of the Northeast, it is locally common in specific areas of Cape Cod National Seashore, most notably in an extensive sand-dune ecosystem known as the Province Lands located at the northern terminus of the Cape Cod peninsula. During 2005 and 2006, we conducted larval trapping surveys at 102 wetlands in the Province Lands primarily to: 1) identify breeding wetlands and 2) to assess breeding habitat use and preferences of S. holbrookii with respect to a suite of selected abiotic and biotic covariates. We captured S. holbrookii larvae at 140/652 (~21.5%) trap locations and 41/102 (~40.2%) wetlands sampled. Model results identified a number of additional habitat covariates that exhibited a statistically significant relationship with larval S. holbrookii abundance including: the percent cover of 1) cranberry (positive relationship), and 2) woody shrub (positive relationship) at a 4m radius from the trap location; 3) percent woody shrub cover at the wetland-scale (negative relationship); 4) percent canopy cover at the wetland-scale (positive relationship); 5) wetland pH (positive relationship); 6) distance to the closest paved road (positive relationship); and the kernel density cover of 7) deciduous shrubland edge (positive relationship), 8) deciduous shrubland (negative relationship), 9) pine (positive relationship), and 10) open dune (positive relationship) in the uplands surrounding the study wetlands. During 2006 and 2008 we radio-tracked adult S. holbrookii in the Province Lands using surgically implanted radio-transmitters to: 1) describe movement patterns and estimate home range sizes, and 2) assess upland habitat preferences of S. holbrookii. We tracked 19/20 individuals (11 males and 8 females) and 12/20 individuals (7 males and 5 females) for at least the first 30 and 100 days post-surgery, respectively, during 2006. During 2008 we successfully tracked 15/25 individuals (10 males and 5 females) and 5/25 individuals (3 males and 2 females) for at least the first 30 and 100 days post-surgery, respectively. In addition to results obtained on a suite of movement attributes we identified a number of upland habitat preferences for S. holbrookii. Use locations were: 1) closer to the nearest deciduous shrub edge, 2) had a greater percent cover of ground-running pitch pine branches at a 1 m scale, 3) had a greater percent cover of deciduous shrubs at a 1 m scale, and 4) had a greater percent cover of reindeer lichen at a 5 m scale. Results from this research provide much needed empirical results on these critical life history attributes related to the movement and breeding ecology of S. holbrookii. These results will aid biologists and Park management staff at Cape Cod National Seashore in more effectively employing conservation and management strategies aimed at enhancing the long-term persistence probability of this regionally rare species in the Province Lands.

Atrazine is one of the most widely applied herbicides and is commonly detected in surface and groundwater samples around Australia. Reproductive and developmental abnormalities in amphibians have reportedly been linked to atrazine exposure. However, the use of Australian native frogs in understanding these effects is limited. This study investigated the effects of atrazine alone and also in combination with a common secondary stressor, elevated salinity, to an Australian non-native anuran species, the cane toad, Rhinella marina and a native species, the striped marsh frog, Limnodynastes peronii, at both acute and chronic level. This study also explored the longer-term effects of atrazine to the cane toad to determine the impacts on its development and reproduction. Variations in larval sensitivities to acute concentrations of atrazine were first determined at different larval developmental stages in both R. marina and L. peronii. The static acute test design involved six nominal concentrations of atrazine, including control, solvent control, 3, 6, 12, and 24 mg/L. Gosner stages (GS) 22–23 as hatchlings, GS 25–26, 28–29, 32–33 as pre-metamorphic, GS 36–37 as prometamorphic and GS 40–41 as metamorphic climax stages of cane toads and the first four sets of Gosner stages (GS 22–33) of striped marsh frogs were exposed to atrazine treatments for 96 hours. Results showed that late larval stages were more sensitive than early stages and significant differences (P ˂ 0.05) occurred in sensitivities across different pre-metamorphic larval stages in both test species. The striped marsh frog showed greater sensitivity to atrazine than the introduced cane toad. In both experimental species, GS 28–29 showed the best response regarding the iv concentration-dependent increase in sensitivities to atrazine compared to other larval stages. To determine the chronic effects of atrazine, GS 28–29 of both test species were exposed to five nominal concentrations of atrazine, including control, solvent control, 0.1, 1.0 and 10 µg/L for 21 days. Results revealed no significant effects from atrazine exposure to survivorship, snout vent length, body weight and development among treatments in both species. Only a small percentage of exposed animals exhibited kinky tail and gonadal anomalies in L. peronii and a low percentage of atrazineexposed cane toads showed both limb and tail anomalies. Interestingly, both species had a female biased sex ratio in all treatments. No significant effect of atrazine on gross and histological morphology of thyroid glands in R. marina was noted; however, no thyroid analysis was performed on L. peronii. In order to evaluate the longer-term effects of atrazine on cane toad reproduction, GS 29–31 of R. marina was exposed to four concentrations of atrazine, including solvent control, 0.1, 4 and 11 µg/L until completion of metamorphosis. The metamorphs obtained from atrazine exposure were reared until their sexual maturity without further atrazine exposure. A number of attempts at breeding, including natural breeding, injecting with Luteinizing Hormone Releasing Hormone (LHRH) and lucrin (synthetic LHRH) were performed; however, no offspring were obtained. Morphological abnormalities, survival, fecundity, erythrocytic nuclear abnormalities, and gonadal and liver histology were analysed to evaluate the potential effects of atrazine on subsequent generations and also to examine the possible reason for the unsuccessful breeding. Results indicated that a concentration of 0.1 µg/L caused significant mortality, and 4 µg/L produced the highest incidence of limb abnormalities and also v decreased the hepato-somatic index during post-exposure period. No significant erythrocytic nuclear abnormalities or liver malformations were detected; however, gonadal histology showed a relatively high percentage of animals containing gonadal anomalies in all atrazine-treated males, except at 0.1 µg/L. To explore the combined effects of atrazine and a common secondary stressor, both R. marina and L. peronii were exposed to mixtures of atrazine and increased salinity. At acute exposure, GS 25–26 of both test species were exposed to salinity alone (0 to 8% sea water) and also in combination with atrazine (0 to 24 mg/L) for 96 hours. Results revealed no acute effects of elevated salinity on either test species; however, cane toad tadpoles demonstrated greater sensitivity to the mixture of atrazine and salinity than atrazine alone, while L. peronii indicated no differences in sensitivity. For chronic exposure, GS 28–29 of both R. marina and L. peronii were firstly exposed to a range of salinity (0 to 8% sea water) alone for 21 days. Results showed that 2% to 8% sea water caused significant effects on growth and development in L. peronii, but not in R. marina. Secondly, five treatments including control, solvent control, 0.1, 1 and 10 µg/L of atrazine each mixed with 8% sea water were used to expose the GS 28–29 for 21 days. Significant effects were obtained on the growth and duration of metamorphosis in L. peronii from mixture treatments, but not in R. marina. Overall, it can be concluded that inter- and intra-species variations in sensitivities to atrazine may occur in anurans residing in Australia and native species may exhibit greater sensitivity to atrazine than the introduced cane toad. Interestingly, although low concentrations of atrazine may not cause any noteworthy adverse effects on the developing R. marina and L. peronii during the larval exposure period; however, the longer-term study with R. marina showed that deleterious impacts may occur during vi the post-exposure period, which may cause adverse effects on the fitness and reproduction of R. marina. This may ultimately affect the population of this species. Longer-term testing with native species is required to determine the potential effects of atrazine on these populations. From the mixture of atrazine and salinity studies, it can be suggested that elevated salinity may pose serious hazards to Australian native anurans. The similar responses obtained from chronic exposure to atrazine in both R. marina and L. peronii may raise the possibility of the cane toad as an indicator for native anurans; nevertheless, further studies are required to establish this.

The cane toad's (Chaunus [Bufo] marinus) introduction in 1935 into Australia has had tremendous consequences for the native wildlife which inhabit the area of invasion. Much effort has recently been concentrated on minimizing the impact of the toad, especially along the invasion front in the Northern Territory and Western Australia. Efforts have focused on implementing short-term management solutions while long term solutions are being developed. Trapping has proved useful in the Northern Territory, and there is strong interest in improving the efficiency of this technique. Current traps use fluorescent lights which improve trapping success, but the mechanism of this has not been documented, and the efficacy of alternative light sources has not been investigated. I investigated the reactions of toads to four different light sources (incandescent, white fluorescent, yellow fluorescent and ultraviolet lights). My results indicate that toads are not attracted to any of these light sources, and white fluorescent and incandescent lights actively repel toads. This suggests that the attractiveness of lighted traps is due to the insects that are attracted by lights, which the cane toads feed upon. Yellow fluorescent and ultraviolet lights neither attract nor repel toads; given this result, and the fact that toads are apparently attracted to traps by the insects that lights attract, it is likely that using UV lights on traps would increase their effectiveness. Previous research indicated that both male and female cane toads are attracted to conspecific mating calls, and that playing back toad calls in the vicinity of traps can improve their efficiency. In other species, it has been established that manipulating certain aspects of calls can produce superstimuli calls that are more attractive than any natural call. Playbacks of a superstimulus might provide even greater efficiency increases for toad traps. I recorded and analyzed the calls of toads in nature to determine the mean and range of dominant frequency, pulse rate, and call length. I evaluated the responses of toads to manipulated calls with characteristics just outside the natural range for dominant frequency (high and low) and pulse rate (high and low). Both male and female toads were more attracted to calls with high pulse rates than they were to natural calls. The responses of females to manipulated calls also depended on their reproductive status. Based on my findings, it appears that the efficiency of traps could be substantially improved by using ultraviolet lights instead of white fluorescent lights, and by adding playbacks of high pulse rate advertisement calls. Validating these suggestions will require field experiments, which should be undertaken as soon as possible.

This study examined the effects of temperature and chronic hypoxia (CH) on pH/CO2- sensitive fictive breathing, and central pH/CO2 chemosensitivity, in cane toads (Bufo marinus). Toads were exposed to CH (10% or 15% O2) or control conditions (21% O2) for 10 days at either room temperature (controls), 10°C or 30°C following which in vitro brainstem-spinal cord preparations were used to examine central pH/CO2-sensitive fictive breathing (i.e., motor output from respiratory nerves which is the neural correlate of breathing). A reduction in artificial cerebral spinal fluid (aCSF) pH increased fictive breathing frequency (fR) and total fictive ventilation (TFV). Cold temperature reduced and hot temperature increased fR and TFV under control conditions. CH attenuated fictive breathing independently of temperature. Additional experiments in which the aCSF temperature was varied indicate that the effects of temperature acclimation result from neural plastic changes within respiratory control centres in the brain.

The impact of the eggs, hatchlings and tadpoles of the introduced cane toad, Bufo marinus, on native aquatic fauna in northern Queensland, Australia was investigated using a series of replicated laboratory and artificial pond experiments. Specifically, the project investigated: (1) the toxic effects of Bufo on native aquatic species, (2) predation by B. marinus tadpoles on native aquatic species, (3) competition between B. marinus tadpoles and native aquatic species, and (4) higher order effects produced by Bufo on other trophic interactions within native aquatic animal assemblages. The toxic effects of Bufo on native aquatic fauna were always associated with the consumption of Bufo; there was no evidence that toxins are released from Bufo into solution. Native aquatic species exhibited considerable inter- and intraspecific variation in their susceptibility to B. marinus toxins. Bufo were highly toxic to certain species but were non-toxic to others. Interspecific variation in toxic effects was not related to the number of Bufo ingested, and there was no clear pattern of distribution of vulnerability among species within higher taxa. Intraspecific variation in responses to toxins may result from (1) differences in the number of Bufo ingested by individuals, (2) individual variation in resistance to B. marinus toxins, or (3) individual variation in toxicity of Bufo. Two "susceptible" native aquatic taxa (fish and anuran larvae) were chosen for detailed studies. Native fish (barramundi: Lates ca/carifer; sooty grunter: Hephaestus fuliginosus) usually learned with minimal trauma to avoid B. marinus tadpoles. Populations of these species are therefore unlikely to experience significant declines in water bodies where they co-occur with Bufo. Anuran larvae, however, exhibited considerable interspecific variation in their ability to detect and avoid B. marinus toxins. Artificial pond experiments demonstrated that populations of species which have limited ability to detect and avoid B. marinus toxins (Litoria bicolor, L. nigrofrenata, Limnodynastes ornatus) experienced significant increases in mortality when exposed to Bufo. However, the toxic effects of Bufo on L. ornatus tadpoles indirectly facilitated the survival of eggs and hatchlings of later breeding native anurans by reducing the intensity of predation on these early life history stages by L. ornatus tadpoles. Bufo tadpoles were not significant predators of native anuran eggs, hatchlings or tadpoles, but did compete with native tadpoles (L. ornatus). The outcome of competition between B. marinus tadpoles and L. ornatus tadpoles was determined by their order of introduction into ponds. Generally, each species performed better when added to ponds before the other species, and performed worse when added to ponds after the other species, as compared to when both species were added to ponds simultaneously. However, the toxic effects of B. marinus eggs and hatchlings on L. ornatus tadpoles reversed these competitive priority effects and allowed late breeding Bufo to perform as well as, or better than, conspecifics which were added to ponds prior to L. ornatus. The results demonstrate that B. marinus eggs, hatchlings and tadpoles may have a significant impact on the composition and dynamics of native aquatic communities, and in particular, on native larval anuran communities.

I describe the natural history and reproductive behaviours of the Emei Moustache Toad (Leptobrachium boringii), testing the hypotheses that the species exhibits resource defense polygyny, and that combat, and paternal care lead to the evolution of male-biased sexual size dimorphism. In this study I document combat behaviour and paternal care for the first time in this species. Between February and March of 2011 and 2012, 26 female and 55 male L. boringii from Mount Emei UNESCO World Heritage Site, Sichuan, China, were observed throughout the breeding season. Prior to the breeding season, males grow 10-16 keratinized maxillary spines, which fall off once the season has ended. Throughout this time, males construct and defend aquatic nests where they produce advertisement calls to attract females. In a natural setting, I documented 14 cases involving a total of 22 males where males used their moustaches for aggressive interaction, and nest take over was observed on seven occasions. Despite my predictions, neither male body size nor body condition significantly affects the outcome of an aggressive interaction, though this may be representative of a low sample size. Males were also observed to possess injuries resulting from combat. Combat trials conducted in artificial nests demonstrated heightened aggression from resident males towards intruders. Genetic analysis using microsatellite markers revealed several cases of multiple paternity, both within nest and within clutch, indicating that some alternative male reproductive strategy, such as satellite behaviour is occurring. Larger males were observed to mate more frequently, and in multiple nests, suggesting that females are selecting for larger males, or that larger males are more capable of defending high quality territories. Males showed evidence of paternal care behaviours by remaining with the nests once females had left, moving throughout the nest cleaning, touching the eggs, and blowing bubbles into the centre of the doughnut-shaped egg masses. From this study I conclude that the male biased sexual size dimorphism in L. boringii is likely the result of both combat and paternal care behaviours creating a selection pressure on male body size.

The overall aim of this study was to contribute knowledge to two areas of inquiry in muscle research: one concerned with the molecular mechanism(s) underlying the positive correlation between intracellular glycogen content and skeletal muscle performance and the other with the MHC isoform composition in amphibian skeletal muscle and single muscle fibres. The organism used throughout this study was the cane toad Bufo marinus.