Dissertations / Theses on the topic 'Bufo marinus'

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.

Monoclonal employed antibodies against to investigate the various myosins have been employed to investigate the muscle fibre types in limb and tongue muscles of the Queensland cane toad Bufomarinus. The results of experiments using immunocytochemical techniques suggest that these antibodies can detect twitch fibres of types 1 - 3 as well as slow-graded fibres of type 4. 5 as established by Smith and' Ovalle (1973) in the limb muscles of Xenopus laevis. Evidence is presented to suggest that twitch fibres may contain more than one type of myosin in a single fibre. In addition, limb muscles showed seasonal variation in immunocytochemical staining patterns which could be explained by synthesis of type 2 myosin in type 1 fibres. In young toads, a difference in immunocytochemical reactivity of fibres compared with mature toads was found, suggesting the possible existence of foetal myosin in type 1 fibres in these animals. In addition to this, there is a progressive increase of type 2 myosin synthesises in type 1 fibres as the young toads mature to adulthood. Immunocytochemical data also suggest that tongue muscle fibres can be classified into types 1-3 as in twitch limb muscles. In the hyoglossus muscle, a population of fibres displays a staining pattern not found in the limb muscles, suggesting the existence of a unique type of myosin in these fibres.

The natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) are members of a family of hormones that play an important role in mammalian fluid and electrolyte balance. In the periphery, natriuretic peptides reduce blood volume and subsequently blood pressure by increasing renal natriuresis and diuresis and relaxation of vascular smooth muscle. The actions of natriuretic peptides are mediated via two membrane-linked guanylate cyclase receptors (NPR-GC); natriuretic peptide receptor-A (NPR-A) which has a high affinity for ANP and BNP; and natriuretic peptide receptor-B (NPR-B)which has the greatest affinity for CNP. A third receptor not linked to guanylate cyclase, natriuretic peptide receptor-C (NPR-C) also exists, which binds to ANP, BNP and CNP with a relatively equal affinity, and is involved with clearance of the peptides from the circulation and tissues. The natriuretic peptides are present in the brain and are particularly predominant in cardiovascular and fluid and electrolyte regulating areas such as the anteroventral third ventricle (AV3V) region. This distribution has led to the suggestion natriuretic peptides play a neuromodulatory role in the central control of fluid homeostasis. Natriuretic peptides in the brain have been observed to inhibit the release of other fluid and electrolyte regulating hormones such as arginine vasopressin (AVP) and angiotensin II (AII). Natriuretic peptides have also been identified in the non-mammalian vertebrates although information regarding the distribution of the peptides and their receptors in the non-mammalian brain is limited. In amphibians, immunohistochemical studies have shown that natriuretic peptides are highly concentrated in the preoptic region of the brain, an area believed to be analogous to the A\T3\ region in mammals, which suggests that natriuretic peptides may also be involved in central fluid and electrolyte regulation in amphibians. To date, CNP is the only natriuretic peptide that has been isolated and cloned from the lower vertebrate brain, although studies on the distribution of CNP binding sites in the brain have only been performed in one fish species. Studies on the distribution of ANP binding sites in the lower vertebrate brain are similarly limited and have only been performed in one fish and two amphibian species. Moreover, the nature and distribution of the natriuretic peptide receptors has not been characterised. The current study therefore, used several approaches to investigate the distribution of natriuretic peptides and their receptors in the brain of the amphibian Bufo marinus. The topographical relationship of natriuretic peptides and the fluid and electrolyte regulating hormone arginine vasotocin was also investigated, in order to gain a greater understanding of the role of the natriuretic peptide system in the lower vertebrate brain. Immunohistochemical studies showed natriuretic peptides were distributed throughout the brain and were highly concentrated in the preoptic region and interpeduncular nucleus. No natriuretic peptide-like immunoreactivity (NP-IR) was observed in the pituitary gland. Arginine vasotocin-like immunoreactivity (AvT-IR) was confined to distinct regions, particularly in the preoptic/hypothalamic region and pituitary gland. Double labelling studies of NP-JR and AvT-IR showed the peptides are not colocalised in the same neural pathways. The distribution of natriuretic peptide binding sites using the ligands 125I-rat ANP (125I-rANP) and 125I-porcine CNP (125I-pCNP) showed different distributions in the brain of B. marinus. The specificity of binding was determined by displacement with unlabelled rat ANP, porcine CNP and C-ANF, an NPR-C specific ligand. 125I-rANP binding sites were broadly distributed throughout the brain with the highest concentration in pituitary gland, habenular, medial pallium and olfactory region. Minimal 125I-rANP binding was observed in the preoptic region. Residual 125I-rANP binding in the presence of C-ANF was observed in the olfactory region, habenular and pituitary gland indicating the presence of both NPR-GC and NPR-C in these regions. 125I-pCNP binding was limited to the olfactory region, pallium and posterior pituitary gland. All 125I-pCNP binding was displaced by C-ANF which suggests that CNP in the brain of B. marinus binds only to NPR-C. Affinity cross-linking and SDS-PAGB demonstrated two binding sites at 136 kDa and 65 kDa under reducing conditions. Guanylate cyclase assays showed 0.1 µM ANP increased cGMP levels 50% above basal whilst a 10-fold higher concentration of CNP was required to produce the same result. Molecular cloning studies revealed a 669 base pair fragment showing 91% homology with human and rat NPR-A and 89% homology with human, rat and eel NPR-B. A 432 base pair fragment showing 67% homology to the mammalian NPR-C and 58% homology with eel NPR-D was also obtained. The results show natriuretic peptides and their receptors are distributed throughout the brain of B. marinus which indicates that natriuretic peptides may participate in a range of regulatory functions throughout the brain. The potential for natriuretic peptides to regulate the release of the fluid and electrolyte regulating hormone AVT also exists due to the high number of natriuretic peptide binding sites in the posterior pituitary gland. At least two populations of natriuretic peptide receptors are present in the brain of B. marinus, one linked to guanylate cyclase and one resembling the mammalian clearance receptor. Furthermore, autoradiography and guanylate cyclase studies suggest ANP may be the major ligand in the brain of B. marinus, even though CNP is the only natriuretic peptide that has been isolated from the lower vertebrate brain to date.

Three aspects of renal function were measured in the toad, Bufo marinus (N=lO): (1) effect of rate of blood volume expansion on renal functions (UFR; GFR; urine and plasma ion concentrations; and ion excretion rates), (2) effect of hypo- and hyperosmotic blood volume expansions on renal functions, and (3) role of GFR and tubular processes in the differential response of UFR under different osmotic expansion stresses. Renal responses to differential rates of blood volume expansion have not been investigated in amphibians. Rate responses will be analyzed considering effects: ( 1) during infusion (neural, or, short term regulation of extracellular fluid volume) and (2) post infusion (hormonal, or, long term regulation of extracellular fluid volume). Volume expansions were administered with hypoosmotic (0.4%) saline and hyperosmotic (1.4%) saline, and ranged in rate from 4.0 to 20.6 ml/kg/min. This protocol is designed to present volume regulatory mechanisms with increased volume stimuli and different osmotic stimuli. Overall, infusion rate had no significant effects on renal responses measured: urine flow rate (UFR); glomerular filtration rate (GFR); urine and plasma ion concentrations; natriuresis; or kaliuresis. This was true for the infusion period and for the observed post infusion period (90 min). Rate was correlated with GFR in the hypoosmotic group (r=0.30, p=0.04) and natriuresis in the hyperosmotic group (r=0.34, p=0.03). A significant positive correlation was observed between UFR and GFR. Relative to treatment, UFR differed significantly; GFR response was inherently similar despite differences at individual intervals, indicating UFR differences between the treatments is due to tubular processes. Responses to hypoosmotic infusion included a significant diuresis, natriuresis, and a decreased urine sodium concentration, relative to hyperosmotic infusion. At low UFRs the hyperosmotic group produced urine relatively concentrated in sodium. Urine sodium concentration and UFR were positively correlated in the hypoosmotic infusion group -- at high UFRs, kidneys were unable to produce a dilute urine.

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.

Currently published data on the role of the lymphatic system in amphibians are inadequate and contradictory. Estimates of the rate of formation of lymph and the role of the lymph hearts in returning this fluid to the circulation are not based on actual volume determinations but rather estimates derived from changes in hematocrit using published values of plasma and blood volume. The lymph hearts are known to be vital to the maintenance of normal fluid compartment physiology and to increase their rate of activity during episodes of hypovolemic stress. Yet, significant redistribution of body fluids following hemorrage appears to occur in animals without lymph hearts. In this study, plasma and blood volumes were determined by the dye dilution technique using injected Evan's blue dye to label the plasma. Eight intact and 6 animals with their lymph hearts destroyed were hemorrhaged to 78% and 75% of their initial blood volumes, respectively. Changes in blood volume were measured following the hemorrhage by analysis of Evan's blue washout and hemodilution.

The present study documents that baroreceptors located in the pulmocutaneous artery (PCA) are key components in control of lymph heart rate in amphibians. A negative feedback control loop exists between arterial pressure and lymphatic heart rate. The recurrent laryngeal nerve (rLN), which innervates the PCA baroreceptors, transmits information on arterial pressure to integration centers in the central nervous system. Lymphatic heart rate (LHR) is reduced as a result of increases in arterial pressure. This loop was determined using three experimental protocols. First, the correlation between LHR reduction and hormonally induced vasoconstriction was determined. Increases in arterial pressure due to pressor actions of angiotensin II and arginine vasotocin at high concentrations was negatively correlated to LHR. Second, lymphatic heart rate changes due to natural increases in arterial pressure were compared to rate changes due to increase in arterial pressure after bilateral denervation of the rLN. Post-denervation LHR was not affected by natural increase in arterial pressure prior to the establishment of a new resting arterial pressure. Increase in arterial pressure due to administration of vasoconstricting hormones was negatively correlated with LHR following denervation. Third, the effect on LHR due to direct stimulation of the rLN was studied. Stimulation of the rLN caused LHR to stop without increases in arterial pressure. Presumably, this negative feedback loop is present to limit fluid return to the cardiovascular system from the lymphatic system during periods of acute hypertension. Reduction in the return of lymph volume to the cardiovascular system could eliminate potential damage to pulmonary tissues due to high arterial pressures.

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.

The relative contribution of arteriovenous oxygen (A-V O2) difference to thermally-induced and activity-induced changes in metabolic rate (VO2) were delineated in the toad, B. marinus. In conjunction, the influence of rest and activity, temperature, and removal of the pericardia! sac on separation efficiency (Es) in the anuran ventricle was determined by direct measurement of arterial and venous percent oxygen saturation (% saturation), using a microsampling technique.

The concept of maximal oxygen consumption (VO2 max) implies that there is a limit to the amount of oxygen that can be consumed by an organism in a given time. Many attempts have been made to delineate exactly what factor or factors are limiting to VO2 max. Current models focus on the steps over which oxygen must traverse in order to get from the ambient air to the mitochondria. However, no model has addressed the issue of whether or not carbon dioxide flux may impose a limit to VO2 max. Impedance of carbon dioxide flux may have a deleterious effect on intracellular pH, which in turn, may effect mitochondrial oxidative performance.

Le rein joue un role central dans le controle de l'homeostasie du milieu interieur. Il permet d'assurer la balance entre les entrees et sorties de differents ions, incluant le sodium (na), le potassium (k) et participe egalement au controle de l'equilibre acido-basique. Deux enzymes membranaires interviennent au premier plan. Il s'agit de la na,k-atpase et de la h,k-atpase renale. La na,k-atpase maintient le gradient electrochimique transmembranaire du na et du k. La presence d'une h,k-atpase specifiquement renale est une decouverte plus recente. Nous avons caracterise au point de vue moleculaire et fonctionnel la na,k-atpase et h,k-atpase exprimees dans la vessie du crapaud bufo marinus. La vessie du crapaud est consideree comme l'equivalent amphibien du canal collecteur cortical de mammifere, segment implique dans l'excretion finale du na et du k dans l'urine. L'utilisation de l'ovocyte comme systeme d'expression et la mise au point de tests fonctionnels adequats ont permis de mieux comprendre le role respectif des differentes sous-unites alpha et beta impliquees dans la formation d'un heterodimere fonctionnel. D'autre part, la caracterisation moleculaire et fonctionnelle de la h,k-atpase renale devrait permettre de mieux comprendre le role de cette enzyme dans le processus d'acidification urinaire ou la reabsorption de k

[Truncated abstract] This study describes the results of a four and a half year investigation examining local regulation of ion transport through pumping epithelial cells. The study focussed on the standard isolated toad skin preparation, made famous by Hans Ussing. Originally, the objective was to perform some simple manipulations on the isolated toad skin, a standard and well-tested epithelial layer, which, according to the literature, was a well-behaved and stable preparation. The purpose of doing these toad skin experiments was to gain familiarity with the experimental techniques, such as measuring the open-circuit voltage (Voc) and the short-circuit current (Isc) across an epithelium. In the process, the experimental information that was obtained was to assist in the development and refinement of a mathematical model of a single pumping epithelial cell . . . Finally, it should be emphasised the toad skin was a convenient tissue model for exploring more general issues such as: (i) how pumping epithelial cells may adjust to changes in the extracellular environment by locally regulating their membrane conductances; (2) how the topology of a cell can influence its function (i.e. the topology can determine whether a cell is optimised for salt transport or water transport). (3) how different cells, with different functions, may be positioned in apposition in a pumping epithelial tissue so that gradients generated by one cell type can be utilised by another. From a broader perspective, it is likely that such issues are also applicable to other pumping epithelia, and ultimately, may assist in understanding how these epithelia function.

Surfactants are one of the more ubiquitous contaminants in aquatic systems. Their importance as toxic components of pesticide formulations has, however, been largely overlooked. Amphibians particularly, as inhabitants of shallow, temporary and often lentic aquatic environments may be at risk from exposure to these chemicals when they enter aquatic systems. This thesis presents data on the toxicity of surfactants to amphibians. Several experimental exposures were conducted with embryo-larval, tadpole and adult developmental stages of the Australian species- Crinia insignifera, Helcioporus eyrei, Limnodynastes dorsalis and Litoria moorei and the exotic species- Bufo marinus and Xenopus laevis. Animals were variously exposed to glyphosate formulations that contain a high proportion of nonionic surfactants, or commercial pesticide wetting agents (alcohol alkoxylate and nonylphenol ethoxylate (NPE) surfactants). Feeding stage tadpoles of C.insignifera, H. eyrei, L. dorsalis and L. moorei were exposed to three commercial glyphosate formulations, glyphosate isopropylamine and glyphosate acid in static-renewal acute toxicity tests. The 48-h LC50 values for Roundup Herbicide (MON 2139) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei ranged between 8.1 and 32.2 mg/L (2.9 and 11.6 mg/L glyphosate acid equivalent (ae)), while the 48-h LC50 values for Roundup Herbicide tested against adult and newly metamorphosed C. insignifera ranged from 137-144 mg/L (49.4-51.8 mg/L ae).Touchdown Herbicide (4 LC-E) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei was slightly less toxic than Roundup with 48-h LC50 values ranging between 27.3 and 48.7 mg/L (9.0 and 16.1 mg/L ae). Roundup Biactive (MON 77920) was practically non-toxic to tadpoles of the same four species producing 48-h LC50 values of 911 mg/L (328 mg/L ae) for L. moorei and >1000 mg/L (>360 mg/L ae) for C. insignifera, H. eyrei and L. dorsalis. Glyphosate isopropylamine was practically non-toxic producing no mortality amongst tadpoles of any of the four species over 48 h, at concentrations between 503 and 684 mg/L (343 and 466 mg/L ae). The toxicity of technical grade glyphosate acid (48-h LC50, 81.2-121 mg/L) is likely to be due to acid intolerance. Feeding stage tadpoles of B. marinus, X laevis, C. insignifera, H.eyrei, L. dorsalis and L. moorei were exposed to NPE and alcohol alkoxylate in static renewal acute toxicity tests. All species exhibited non-specific narcosis following exposure to both these surfactants. The 48-h EC50 values for NPE ranged between 1.1 mg/L (mild narcosis) and 12.1 mg/L (full narcosis). The 48-h EC50 values for alcohol alkoxylate ranged between 5.3 mg/L (mild narcosis) and 25.4 mg/L (full narcosis). Xenopus laevis was the most sensitive species tested. The sensitivity of the other five species was size dependent with larger species displaying greater tolerance. Replicate acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius over 96 hours indicated that the narcotic effects were not particularly time dependant.The mean 24, 48, 72 and 96-h EC50 (mild narcosis) were 3.6, 3.7, 3.5 and 3.5 mg/L respectively. The mean 24, 48, 72, and 96-h EC50 (full narcosis) values were 4.0, 4.1, 4.2 and 4.0 respectively. Acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius under conditions of low dissolved oxygen (0.8-2.3 mg/L) produced a two to threefold increase in toxicity. The 12-h EC50 values ranged from 1.4 to 2.2 mg/L. The embryotoxicity of NPE was determined in X. laevis, L. adelaidensis and C. insignifera using a Frog Embryo Teratogenesis assay-Xenopus (FETAX). The 96-h LC50, EC50 and MCIG (LOEC) values for X. laevis were 3.9 to 5.4 mg/L, 2.8 to 4.6 mg/L and 1.0 to 3.0 mg/L respectively. The 140-h LC50, ECSO and MCIG values for L. adelaidensis were 9.2 mg/L, 8.8 mg/L and 5.1 to 6.0 ing/L respectively. The 134-h LC50, EC50 and MCIG values for C. insignifera were 6.4 mg/L, 4.5 mg/L and 4.0 mg/L respectively. Teratogenicity indices for the three species ranged between 1.0 and 1.6 indicating either no or low teratogenicity. Xenopus laevis was the more sensitive of the three species and the only species that displayed indisputable terata. The acute toxicity data indicated that the amphibian species tested were of similar sensitivity to fish and some invertebrates. Developmental retardation and oestrogenic effects following exposure to nonylphenol ethoxylate were indicated by sublethal toxicity tests. Crinia insignifera embryos were exposed during early embryogenesis to sublethal concentrations of NPE.Exposure to NPE did not affect either weight nor size (snout-vent length) at metamorphosis. Exposure to 5.0mg/L NPE resulted in a significant delay in the time required to reach metamorphosis. Also, exposure to 3.0 mg/L NPE for the first 6 days of embryonic development or exposure to 5.0 mg/L NPE from day 2 to day 6 resulted in a statistically significant predominance in the female phenotype amongst metamorphosing froglets. Exposure for the first five days to 1.5 ing/L or 3.0 mg/L NPE had no effect on sex ratio. The results indicated that exposure to NPEs has endocrine disruptive effects in this species and that a narrow window of susceptibility exists for the induction of predominantly female phenotype. This study has also followed the degradation of a mixture of NPE oligomers and the concomitant formation of individual oligomers in static die-away tests with and without illumination in freshwater. Over 33 days in darkness there was a progressive and complete loss of long chain oligomers (NPEO(subscript)8-17), transient increases and subsequent loss of short to medium chain oligomers (NPE0(subscript)4-7), and large persistent increases (approximately 1000%) in short chain oligomers (NPE0(subscript)1-3). In the presence of illumination, biodegradation was retarded and heterotrophic bacterial proliferation was inhibited. After 33 days there was complete loss of long chain oligomers (NPE0(subscript)9-17), incomplete loss of medium chain oligomers (NPE0(subscript)6.8) and increases in short chain oligomers (NPE0(subscript)1-5).This thesis discusses the importance of persistent metabolites of NPE degradation as it pertains to the habitat, developmental time frame and ecology of amphibians. Degradation of NPE is likely to occur over a time frame that is longer than that required for complete embryogenesis and metamorphosis of many species of amphibians, and may easily encompass those critical stages of development during which oestrogenic metabolites can affect development.

Surfactants are one of the more ubiquitous contaminants in aquatic systems. Their importance as toxic components of pesticide formulations has, however, been largely overlooked. Amphibians particularly, as inhabitants of shallow, temporary and often lentic aquatic environments may be at risk from exposure to these chemicals when they enter aquatic systems. This thesis presents data on the toxicity of surfactants to amphibians. Several experimental exposures were conducted with embryo-larval, tadpole and adult developmental stages of the Australian species- Crinia insignifera, Helcioporus eyrei, Limnodynastes dorsalis and Litoria moorei and the exotic species- Bufo marinus and Xenopus laevis. Animals were variously exposed to glyphosate formulations that contain a high proportion of nonionic surfactants, or commercial pesticide wetting agents (alcohol alkoxylate and nonylphenol ethoxylate (NPE) surfactants). Feeding stage tadpoles of C.insignifera, H. eyrei, L. dorsalis and L. moorei were exposed to three commercial glyphosate formulations, glyphosate isopropylamine and glyphosate acid in static-renewal acute toxicity tests. The 48-h LC50 values for Roundup Herbicide (MON 2139) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei ranged between 8.1 and 32.2 mg/L (2.9 and 11.6 mg/L glyphosate acid equivalent (ae)), while the 48-h LC50 values for Roundup Herbicide tested against adult and newly metamorphosed C. insignifera ranged from 137-144 mg/L (49.4-51.8 mg/L ae).
Touchdown Herbicide (4 LC-E) tested against tadpoles of C. insignifera, H. eyrei, L. dorsalis and L. moorei was slightly less toxic than Roundup with 48-h LC50 values ranging between 27.3 and 48.7 mg/L (9.0 and 16.1 mg/L ae). Roundup Biactive (MON 77920) was practically non-toxic to tadpoles of the same four species producing 48-h LC50 values of 911 mg/L (328 mg/L ae) for L. moorei and >1000 mg/L (>360 mg/L ae) for C. insignifera, H. eyrei and L. dorsalis. Glyphosate isopropylamine was practically non-toxic producing no mortality amongst tadpoles of any of the four species over 48 h, at concentrations between 503 and 684 mg/L (343 and 466 mg/L ae). The toxicity of technical grade glyphosate acid (48-h LC50, 81.2-121 mg/L) is likely to be due to acid intolerance. Feeding stage tadpoles of B. marinus, X laevis, C. insignifera, H.eyrei, L. dorsalis and L. moorei were exposed to NPE and alcohol alkoxylate in static renewal acute toxicity tests. All species exhibited non-specific narcosis following exposure to both these surfactants. The 48-h EC50 values for NPE ranged between 1.1 mg/L (mild narcosis) and 12.1 mg/L (full narcosis). The 48-h EC50 values for alcohol alkoxylate ranged between 5.3 mg/L (mild narcosis) and 25.4 mg/L (full narcosis). Xenopus laevis was the most sensitive species tested. The sensitivity of the other five species was size dependent with larger species displaying greater tolerance. Replicate acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius over 96 hours indicated that the narcotic effects were not particularly time dependant.
The mean 24, 48, 72 and 96-h EC50 (mild narcosis) were 3.6, 3.7, 3.5 and 3.5 mg/L respectively. The mean 24, 48, 72, and 96-h EC50 (full narcosis) values were 4.0, 4.1, 4.2 and 4.0 respectively. Acute toxicity tests with B. marinus exposed to NPE at 30 degrees celsius under conditions of low dissolved oxygen (0.8-2.3 mg/L) produced a two to threefold increase in toxicity. The 12-h EC50 values ranged from 1.4 to 2.2 mg/L. The embryotoxicity of NPE was determined in X. laevis, L. adelaidensis and C. insignifera using a Frog Embryo Teratogenesis assay-Xenopus (FETAX). The 96-h LC50, EC50 and MCIG (LOEC) values for X. laevis were 3.9 to 5.4 mg/L, 2.8 to 4.6 mg/L and 1.0 to 3.0 mg/L respectively. The 140-h LC50, ECSO and MCIG values for L. adelaidensis were 9.2 mg/L, 8.8 mg/L and 5.1 to 6.0 ing/L respectively. The 134-h LC50, EC50 and MCIG values for C. insignifera were 6.4 mg/L, 4.5 mg/L and 4.0 mg/L respectively. Teratogenicity indices for the three species ranged between 1.0 and 1.6 indicating either no or low teratogenicity. Xenopus laevis was the more sensitive of the three species and the only species that displayed indisputable terata. The acute toxicity data indicated that the amphibian species tested were of similar sensitivity to fish and some invertebrates. Developmental retardation and oestrogenic effects following exposure to nonylphenol ethoxylate were indicated by sublethal toxicity tests. Crinia insignifera embryos were exposed during early embryogenesis to sublethal concentrations of NPE.
Exposure to NPE did not affect either weight nor size (snout-vent length) at metamorphosis. Exposure to 5.0mg/L NPE resulted in a significant delay in the time required to reach metamorphosis. Also, exposure to 3.0 mg/L NPE for the first 6 days of embryonic development or exposure to 5.0 mg/L NPE from day 2 to day 6 resulted in a statistically significant predominance in the female phenotype amongst metamorphosing froglets. Exposure for the first five days to 1.5 ing/L or 3.0 mg/L NPE had no effect on sex ratio. The results indicated that exposure to NPEs has endocrine disruptive effects in this species and that a narrow window of susceptibility exists for the induction of predominantly female phenotype. This study has also followed the degradation of a mixture of NPE oligomers and the concomitant formation of individual oligomers in static die-away tests with and without illumination in freshwater. Over 33 days in darkness there was a progressive and complete loss of long chain oligomers (NPEO(subscript)8-17), transient increases and subsequent loss of short to medium chain oligomers (NPE0(subscript)4-7), and large persistent increases (approximately 1000%) in short chain oligomers (NPE0(subscript)1-3). In the presence of illumination, biodegradation was retarded and heterotrophic bacterial proliferation was inhibited. After 33 days there was complete loss of long chain oligomers (NPE0(subscript)9-17), incomplete loss of medium chain oligomers (NPE0(subscript)6.8) and increases in short chain oligomers (NPE0(subscript)1-5).
This thesis discusses the importance of persistent metabolites of NPE degradation as it pertains to the habitat, developmental time frame and ecology of amphibians. Degradation of NPE is likely to occur over a time frame that is longer than that required for complete embryogenesis and metamorphosis of many species of amphibians, and may easily encompass those critical stages of development during which oestrogenic metabolites can affect development.

The reproductive and larval ecology of the introduced species Bufo marinus was studied at two breeding sites in tropical northern Australia. Reproductive activity, recruitment patterns, larval growth and survival, and predation by conspecifics and by insect, crustacean and fish species were looked at in detail. Reproduction at both sites occurred from December to June. Recruitment from reproductive events was sporadic. It was not concentrated during any particular period of the breeding season. During breeding periods, individuals at oviposition sites were predominantly males, with a population sex ratio of 10:1. Less than 30 percent of the estimated male population and 5 percent of the estimated female population were active (found in transect searches) on any one night during the breeding period. The proportion of active individuals in amplexus at any time was low. Total numbers of active individuals declined by the end of the dry season and sex ratios approached 1:1. No breeding was observed during the late dry season although histological examination of gonads indicated that reproductively active males and females (with mature sperm and ova) were present all year round. Sampling of larval populations indicated that densities recorded at both sites varied temporally and spatially. High variance-mean ratios for sample densities indicated that larval distributions within transects were aggregated. Peak densities were typically between 600 to 800 per m². Larval density was used to determine population size but estimates showed no consistent association with recruitment of eggs from observed breeding. Experimental studies indicated that during the embryonic stages (Gosner stages 1 to 24) predator-free survival was approximately 70 percent (90 percent survival/day) over a mean developmental period of 72 hours. Highest mortality occurred between three days (hatching) and twelve days (stage 25) after laying. Mean larval periods of 22 to 54 days were recorded. Shorter larval periods were associated with lower larval density. Survival to metamorphosis ranged from 0.1 to 10 percent (97 percent mean survival/day). Survival and growth rates in most experiments were density dependent. Growth of pre-hatching stages was linear but growth was exponential or decreasing exponential during the tadpole stages, controlled either by temperature or density. Growth of larvae is food limited, with advantages to older larvae demonstrated in an artificial pond experiment with two overlapping cohorts of different age. Growth was reduced for both cohorts but the effect was greater for younger cohorts. Individuals that survived egg predation by older cohorts grew larger because of reduced competition. Egg predation by older cohorts of Bufo larvae appeared to be a major source of mortality. Intraspecific predation was restricted to pre-swimming embryo stages and greatly reduced pre-hatching survival in field and experimental populations (1.67 percent mean survival/day). No other predators of eggs were recorded. Few species were active predators of hatched tadpoles. Only two of eleven potential predators examined in feeding rate experiments consumed tadpoles. These were an adult dytiscid beetle, Homeodytes scutellaris (0.28 tadpoles/hour), and a freshwater crayfish, Cherax quadricarinatus (0.48 tadpoles/hour). None of the native fish species examined successfully consumed eggs or larvae. A predation experiment was conducted in experimental ponds with aeschnid dragonfly larvae, Hemianax papuensis, and high and low densities of Bufo larvae. Predation significantly reduced survival of larvae at both densities and growth was significantly lower for larvae in predator treatments at high density. Slower growth for larvae in predator treatments probably resulted from larvae altering their foraging behaviour in response to the presence of predators. Tadpoles appeared to be less conspicuous in predator treatments. In predator treatments with a low initial density of Bufo, initial predation decreased densities to a level where encounter rates with predators were low and tadpole food resources were probably not limiting. Final mass and larval period for tadpoles at low density in predator treatments were similar to controls. Larval population size is most likely to be influenced by differences in cohort overlap resulting from adult breeding phenology. A small overlap will result in generations with narrow size differences and will produce increased competition between cohorts. A large overlap may result in large size differences in cohorts and may lead to heavy predation of eggs by older cohorts. Invertebrate predators are likely to affect the survival of larval Bufo marinus populations during colonisation and periods of habitat deterioration.

Aspects of the ecology of the terrestrial stage of Bufo marinus from two populations in the wet-dry tropics of north-eastern Australia, one at Calvert Hills in the Gulf of Carpentaria of Northern Territory, and the other in the Townsville region on the north coast of Queensland, were examined and compared. The stages of the toad life cycle examined ranged from small metamorphs, that had only recently emerged from their natal pools, through to large breeding adults. Considerable variation in the growth and survival rates, activity patterns, individual toad size, body condition and shelter requirements were found between and within these two populations. Once B. marinus entered the terrestrial component of its life cycle, ie. emergence onto land at approximately 8mm in length, individual metamorphs were faced with a series of challenges to their survival and growth through to sub-adult size (30mm). The major obstacles to a metamorph's survival through this stage included stresses from dehydration and from high densities of metamorphs present at the water's edge shortly after the peak breeding period in the wet season. Metamorphs remained close to their natal pond until they were large enough to forage without hydric risks. During this time daily survival rate was restricted by environmental factors such as high temperatures. However, metamorph growth rates were dependant on the density of metamorphs, with higher densities reducing the time required to reach juvenile size, ie. approximately 30mm. Once metamorphs obtain this size it appears that they have an increased chance of survival. Toads above 30mm were subject to a long-term mark-recapture study at Calvert Hills and Townsville. This technique allowed for several aspects of toad population ecology to be examined including growth rates from sub-adult size and seasonal variations in activity patterns, sex ratio, and body size and condition within a given area alongside a water source. Growth rates of B. marinus were documented from first emergence from water through to adult breeding size. Their rate of growth was shown to be higher than previously reported for the species in endemic areas and other species of Bufo. Toads at Calvert Hills grew faster than those at Townsville, and attained adult size within one year. A shorter, hotter wet season, corresponding to the period of highest food availability, accounted for the higher growth rates and increased body condition shown by toad populations at Calvert Hills compared to Townsville populations. Toad activity patterns were shown to vary according to age, size, and seasonal conditions. When toads first emerge from the water (approximately 8mm) and commence their terrestrial stage their activity was centred at the water's edge. With growth and decreased hydric stresses metamorphs gradually foraged further away from water. The activity of sub-adult and adult toads was influenced by the time since rain periods, such as the wet season. Except for breeding periods, male and female toads generally centred their activity patterns away from water during the wet season especially after recent rain. As time since rain increased, toad activity switched back to the edge of a water source probably as a response to increasing hydric stresses. The number of toads active at a water source was highest at both locations at the commencement of the dry season thus reflecting the influx of smaller cohorts from wet season breeding activity and the requirement for toads to rehydrate because of the onset of hot, dry conditions. Toad size at a water source was largest during the wet season indicating that larger male and female toads invested more energy into breeding than smaller toads. The number of toads at the water's edge was therefore in favour of males during all seasons and at both locations. A male-biased sex ratio probably reflected behavioural differences between the sexes rather than actual variation in densities. For instance, during the wet season, females spent considerable time foraging away from water and only came to the water's edge, when gravid, to breed. The presence of some male toads at a water source throughout the breeding season provided them with a selective breeding advantage whenever female toads came to the water to breed. The body condition of toads declined into the dry season corresponding with low food availability and dehydration stresses. Furthermore, female body condition demonstrated marked variation by increasing rapidly during favourable conditions, but declining after the wet season. This type of variation reflected energy input during the wet season followed by a sharp decline in condition after egg deposition. Climatic differences explained much of the variation in toad population ecology, such as growth and survival rates, activity patterns and body size and condition, between the two locations. Although both study regions were located within the wet-dry tropics of northern Australia, the dry season at Townsville was less harsh than that experienced at Calvert Hills and was often punctuated with rain periods. In contrast, the length of the wet season at Calvert Hills was shorter and usually consisted of less rain while the dry season was long and characterised by high temperatures. The climatic variation between the two locations led to variations in the length and timing of the breeding season, rate of growth, and survival through the dry season. The long, hot dry season at Calvert Hills put severe hydric pressures on toads. A critical requirement for toads was therefore the selection of a favourable shelter site, especially during periods of inactivity when nightly temperatures were low. Favourable shelter sites have high relative soil moisture, high temperatures, and the presence of other toads. Favoured shelters are an essential resource especially during the dry season, and once found by toads, are returned to repeatedly while conditions within the shelter remain favourable and those outside are unfavourable.

Thesis (Ph. D.) -- University of Tennessee, Knoxville, 2005.
Title from title page screen (viewed on Sept. 6, 2005). Thesis advisor: Arthur C. Echternacht. Thesis advisior: Christine R. B. Boake. Document formatted into pages (xvi, 151 p. : ill. (some col.)). Vita. Includes bibliographical references.

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.

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.

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.