Thimet oligopeptidase (THOP 1) distribution in cane toad (Bufo Marinus, Linnaeus, 1758) brain.

Thimet oligopeptides (THOP 1) is a metal-dependent peptidase involved in the metabolism of neuropeptides and the presentation of peptides via MHC-1. It has been shown to play a role in the regulation of protein-protein interactions and the metabolism of intracellular peptides. THOP 1 is associated with important biological processes such as metabolism and neurodegenerative diseases, among others. The objective of this study is to elucidate the distribution of THOP 1 in the Bufo marinus brain. The analysis of THOP 1 amino acid sequences indicates that they have been conserved throughout evolution, with significant homology observed across various phyla. When comparing amphibians with other species, more than 70% identity can be identified. Immunohistochemistry analysis of the toad's brain has demonstrated that the enzyme has a ubiquitous distribution, consistent with previous findings in mammals. THOP 1 can be found in important areas of the brain, such as bulb, thalamic nuclei, striatum, hypothalamus, and among others. Nonetheless, THOP 1 is consistently localized within the nucleus, a pattern also observed in the rat brain. Therefore, based on these results, the toad appears to be an excellent model for studying the general biology of THOP 1, given the substantial homology of this enzyme with mammals and its similarity in distribution within the brain.

Vasoconstrictor and hemodynamic effects of a methanolic extract from Rhinella marina toad poison.

Rhinella marina toad is abundant in Brazil. Its poison contains cardiac glycosides called bufadienolides, which are extensively investigated for their bioactivity. Our aim was to characterize the vasoactivity of Rhinella marina poison (RmP) on the aorta of male Wistar rats. For this, the RmP was first collected and processed to obtain an alcoholic extract. To determine cardiovascular effects of RmP, we performed in vivo tests by administering RmP intravenously in doses of 0.1-0.8 mg/kg. Vascular reactivity was also performed through concentration-response curves to RmP (10 ng/mL to 200 µg/mL) in aortic segments with and without endothelium. RmP induced a concentration-dependent contraction in rat aorta which was partly endothelium-mediated. Nitric oxide contributes with this response in view that incubation with L-NAME increased the contractile response. Additionally, treatment with indomethacin [cyclooxygenase, (COX) inhibitor], nifedipine (L-type voltage-gated calcium channels blocker), and BQ-123 (ETA receptors antagonist) decreased maximum response, and ketanserin (5-HT2 receptors antagonist) decreased pEC50, suggesting active participation of these pathways in the contractile response. On the other hand, apocynin (NADPH oxidase inhibitor) did not alter contractility. Incubation with prazosin (α1-adrenergic receptor antagonist) abolished the contractile response, suggesting that the RmP-induced contraction is dependent on the adrenergic pathway. In the Na+/K+ ATPase protocol, a higher Emax was observed in the RmP experimental group, suggesting that RmP potentiated Na+/K+ATPase hyperpolarizing response. When this extract was injected (i.v.) in vivo, increase in blood pressure and decrease in heart rate were observed. The results were immediate and transitory, and occurred in a dose-dependent manner. Overall, these data suggest that the poison extract of R. marina toad has an important vasoconstrictor action and subsequent vasopressor effects, and its use can be investigated to some cardiovascular disorders.

Identification of Na+/K+-ATPase α/ß isoforms in Rhinella marina tissues by RNAseq and a molecular docking approach at the protein level to evaluate α isoform affinities for bufadienolides.

Na+/K+-ATPase (NKA) function is inhibited by Bufadienolides (BD), a group of cardiotonic steroids (CTS) primarily produced by anurans of the Bufonidae family, such as Rhinella marina. This study characterized the presence of α and ß NKA subunit isoforms in R. marina via RNAseq in four tissues: oocytes, skin, heart, and skeletal muscle. Transcripts encoding three α-like isoforms (α1, α2, α3) and three ß-like isoforms (ß1, ß2, ß4) were identified. The amino acid sequence of α1-like isoform shared 99.4% identity with the α1 isoform previously published for R. marina. Sequences for α2, α3, and ß4 from R. marina were previously unavailable. The first extracellular loop in the α2-like isoform in R. marina showed similar substitutions to those found in their susceptible homologues in other taxa (L/Q111T and S119T); in contrast, this same loop in α3-like isoform showed similar substitutions (Q111L and G120R) to those reported for toad-eating animals such as snakes, which suggests relatively lower affinity for CTS. Docking results showed that all three α-like isoforms identified in R. marina transcriptomes have low affinity to CTS compared to the susceptible α1 isoform of Sus scrofa (pig), with α1-like isoform being the most resistant. The tissue-specific RNAseq results showed the following expression of NKA α-like and ß-like subunit isoforms: Oocytes expressed α1 and ß1; skin α1, ß1, and low levels of ß2; heart α1, α3, and ß1; skeletal muscle α1, ß4, with low levels of α2, α3, and ß1. R. marina could be used as an important model for future structural, functional and pharmacological studies of NKA and its isoforms.

Predicting in vivo absorption of chloramphenicol in frogs using in vitro percutaneous absorption data.

BACKGROUND: Infectious disease, particularly the fungal disease chytridiomycosis (caused by Batrachochytrium dendrobatidis), is a primary cause of amphibian declines and extinctions worldwide. The transdermal route, although offering a simple option for drug administration in frogs, is complicated by the lack of knowledge regarding percutaneous absorption kinetics. This study builds on our previous studies in frogs, to formulate and predict the percutaneous absorption of a drug for the treatment of infectious disease in frogs. Chloramphenicol, a drug with reported efficacy in the treatment of infectious disease including Batrachochytrium dendrobatidis, was formulated with 20% v/v propylene glycol and applied to the ventral pelvis of Rhinella marina for up to 6 h. Serum samples were taken during and up to 18 h following exposure, quantified for chloramphenicol content, and pharmacokinetic parameters were estimated using non-compartmental analysis. RESULTS: Serum levels of chloramphenicol reached the minimum inhibitory concentration (MIC; 12.5 µg.mL- 1) for Batrachochytrium dendrobatidis within 90-120 min of exposure commencing, and remained above the MIC for the remaining exposure time. Cmax (17.09 ± 2.81 µg.mL- 1) was reached at 2 h, while elimination was long (t1/2 = 18.68 h). CONCLUSIONS: The model, based on in vitro data and adjusted for formulation components and in vivo data, was effective in predicting chloramphenicol flux to ensure the MIC for Batrachochytrium dendrobatidis was reached, with serum levels being well above the MICs for other common bacterial pathogens in frogs. Chloramphenicol's extended elimination means that a 6-h bath may be adequate to maintain serum levels for up to 24 h. We suggest trialling a reduction of the currently-recommended continuous (23 h/day for 21-35 days) chloramphenicol bathing for chytrid infection with this formulation.

Variation in size and shape of toxin glands among cane toads from native-range and invasive populations.

If optimal investment in anti-predator defences depends on predation risk, invading new regions (and thus, encountering different predators) may favour shifts in that investment. Cane toads offer an ideal system to test this prediction: expensive anti-predator toxins are stored mainly in parotoid glands whose dimensions are easy to measure, and toad invasions have changed the suites of predators they encounter. Although plasticity may influence parotoid morphology, comparisons between parents and progeny revealed that gland dimensions were highly heritable. That heritability supports the plausibility of an evolved basis to variation in gland dimensions. Measurements of 3779 adult toads show that females have larger glands than males, invasive populations have larger glands than in the native-range, and that parotoid sexual size dimorphism varies strongly among invaded areas. Geographic variation in parotoid morphology may be driven by predation risk to both adult toads and offspring (provisioned with toxins by their mother), with toxins allocated to eggs exacerbating the risk of cannibalism but reducing the risk of interspecific predation. Investment into chemical defences has evolved rapidly during the cane toad's international diaspora, consistent with the hypothesis that organisms flexibly adjust resource allocation to anti-predator tactics in response to novel challenges.

The Insecticidal Activity of Rhinella schneideri (Werner, 1894) Paratoid Secretion in Nauphoeta cinerea Cocroaches.

Rhinella schneideri is a common toad found in South America, whose paratoid toxic secretion has never been explored as an insecticide. In order to evaluate its insecticidal potential, Nauphoeta cinerea cockroaches were used as an experimental model in biochemical, physiological and behavioral procedures. Lethality assays with Rhinella schneideri paratoid secretion (RSPS) determined the LD50 value after 24 h (58.07µg/g) and 48 h exposure (44.07 µg/g) (R2 = 0.882 and 0.954, respectively). Acetylcholinesterase activity (AChE) after RSPS at its highest dose promoted an enzyme inhibition of 40%, a similar effect observed with neostigmine administration (p < 0.001, n= 5). Insect locomotion recordings revealed that RSPS decreased the distance traveled by up to 37% with a concomitant 85% increase in immobile episodes (p < 0.001, n = 36). RSPS added to in vivo cockroach semi-isolated heart preparation promoted an irreversible and dose dependent decrease in heart rate, showing a complete failure after 30 min recording (p < 0.001, n ≥ 6). In addition, RSPS into nerve-muscle preparations induced a dose-dependent neuromuscular blockade, reaching a total blockage at 70 min at the highest dose applied (p < 0.001, n ≥ 6). The effect of RSPS on spontaneous sensorial action potentials was characterized by an increase in the number of spikes 61% (p < 0.01). Meanwhile, there was 42% decrease in the mean area of those potentials (p < 0.05, n ≥ 6). The results obtained here highlight the potential insecticidal relevance of RSPS and its potential biotechnological application.

Can models of percutaneous absorption based on in vitro data in frogs predict in vivo absorption?

The primary aim of in vitro testing of chemicals delivered via the percutaneous route is to predict the absorption that would ensue if exposure occurred in live animals. While there is mounting evidence that in vitro diffusion studies in mammalian skin can provide valid information regarding likely in vivo absorption, little is known whether such a correlation exists between in vitro diffusion testing and in vivo blood levels in amphibians. The current study used previously-reported in vitro absorption data for caffeine, benzoic acid, and ibuprofen across isolated skin from the cane toad (Rhinella marina) to produce a series of linear mixed-effect models of the absorption parameters flux and permeability coefficient (Kp). Models investigated the relative impacts of animal weight, physicochemical characteristics of the applied chemical (logP or molecular weight), and site of application. The top models were then used to predict the flux, Kp and serum concentrations of the same three model chemicals. Finally, the absorption of these chemicals was determined in live cane toads, and results compared to the model predictions. LogP and site of application were included in all top models. In vivo absorption rates were lower than predicted for all chemicals, however, the models provided reasonable predictions of serum concentration, with factors of difference (FOD) ranging from 2.5-10.5. Ibuprofen, the chemical with the highest relative lipophilicity, had the poorest predictive performance, consistently having the highest FOD for all predictions. This report presents the first models of percutaneous absorption in an amphibian. These models provide a basic method to establish the approximate in vivo absorption of hydrophilic and moderately-lipophilic chemicals through frog skin, and could therefore be used to predict absorption when formulating such chemicals for treatment of disease in frogs, or for risk-assessments regarding chemical pollutants in frog habitats.

Innate immunity of Florida cane toads: how dispersal has affected physiological responses to LPS.

Physiological tradeoffs occur in organisms coping with their environments, which are likely to increase as populations reach peripheries of established ranges. Invasive species offer opportunities to study tradeoffs that occur, with many hypotheses focusing on how immune responses vary during dispersal. The cane toad (Rhinella marina) is a well-known invasive species. Populations near the expanding edge of the Australian invasion have altered immune responses compared to toads from longer-established core populations, although this has not been well-documented for Florida populations. In this study, cane toads from a northern edge [New Port Richey (NPR)] and southern core (Miami) population in Florida were collected and injected with lipopolysaccharide (LPS) to compare immune responses. Core population individuals injected with LPS showed greater metabolic increases compared to their baseline rates that were higher compared to those from the edge population. In addition, LPS-injected core individuals had different circulating leukocyte profiles compared to saline-injected cane toads while edge individuals did not. There was a significant interaction between plasma bacteria-killing capability (BKA) and treatment, such that BKA decreased with time in saline compared to LPS-injected individuals, and saline-injected toads from the edge population had lower BKA compared to LPS-injected edge toads at 20 h post-injection. There was also a significant interaction between location and time on circulating corticosterone (CORT) levels following injections with saline or LPS, with CORT decreasing more with time in core population toads. The differential CORT response indicates that differential stress responses contribute to the tradeoffs observed with immunity and dispersal.

Effects of skin region and relative lipophilicity on percutaneous absorption in the toad Rhinella marina.

Owing to the dynamic interaction between frog skin and the environment, xenobiotics in frog habitats are of particular concern, and knowledge of percutaneous absorption in frog skin is necessary for risk-mitigation purposes. Baseline transdermal kinetics in adult aquatic and arboreal frog species have recently been reported; however, there is little information regarding absorption kinetics in adult terrestrial species. The present study investigated the in vitro absorption kinetics of 3 model chemicals-caffeine, benzoic acid, and ibuprofen-through different skin regions in the terrestrial toad Rhinella marina. Caffeine flux was consistently higher than that of the other 2 chemicals (p < 0.001), whereas the fluxes of the moderately and highly lipophilic chemicals (benzoic acid and ibuprofen) were similar, regardless of skin region. When considering individual chemicals, caffeine demonstrated increased flux through the ventral pelvic skin compared with the ventral thoracic or dorsal skin regions. Flux did not differ between skin regions for either benzoic acid or ibuprofen. These findings have implications for management of environmental contamination in frog habitats, as many environmental xenobiotics are of moderate to high lipophilicity and would be expected to be equally absorbed from all skin surfaces in terrestrial toads. Environ Toxicol Chem 2019;38:361-367. © 2018 SETAC.

Climatic modulation of neurotransmitter release in amphibian neuromuscular junctions: role of dynorphin-A.

Amphibian neuromuscular junctions (NMJs) become relatively more silent during the dry winter season in Australia. During the dry, calcium sensitivity is reduced, whereas calcium dependence remains unchanged. Endogenous opioid peptides play an important role in the regulation of the physiological functions of active and dormant vertebrates. Previous findings suggest that dynorphin-A is more potent than other opiates in decreasing evoked neurotransmission in amphibian NMJs. Dynorphin-A has been shown not to alter the amplitude or the frequency of miniature quantal neurotransmitter release. In the present study, we report that dynorphin-A exerted a more pronounced inhibitory effect on evoked neurotransmitter release during the dry (hibernating period) when compared with the wet (active period) season. Dynorphin-A increased the frequency and decreased the amplitude of miniature neurotransmitter release only at relatively high concentration during the dry season. In the present study, we propose that dynorphin-A suppresses evoked neurotransmitter release and thus contraction of skeletal muscles, while allowing subthreshold activation of the NMJ by miniature neurotransmission, thus preventing any significant neuromuscular remodeling. The inhibitory effect of dynorphin-A on evoked transmitter release is reduced by increasing the extracellular calcium concentration.

Application of Focused Ultrasound-Assisted Extraction for the Quantification of Persistent Organic Pollutions in Liver Tissue of Giant Toad (Rhinella marina).

A simple and rapid focused ultrasound extraction method was developed for the determination of Persistent Organic Pollutants (POPs) in liver tissue obtained of giant toad (Rhinella marina) using a gas chromatography coupled to a mass detector with electron impact ionization. The performed method for POPs, was validated in fortified matrix, showing linearity from the LOQ up to 100 ng/mL; LODs and LOQs for each compound were between 1.7 and 4.8 and 3.5-7.5 ng/mL, respectively. Recovery rates were among 79%-116% for POPs determined. Finally, the method was applied in liver samples of giant toads found in a malarial area in Mexico. The sensitivity of the proposed method was good enough to ensure reliable determination of target analytes at concentration levels commonly found in this kind of samples.

Quantification of bufadienolides in the poisons of Rhinella marina and Rhaebo guttatus by HPLC-UV.

Bufadienolides are the main active compounds in the Bufonidae family of frogs. Recent studies have demonstrated cytotoxic and/or antitumor activity in these molecules. A HPLC-DAD method was developed and validated to quantify three bufadienolides (telocinobufagin, marinobufagin and bufalin) in ethyl acetate extracts of the cane toad poison frogs and smooth-sided toad. The chromatographic analysis was performed on Phenomenex Luna C18 (250.0 × 4.6 mm, 5 µm), using gradient elution with acetonitrile and water, at a flow rate of 1.0 mL min(-1) and detection at 296 nm. The method showed linearity (r > 0.999) and adequate recovery values (86%-111%). The limits of quantification of bufadienolides were 7.4 µg mL(-1) for telocinobufagin, 4.2 µg mL(-1) for marinobufagin and 4.0 µg mL(-1) for bufalin. Intraday and interday values of the method were evaluated and presented standard deviation values lower than 5%. The method was successfully applied to quantify the bufadienolides in the venom extract of the cane toad, which showed a content of 60% of marinobufagin. The same method was not selective for the venom extract of the Rhaebo guttatus, despite being linear, accurate and precise, requiring the development of a technique that presents a greater selectivity.

Living up to its name? The effect of salinity on development, growth, and phenotype of the "marine" toad (Rhinella marina).

The highly permeable integument of amphibians renders them vulnerable to chemical characteristics of their environment, especially during the aquatic larval stage. As the cane toad (Rhinella marina, Bufonidae) invades southwards along the east coast of Australia, it is encountering waterbodies with highly variable conditions of temperature, pH, and salinity. Understanding the tolerance of toads to these conditions can clarify the likely further spread of the invader, as well as the adaptability of the species to novel environmental challenges. We measured salinity in waterbodies in the field and conducted laboratory trials to investigate the impacts of salinity on toad viability. Eggs and tadpoles from the southern invasion front tolerated the most saline conditions we found in potential spawning ponds during surveys [equivalent to 1200 ppm (3.5 % the salinity of seawater)]. 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. Our study shows that the egg and larval life stages of cane toads can tolerate wide variation in the salinity of natal ponds and that this aspect of waterbody chemistry is likely to facilitate rather than constrain continued southward expansion of the toad invasion front in eastern Australia.

Effects of acute restraint stress, prolonged captivity stress and transdermal corticosterone application on immunocompetence and plasma levels of corticosterone on the cururu Toad (Rhinella icterica).

Glucocorticoid steroids modulate immunocompetence in complex ways with both immunoenhancing and immunosuppressive effects in vertebrates exposed to different stressors. Such bimodal effects have been associated with variation in duration and intensity of the stress response. Given that natural populations have been exposed to a multitude of stressors, a better understanding of the functional association between duration and intensity of the stress response, the resulting changes in glucocorticoid plasma levels and their impact on different aspects of immunocompetence emerges as a cornerstone for vertebrate conservation strategies. We investigated the effects of a restraint challenge (with and without movement restriction), long-term captivity, and transdermal corticosterone application on plasma levels of corticosterone (hereinafter referred to as CORT) and different parameters of innate immunocompetence in the male cururu toads (Rhinella icterica). We show that for R. icterica restraint for 24h proved to be a stressful condition, increasing CORT by 3-fold without consistent immunological changes. However, the application of a more intense stressor (restraint with movement restriction), for the same period, potentiated this response resulting in a 9-fold increase in CORT, associated with increase Neutrophil/Lymphocyte ratio (N:L) and a lower bacterial killing ability (BKA). Transdermal application of corticosterone efficiently mimics repeated acute stress response events, without changing the immune parameters even after 13 days of treatment. Interestingly, long-term captivity did not mitigate the stress response, since the toads maintained 3-fold increased CORT even after 3 months under these conditions. Moreover, long-term captivity in the same condition increased total leukocyte count (TLC) and generated an even greater decrease in BKA, suggesting that consequences of the stress response can be aggravated by time in captivity.

Serum and hepatic vitamin A levels in captive and wild marine toads (Bufo marinus).

The captive breeding program for the endangered Puerto Rican crested toad (Peltophryne [Bufo] lemur) has been hampered by an undiagnosed condition called "Brown Skin Disease" (BSD). Toads develop widespread skin darkening, skin thickening and abnormal shedding and eventually succumb to a chronic loss of viability. This project evaluated the marine toad (Bufo marinus) as a model for the PRCT, examining vitamin A deficiency as a potential cause of BSD. Wild caught marine toads had significantly higher liver vitamin A concentrations (61.89 ± 63.49 µg/g) than captive born marine toads (0.58 ± 0.59 µg/g); P<0.001). A significant difference in serum vitamin A concentration was found between the captive and wild caught toads (P=0.013) and between the low vitamin A-fed and wild caught toads (P=0.004), when controlling for liver vitamin A concentrations. After captive toads were treated with topical and/or oral vitamin A, their hepatic vitamin A concentrations were similar to those of the wild toads, averaging 48.41 ± 37.03 µg/g. However, plasma vitamin A concentrations pre- and post-vitamin A supplementation did not differ statistically. We concluded that plasma vitamin A concentrations do not provide a linear indication of liver/body vitamin A status, and that both topical and oral supplementation with an oil-based vitamin A formulation can increase liver stores in amphibians. No evidence of BSD or other signs of deficiency were noted in the marine toads, although this feeding trial was relatively short (127 days). To date, clinical, pathological and research findings do not support vitamin A deficiency as a primary factor underlying BSD.

Adrenocortical stress responses influence an invasive vertebrate's fitness in an extreme environment.

Continued range expansion into physiologically challenging environments requires invasive species to maintain adaptive phenotypic performance. The adrenocortical stress response, governed in part by glucocorticoid hormones, influences physiological and behavioural responses of vertebrates to environmental stressors. However, any adaptive role of this response in invasive populations that are expanding into extreme environments is currently unclear. We experimentally manipulated the adrenocortical stress response of invasive cane toads (Rhinella marina) to investigate its effect on phenotypic performance and fitness at the species' range front in the Tanami Desert, Australia. Here, toads are vulnerable to overheating and dehydration during the annual hot-dry season and display elevated plasma corticosterone levels indicative of severe environmental stress. By comparing unmanipulated control toads with toads whose adrenocortical stress response was manipulated to increase acute physiological stress responsiveness, we found that control toads had significantly reduced daily evaporative water loss and higher survival relative to the experimental animals. The adrenocortical stress response hence appears essential in facilitating complex phenotypic performance and setting fitness trajectories of individuals from invasive species during range expansion.

Analytical aspects of marinobufagenin.

Marinobufagenin (MBG), a steroid compound belonging to the bufadienolide cardiac inotropes, is a molecule enjoying a growing interest in the early diagnostic of volume expansion-mediated hypertensive states. This endogenous mammalian cardiotonic and natriuretic bufadienolide (characterized by vasoconstrictive activities) inhibits the α1 isoform of Na(+), K(+)-ATPase, implicating it in series of pathophysiological circumstances such as volume-expansion, essential hypertension and preeclampsia. Indeed, the enhanced production of MBG in preeclamptic patients has been confirmed in several studies, leading us to consider MBG as a biomarker for preeclampsia. The main source for MBG is located in the parotid and skin gland secretions of the toad Bufo marinus in which MBG is the major steroid cardiotonic component. This review emphasizes the key role of analytical development for dosage methods of MBG in biofluids, in the emergence of future perspectives in the diagnostic and therapeutic fields of preeclampsia (e.g. to investigate the biosynthetic origin of MBG and to better understand its implications).

Seasonal changes and regulation of the glucocorticoid receptor in the testis of the toad Rhinella arenarum.

Several studies indicate that wild free-living vertebrates seasonally regulate plasma glucocorticoids. However, not only glucocorticoids but also the amount of receptors is important in determining biological responses. In this context, seasonal regulation of glucocorticoid receptor (GR) is crucial to modulate the response to glucocorticoids. Rhinella arenarum is an anuran exhibiting seasonal variations in plasma glucocorticoids and also in the number of binding sites (B(max)) of the testicular cytosolic GR. In this work, we evaluated if the annual pattern of GR protein in the testis varies seasonally and, by an in vitro approach, the role of glucocorticoids, androgens, and melatonin in the regulation of the GR B(max) and protein level. For this purpose, testes were treated with two physiological concentrations of melatonin (40 and 200 pg/ml), with or without luzindole (melatonin-receptor antagonist); with testosterone, cyanoketone (inhibitor of steroidogenesis) or casodex (androgen-receptor antagonist); or with dexamethasone or RU486 (GR antagonist). After treatments, B(max) and protein level were determined by the binding of [(3)H]dexamethasone and Western blot, respectively. Results showed that GR protein decreases in the winter. The in vitro treatment with melatonin produced a biphasic effect on the B(max) with the lowest concentration decreasing this parameter by a receptor-mediated mechanism. However, melatonin had no effect on the GR protein level. Conversely, a high concentration of dexamethasone up-regulated the GR protein and androgens neither changed the B(max) nor the protein level. These findings suggest that seasonal changes in plasma melatonin and glucocorticoids modulate the effect of glucocorticoids in the testis of R. arenarum.

Aerobic scope and cardiovascular oxygen transport is not compromised at high temperatures in the toad Rhinella marina.

Numerous recent studies convincingly correlate the upper thermal tolerance limit of aquatic ectothermic animals to reduced aerobic scope, and ascribe the decline in aerobic scope to failure of the cardiovascular system at high temperatures. In the present study we investigate whether this 'aerobic scope model' applies to an air-breathing and semi-terrestrial vertebrate Rhinella marina (formerly Bufo marinus). To quantify aerobic scope, we measured resting and maximal rate of oxygen consumption at temperatures ranging from 10 to 40°C. To include potential effects of acclimation, three groups of toads were acclimated chronically at 20, 25 and 30°C, respectively. The absolute difference between resting and maximal rate of oxygen consumption increased progressively with temperature and there was no significant decrease in aerobic scope, even at temperature immediately below the lethal limit (41-42°C). Haematological and cardiorespiratory variables were measured at rest and immediately after maximal activity at benign (30°C) and critically high (40°C) temperatures. Within this temperature interval, both resting and active heart rate increased, and there was no indication of respiratory failure, judged from high arterial oxygen saturation, P(O2) and [Hb(O2)]. With the exception of elevated resting metabolic rate for cold-acclimated toads, we found few differences in the thermal responses between acclimation groups with regard to the cardiometabolic parameters. In conclusion, we found no evidence for temperature-induced cardiorespiratory failure in R. marina, indicating that maintenance of aerobic scope and oxygen transport is unrelated to the upper thermal limit of this air-breathing semi-terrestrial vertebrate.

Exploiting intraspecific competitive mechanisms to control invasive cane toads (Rhinella marina).

If invasive species use chemical weapons to suppress the viability of conspecifics, we may be able to exploit those species-specific chemical cues for selective control of the invader. Cane toads (Rhinella marina) are spreading through tropical Australia, with negative effects on native species. The tadpoles of cane toads eliminate intraspecific competitors by locating and consuming newly laid eggs. Our laboratory trials show that tadpoles find those eggs by searching for the powerful bufadienolide toxins (especially, bufogenins) that toads use to deter predators. Using those toxins as bait, funnel-traps placed in natural waterbodies achieved near-complete eradication of cane toad tadpoles with minimal collateral damage (because most native (non-target) species are repelled by the toads' toxins). More generally, communication systems that have evolved for intraspecific conflict provide novel opportunities for invasive-species control.