Stress and immunity: Field comparisons among populations of invasive cane toads in Florida.

Cane toads (Rhinella marina) were introduced worldwide and have become invasive in multiple locations, representing a major driver of biodiversity loss through competition (food, shelter, territory), predation, and the poisoning of native species. These toads have been used in Australia as a model for studies concerning invasion biology and ecoimmunology, as longer-established (core) and invasion front (edge) populations show altered stress and immune response profiles. Although cane toads were also introduced into the United States in the 1950s, these patterns have yet to be evaluated for the populations spanning Florida. Toads introduced into Florida have dispersed primarily northward along a latitudinal gradient, where they encounter cooler temperatures that may further impact stress and immune differences between core and edge populations. In this study, we sampled cane toads from nine different locations spanning their invasion in Florida. Cane toads from southern populations showed higher plasma bacterial killing ability and natural antibody titers than the toads from the northern populations, indicating they have a better immune surveillance system. Also, southern toads were more responsive to a novel stressor (1 hr restraint), showing a higher increase in corticosterone levels. These results indicate that possible trade-offs have occurred between immune and stress responses as these toads have become established in northern cooler areas in Florida.

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.

Mud in the blood: Novel potent anticoagulant coagulotoxicity in the venoms of the Australian elapid snake genus Denisonia (mud adders) and relative antivenom efficacy.

Due to their potent coagulotoxicity, Australian elapid venoms are unique relative to non-Australian members of the Elapidae snake family. The majority of Australian elapids possess potent procoagulant venom, while only a few species have been identified as possessing anticoagulant venoms. The majority of research to-date has concentrated on large species with range distributions overlapping major city centres, such as brown snakes (Pseudonaja spp.) and taipans (Oxyuranus spp.). We investigated the venom from the poorly studied genus Denisonia and documented anticoagulant activities that were differentially potent on amphibian, avian, and human plasmas. Both species were potently anticoagulant upon amphibian plasma, consistent with these snakes preying upon frogs as their primary food source. While D. devisi was only relatively weakly active on avian and human plasma, D. maculata was potently anticoagulant to amphibian, avian, and human plasma. The mechanism of anticoagulant action was determined to be the inhibition of prothrombin activation by Factor Xa by blocking the formation of the prothrombinase complex. Fractionation of D. maculata venom followed by MS sequencing revealed that the toxins responsible were Group I phospholipase A2. As no antivenom is produced for this species or its near relatives, we examined the ability of Seqirus Australian snake polyvalent antivenom to neutralise the anticoagulant effects, with this antivenom shown to be effective. These results contribute to the body of knowledge regarding adaptive evolution of venom, revealing a unique taxon-specific anticoagulant effect for D. devisi venom. These results also reveal the potential effects and mechanisms behind envenomation by the potently acting D. maculata venom on human plasma, while the discovery of the efficacy of an available antivenom provides information crucial to the design of snakebite management strategies.

Testing for Short- and Long-Term Thermal Plasticity in Corticosterone Responses of an Ectothermic Vertebrate.

Phenotypic plasticity, broadly defined as the capacity of one genotype to produce more than one phenotype, is a key mechanism for how animals adapt to environmental (including thermal) variation. Vertebrate glucocorticoid hormones exert broad-scale regulation of physiological, behavioral, and morphological traits that influence fitness under many life-history or environmental contexts. Yet the capacity for vertebrates to demonstrate different types of thermal plasticity, including rapid compensation or longer acclimation in glucocorticoid hormone function, when subject to different environmental temperature regimes remains poorly addressed. Here, we explore whether patterns of urinary corticosterone metabolites respond (i.e., evidence of acclimation) to repeated short-term and sustained long-term temperature exposures in an amphibian, the cane toad (Rhinella marina). In response to three repeated short (30-min) high-temperature (37°C) exposures (at 10-d intervals), toads produced urinary corticosterone metabolite responses of sequentially greater magnitude, relative to controls. However, toads subjected to 4 wk of acclimation to either cool (18°C)- or warm (30°C)-temperature environments did not differ significantly in their urinary corticosterone metabolite responses during exposure to a thermal ramp (18°-36°C). Together, these results indicate that adult toads had different, including limited, capacities for their glucocorticoid responses to demonstrate plasticity to different regimes of environmental temperature variation. We advocate further research as necessary to identify plasticity, or lack thereof, in glucocorticoid physiology, to better understand how vertebrates can regulate organismal responses to environmental variation.

Genetic damage in Rhinella marina populations in habitats affected by agriculture in the middle region of the Sinú River, Colombia.

Contamination with pesticide residues affects the environmental health of agroecosystems, especially the amphibian fauna that lives in these environments. The objective of the present study was to determine pesticides concentrations in sediments of agroecosystems and to evaluate genetic damage in Rhinella marina populations living in these zones. A total of 91 individuals were collected, 51 in the group exposed in different areas of the middle region of the Sinú River (Irrigation District of Mocari 16, Irrigation District of Aguas Negras 21, Irrigation District of Cerete 14) and 40 in a control group; at the same time, 36 subsamples of sediments were taken at each sampled station to determine pesticides organochlorine by means of chromatography coupled with ISQ Thermo Scientific mass spectrometer. The micronucleus test was applied in erythrocytes of the individuals collected. Results showed the presence of persistent organochlorine pesticides (POPs) in the sediment samples (p,p'-DDT, p,p'-DDE, and p,p'-DDD) of agricultural soils. Two individuals were registered with abnormalities in their limbs at the Mocari station, representing 12.5% of the morphological malformations to this sector. Micronucleus analysis revealed statistically significant genetic damage in exposed individuals (Mocari 9.87 ± 5.1, Cerete 7.7 ± 1.7, Aguas Negras 5.6 ± 3.6) with respect to the control group (2.4 ± 1.9) (p < 0.05). Spearman correlation analysis revealed a positive association between genetic damage and POP concentrations (p < 0.05). In addition, cellular alterations such as nuclear buds, and pyknosis (cell death), were statistically significant in the exposed group compared to the control group (p < 0.05). This study suggests that there is evidence for morphological and genotoxic effects in R. marina populations inhabiting areas influenced by agriculture, possibly associated with the presence of p,p'-DDT, p,p'-DDD, and p,p'-DDE.

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.

Changes in serum and urinary corticosterone and testosterone during short-term capture and handling in the cane toad (Rhinella marina).

Non-invasive endocrine monitoring with minimally invasive biological samples, such as urine, is being used widely for conservation biology research on amphibians. Currently, it is unknown how closely urinary measurements correspond with the traditional serum hormone measurements. We compared urinary and serum concentrations of corticosterone (CORT) and testosterone (T) in adult male cane toads (Rhinella marina) using a standard capture and handling (short-term stressor) protocol. Free-living male cane toads were captured and sampled for baseline urine (0h) with a second urine sample taken at 0.5h and hourly between 1 and 8h. A single blood sample was collected from each toad after the final urine sampling and capture handling. The mean serum CORT concentration increased between 0 and 0.5h, reaching the highest level between 6 and 8h. The mean urinary CORT concentration increased with a lag-time of 1h and continued to increase up to 8h. The mean level of serum T decreased between 0 and 7h and increased between 7 and 8h. Mean urinary T concentration decreased with a lag-time of 0.5h. Urinary T levels did not change between 4 and 8h. Mean serum T levels reached 50% of the original 0h value at 1h while mean serum CORT levels reached 200% of the original 0h value within 0.5h. Mean urinary T levels reached 50% of the original 0h value within 3h while mean urinary CORT levels reached 200% of the original 0h value within 3h. The inter-individual variation in baseline serum and urinary CORT and T levels were highly comparable, suggesting that baseline urine sample provides a reliable indicator of the physiological status of the animal. Overall, the results have demonstrated that urine sampling and standard capture handling protocol provide reliable measures of baseline corticosterone and testosterone, as well as short-term stress hormone responses in amphibians.

Blood lead levels, δ-ALAD inhibition, and hemoglobin content in blood of giant toad (Rhinella marina) to assess lead exposure in three areas surrounding an industrial complex in Coatzacoalcos, Veracruz, Mexico.

The Coatzacoalcos Region in Veracruz, Mexico houses one of the most important industrial complexes in Mexico and Latin America. Lead is an ubiquitous environmental pollutant which represents a great risk to human health and ecosystems. Amphibian populations have been recognized as biomonitors of changes in environmental conditions. The purpose of this research is to measure exposure to lead and evaluate hematological and biochemical effects in specimens of giant toads (Rhinella marina) taken from three areas surrounding an industrial complex in the Coatzacoalcos River downstream. Lead levels in toads' blood are between 10.8 and 70.6 µg/dL and are significantly higher in industrial sites. We have found a significant decrease in the delta-aminolevulinic acid dehydratase (δ-ALAD) activity in blood from 35.3 to 78 % for the urban-industrial and industrial sites, respectively. In addition, we have identified a strong inverse relationship between the δ-ALAD activity and the blood lead levels (r = -0.84, p < 0.001). Hemoglobin and mean corpuscular hemoglobin levels, as well as the condition factor, are found to be lower at industrial sites compared with the reference sites. Our results suggest that the R. marina can be considered a good biomonitor of the δ-ALAD activity inhibition and hematological alterations at low lead concentrations.

Corticosterone-immune interactions during captive stress in invading Australian cane toads (Rhinella marina).

Vertebrates cope with physiological challenges using two major mechanisms: the immune system and the hypothalamic pituitary-adrenal axis (e.g., the glucocorticoid stress response). Because the two systems are tightly integrated, we need simultaneous studies of both systems, in a range of species, to understand how vertebrates respond to novel challenges. To clarify how glucocorticoids modulate the amphibian immune system, we measured three immune parameters and plasma corticosterone (CORT), before and after inflicting a stressor (capture and captive confinement) on introduced cane toads (Rhinella marina) near their invasion front in Australia. Stress increased CORT levels, decreased complement lysis capacity, increased leukocyte oxidative burst, and did not change heterologous erythrocyte agglutination. The strength of the CORT response was positively correlated with leukocyte oxidative burst, and morphological features associated with invasiveness in cane toads (relative leg length) were correlated with stress responsiveness. No immune parameter that we measured was affected by a toad's infection by a parasitic nematode (Rhabdias pseudosphaerocephala), but the CORT response was muted in infected versus uninfected toads. These results illustrate the complex immune-stress interactions in wild populations of a non-traditional model vertebrate species, and describe immune adaptations of an important invasive species.

Resting respiratory behavior in minimally instrumented toads--effects of very long apneas on blood gases and pH.

Resting respiratory behavior of Bufo marinus in minimally instrumented toads is described for a period of 24 hours in which the animals are left undisturbed. Torpor-related long apneas are described and their implications for blood gas levels are investigated. Results show that the resting ventilation rate of Bufo marinus is much lower than that reported so far. Levels of arterial oxygen, carbon dioxide, and pH are monitored during artificial long apneas induced by anesthesia. The toads showed an unexpected ability to unload carbon dioxide by non-respiratory means, even while being kept on dry plastic box with no access to water. Oxygen arterial partial pressure dropped to very low levels after one hour of apnea. This suggests that these animals may endure very well severe hypoxia for long periods of time while in torpor.

An improved enzyme linked immunosorbent assay for detection of anti-ranavirus antibodies in the serum of the giant toad (Bufo marinus).

An improved ranavirus antibody ELISA (R Ab ELISA) for the specific detection of anti-ranavirus antibodies in toad sera was developed. Sheep anti-epizootic haematopoietic necrosis virus (EHNV) was used as the antigen-capture antibody. EHNV was used as the antigen and sera from field and challenged toads were used to detect the virus. Rabbit anti-toad IgG and IgM were used to detect bound toad antibody. Pre-absorption of toad sera with a monoclonal antibody, raised against the 50 kDa EHNV protein, improved the specificity of the technique. A blocking ELISA, immunofluorescence and immuno-electron microscopy were used to confirm the validity of the ELISA. The assay has potential use in screening sera from Bufo marinus for the presence of antibodies against ranaviruses and to facilitate understanding of the humoral immunological response in toads during virus infection.

Heterogeneity and lability of endogenous digitalis-like substances in the plasma of the toad, Bufo marinus.

Three major groups of endogenous digitalis-like substances (EDLS) have been identified in the plasma of the toad, Bufo marinus. One group of compounds, present in fresh plasma, is composed of chromatographically homogeneous polar conjugates, principally bufadienolide 3-sulfates, which exhibit relatively weak Na(+)-K(+)-adenosinetriphosphatase (ATPase) inhibitory activity. A second and larger group of compounds, also found in fresh plasma, includes chromatographically heterogeneous conjugates, which are effective inhibitors of Na(+)-K(+)-ATPase; these compounds possess properties similar to those of bufotoxins. The third group of EDLS consists of free unconjugated bufadienolides, which are also effective Na(+)-K(+)-ATPase inhibitors. These unconjugated bufadienolides are present in relatively low concentrations in fresh toad plasma, but appreciable quantities are enzymatically generated from conjugates (believed to consist principally of bufotoxins) during the in vitro incubation of plasma. We suggest that the extent to which circulating polar EDLS are enzymatically deconjugated in vivo may be important in the regulation of the digitalis-sensitive Na(+)-K(+)-ATPase of toad brain, the only known digitalis-sensitive Na(+)-K(+)-ATPase in the toad.

Acid-base-electrolyte balance responses of Bufo marinus to aminoglutethimide, corticosterone, and aldosterone during hypercapnia.

Experiments were conducted to test the hypothesis that one or more interrenal steroids are active in regulatory responses to respiratory acidosis in the toad, Bufo marinus. Toads were divided into four experimental groups. The first group received sham injections. The second group received 1-3 mg of aminoglutethimide (AG) every 8 hr. AG inhibits the conversion of cholesterol to pregnenolone, thus inhibiting all steroid hormone synthesis. The third group received AG + 5 micrograms of aldosterone on the same schedule. The fourth group received AG + 25 micrograms of corticosterone on the same schedule as the other groups. All four groups were subjected to hypercapnia using 5% CO2 to induce a respiratory acidosis. The sham-operated animals displayed the normal compensatory pattern of producing a metabolic alkalosis (elevated plasma HCO3-) after 24 hr. AG-treated toads failed to elevate plasma HCO3-. Administration of interrenal steroids produced compensation in varying degrees. Aldosterone produced a small compensation while corticosterone produced a compensation similar to that seen in sham-operated animals. Analysis of steroid titers in toad plasma during hypercapnia showed that Bufo marinus does not elevate aldosterone during respiratory acidosis, but that corticosterone is elevated. AG blocked the corticosterone elevation, however. AG also produced a hyponatremia that was corrected with aldosterone or corticosterone. Normocapnic controls showed that AG does not produce deleterious effects on pH or blood gases in toads in the absence of a respiratory acidosis. We conclude that corticosterone is important in acid-base regulatory responses to respiratory acidosis in this amphibian.

Detergent-based isolation of marginal bands of microtubules from nucleated erythrocytes.

We have developed a new, detergent-based method for the isolation of marginal bands (MBs) of microtubules from non-mammalian vertebrate erythrocytes. The critical step in MB isolation is selective removal of the "membrane skeleton" network (MS), within which the MB is enclosed. To test potential MS solubilizing agents systematically, we prepared dogfish (Mustelus canis) erythrocyte cytoskeletons in the presence of protease inhibitors and stored them at -20 degrees C in medium containing 50% glycerol and 10 microM taxol to stabilize the MB. Using this as a standard starting material, we found that low concentrations of sodium dodecyl sulfate (SDS) (0.025-0.1%) in the presence of Triton X-100 (0.1-0.4%) released both MBs and nuclei intact from cytoskeletons. Either detergent alone was ineffective. MB release from cytoskeletons was blocked by excess Triton X-100 and slowed by glycerol, and this was useful for stopping the release reaction during quantitative time-course studies. Most MBs (greater than 90%) were liberated from cytoskeletons in 5 to 30 min, depending upon detergent concentrations and other conditions, and they were sufficiently stable for mass isolation by differential centrifugation. Added standard proteins were not proteolyzed during MB release, nor was release blocked by protease inhibitors, indicating that endogenous proteases were not involved. As observed in thin sections and negatively stained whole mounts (transmission electron microscopy) and in critical-point dried preparations (scanning electron microscopy), the isolated MBs consisted principally of bundled microtubules, with some additional adhering material. SDS-polyacrylamide gel electrophoresis showed the isolated MBs to be composed primarily of four tubulin region polypeptides, with the same stoichiometry as in the whole cytoskeleton. As determined by immunofluorescence microscopy, isolated MBs bound antibody to both chicken brain and erythrocyte tau, in addition to anti-tubulin. Thus, proteins of the tau family may be involved in bundling of MB microtubules. Unlike previous MB isolation methods, that employed here is applicable to erythrocytes of diverse species, including the marine toad (Bufo marinus) and the chicken (Gallus domestica), both of which should be of value for comparative studies.

Effects of activity, hemorrhage, and dehydration on plasma catecholamine levels in the marine toad (Bufo marinus).

Resting plasma epinephrine and norepinephrine levels were 13.1 and 2.1 nmol liter-1 for the marine toad (Bufo marinus). Plasma catecholamine levels increased during enforced activity by five- to sixfold. Marine toads are remarkably tolerant of graded hemorrhagic loss of blood (over 10% mass loss). Plasma catecholamine levels did not increase at moderate blood loss, but increased substantially when cardiovascular variables (blood pressure, blood flow) were compromised and peripheral resistance was increased. Plasma catecholamine levels did not increase with dehydrational mass loss until a 15-20% loss of mass. The increase in plasma catecholamine concentration was correlated with an increase in vivo vascular resistance. Vascular resistance measured in vitro was unaltered at physiological catecholamine concentrations, although systemic resistance increased at pharmacological concentrations. The lack of effects of adrenalectomy on plasma catecholamine levels suggests that nerve terminal release, rather than adrenal secretion, may be the primary source of circulating catecholamines. We therefore suggest that circulating catecholamine levels are not an important endocrinological mechanism for defense of activity blood pressure, at least until it is compromised to the resting value.

Plasma steroid concentrations change in response to sexual behavior in Bufo marinus.

Steroid hormone concentrations change in response to social or environmental stimuli in many vertebrates. To test this phenomenon in an amphibian, we examined plasma androgen (A) and corticosterone (B) concentrations in male marine toads (Bufo marinus), a tropical species exhibiting intermale competition, amplectic clasping of females, and bouts of breeding behavior following rains. When males clasped females for 0, 1, 2, or 3 hr, plasma A concentrations were significantly and positively correlated with hours spent in amplexus. In field-sampled males, plasma A concentrations were higher in amplexing males than in single males. Among single males those found closer to breeding ponds had higher A concentrations than those more distant. These data support the hypothesis that sexual stimuli enhance plasma A concentrations in this amphibian. In 3-hr experimental tests and field-sampled males, B concentrations were higher in amplexing than in single males. Unlike some amphibians, short-term elevations of B apparently are not associated with decreased reproductive function. However, as in other amphibians in which high B concentrations are associated with stress-induced inhibition of reproduction, after 48-72 hr in captivity male toads showed high B concentrations and low plasma androgen concentrations. The bursts of sexual activities exhibited by B. marinus following heavy rains were associated with no changes in A concentration and with increased B concentration.