Baseline and stress-induced steroid plasma levels and immune function vary annually and are associated with vocal activity in male toads (Rhinella icterica).

Theoretical models predict that elevated androgen and glucocorticoid levels in males during the reproductive season promote immunosuppression. However, some studies report decreased stress response during this season. This study investigated annual variation in plasma corticosterone and testosterone levels, plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) in free-living male toads (Rhinella icterica). Toads were sampled in the field (baseline) and 1 h-post restraint over five months, and we considered the occurrence of vocal activity. Baseline corticosterone, testosterone, and BKA showed higher values during the reproductive period, specifically in calling male toads. The NLR was similar throughout the year, but higher values were observed in calling toads. Moreover, baseline NLR and BKA were positively correlated with both testosterone and corticosterone, suggesting higher steroid levels during reproduction are associated with enhanced cellular and humoral immunity. Despite fluctuation of baseline values, post-restraint corticosterone levels remained uniform over the year, indicating that toads reached similar maximum values throughout the year. Testosterone levels decreased following restraint before one specific reproductive period but increased in response to restraint during and after this period. Meanwhile, BKA decreased due to restraint only after the reproductive period, indicating immune protection and resilience to immunosuppression by stressors associated with steroid hormones during reproduction. Our results show that baseline and stress-induced hormonal and immune regulation varies throughout the year and are associated with vocal activity in R. icterica males, indicating a possible compromise between steroids and immune function in anuran males.

Dehydration followed by restraint sustains high circulating corticosterone and improves immunity in toads.

Amphibians are suffering population declines due to a variety of factors such as increased ultraviolet radiation, climate change, habitat loss, pathogens, and pollution, or a combination of these. Such changes are associated with a reduction in the availability of water, exposing these animals to a greater risk of desiccation. In this context, understanding how dehydration can modulate the hypothalamic-pituitary-interrenal axis (HPI) and the immune response is an imperative question to predict how stressors can affect amphibian species. We investigated whether dehydration promotes long-lasting effects on toads' ability to respond to a consecutive stressor (restraint) even if the toads are allowed to rehydrate, as well as its effects on the immune function. We also tested the hypothesis that the toads showing more severe dehydration would exhibit lower responsiveness to restraint challenge, even if the animals were allowed to rehydrate. Individuals of R. ornata were dehydrated mildly and severely. Thereafter, they were submitted to a restraint stress challenge for 1 and 24 h. Our results show that dehydration increased hematocrit and CORT in R. ornata toads. The restraint induced an acute stress response in fully hydrated toads (increased CORT and neutrophil: lymphocyte ratio). Otherwise, restraint in moistened cloth bags allowed full rehydration in previously dehydrated toads and did not induce an additional increase in CORT, but those toads sustained elevated CORT up to 24 h of restraint. Also, these animals showed increased neutrophil: lymphocyte ratio and the phagocytic activity of blood cells, even when they rehydrated during restraint. These results point to a continuous activation of the HPA during dehydration and subsequent restraint, even when they recovered from the dehydration state. Also, acute stressors seem to promote immune cell redistribution and augmentation of immune cellular function in R. ornata toads.

Corticosterone treatment results in fat deposition and body mass maintenance without effects on feeding behaviour or immunity in female lizards (Tropidurus catalanensis).

Throughout life, animals must maintain homeostasis while coping with challenging events. The period after reproduction can be challenging for oviparous females to maintain homeostasis since they direct most of their energy stores to vitellogenesis, possibly increasing the vulnerability to stressors. Changes in glucocorticoids' (GC) secretion promote various behavioural and physiological adjustments daily and to restore balance after facing stressors. However, when GC are elevated for extended periods, which usually occurs in response to chronic exposure to stressors, they can affect feeding behaviour and suppress the immune function. We aim to elucidate the effects of chronic corticosterone (CORT) exposure on feeding behaviour, body condition and immune function in female lizards, Tropidurus catalanensis, in the post-reproductive period. Thirty animals were divided into three groups: 1. Control (no experimental procedure performed); 2. Empty Implant (animals implanted with empty silastic tube); and 3. CORT Implant (animals implanted with silastic tube filled with CORT, with a chronic continuous release for at least a week). CORT plasma levels feeding behaviour, body condition (body index [BI] and fat index [FI]), leukocyte count, and several immune function variables (bacterial killing ability [BKA], hemagglutination titer, phytohemagglutinin [PHA] immune challenge and leukocyte count) were evaluated. After implantation, CORT treated animals maintained stable body mass through the experiment, while Control and Empty Implant groups displayed weight loss. In the CORT treated animals, there was also a positive relation between BI and FI, and higher FI when compared to groups 1 and 2. No effects of CORT were observed on feeding behaviour nor on the immune function.

Baseline and stress-induced changes in plasma bacterial killing ability against gram-negative bacteria are partially mediated by the complement system in Rhinella diptycha toads.

The plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads.

Elevated corticosterone levels are associated with increased immunocompetence in male toads, both when calling and under experimental conditions.

Circulating androgens can influence immune responses and sexual traits in male vertebrates. However, in the last 20 years, glucocorticoids have also been implicated as a possible source of variation in male sexual traits and immunocompetence. In this context, we studied the relations between male vocal mating display, immunity, androgens, and glucocorticoids in the explosive breeding toad Rhinella granulosa. In the field, males with high calling effort display either high- or low-corticosterone (CORT) plasma levels, but only males with both high calling effort and high CORT plasma levels showed high bacterial killing ability (BKA), suggesting that the acute CORT elevation can be immunostimulatory. CORT treatments increased BKA in laboratory experiments, confirming the functional relationship observed in the field. However, toads treated with a low dose of CORT increased BKA for 10 h after the treatment, while toads that received a high dose increased BKA for only 1 h after the treatment. These results indicate that different CORT doses can result in temporal differences in the immune response. We did not find any relationship between calling effort, immune response (BKA and PHA swelling response), and testosterone plasma levels in the field, or any effects of testosterone treatment on immunocompetence. Our results suggest a complex relationship between calling effort and immunity, mediated by CORT plasma levels.

Hormonal daily variation co-varies with immunity in captive male bullfrogs (Lithobates catesbeianus).

Almost all physiological processes within the organism, including immune parameters and hormones, follow a circadian rhythm. These daily fluctuations are often observed in free-living organisms; however, little is known regarding hormonal and immune daily variations in anurans, particularly under laboratory conditions. This study aimed to investigate the hormonal and immune daily variation in captive-bred Bullfrogs (Lithobates catesbeianus) under constant conditions (21 °C and 12:12 LD cycle). Our results showed a daily variation for plasma corticosterone (CORT), testosterone (T), and melatonin (MEL), as well as for blood leukocyte profile, phagocytic activity, and plasma bacterial killing ability (BKA). Hormonal profile and immune activity were higher at the dark when compared with the light phase; however, monocytes and lymphocytes followed the opposite pattern. Moreover, CORT was positively correlated with phagocytosis percentage of blood cells, BKA, and monocytes, while MEL and T showed a positive correlation with PP. Our results demonstrate the daily covariation of different immune variables and immunomodulatory hormones. These 24 h-day variations and covariation certainly have broad implications and need to be considered for better understanding anuran physiology both in the context of laboratory and field studies.

Challenges of a novel range: Water balance, stress, and immunity in an invasive toad.

Species introduced by human activities can alter the normal functioning of ecosystems promoting negative impacts on native biodiversity, as they can rapidly expand their population size, demonstrating phenotypic plasticity and possible adaptive capacity to novel environments. Twenty years ago, the guttural toad, Sclerophrys gutturalis, was introduced to a peri-urban area of Cape Town, with cooler and drier climatic characteristics than its native source population, Durban, South Africa. Our goal was to understand the phenotypic changes, in terms of physiology and immunity, of populations in native and novel environments. We evaluated body index (BI), field hydration level, plasma corticosterone levels (CORT), proportion of neutrophils: lymphocytes (N: L), plasma bacterial killing ability (BKA), and hematocrit (HTC) in the field, and after standardized stressors (dehydration and movement restriction) in males from the native and invasive populations. Toads from the invasive population presented lower BI and tended to show a lower field hydration state, which is consistent with living in the drier environmental conditions of Cape Town. Additionally, invasive toads also showed higher BKA and N:L ratio under field conditions. After exposure to stressors, invasive animals presented higher BKA than the natives. Individuals from both populations showed increased CORT after dehydration, an intense stressor for these animals. The highest BKA and N:L ratio in the field and after submission to stressors in the laboratory shows that the invasive population has a phenotype that might increase their fitness, leading to adaptive responses in the novel environment and, thus, favoring successful dispersion and population increase.

Biomarker-based assessment of the muscle maintenance and energy status of anurans from an extremely seasonal semi-arid environment, the Brazilian Caatinga.

Strongly seasonal environments pose challenges for performance and survival of animals, especially when resource abundance seasonally fluctuates. We investigated the seasonal variation of key metabolic biomarkers in the muscles of males from three species (Rhinella jimi, R. granulosa and Pleurodema diplolister) of anurans from the drastically seasonal Brazilian semi-arid area, Caatinga. We examined the expression of proteins regulating energy turnover (AMP-activated protein kinase [AMPK] and protein kinase B [AKT]), protein synthesis and homeostasis (total and phosphorylated eukaryotic initiation factor 2α [eIF2α and p-eIF2α] and chaperone proteins [HSP 60, 70, and 90]) in muscles predominantly related to reproduction and locomotion. Cytochrome c oxidase (COX) activity was also assessed as an index of the muscle aerobic capacity. The expression pattern of metabolic biomarkers indicates that the maintenance of muscular function is regulated in a species-specific manner during the drastic seasonal variation. Rhinella jimi and R. granulosa that remain active during the drought appear to maintain muscles through more energy expensive pathways including elevated protein synthesis, while the aestivating P. diplolister employs energy conservation strategy suppressing protein synthesis, decreasing chaperone expression and increasing expression of AMPK. Two (P. diplolister and R. granulosa) of the three studied species activate cell survival pathways during the drought likely to prevent muscle atrophy, and all three studied species maintain the muscle aerobic capacity throughout the year, despite the resource limitation. These strategies are important considering the unpredictability of the reproductive event and high demand on muscular activity during the reproductive season in these amphibians. SUMMARY STATEMENT: We studied seasonal variation of key metabolic biomarkers in the muscles of anurans that experience drastic variation in environmental conditions and differ in seasonal activity patterns.

Effects of dehydration on thermoregulatory behavior and thermal tolerance limits of Rana catesbeiana ().

Predicting the effects of high environmental temperatures and drought on populations requires understanding how these conditions will influence the thermoregulatory behavior and thermal tolerance of organisms. Ectotherms show proportional (fine-tuned) and all-or-none (abrupt) responses to avoid overheating. Scattered evidence suggests that dehydration alters these behavioral responses and thermal tolerance, but these effects have not been evaluated in an integrative manner. We examined the effects of hydration level on the behavioral thermoregulation and behavioral and physiological thermal limits of the "bullfrog" (Rana catesbeiana), a well-studied and important invasive species. To examine the effects of dehydration on proportional responses, we compared the Preferred Body Temperatures (PBT) of frogs with restricted and unrestricted access to water. To assess the effect of dehydration on all-or-none responses, we measured and compared the Voluntary Thermal Maximum (VTMax) at different hydration levels (100%, 90%, 80% of body weight at complete hydration). Finally, to understand the effect of dehydration on physiological thermal tolerance, we measured the Critical Thermal Maximum (CTMax) of frogs at matched hydration levels. PBT, VTMax, and CTMax all decreased in response to higher dehydration levels. However, bullfrogs changed their PBT more than their VTMax or CTMax in response to dehydration. Moreover, some severely dehydrated individuals did not exhibit a VTMax response. We discuss the implications of our results in the context of plasticity of thermoregulatory responses and thermal limits, and its potential application to mechanistic modeling.

Acute stress, steroid plasma levels, and innate immunity in Brazilian toads.

Stress from habitat fragmentation has been shown to impact amphibian declines. Studies from a variety of vertebrates indicate that stressed animals exhibit an acute increase in circulating plasma glucocorticoid (GC) levels and consequent immunomodulation. To further explore the relationship between GCs and immunity, we subjected three species of newly captured Brazilian toads, Rhinella ornata, R. icterica and R. schneideri to restraint with or without movement restriction (maintenance in a moistened cloth bag vs. maintenance in a bin) for 24 h. We compared various parameters from baseline (field conditions) with values after restraint, including those associated with stress (corticosterone [CORT] plasma levels), and the neutrophil/lymphocyte ratio [N:L ratio]), potential reproduction (testosterone [T] plasma levels), and innate immunity (bacterial killing ability [BKA]). General responses to the restraint challenge (baseline vs. restraint) included increased CORT levels and N:L ratio, and decreased T levels and BKA. Additionally, CORT levels and N:L ratio tended to increase more from restraint with movement restriction than to restraint without movement restriction, indicating toads showed increased stress response to the more intense stressor. All variables showed interspecific variation at baseline conditions: R. ornata had higher CORT levels when compared to the other two species, while R. icterica had the highest BKA values. After restraint (with or without movement restriction), R. ornata displayed higher values for T and N:L ratio, and showed higher CORT values after restraint without movement restriction; however, the CORT values were similar among species after restraint with movement restriction. In terms of immunity, in response to restraint, BKA was different among species only after restraint with movement restriction, with R. schneideri showing the lowest BKA values. Our results show that restraint increases common markers of the stress response, and could reduce potential reproduction and innate immune responses in toads from all studied species. Our results also showed variation at the interspecific level, with the amplitude of change in the studied variables being consistent and more pronounced following restraint with movement restriction for the three-studied species.

Immunomodulation by testosterone and corticosterone in toads: Experimental evidences from transdermal application.

Androgens and glucocorticoids play important roles in vertebrate's reproduction and display complex immunomodulatory functions that may affect survival. In anurans, testosterone and corticosterone are correlated to sexual behavior, reproduction, and immune function. Male toads (Rhinella jimi) were treated with acute doses of testosterone (T) and corticosterone (CORT) and immune variables (plasma bacterial killing ability [BKA], swelling after phytohemagglutinin [PHA] injection and the time point of maximum PHA swelling response) were measured. Transdermal T and CORT application increased androgen (T-DHT) and CORT plasma levels after 1 h of treatment, respectively, without a dose-specific effect. Transdermal T treatment did not affect BKA or PHA swelling response. Individuals treated with transdermal CORT showed an earlier maximum PHA swelling response and a tendency of lower BKA 15 h after treatment. Our results indicated that an acute experimental increase of CORT plasma levels diminished time for inflammatory resolution and suppressed non-cellular innate response.

Thermal sensitivity of innate immune response in three species of Rhinella toads.

Immune activity is temperature-dependent and strongly related to thermal biology in ectotherms. Eurythermic, vs stenothermic, species commonly show a broader range of immune responses. Furthermore, the development of behavioral fever in ectotherms is correlated with improved immune function. Although amphibians generally show restricted capacity for thermoregulation in the field, behavioral fever has been documented in the laboratory for several anurans. However, the match between behavioral fever and improved immune response at fever thermal preferendum has still to be determined in these animals. In this study, we investigate the thermal sensitivity of the innate immune response, as measured by the plasma bacterial killing ability (BKA) against Aeromonas hydrophila, in three species of toads from genus Rhinella (R. schneideri, R. icterica and R. ornata) during their breeding season. Moreover, we show lipopolysaccharide-induced behavioral fever for R. ornata. The three species of toads showed an inverted U-shaped pattern of thermal sensitivity regarding BKA, with a high efficiency of immune response at temperatures around their thermal preferendum. The results partially corroborate the hypothesis that immune function is maximized at fever thermal preferendum, given that two of the species showed a maximal BKA performance temperature closer to fever than their normal thermal preferendum. Toads also showed an eurythermic pattern of immune response (large temperature breadth of BKA performance ≥95%; B95) during the breeding season. This large B95 encompasses much of the ecologically relevant temperatures, with the exception of those exhibited by two species that maintain activity during winter. Lastly, BKA was commonly suppressed at 37 °C, highlighting the importance of choosing ecologically relevant temperatures when conducting in vitro immunological tests.

Associations of water balance and thermal sensitivity of toads with macroclimatic characteristics of geographical distribution.

Interspecific variation in patterns of geographical distribution of phylogenetically related species of amphibians might be related to physiological adaptation to different climatic conditions. In this way, a comparative study of resistance to evaporative water loss, rehydration rates and sensitivity of locomotor performance to variations on hydration level and temperature was performed for five species of Bufonidae toads (Rhinella granulosa, R. jimi, R. ornata, R. schneideri and R. icterica) inhabiting different Brazilian biomes. The hypotheses tested were that, when compared to species inhabiting mesic environments, species living at hot and dry areas would show: (1) greater resistance to evaporative water loss, (2) higher rates of water uptake, (3) lower sensitivity of locomotor performance to dehydration and (4) lower sensitivity of locomotor performance at higher temperatures and higher sensitivity of locomotor performance at lower temperatures. This comparative analysis showed relations between body mass and interspecific variation in rehydration rates and resistance to evaporative water loss in opposite directions. These results might represent a functional compensation associated with relatively lower absorption areas in larger toads and higher evaporative areas in smaller ones. Moreover, species from the semi-arid Caatinga showed locomotor performance less sensitive to dehydration but highly affected by lower temperatures, as well greater resistance to evaporative water loss, when compared to the other species from the mesic Atlantic Forest and the savannah-like area called Cerrado. These results suggest adaptation patterns to environmental conditions.

ACTH modulation on corticosterone, melatonin, testosterone and innate immune response in the tree frog Hypsiboas faber.

The modulation exerted by glucocorticoids in physiological responses to stressors is essential for maintaining short-term homeostasis. However, highly frequent and/or prolonged activation of the hypothalamic-pituitary-adrenal/interrenal axis may inhibit processes that are important to long-term fitness and health, including reproduction and immunocompetence. The present study evaluates the response to adrenocorticotropic hormone (ACTH) injection in the adult male tree frog, Hypsiboas faber, as indicated by levels of plasma corticosterone (CORT), plasma testosterone (T), ocular melatonin (MEL), hematocrit and immune functioning (total leukocyte count and bacterial killing ability against Escherichia coli). All levels were measured 1, 3 and 6h after treatment. ACTH increased CORT levels whilst decreasing T and MEL levels at 1h post-treatment. 6h after ACTH injection, hematocrit and MEL levels increased. ACTH treatment did not significantly modulate the immune measures over the time-range sampled. The hormonal changes observed in response to ACTH treatment suggest that stressors could act as inhibitors of reproductive activity, as well as differentially modulating melatonin levels at different time-points.

Breeding under unpredictable conditions: Annual variation in gonadal maturation, energetic reserves and plasma levels of androgens and corticosterone in anurans from the Brazilian semi-arid.

Anurans living in arid and semi-arid habitats are subjected to unpredictable rain patterns. Consequently, they should be prepared to reproduce at the onset of rain events. We investigated the covariation between calling behavior, testicular maturation, abdominal fat body index (FBI), plasma levels of androgens (T-DHT) and corticosterone (CORT) of males from three species of anurans in the Brazilian semi-arid during the reproductive period and drought. One of these species aestivates during the drought, while the other two species remain foraging. Although the three species display different behavioral strategies during the dry period, they present the same general reproductive patterns. T-DHT levels on the plasma and germinative cyst diameters were higher during the reproductive and breeding period compared to the drought. Additionally, the germinative cysts had all cell stages including sperm bundles during the dry season, however, it was only during the breeding event that free spermatozoa were found in the cyst lumen. These results suggest that these species present the reproductive pattern typical of desert anurans, consisting of opportunistic breeders that reproduce when triggered by a rain stimulus. Rhinella jimi and Pleurodema diplolister had higher CORT when males were calling. Moreover, Rhinella granulosa and P. diplolister showed lower FBI during breeding event, when males were calling. The high levels of CORT and lower FBI during reproductive period are associated, indicating that CORT modulates the recruitment of energy stores to prepare and maintain reproduction, particularly the expensive calling effort.

Calling rate, corticosterone plasma levels and immunocompetence of Hypsiboas albopunctatus.

During the breeding season, male anuran amphibians produce advertisement calls. Androgens play a permissive role in the activation of calling activity, which is often positively correlated to androgen plasma levels and testes mass. Additionally, calling effort is also correlated to corticosterone plasma levels (hereinafter referred to as CORT), which is associated with the mobilization of energy substrates to sustain the high energy flux associated with this activity. However, high CORT also has many immunosuppressive effects and might interfere with reproduction. Consequently, CORT might mediate a compromise between reproductive effort and immunocompetence in anurans. In the present study, we investigated the relationship between calling rate, immunocompetence, and CORT in Hypsiboas albopunctatus, a midsize anuran occurring in South America. To understand these relationships, we conducted focal observations of calling behavior, followed by blood collection for CORT measurements and evaluation of some immune parameters. Our results showed that individuals with larger testes had higher calling rates, and those with higher calling rates showed lower cell-mediated immune response (swelling response to phytohaemagglutinin), although these relationships were not mediated by CORT. Furthermore, males calling early in the evening showed high CORT, and individuals with lower body condition index had higher CORT. We conclude that calling activity shows a cost in terms of cellular immune response in H. albopunctatus, but this compromise does not appear to be mediated by glucocorticoid plasma levels.

Testosterone immunomodulation in free-living and captive Rhinella icterica male toads.

Testosterone (T) regulates immune function, with both immunostimulatory and immunosuppressive effects on several vertebrates. We investigated the covariation between plasma T and corticosterone (CORT) levels and immunity (plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR)) in free-living Rhinella icterica male toads inside and outside the reproductive season. We found an overall positive correlation between steroids and immune traits, with toads during the reproductive season displaying increased T, CORT and BKA. We also investigated the T transdermal application effects on T, CORT, phagocytosis of blood cells, BKA and NLR in captive toads. Toads were treated with T (1, 10 or 100 µg) or vehicle (sesame oil) for eight consecutive days. Animals were bled on the first and eighth days of treatment. Increased plasma T was observed on the first and last day of T-treatment, while increased BKA was observed following all T doses on the last day, with a positive correlation between T and BKA. Plasma CORT, NLR and phagocytosis increased on the last day for all T-treated and vehicle groups. Overall, we demonstrated a positive covariation between T and immune traits in the field and T-induced augmented BKA in captive toads, indicating a T immunoenhancing effect in R. icterica males. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Contaminated meal intake (Aeromonas hydrophila) does not elicit hormonal or immune modulation in bullfrogs (Lithobates catesbeianus).

The gastrointestinal tract (GIT) is colonized by resident microbiota but contact with foreign microbiota during feeding can impair GIT functions. During meal digestion, several vertebrates modulate the systemic immune function and immunoregulatory hormones concentration. However, in ectothermic animals, it is not known if this hormonal and immune modulation during the postprandial period is affected by the presence of pathogenic microbiota in the food. This study aimed to investigate the effects of contaminated meal ingestion on hormonal and innate immune responses in bullfrogs (Lithobates catesbeianus). Bullfrogs were divided into three treatments: fed three times with sterilized fish feed (control group), fed twice with sterilized fish feed and once with fish feed containing live bacteria (Aeromonas hydrophila, 109 UFC/mL), and fed three times with fish feed containing live bacteria. Blood and GIT tissues were collected 24 h after treatments to measure plasma and tissue corticosterone levels, NL ratio, and plasma bacterial killing ability. The ingestion of contaminated meal did not affect the hormonal and immune parameters. In conclusion, ingestion of contaminated food was not capable of intensifying the hypothalamic-pituitary-interrenal axis activation and the consequent hormonal and immune responses observed after feeding in bullfrogs. However, our results suggest that the ingestion of three contaminated meals tended to decrease stomach corticosterone levels (nonstatistically significant), possibly contributing to preventing the transmigration of the bacteria to organs outside the GIT.

Vocal and territorial behavior in the Smith frog (Hypsiboas faber): relationships with plasma levels of corticosterone and testosterone.

The possible trade-off between the roles of glucocorticoids as facilitators of energy substrate mobilization and neural inhibitors of sexual behavior during breeding season is under debate. We studied the relationship between calling and territorial behavior with plasma levels of corticosterone (CORT) and plasma levels of testosterone (T) across the breeding season of Hypsiboas faber, a large and territorial Neotropical treefrog. We investigated these relationships through focal observations of males calling naturally, followed by blood sampling for hormonal radioimmunoassay. We additionally used an experimental approach, which consisted of broadcasting recorded advertisement calls for 10 min to simulate an invasion in the territory of the focal subjects, followed by behavioral observation and blood sampling for hormonal radioimmunoassay. Results showed a pattern of co-variation between CORT and T across the breeding season. Furthermore, individual variation in CORT and T was related to different aspects of behavior: individuals with higher CORT showed higher calling rates, and individuals with higher steroid levels, mainly T, showed higher responsivity to social stimulation by other males in the chorus. Experimental simulation of territorial intrusion by using playback of advertisement calls of this species did not elicit consistent changes in agonistic behavior and CORT, but decreased T in focal males.

Heat-Induced Hatching of Red-Eyed Treefrog Embryos: Hydration and Clutch Structure Increase Behavioral Thermal Tolerance.

Climate change is increasing both environmental temperatures and droughts. Many ectotherms respond behaviorally to heat, thereby avoiding damage from extreme temperatures. Within species, thermal tolerance varies with factors such as hydration as well as ontogenetic stage. Many tropical anurans lay terrestrial eggs, relying on environmental moisture for embryonic development. These eggs are vulnerable to dehydration, and embryos of some species can hatch prematurely to escape from drying eggs. Warmer temperatures can accelerate development and thus hatching, but excess heat can kill embryos. Thus, we hypothesize that embryos may show a behavioral thermal tolerance limit, hatching prematurely to avoid potentially lethal warming. If so, because warming and drying are often associated, we hypothesize this limit, measurable as a voluntary thermal maximum, may depend on hydration. We manipulated the hydration of the terrestrial eggs of Agalychnis callidryas, in intact clutches and egg-groups isolated from clutch jelly, then warmed them to assess if embryos hatch early as a behavioral response to high temperatures and whether their thermal tolerance varies with hydration or surrounding structure. We discovered that heating induces hatching; these embryos show a behavioral escape-hatching response that enables them to avoid potentially lethal warming. Hydrated eggs and clutches lost more water and warmed more slowly than dehydrated ones, indicating that hydration buffers embryos from environmental warming via evaporative cooling. Embryos in hydrated clutches tolerated greater warming before hatching and suffered higher mortality, suggesting their behavioral Thermal Safety Margin is small. In contrast, lower thermal tolerance protected dry embryos, and those isolated from clutch jelly, from lethal warming. Heat-induced hatching offers a convenient behavioral assay for the thermal tolerance of terrestrial anuran embryos and the interactive effects of warming and dehydration at an early life stage. This work expands the set of threats against which embryos use hatching in self-defense, creating new opportunities for comparative studies of thermal tolerance as well as integrative studies of self-defense mechanisms at the egg stage.

El cambio climático está aumentando tanto las temperaturas ambientales como las sequías. Muchos ectotermos responden conductualmente al calor, evitando así los daños por temperaturas extremas. Dentro de las especies, la tolerancia térmica varía con factores como la hidratación y la ontogenia. Muchos anuros tropicales depositan huevos terrestres que dependen de la humedad ambiental para el desarrollo embrionario. Estos huevos son vulnerables a la deshidratación y los embriones de algunas especies pueden eclosionar prematuramente para escapar de la desecación de los huevos. Las temperaturas más cálidas pueden acelerar el desarrollo y, por lo tanto, la eclosión, pero el exceso de calor puede matar a los embriones. Por lo tanto, planteamos la hipótesis de que los embriones pueden mostrar un límite de tolerancia térmica conductual, eclosionando prematuramente para evitar un calentamiento potencialmente letal. Si es así, debido a que el calentamiento y la desecación a menudo están asociados, planteamos la hipótesis de que este límite, medible como una tolerancia térmica voluntaria, puede depender de la hidratación. Manipulamos la hidratación de los huevos terrestres de Agalychnis callidryas, en posturas intactas y grupos de huevos aislados de la gelatina de la postura. Luego, los calentamos para evaluar si los embriones eclosionan temprano como respuesta conductual a las altas temperaturas y si su tolerancia térmica varía con la hidratación o la estructura circundante. Descubrimos que el calentamiento induce la eclosión. Estos embriones muestran una respuesta conductual de eclosión de escape que les permite evitar un calentamiento potencialmente letal. Los huevos y las posturas hidratadas perdieron más agua y se calentaron más lentamente que los deshidratados, lo que indica que la hidratación protege a los embriones del calentamiento ambiental a través del enfriamiento por evaporación. Los embriones en posturas hidratadas toleraron un mayor calentamiento antes de la eclosión y sufrieron una mayor mortalidad, lo que sugiere que su margen de seguridad térmica conductual es pequeño. En contraste, la tolerancia térmica más baja protegió a los embriones deshidratados, y a los aislados de la gelatina, del calentamiento letal. La eclosión inducida por calor ofrece un ensayo de comportamiento conveniente para la tolerancia térmica de los embriones terrestres de anuros y los efectos integrados del calentamiento y la deshidratación en una etapa temprana de la vida. Este estudio amplía el conjunto de amenazas contra las cuales los embriones usan la eclosión en defensa propia, creando nuevas oportunidades para estudios comparativos de tolerancia térmica, así como estudios integradores de mecanismos de autodefensa en etapa de huevo.

As mudanças climáticas estão aumentando as temperaturas ambientais e as secas. Muitos ectotermos respondem comportamentalmente ao calor, evitando danos devido a temperaturas extremas. Dentro das espécies, a tolerância térmica varia com fatores como a hidratação e a ontogenia. Muitos anuros tropicais depositam ovos terrestres que dependem da umidade ambiental para o desenvolvimento embrionário. Esses ovos são vulneráveis à desidratação, e os embriões de algumas espécies podem eclodir prematuramente para escapar da dessecação dos ovos. Temperaturas mais altas podem acelerar o desenvolvimento e, portanto, a eclosão, mas o excesso de calor pode matar os embriões. Portanto, propomos a hipótese de que os embriões podem mostrar um limite de tolerância térmica comportamental, eclodindo prematuramente para evitar o aquecimento potencialmente letal. Nesse caso, como o aquecimento e a dessecação são frequentemente associados, propomos a hipótese de que esse limite, mensurável como uma tolerância térmica voluntária, pode depender da hidratação. Manipulamos a hidratação dos ovos terrestres de Agalychnis callidryas, em desovas completas e grupos de ovos isolados da geleia da desova. Depois, aquecemos os ovos para avaliar se os embriões eclodem cedo como uma resposta comportamental a altas temperaturas, e se sua tolerância térmica varia com a hidratação ou a estrutura circundante. Descobrimos que o aquecimento induz a eclosão. Esses embriões mostram uma resposta comportamental de eclosão que lhes permite evitar o aquecimento potencialmente letal. Ovos e desovas hidratadas perderam mais água e se aqueceram mais lentamente que os desidratados, indicando que a hidratação protege os embriões do aquecimento ambiental através do resfriamento por evaporação. Os embriões em desovas hidratadas toleraram maior aquecimento antes da eclosão e sofreram maior mortalidade, sugerindo que sua margem de segurança térmica comportamental é menor. Por outro lado, a menor tolerância térmica protegeu os embriões desidratados e os isolados da geleia, do aquecimento letal. A eclosão induzida pelo calor oferece um teste comportamental conveniente para a estimativa da tolerância térmica de embriões de anuros terrestres e dos efeitos integrados do aquecimento e da desidratação em um estágio inicial da vida. Este estudo expande o conjunto de ameaças contra as quais os embriões usam a eclosão como defesa, criando novas oportunidades para estudos comparativos de tolerância térmica, além de estudos integrativos dos mecanismos de autodefesa no estágio de ovo.