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'.

Introduction to the special issue Amphibian immunity: stress, disease and ecoimmunology.

Amphibian populations have been declining worldwide, with global climate changes and infectious diseases being among the primary causes of this scenario. Infectious diseases are among the primary drivers of amphibian declines, including ranavirosis and chytridiomycosis, which have gained more attention lately. While some amphibian populations are led to extinction, others are disease-resistant. Although the host's immune system plays a major role in disease resistance, little is known about the immune mechanisms underlying amphibian disease resistance and host-pathogen interactions. As ectotherms, amphibians are directly subjected to changes in temperature and rainfall, which modulate stress-related physiology, including immunity and pathogen physiology associated with diseases. In this sense, the contexts of stress, disease and ecoimmunology are essential for a better understanding of amphibian immunity. This issue brings details about the ontogeny of the amphibian immune system, including crucial aspects of innate and adaptive immunity and how ontogeny can influence amphibian disease resistance. In addition, the papers in the issue demonstrate an integrated view of the amphibian immune system associated with the influence of stress on immune-endocrine interactions. The collective body of research presented herein can provide valuable insights into the mechanisms underlying disease outcomes in natural populations, particularly in the context of changing environmental conditions. These findings may ultimately enhance our ability to forecast effective conservation strategies for amphibian populations. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

The Impacts of Transdermal Application of Corticosterone on Toad (Rhinella icterica) Immunity.

Recent studies have shown that acute physiological increases in endogenous glucocorticoid levels have immunostimulatory effects. Although post-acute stress immunosuppressive effects have also been described, the difference between enhancing and suppressing the immune response seems mediated by the stressor's duration, intensity, and the immune component under analysis. To elicit physiologically relevant corticosterone levels that can be found in Rhinella icterica toads after stressful events (e.g., restraint or captivity) and understand how acute increased glucocorticoid levels of different intensities affect corticosterone and testosterone plasma levels and immune parameters (in vitro plasma bacterial killing ability, neutrophil-to-lymphocyte ratio, and in vivo phagocytosis of peritoneal leukocytes), we submitted toads to the transdermal application of two corticosterone doses (1 and 10 µg). Corticosterone transdermal application increased corticosterone plasma levels with different intensities: 3 times for 1 µg and fourteen times for 10 µg, compared to the vehicle, and the neutrophil-to-lymphocyte ratio increased regardless of the corticosterone dose. However, there was no effect on testosterone levels and bacterial killing ability. Interestingly, both corticosterone doses promoted immunosuppression, decreasing peritoneal leukocytes' phagocytosis activity by 60% for toads receiving the dose of 1µg and 40% for those receiving 10 µg. Our results show the complexity of the relationship between increased corticosterone levels and immunomodulation. The different corticosterone doses promoted increases of distinct magnitudes in corticosterone plasma levels, with the less intense increase in corticosterone levels generating greater cell-mediated immunosuppression. Future studies using different corticosterone doses to achieve and compare physiological vs. pharmacological hormone levels are imperative to understanding these interrelationships between corticosterone and immune response.

Immunoendocrinology and Ecoimmunology in Brazilian Anurans.

This paper reviews several aspects of immunoendocrinology and ecoimmunology in Brazilian species of anurans under investigation for more than a decade, including (1) patterns of annual covariation of circulating steroids, calling behavior and innate immunity, (2) endocrine and immune correlates of calling performance, (3) behavioral and physiological correlates of parasite load, (4) thermal sensitivity of immune function, and (5) endocrine and immunomodulation by experimental exposure to acute and chronic stressors, as well as to endocrine manipulations and simulated infections. Integrated results have shown an immunoprotective role of increased steroid plasma levels during reproductive activity in calling males. Moreover, a higher helminth parasite load is associated with changes in several behavioral and physiological traits under field conditions. We also found anuran innate immunity is generally characterized by eurythermy, with maximal performance observed in temperatures close to normal and fever thermal preferendum. Moreover, the aerobic scope of innate immune response is decreased at fever thermal preferendum. Experimental exposure to stressors results in increased corticosterone plasma levels and immune redistribution, with an impact on immune function depending on the duration of the stress exposure. Interestingly, the fate of immunomodulation by chronic stressors also depends in part on individual body condition. Acute treatment with corticosterone generally enhances immune function, while prolonged exposure results in immunosuppression. Still, the results of hormonal treatment are complex and depend on the dose, duration of treatment, and the immune variable considered. Finally, simulated infection results in complex modulation of the expression of cytokines, increased immune function, activation of the Hypothalamus-Pituitary-Interrenal axis, and decreased activity of the Hypothalamus-Pituitary-Gonadal axis, as well as reduced melatonin plasma levels, suggesting that anurans have a functional Immune-Pineal axis, homologous to that previously described for mammals. These integrated and complementary approaches have contributed to a better understanding of physiological mechanisms and processes, as well as ecological and evolutionary implications of anuran immunoendocrinology.

Short-term stressors and corticosterone effects on immunity in male toads (Rhinella icterica): A neuroimmune-endocrine approach.

In the last decades, it is growing the idea that stress-induced immunomodulation is bimodal: with acute stress associated with enhancing effects while chronic stress with suppressive effects. However, the immune-endocrine interactions and its implications are often overlooked in ectotherms. We investigated the impact of corticosterone (CORT) treatment and short-term stressors on CORT plasma levels and the immunity of male toads (Rhinella icterica), using three distinct protocols: restraint, immune challenge (with lipopolysaccharide, LPS), and CORT transdermal application (TA). Our results showed increased CORT and neutrophil: lymphocyte ratio (NLR) regardless of the stress input (restraint, LPS challenge) or CORT TA. In the meantime, the bacterial killing ability (BKA) was not affected by any treatment, suggesting this immune parameter might be a more constitutive and robust response. Interestingly, the cellular immune response showed distinct patterns. Increased phagocytosis of blood leukocytes and phytohemagglutinin edema followed LPS and CORT TA (15 â€‹µg), respectively. In contrast, the phagocytosis of peritoneal leukocytes decreased after CORT TA (1 and 10 â€‹µg), indicating that short-term increases in CORT levels might impair local immune function. Such differences in cellular immunity might also be associated with CORT doses or the interaction between CORT and other immune mediators, such as melatonin, testosterone, and cytokines. Overall, our results highlight the immune-enhancing effects of the acute stress response and CORT TA, and the complexity of the immune-endocrine interaction in anurans. It also highlights the relevance of investigating distinct contexts for CORT increase arising from different situations, as well as diverse immune components for a better understanding of the stress-induced immunomodulation.

LPS-induced immunomodulation and hormonal variation over time in toads.

The inflammatory response is a complex process that relies on interactions among multiple endocrine and immune modulators. Studies incorporating time-related and integrative endocrine and immune responses to an immune challenge might shed light on the characterization of the phases of the inflammatory response in anurans. The present study investigated time-related changes (1, 3, 6, and 18 h post-challenge) in plasma corticosterone (CORT), melatonin (MEL) and testosterone (T) levels, phagocytosis percentage (PP), plasma bacterial killing ability (BKA), and neutrophil to lymphocyte ratio (NLR) following a lipopolysaccharide (LPS) immune challenge in Rhinella diptycha toads. Our results showed the response to LPS injection was characterized by increased CORT, PP, BKA, and NLR, with a concomitant decrease in plasma MEL and T. Increased CORT was more pronounced at 6 and 18 h, while increased NLR was observed only 18 h post-LPS injection. Meanwhile, plasma MEL and T decreased independently of the time post-LPS injection. Additionally, toads in better body condition showed higher BKA and PP in the LPS-treated group, regardless of the time postinjection. Our results show that toads (R. diptycha) were sensitive to the LPS challenge, mounting an inflammatory response, which started quickly (after 1 h) and developed over time and was influenced by body condition. These results demonstrate a time-related hormonal and immune variation as a consistent pattern of activation of the immune system, as well as of hypothalamic-pituitary-adrenal/interrenal and immune-pineal axes following an immune challenge more deeply studied in mammals, suggesting the evolutionary conservation of the regulatory mechanisms for tetrapod vertebrates.

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.

Time-related immunomodulation by stressors and corticosterone transdermal application in toads.

Immune responses have been mostly studied at a specific time in anuran species. However, time-changes related to immunomodulation associated with glucocorticoid (GC) alterations following stressors and GC treatment are complex. The present study describes time-related changes in immune response and corticosterone (CORT) plasma levels following restraint challenge, short, mid and long-term captivity, and CORT exogenous administration by transdermal application (TA) in Rhinella ornata toads. We observed increased neutrophil: lymphocyte ratios after restraint challenge and CORT TA, without changes following short and mid-term captivity. Plasma bacterial killing ability was sustained in all treatments, except long-term captivity, with decreased values after 90 days under such conditions. Phagocytic activity of peritoneal cells increased after mid-term captivity, and the phytohemagglutinin swelling response was impaired in those animals treated with CORT TA for 20 consecutive days. Plasma CORT levels increased or were sustained after restraint challenge (depending on initial values), decreased following mid and long-term captivity (for those animals showing high CORT in the field) and increased after 20 days of CORT TA. By performing assessments of time-changes in immune processes and CORT plasma levels in R. ornata, we demonstrate immuno-enhancing effects following restraint, short and mid-term stressors, while long-term stressors and CORT TA promoted immunosuppression in these toads.

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.

Interplay among steroids, body condition and immunity in response to long-term captivity in toads.

Stressful experiences can promote harmful effects on physiology and fitness. However, stress-mediated hormonal and immune changes are complex and may be highly dependent on body condition. Here, we investigated captivity-associated stress effects, over 7, 30, 60, and 90 days on plasma corticosterone (CORT) and testosterone (T) levels, body index, and innate immunity (bacterial killing ability and phagocytosis of peritoneal cells) in toads (Rhinella icterica). Toads in captivity exhibited elevated CORT and decreased T and immunity, without changes in body index. The inter-relationships between these variables were additionally contrasted with those obtained previously for R. schneideri, a related species that exhibited extreme loss of body mass under the same captive conditions. While T and phagocytosis were positively associated in both species, the relationship between CORT and bacterial killing ability was dependent on body index alterations. While CORT and bacterial killing ability were positively associated for toads that maintained body index, CORT was negatively associated with body index in toads that lost body mass over time in captivity. In these same toads, body index was positively associated with bacterial killing ability. These results demonstrate that steroids-immunity inter-relationships arising from prolonged exposure to a stressor in toads are highly dependent on body condition.

Differential gene expression to an LPS challenge in relation to exogenous corticosterone in the invasive cane toad (Rhinella marina).

The cane toad (Rhinella marina) is an invasive amphibian in several parts of the world. Much of the research performed on assessing the dispersal potential of invasive species has focused immunity. Invaders are predicted to rely less on pro-inflammatory immunity, allowing them to allocate energy to dispersal. Elevated stress may play a role in regulation of immune responses used by invasive species. RNA sequencing of spleen tissue from cane toads subjected to an acute LPS challenge revealed genes coding for cytokines involved in typical innate responses such as phagocytic cell recruitment, extravasation, inflammation, and lymphocyte differentiation were significantly upregulated, while toads receiving transdermal application of corticosterone in addition to an LPS injection showed downregulation of genes involved with cell mediated immunity. These results indicate hormonal changes associated with acute stress may alter investment into mounting cell-mediated or humoral responses while allowing for prolonged phagocytic innate responses in this invasive species.

Captivity effects on immune response and steroid plasma levels of a Brazilian toad (Rhinella schneideri).

Stressors can increase plasma glucocorticoid (GC) levels and decrease plasma androgen levels in different species of vertebrates. GCs can have immune-enhancing or immunosuppressive effects, which are dependent upon stress duration and intensity. The worldwide decline in amphibian populations is strongly linked to an array of different stressors. The impacts of stress on GCs, androgens, and the immune response are important to clarify and should lead to the better development of conservation strategies. The present study in adult male toads of Rhinella schneideri investigated the effects of captivity (7, 30, and 60 days) on plasma corticosterone (CORT) and plasma testosterone (T), as well as innate immune responses, specifically humoral and cell mediated responses, as indicated by bacterial killing ability (BKA) and phagocytosis by peritoneal cells, respectively. Captivity increased CORT threefold and decreased T versus controls. CORT maintained a threefold elevation throughout the captivity period, while body mass and T gradually decreased with time in captivity. BKA was lower at day 30, versus days 7 and 60, while peritoneal cell phagocytic efficiency decreased after day 30, remaining low at day 60. Moreover, phagocytosis efficiency was positively associated with T and body condition, suggesting that the effects of chronic stress on reproductive potential and immune response might be associated with the state of energetic reserves.

Corticosterone transdermal application in toads (Rhinella icterica): Effects on cellular and humoral immunity and steroid plasma levels.

Previous studies in a variety of vertebrates show stressed animals to exhibit acute increases in glucocorticoid levels, with consequences for immune modulation. Chronically elevated glucocorticoid levels are mostly associated with immunosuppression. However, there is still a paucity of knowledge regarding the amphibian physiological reaction to short- and long-term stress conditions, including glucocorticoid effects on the immune response. In order to better clarify the relationship between glucocorticoids and immune response, newly captured Brazilian toads (Rhinella icterica) (1 week in captivity) were subjected to a daily transdermal application (TA), of corticosterone or vehicle, for 30 consecutive days. Measures were made on the first day (acute stressor) and last day (chronic stressor), at timepoints 1, 6, and 12 hr post TA. A number of variables were analyzed: corticosterone plasma levels (CORT); neutrophil/lymphocyte ratio (N:L); testosterone plasma levels (T); innate immune response, as indicated by bacterial killing ability (BKA); and whole blood phagocytosis. The corticosterone TA only simulated hormonal changes associated with the acute stress response, even after 30 consecutive days of the treatment, with the increased CORT in response after corticosterone TA being evident only 1 hr postapplication and not thereafter. The general responses to corticosterone TA included increased CORT and N:L at first day of the treatment, and increased CORT and phagocytosis on the last day of the treatment. A decrease in T and BKA associated with the time in captivity was also evident, suggesting that captivity may be a chronic stressor for these toads.

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.

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.

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.

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.