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Melatonin is a hormone known as an endogenous temporal marker signaling the dark phase of the day. Although the eyes seem to be the main site of melatonin production in amphibians, little information is available about the natural variation in ocular melatonin levels and its modulation following immune stimulation. We investigated the daily variation of plasma and ocular melatonin levels in bullfrogs (Lithobates catesbeianus) and their modulation following an immune stimulation with lipopolysaccharide (LPS) in yellow cururu toads (Rhinella icterica). For the daily variation, bullfrogs were bled and then euthanized for eye collection every 3 h over 24 h to determine plasma and ocular melatonin levels. We found a positive correlation between ocular and plasma melatonin levels, with maximum values at night (22 h) for both plasma and the eyes. For immune stimulation, yellow cururu toads received an intraperitoneal injection of LPS or saline solution during the day (10 h) or at night (22 h). Two hours after injection, toads were bled and euthanized for eye collection to obtain plasma and ocular melatonin levels. In addition, the liver and bone marrow were collected to investigate local melatonin modulation. Our results demonstrate that retinal light-controlled rhythmic melatonin production is suppressed while liver and bone marrow melatonin levels increase during the inflammatory assemblage in anurans. Interestingly, the LPS injection decreased only ocular melatonin levels, reinforcing the central role of the eyes (i.e., retina) as an essential organ of melatonin production, and a similar role to the pineal gland during the inflammatory response in amphibians. Together, these results point to a possible immune-pineal-ocular axis in amphibians, yet to be fully described in this group., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

The red-leg syndrome in amphibians is a condition commonly associated with the bacteria Aeromonas hydrophila and has led to population declines. However, there is little information concerning the inflammatory assemblage in infected anurans. We evaluated immune and endocrine alterations induced by stimulation with heat-killed A. hydrophila injected in Rhinella diptycha toads. Control animals were not manipulated, while the others were separated into groups that received intraperitoneal injection of 300 μl of saline or heat-killed bacteria: groups A1 (3 × 10 7 cells), A2 (3 × 10 8 cells), and A3 (3 × 10 9 cells). Animals were bled and euthanized six hours post-injection. We evaluated neutrophil: lymphocyte ratio (NLR), plasma bacterial killing ability (BKA), testosterone (T), melatonin (MEL), and corticosterone (CORT) plasma levels. Heat-killed A. hydrophila increased CORT and NLR, and decreased MEL, especially at higher concentrations. There was no effect of treatment on T and BKA. We then selected the saline and A3 groups to conduct mRNA expression of several genes including glucocorticoid receptor (GR), toll-like receptor-4 (TLR-4), interferon-γ (IFN-γ), interleukin (IL)-1β, IL-6, and IL-10. We found higher expression of IL-6, IL-1β, IL-10, and IFN-γ in group A3 compared to the saline group. These results indicate the beginning of an inflammatory assemblage, notably at the two highest concentrations of bacteria, and give a better understanding of how anurans respond to an infection within an integrated perspective, evaluating different physiological aspects. Future studies should investigate later phases of the immune response to elucidate more about the inflammation in amphibians challenged with A. hydrophila., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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'.

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 inflammatory response comprises highly orchestrated events that are conserved amongst vertebrate groups. Hepatic and splenic cytokines are major mediators of the systemic inflammatory processes. However, the liver is still neglected as an immune organ in amphibians. This study reports organ-dependent gene expression using an anuran model. We tracked mRNA levels of immune proteins [C1s (subcomponent S of the complement protein 1), IFN-γ, IL-1β, IL-6, and IL-10] at four time-points (1 h, 3 h, 6 h, and 18 h post-injection) in spleens and livers of intraperitoneal LPS-challenged (2 mg/kg) adult male toads (Rhinella diptycha) using independent samples. We found acute C1s up-regulation in the liver 1 h post-injection, with no treatment effect in the spleen. The LPS injection did not show any effect in splenic IFN-γ gene expression while eliciting only a marginal effect in the hepatic tissue. IL-1β was up-regulated in both organs, with the liver initially displaying early expression (1 h and 3 h) and the spleen taking over late expression (18 h). Both organs exhibited similar patterns for IL-6, with early up-regulation (1 h and 3 h) and late peak (18 h). Although IL-10 was early detected and up-regulated only in the liver, both organs showed up-regulation in 6 h and 18 h post-injection. Our results show an exclusive hepatic prominence in complement protein expression during the acute-phase response. Furthermore, hepatic pro-inflammatory cytokine expression was more pronounced in earliest time-points, while the spleen offers a slower and more consistent response overall. Our data provide an organ-integrative outlook into the initial hours of the inflammation in amphibians, confirming the liver's pivotal role as a regulator in the acute-phase of the inflammatory response in amphibians., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Inflammation comprises alterations in glucocorticoids (in amphibians, corticosterone-CORT) and melatonin (MEL) levels, two hormones with immunomodulatory effects on cytokine production in several vertebrates. Cytokines mediate inflammation progress differently depending on their function. While some are secreted during the acute phase of the immune response, others prevail during the resolution phase. Major efforts have been made to understand the interaction of endocrine mediators and cytokine production in endotherms, but little is known for ectotherms so far. Characterizing the stages of inflammation and their interplay with endocrine mediators is crucial for an assertive and integrative approach to amphibian physiology and ecoimmunology. Herein, we investigated CORT and MEL plasma levels as well as splenic cytokine (IL-1β, IL-6, and IL-10) mRNA levels during the progression of the inflammatory response in toads (Rhinella diptycha) in four time-points (1, 3, 6, and 18 h) after an immune challenge with lipopolysaccharide (LPS) using independent samples. Toads were responsive to LPS, with all hormones and cytokines affected by LPS. IL-1β and IL-6 were up-regulated after 1 h, but IL-1β decreased right after 3 h, while IL-6 sustained up-regulation throughout all time-points. IL-10 had not been detected until 6 h post-LPS-stimulation, when it showed up-regulation, along with a CORT increase at the same time-point. After 18 h, CORT levels were still high, and IL-1β was up-regulated again, along with up-regulated IL-6 and an IL-10 decrease. We also found positive correlations between IL-1β with IL-6 for LPS and saline groups. LPS-treated individuals showed an overall decrease in MEL plasma levels compared to saline counterparts. Our results showcase the early endocrine and molecular events of the amphibian immune response. We also report activation of the hypothalamus-pituitary-interrenal (HPI) axis during inflammation and increasing evidence for an immune-pineal axis to be described in amphibians., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

Glucocorticoids and melatonin display immunomodulatory functions, with both immune-stimulatory and suppressor effects, depending on the context. While their immune properties are well-explored in mammals, there are still few studies on this immune-endocrine interaction in an inflammatory context in amphibians, all of them under captivity conditions, which can constitute a stressor for these animals. Evaluating how amphibians react to an immune challenge in the field would reveal relevant information regarding how immune-physiological parameters are modulated in natural conditions. This study aimed to investigate the effects of lipopolysaccharide (LPS) injection in male toads (Rhinella icterica) recently captured in their natural habitat in the Atlantic Forest at two different times of the day. We evaluated: splenic cytokines mRNA (interleukin [IL]-1β, IL-6, IL-10, interferon-γ) and complement system protein (C1s), plasma bacterial killing ability (BKA), plasma corticosterone (CORT), melatonin (MEL), and testosterone (T) levels, and neutrophil to lymphocyte ratio (NLR), two hours post-injections. LPS-injection increased NLR, the gene expression of IL-1β, and less evidently CORT levels independently of the time of the day. These results evidence LPS-induced inflammation, similarly observed in toads in captivity. Saline and LPS-injected toads showed a positive correlation between IL-1β and IL-6, both cytokines with pro-inflammatory effects. Also, CORT was negatively associated with T, suggesting inhibition of the hypothalamus-pituitary-gonadal axis in the LPS-stimulated group. Our results are associated with the first stage of the inflammatory assemblage. Studies evaluating further steps of this process might lead to a better understanding of the immune-endocrine relations in amphibians., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The immune-endocrine interactions following an immune challenge have been demonstrated in amphibians. When considering immune challenges, the immune-endocrine implications can vary with the injection time (day or night), a pattern not explored in amphibians. We investigated the immune response following a lipopolysaccharide - LPS injection, measured as plasma bacterial killing ability - BKA, phagocytosis of blood cells - PP, and neutrophil to lymphocyte ratio - NLR, splenic proinflammatory cytokines mRNA (IL-1β and IL-6), and also endocrine mediators (corticosterone - CORT and melatonin - MEL plasma levels) in Rhinella icterica adult male toads injected at day (10 am) or night (10 pm). LPS induced increases in CORT, NLR, PP, and IL-1β mRNA compared with amphibian phosphate-buffer saline-injected individuals. For plasma CORT, the response was more pronounced during the night. While for the PP and IL-1β mRNA, the effect was more evident during the day. For NLR, the increase happened at both times, day and night, in the LPS-injected toads. Meanwhile, no changes were observed in BKA, IL-6 mRNA, and MEL levels. Overall, our results demonstrated an LPS-induced inflammatory response in R. icterica toads, characterized by higher PP, NLR, and IL-1β mRNA, followed by activation of the hypothalamic-pituitary-interrenal axis (higher CORT levels). The time in which the toads received the LPS injection affected the endocrine and immune mediators. The higher CORT and lower inflammatory response at night suggested a potential functional interaction between CORT and immune reactivity associated with the differences in night vs. day in R. icterica toads. These results highlight the relevance of investigating different injection times and mechanistic pathways to understand LPS-induced immunomodulation in anurans., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Mining is one of the main activities that drive the economy of Brazil. Mining activity is associated with risk of contamination of environment and local fauna by metals. Amphibians have a life cycle that requires a transition between aquatic and terrestrial environments, increasing their vulnerability to metal contamination in the water and substrate. Metals are ubiquitous, with high bioaccumulative and biomagnifying potential, and may lead to immune and endocrine disruption. In this study, we analyzed two different components of the innate immune response, bacterial killing ability (BKA) and phytohemagglutinin edema (PHA), and two stress biomarkers, corticosterone plasma levels (CORT) and the neutrophil to lymphocyte ratio (N:L), of toads (Rhinella diptycha) living in places contaminated by metals. Blood samples were collected pre- and post-restraint (1h), followed by an immune challenge with PHA and tissue collection (liver, spleen, and kidneys). Toads liver metal bioaccumulation did not correlate with the immune response or stress biomarkers. Post-restraint, animals had increased CORT and reduced BKA, independently of the collection site, and these variables were not correlated with liver metal bioaccumulation. Interestingly, toads with the larger spleen (immune organ) showed increased N:L post-restraint and greater edema after the PHA challenge. Our results indicate that toads living in metal-contaminated environments responded to acute stressor, activating the hypothalamic-pituitary-interrenal axis and the immune response. Keep tracking the physiological variables of these animals and the presence of metals in the environment and tissues should provide valuable health status indicators for the population, which is vital for proposing amphibian conservation strategies in these areas., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Glucocorticoids and melatonin (MEL) show integrated and complex immunomodulatory effects, mostly described for endotherms, yet underexplored in amphibians. In this context, the RT-qPCR of molecules mediating inflammatory processes in amphibians is a valuable tool to explore the relationships among molecular biology, endocrine mediators, and immune response in these animals. In this study, toads ( Rhinella diptycha ) received an intraperitoneal saline injection or lipopolysaccharide (LPS; 2 mg/kg). Six hours post-injection, we analyzed plasma corticosterone (CORT) and MEL levels and pro- and anti-inflammatory molecules (IL-1β, IL-6, IL-10, IFN-γ, and C1s). We found increased CORT and decreased MEL levels in response to LPS. Also, IL-1β, IL-6, and IL-10 were upregulated in LPS-injected toads compared with saline-injected toads. Overall, our results demonstrate an LPS-induced inflammatory response with endocrine and immune modulation in R. diptycha toads, exhibiting expected patterns for an inflammatory stimulus within this time frame (6 h post-injection). Toads were responsive to LPS by secreting different cytokines, such as proinflammatory cytokines IL-1β and IL-6, related to immune cell attraction to inflammatory sites and the anti-inflammatory cytokine IL-10, which limits the rate of leukocyte infiltration, inflammation, and downregulates the expression of proinflammatory cytokines. Increased circulating CORT levels are probably associated with the activation of the hypothalamus-pituitary-interrenal axis by the LPS and the endocrine actions of IL-6. Furthermore, decreased circulating MEL levels are likely due to inhibited MEL secretion by the pineal gland by inflammatory stimuli, indicating the activation/existence of the immune-pineal axis in amphibians., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology.)

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., (© 2021 Wiley Periodicals LLC.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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 ​μg), respectively. In contrast, the phagocytosis of peritoneal leukocytes decreased after CORT TA (1 and 10 ​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., Competing Interests: The authors have no competing interests to declare., (© 2021 The Authors.)

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., (© 2020 Wiley Periodicals LLC.)

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.

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., Competing Interests: The authors have declared that no competing interests exist.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (© 2017 Wiley Periodicals, Inc.)

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., (© 2017 Wiley Periodicals, Inc.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We study a modified version of the stochastic susceptible-infected-refractory-susceptible (SIRS) model by employing a nonlinear (exponential) reinforcement in the contagion rate and no diffusion. We run simulations for complete and random graphs as well as d-dimensional hypercubic lattices (for d=3,2,1). For weak nonlinearity, a continuous nonequilibrium phase transition between an absorbing and an active phase is obtained, such as in the usual stochastic SIRS model [Joo and Lebowitz, Phys. Rev. E 70, 036114 (2004)]. However, for strong nonlinearity, the nonequilibrium transition between the two phases can be discontinuous for d>or=2, which is confirmed by well-characterized hysteresis cycles and bistability. Analytical mean-field results correctly predict the overall structure of the phase diagram. Furthermore, contrary to what was observed in a model of phase-coupled stochastic oscillators with a similar nonlinearity in the coupling [Wood, Phys. Rev. Lett. 96, 145701 (2006)], we did not find a transition to a stable (partially) synchronized state in our nonlinearly pulse-coupled excitable elements. For long enough refractory times and high enough nonlinearity, however, the system can exhibit collective excitability and unstable stochastic oscillations.

We study the response properties of d-dimensional hypercubic excitable networks to a stochastic stimulus. Each site, modeled either by a three-state stochastic susceptible-infected-recovered-susceptible system or by the probabilistic Greenberg-Hastings cellular automaton, is continuously and independently stimulated by an external Poisson rate h. The response function (mean density of active sites rho versus h) is obtained via simulations (for d=1,2,3,4) and mean-field approximations at the single-site and pair levels (for all d). In any dimension, the dynamic range and sensitivity of the response function are maximized precisely at the nonequilibrium phase transition to self-sustained activity, in agreement with a reasoning recently proposed. Moreover, the maximum dynamic range attained at a given dimension d is a decreasing function of d.