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.

Plasma steroids and immune measures vary with restraint duration in a toad (Rhinella icterica).

Immunoenhancing effects have been widely described following acute stressors in several vertebrates, and valuable contributions have been made from studies on acute stress to understand hormonal-immune interactions. However, most studies focus on hormonal and immune responses after standardized time lapses, neglecting potential influence of duration of exposition to stressor. Herein, we investigate fluctuations of plasma hormone concentrations (corticosterone and testosterone) and immunity (neutrophil to lymphocyte ratio, phagocytosis of blood cells, and plasma bacterial killing ability) in a toad species (Rhinella icterica) in response to six different periods of exposure to restraint stress. We observed increased plasma corticosterone concentrations following restraint in all sampled times (0.5 to 48 h), with the highest values being observed during the first hour (0.5 to 1 h). Restraint-induced increases in the neutrophil to lymphocyte ratio and phagocytosis percentage were observed from the first 0.5 h, gradually increasing after that with the time of restraint. We also observed decreased testosterone plasma concentrations in response to a more prolonged restraint (24 and 48 h). No changes were observed in plasma bacterial killing ability following restraint. Together, our results demonstrate dynamic time-related hormonal and immune changes. These results point to the fact that for some species measuring hormonal and immune variables at single time points following a stressor might work better when preceded by a study of the temporal changes of the response variables to the stimuli applied. Also, time of response needs to be considered when different variables are used as proxies of stress.

Daily and LPS-induced variation of endocrine mediators in cururu toads (Rhinella icterica).

Increased plasma glucocorticoids (corticosterone - CORT, in amphibians) and melatonin (MEL) are associated with the daily activity phase and with environmental darkness, respectively. Besides, CORT and MEL also play pivotal immunomodulatory roles in several vertebrates. Herein we described the daily profile of plasma MEL and CORT for Rhinella icterica toads in captivity. Thereafter, we investigated the effects of lipopolysaccharide (LPS)-induced systemic inflammation on the production of CORT and MEL in the R. icterica. Captive toads showed CORT and MEL diurnal variation typical of nocturnal species, with increased values for CORT at ZT12 (18 h) and MEL peak at ZT18 (24 h). LPS-induced hormonal changes included increased plasma CORT and decreased ocular and plasma MEL when compared to those from toads treated with saline 2 h post-injection. Our results demonstrated the presence of a diurnal CORT and MEL variation in toads. We also showed the crosstalk between CORT and MEL during the toad's systemic inflammation in response to an immune challenge with LPS. Additionally, our results demonstrated that anuran eyes' MEL production might be regulated during the inflammatory processes.

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.

Dehydration as a stressor in toads (Rhinella ornata).

Availability of a humid environment is essential for amphibians to carry out their activities and most species are characterized by low resistance to evaporative water loss. Moreover, dehydration severely compromises amphibian locomotor and foraging performance, representing a relevant selective factor modulating the evolution of its integrative phenotype. In this way, we hypothesized that dehydration is a stressor for toads, inducing a stress response comparable to that elicited by another commonly used stress protocol: restraint challenge. We evaluated changes in plasma levels of corticosterone (CORT), hematocrit (Hct), and neutrophil:lymphocyte (N:L) ratio in adult males of Rhinella ornata, experimentally submitted to different levels of hydration (100%, 90%, and 80% of standard body mass) and to restraint challenge. Our results showed that dehydrating toads by 10% increase CORT to levels equivalent to that obtained by restraint. Moreover, toads dehydrated by 20% show a more pronounced increase in CORT, along with increased Hct and N:L ratio. In this way, we corroborated the hypothesis that dehydration triggers a pronounced stress response in R. ornata.

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.

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.