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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 plasma bacterial killing ability (BKA) is modulated by the stress response in vertebrates, including amphibians. The complement system is an effector mechanism comprised of a set of proteins present in the plasma that once activated can promote bacterial lysis. Herein, we investigated whether changes in plasma BKA as a result of the acute stress response and an immune challenge are mediated by the complement system in Rhinella diptycha toads. Additionally, we investigated whether the observed changes in plasma BKA are associated with changes in plasma corticosterone levels (CORT). We subjected adult male toads to a restraint or an immune challenge (with three concentrations of Aeromonas hydrophila heat inactivated), and then evaluated the plasma BKA against A. hydrophila, in vitro. We determined the complement system activity on plasma BKA, by treating the plasma (baseline, 1 h and 24 h post-restraint, and after the immune challenge) with ethylenediaminetetraacetic acid, heat, or protease. Our results showed increased CORT 1 h and 24 h after restraint and decreased plasma BKA 24 h post-restraint. The inhibitors of the complement system decreased the plasma BKA compared with untreated plasma at all times (baseline, 1 h, and 24 h after restraint), demonstrating that the plasma BKA activity is partially mediated by the complement system. The immune challenge increased CORT, with the highest values being observed in the highest bacterial concentration, compared with control. The plasma BKA was not affected by the immune challenge but was demonstrated to be partially mediated by the complement system. Our results demonstrated that restraint and the immune challenge activated the hypothalamus-pituitary-interrenal axis, by increasing plasma CORT levels in R. diptycha. Also, our results demonstrated the complement system is participative in the plasma BKA for baseline and post-stress situations in these toads., Competing Interests: Declaration of competing interest The authors have no competing interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Amphibians are suffering population declines due to a variety of factors such as increased ultraviolet radiation, climate change, habitat loss, pathogens, and pollution, or a combination of these. Such changes are associated with a reduction in the availability of water, exposing these animals to a greater risk of desiccation. In this context, understanding how dehydration can modulate the hypothalamic-pituitary-interrenal axis (HPI) and the immune response is an imperative question to predict how stressors can affect amphibian species. We investigated whether dehydration promotes long-lasting effects on toads' ability to respond to a consecutive stressor (restraint) even if the toads are allowed to rehydrate, as well as its effects on the immune function. We also tested the hypothesis that the toads showing more severe dehydration would exhibit lower responsiveness to restraint challenge, even if the animals were allowed to rehydrate. Individuals of R. ornata were dehydrated mildly and severely. Thereafter, they were submitted to a restraint stress challenge for 1 and 24 h. Our results show that dehydration increased hematocrit and CORT in R. ornata toads. The restraint induced an acute stress response in fully hydrated toads (increased CORT and neutrophil: lymphocyte ratio). Otherwise, restraint in moistened cloth bags allowed full rehydration in previously dehydrated toads and did not induce an additional increase in CORT, but those toads sustained elevated CORT up to 24 h of restraint. Also, these animals showed increased neutrophil: lymphocyte ratio and the phagocytic activity of blood cells, even when they rehydrated during restraint. These results point to a continuous activation of the HPA during dehydration and subsequent restraint, even when they recovered from the dehydration state. Also, acute stressors seem to promote immune cell redistribution and augmentation of immune cellular function in R. ornata toads., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2023. Published by Elsevier Inc.)

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., 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.)

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.)