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.

Day Versus Night Melatonin and Corticosterone Modulation by LPS in Distinct Tissues of Toads (Rhinella Icterica).

Pathogen-associated molecular patterns modulate melatonin (MEL) production in the pineal and extra-pineal sites and corticosterone (CORT) synthesis in the adrenal/interrenal and other tissues. Both MEL and CORT play essential and complex immunomodulatory roles, controlling the inflammatory response. Given that most of what we know about these interactions is derived from mammalian studies, discovering how MEL and CORT are modulated following an immune challenge in anurans would increase understanding of how conserved these immune-endocrine interactions are in vertebrates. Herein, we investigated the modulation of MEL and CORT in plasma vs. local tissues of toads (Rhinella icterica) in response to an immune challenge with lipopolysaccharide (LPS; 2 mg/kg) at day and night. Blood samples were taken 2 hours after injection (noon and midnight), and individuals were killed for tissue collection (bone marrow, lungs, liver, and intestine). MEL and CORT were determined in plasma and tissue homogenates. LPS treatment increased MEL concentration in bone marrow during the day. Intestine MEL levels were higher at night than during the day, particularly in LPS-injected toads. Bone marrow and lungs showed the highest MEL levels among tissues. Plasma MEL levels were not affected by either the treatment or the phase. Plasma CORT levels increased in LPS-treated individuals, with an accentuated increase at night. Otherwise, CORT concentration in the tissues was not affected by LPS exposure. Modulation of MEL levels in bone marrow suggests this tissue may participate in the toad's inflammatory response assembly. Moreover, MEL and CORT levels were different in tissues, pointing to an independent modulation of hormonal concentration. Our results suggest an important role of immune challenge in modulating MEL and CORT, bringing essential insights into the hormone-immune interactions during anuran's inflammatory response.