Currently, effects of increased atmospheric temperature, in the context of ongoing climate change, have been investigated in multiple organisms and levels of biological organization. While there has been a focus on the impacts of increased mean temperature, an emergent and equally important point is the consequences of recurrent exposure to extreme temperature events, simulating heat waves. This study investigated the effects of serial exposure to high temperatures on immune and endocrine variables before and after exposure to an acute secondary stressor in bullfrogs (Lithobates catesbeianus). Adult males were divided into three groups and subjected to three thermal regimes: Control (C; constant 22°C); Experimental 1 (E1; kept at 22°C and exposed to 4 days of 30°C every 16 days); and Experimental 2 (E2; kept at 22°C and exposed to 4 days of 30°C every 6 days). Blood samples were collected on the last day of key extreme heat events. Two weeks after the last extreme heat event, animals were subjected to restraint stress (1h) and sampled again. Blood samples were used to determine neutrophil: lymphocyte ratio, plasma bacterial killing ability, as well as corticosterone and plasma testosterone levels. Overall, we found exposure to extreme heat events did not affect immune and endocrine variables over time. Meanwhile, the previous exposure to extreme heat events modulated the responsiveness to restraint. The amplitude of increased corticosterone plasma levels and neutrophil: lymphocyte ratio in response to restraint decreased with the number of previous exposures to extreme heat events. These results suggest that exposure to extreme climatic events has hidden effects on bullfrog's stress response, expressed as diminished reactive scope to a novel stressor. This represents a highly deleterious facet of climate change since diminished responsiveness prevents proper coping with wildlife challenges.

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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 cytokines (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 18h) 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 1h, but IL-1β decreased right after 3h, while IL-6 sustained up-regulation throughout all time points. IL-10 had not been detected until 6h post-LPS-stimulation, when it showed up-regulation, along with a CORT increase at the same time-point. After 18h, 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 HPI axis during inflammation and increasing evidence for an immune-pineal axis to be described in amphibians.

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