Vania Regina Assis

The timing of an animal's activity affects its survival and reproduction because timing determines the nature of every interaction an organism has with its environment. Although the timing of activity may depend on aspects of the individual (e.g., body mass, reproductive status, experience) or the nature of the environment (e.g., temperature, precipitation, abundance of congeners), we lack studies that evaluate timing of large numbers of conspecifics spanning an array of environmental conditions. We quantified the timing of activity onset (measured as the time when an individual was captured) for 1749 capture events of 716 individual white‐footed mice ( Peromyscus leucopus ) sampled in 2022 and 2023 at eight sites in the National Ecological Observatory Network (NEON) across the upper midwestern and eastern United States. We found strong effects of body mass, sex, and prior capture experience on activity onset. Larger individuals, reproductive females, and individuals with previous capture experience were all captured earlier in the night. Extrinsic factors also influenced activity onset: Animals were captured earlier when temperatures were warmer, later on nights with precipitation, and earlier as the density of congeners increased. Individual traits interacted with extrinsic factors to influence activity onset, as the shift to earlier capture with increasing capture experience was greatest for smaller individuals, and only reproductive females were active earlier (not males or nonreproductive females). Overall, our results indicated a strong role for individual and environmental factors on the timing of activity onset: Individuals modified the timing of activity onset in response to environmental conditions, used experience to potentially increase resource acquisition (earlier activity following initial capture), and shifted activity in more‐competitive environments (earlier activity when density of conspecifics was high). Our results provide broad evidence of learning by wild rodents across a large extent of the species' geographic range, suggesting that use of experience to modify behavior is common for animals in natural environments. Moreover, our results indicate that traits that vary among individuals (e.g., sex, age) and within individuals (experience) are essential for predicting changes in individual behavior as well as potential changes in interactions mediated by individual activity (e.g., competition, predation, and disease dynamics).

Amphibians face threats from emerging bacterial, viral, and fungal pathogens, and their innate immune response plays a key role in early defense. The liver is a central immune organ that produces acute-phase proteins (APPs) and complement system proteins, which are essential for early pathogen neutralization and a positive disease outcome. Here, we investigated the temporal dynamics of hepatic gene expression for APPs [C-reactive protein (CRP), serum amyloid A (SAA), fibrinogen-like protein 1 (FGL1)] and complement components (C1s, C3, C4, C5, C9) in Rhinella diptycha toads injected with lipopolysaccharide (LPS) or saline. Using RT-qPCR at four time points post-injection (1h, 3h, 6h, and 18h), we detected a significant effect of LPS on C1s expression and a treatment x time interaction for C4, C5, and C9, indicating time-dependent activation of different complement proteins. In contrast, we found no evidence for LPS-induced changes in APPs expression. However, several APPs were positively correlated with complement components, suggesting coordinated regulation even in the absence of differential expression. Our findings provide a temporal analysis of hepatic acute-phase responses to LPS in Rhinella diptycha toads, unraveling a complex network of complement system activation. By showing that complement components, rather than APPs, dominate the early hepatic response to a bacterial antigen in a widespread Neotropical toad species, our study provides a temporal and mechanistic baseline for future investigations of amphibian innate immunity and underscores the need for future work to determine how these early pathways respond to different pathogens and contribute to variation in disease outcomes among amphibians.

Synopsis: Amphibians, like other vertebrates, respond to stressors through the activation of the hypothalamic-pituitary-interrenal (HPI) axis, leading to elevated levels of glucocorticoids in the bloodstream. The amphibian HPI axis is functionally analogous to the mammalian hypothalamic-pituitary-adrenal (HPA) axis, coordinating the stress response via glucocorticoid release. Among these, corticosterone (CORT) acts as the principal downstream effector hormone, exerting widespread effects on various physiological systems. As seen in many other vertebrates, physiologically increased CORT levels are commonly associated with immune modulation, which might enhance or suppress the immune response. This immune outcome is influenced by several factors, including the duration and intensity of the stressors, the body condition of individuals, life history, and species-specific traits. Here, we provide a literature review on the role of stressors and CORT in amphibian immunity, including studies conducted in natural environments and controlled settings. These studies involve standardized stress protocols (i.e., restraint, captivity, and exogenous hormone treatment), along with “in vivo” and “in vitro” immune assays. Overall, CORT levels and their effects on immunity are highly variable, yet they do not act in isolation. There is significant interaction between CORT and other hormones, such as testosterone and melatonin, which further influences the immune response in amphibians. This interplay underscores the complexity of the stress-immune relationship and suggests that a holistic approach is essential to fully understand the impact of stressors on amphibian health and conservation.

Mice in the genus Peromyscus are abundant and geographically widespread in North America, serving as reservoirs for zoonotic pathogens, including Borrelia burgdorferi (B. burgdorferi), the causative agent of Lyme disease, transmitted by Ixodes scapularis ticks. While the white‐footed mouse (Peromyscus leucopus (P. leucopus)) is the primary reservoir in the United States, the deer mouse (P. maniculatus), an ecologically similar congener, rarely transmits the pathogen to biting ticks. Understanding the factors that allow these similar species to serve as a poor and competent reservoir is critical for understanding tick‐borne disease ecology and epidemiology, especially as climate change expands the habitats where ticks can transmit pathogens. Our study investigated immunological differences between these rodent species. Specifically, we compared the expression of six immune genes (i.e., TLR‐2, IFN‐γ, IL‐6, IL‐10, GATA‐3, TGF‐β) broadly involved in bacterial recognition, elimination, and/or pathology mitigation in ear biopsies collected by the National Ecological Observatory Network (NEON) as part of their routine surveillance. A principal components analysis indicated that immune gene expression in both species varied in two dimensions: TLR2, IFN‐γ, IL‐6, and IL‐10 (comprising PC1) and TGF‐β and GATA3 (comprising PC2) expression tended to covary within individuals. However, when we analyzed expression differences of each gene singly between species, P. maniculatus expressed more TLR2, IL‐6, and IL‐10 but less IFN‐γ and GATA3 than P. leucopus. This immune profile could partly explain why P. leucopus is a better reservoir for bacterial pathogens such as B. burgdorferi.

Geographic variation in host immunity could have major influences on disease dynamics, including zoonotic forms that affect humans. Such variation in immunity could be driven by variation in climate, either directly or, more likely, indirectly via resource availability. We compared the immune gene expression of wild Peromyscus leucopus mice, the primary reservoir for the bacterium that causes Lyme disease, Borrelia burgdorferi , among eight sites spanning 1,400 km of the northeastern United States. We discovered that climate conditions at sites strongly predicted immunity to the most common zoonotic pathogen in the U.S.: mice from warmer, wetter sites were more prepared to resist B. burgdorferi infections. Our results reveal a novel pathway by which climate change could affect pathogen spillover or zoonotic epidemics generally.

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.

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’.

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’.

Model organisms have long been used to characterize the vertebrate immune system, which is responsible for protecting the host from mechanical and pathogen-induced injuries, pathology, and disease development ( Janeway et al. 2001). As a result, today we have a deep understanding of how vertebrate defensive responses can be mediated by innate (i.e., non-specific) or adaptive (i.e., specific) mechanisms, or a mixture of both. Innate responses involve a generalized activation of immune mechanisms that can operate in response to any infectious agent (i.e., they are actionable to a diverse range of pathogens and parasites; Pham and Schneider 2008). In contrast, adaptive responses entail an activation of a subset of cells and/or systems to defend a host against a precise pathogen. While many of these mechanisms are highly conserved across vertebrate classes, intriguing distinctions have been revealed by research on non-model organisms ( Demas and Nelson 2012). Ecoimmunology, or ecological immunology, emerged as a research area in the late 1990s and early 2000s ( Sheldon and Verhulst 1996; Norris and Evans 2000; Zuk and Stoehr 2002; Brock et al. 2014; Downs et al. 2014). In this field, researchers seek to understand the factors leading to variation in the function of the immune system and how this variation contributes to disease susceptibility in a wide variety of taxonomic groups ( Sheldon and Verhulst 1996; Norris and Evans 2000; Zuk and Stoehr 2002; Brock et al. 2014; Downs et al. 2014). Since its inception, ecoimmunological studies have predominantly focused on birds, with relatively fewer studies focused on other taxonomic groups ( Martin et al. 2003; Ashley et al. 2012; Evans et al. 2015; Haase et al. 2016; Taves et al. 2017; Tieleman 2018). More recently, however, there has been an expansion in the diversity of taxa studied from an ecoimmunological perspective, with studies ranging from plants to invertebrates, ectothermic vertebrates, and non-model (i.e., lab rodents) mammalian species ( Rolff and Siva-Jothy 2003; Zimmerman et al. 2010; Ardia et al. 2012; Fisher et al. 2012; Pijanowski et al. 2015; Titon et al. 2019; Sueiro et al. 2020; Zimmerman 2020). In addition, there has been a closer examination and optimization of the immune methods and techniques that are needed to conduct ecoimmunology studies in diverse organisms (e.g., Claunch et al. 2022).