Introduction to the special issue Amphibian immunity: stress, disease and ecoimmunology

Abstract

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’.

1. Introduction

Amphibian population declines and extinctions have been reported worldwide [1,2]. In addition to environmental changes like temperature shifts, changes in rainfall regimes, loss of habitats, fragmentation, pollution, infectious diseases significantly contribute to this scenario [2–7]. In addition, there are a multitude of documented shifts in behaviour and physiology that occur in native populations dealing with climate change and infectious diseases, which have been associated with alterations in hormonal and immune modulation, possibly leading to systemic failure and death [8–13]. Among physiological changes, alterations in stress-related measures such as increased glucocorticoid levels may have critical implications on disease resistance and host survival [7,9,11,13,14].

One physiological aspect that has been recently observed in some outbreaks is the increase in amphibian plasma glucocorticoid levels, signalling a stress response. In the last decades, studies have been showing that habitat alterations and fragmentation, changes in temperature, and dehydration are associated with increased corticosterone levels, the main glucocorticoid in amphibians [8–10,15–17]. In addition, increased pathogenicity of the fungus Batrachochytrium dendrobatidis (Bd) and ranavirus has been positively correlated with corticosterone levels in some amphibian species [11,18–22], pointing to a possible glucocorticoid influence on the stress-related augmented mortality in diseased individuals.

Stress-induced immunomodulation in vertebrates has been widely investigated in the last decades, with acute stress response being associated with immunoenhancing and chronic stress with immunosuppressive effects [23–26], including in amphibians [27–33]. In addition, the general effects of stressors and their primary mediator (corticosterone) on amphibian immunity have been suggested as a potential driver of amphibian resistance/vulnerability to pathogens and habitat alterations [7,11,18]. Therefore, understanding the health-related implications of stress-induced immunomodulation and its endocrine associations in amphibians under acute/short-term and chronic/long-term conditions will bring a better idea of the consequences of environmental stressors on amphibian immunity. Moreover, determining how and which physiological mediators are involved in natural and anthropogenic-induced stress responses is of great importance for the physiology, ecology, ecoimmunology and conservation biology of amphibians.

The papers in this theme issue focus on what has recently been learned about amphibian immunity and its endocrine and environmental modulation (figure 1). Each study helps us to better define how environmental changes, stress physiology and disease resistance can affect amphibian disease outcome. Each topic covered describes the amphibian immune system and its modulation by critical biotic and abiotic factors (e.g. stress, hormone manipulation, infection, temperature and dehydration). Moreover, the papers in the issue help to identify and describe which elements of the amphibian immune system contribute to amphibian success in a changing environment. In this way, the theme issue adds novelty to the disease ecology, ecoimmunology and conservation biology fields and offers meaningful insights for future directions.

Figure 1.

A representative figure of how the papers in the present issue contribute to the distinct approaches to investigating amphibian immunity. Ig: antibodies; CORT: corticosterone; TESTO: testosterone. (Online version in colour.)

2. Amphibian immune system

Although many aspects of the amphibian immune system have been described in the last decades, most studies have been dedicated to the Xenopus genus [34]. In this issue, [35] describes the current knowledge about the amphibian immune system more widely. The authors describe molecular and cellular characteristics, tissue composition and function in anurans, urodeles and legless caecilians [35]. In addition, they point to several immune aspects related to innate and adaptive responses in amphibians, which are better characterized in Xenopus, axolotl and the giant salamander. The authors also identify areas where the lack of knowledge is particularly critical for a better understanding of infectious diseases such as chytridiomycosis. They also warn about blind reliance on transcriptomics without a reliable genome reference for rapidly evolving immune genes and without support of functional assays and kinetics of immune responses. Bringing detailed information about amphibian macrophage-lineage cells, [36] demonstrated that Xenopus laevis interleukin-34 (IL-34) macrophages and FLT3L dendritic cells share many similarities with colony-stimulating factor-1 (CSF1) macrophages, including transcriptional profiles and functional capacities. The authors also report that the IL34 macrophages and FLT3L dendritic cells have greater MHC class I, but not class II, surface expression, and are better at eliciting mixed leucocyte responses in vitro and generating in vivo memory immune responses against Mycobacterium marinum compared with X. laevis CSF1 macrophages [36].

To determine the main effects of the biotic and abiotic influences on the amphibian immune system under natural or laboratory conditions, different wild/free-living amphibian species have been used [16,27,29,33,37–41]. However, the toolbox for measuring the immune system aspects in free-living amphibian species remains limited. In this theme issue, you will find immune variables that can be measured in amphibian species. To measure cellular immunity in the blood, one can use the number of circulating white blood cells (total leucocyte count), the neutrophil to lymphocyte ratio and the phagocytosis activity of blood cells (mainly monocytes and neutrophils). Also, the inflammatory response and wound-healing processes are in vivo cellular aspects broadly used to measure innate immunity in many contexts in wild and captive amphibians [28,30,41–43]. Likewise, for non-cellular parameters of the immune system, the plasma bacterial killing ability, a measure that determines the in vitro plasma protein's ability to kill a pathogen [44], has been used in several studies [27,30,39,45]. In this issue, highlighting an important aspect of the adaptive immune response, [46] describes ontogenetic differences in IgY levels by showing that post-metamorphic frogs displayed higher IgY antibody levels than tadpoles and metamorphic frogs.

It is worth mentioning that in amphibians, the resident skin microbiota plays an antimicrobial role, helping to eliminate pathogens such as the Bd fungus and the ranavirus [47–51]. This theme issue adds to evidence that the microbiome composition is an essential component of the immune system in tadpoles and adult anurans (X. laevis and Pseudacris regilla) and salamanders (Notophthalmus viridescens) [52–54]. Specifically, [52] demonstrates that experimental reductions of the microbiome, by using antimicrobial treatments, during embryonic and larval stages reduced microbial richness and diversity and altered community composition in tadpoles (X. laevis) prior to metamorphosis, which could lead to increased disease susceptibility in amphibian tadpoles and adults. However, [52] found no evidence of increased susceptibility for Bd in this study. However, the authors propose that developing a gnotobiotic (germ-free) amphibian model system could be a handy tool for future immunological investigations. In addition, inducing resistance against emerging pathogens through prophylaxis is an exciting management strategy that may impact pathogens and their host-associated microbiome [55]. Accordingly, [54] provides evidence that treatment with an increased prophylactic concentration of fungal metabolic products and exposure duration are associated with significant increases in proportions of Bd-inhibitory host-associated bacterial taxa. These results suggest a protective prophylactic-induced shift toward microbiome members that are antagonistic to Bd and imply that exposure to a pathogen (Bd) alters the microbiome to better cope with subsequent encounters with the same pathogen (Bd).

3. Stress-related immunomodulation

Stress-driven immunomodulation has gained more attention in ectothermic vertebrates in the last decades [25,27,39,56–62], with implications on the stress-induced protective and harmful effects on several immune functions. As observed in other vertebrates, the amphibian immune system is subjected to stressors and its endocrine mediators, including glucocorticoid-induced immunomodulation [27,28,31,32,63,64]. Stress-induced immunomodulation has been described when animals are facing environmental changes (e.g. temperature variation, dehydration and pollution) [13,15,65–68] and well-established stress-induced protocols (e.g. restraint and captivity maintenance) [27,29,69–71]. Interestingly, although these aforementioned stressors are distinct, their impact on amphibian immunity is generally favourable when associated with short-term stressors [16,27,30,60], contrasting with harmful effects under long-term stress conditions [28–30]. However, complex stress-induced immunomodulation is described with increased, decreased and/or no stress impacts on anuran immunity [27,28,30,32,45].

Several papers in this issue describe stress-induced immunomodulation and causal effects of experimental increases in corticosterone on anurans. A review, [72], summarizes effects of heat and dehydration on amphibian immunity, highlighting the importance of these two stressors for amphibian survival. Studies suggest that heat and desiccation stress can activate the hypothalamus–pituitary–interrenal axis, resulting in increased corticosterone plasma levels, with possible immune suppression of some innate and lymphocyte-mediated responses. In addition, increased temperatures can alter microbial communities in amphibian skin and gut, resulting in potential dysbiosis that fosters reduced resistance to pathogens [72].

Regarding corticosterone experimental manipulation, [73] in this special issue shows that increase in plasma corticosterone levels by transdermal application for 48 days induced faster wound-healing when compared with controls in bullfrogs (Lithobates catesbeianus), pointing to the immune-enhancing effect of acute daily increases in corticosterone. The authors also show that frogs implanted with corticosterone (1 cm silastic tube filled with powdered corticosterone for 48 to 61 days) tended to heal more slowly than controls (with empty silastic tube), suggesting that the chronic increase in corticosterone can show immunosuppressive effects in these animals [73]. In addition, [53] tested the hypothesis that short- and long-term corticosterone treatment modulates the immune system in a salamander model (N. viridescens). The authors found decreased plasma bacterial killing ability and melanomacrophage centre and lower skin microbiome after long-term treatment (26 days), compared with short-term treatment (5 days), but these changes were not associated with corticosterone treatment. These results demonstrate plasma corticosterone increases may not result in immune changes in the eastern newt, at least in the investigated immune aspects [53].

4. Amphibian infectious diseases

Several infectious diseases affect amphibians [74]; some of the most common include (i) red leg syndrome: bacterial septicaemia commonly caused by Aeromonas hydrophila, resulting in erythema (redness) and oedema (swelling) on the abdomen and ventral part of the posterior members, which may progress to other parts of the body [75,76]. Infected animals may also develop ulcers, open wounds and internal organ damage. In severe cases, it can ultimately lead to death [75,76]. (ii) Chytridiomycosis: caused by the chytrid fungus B. dendrobatidis (Bd) or Batrachochytrium salamandrivorans (Bsal), which affects the skin of amphibians, interfering with their respiration and osmoregulation [51,77,78]. Infected animals can develop skin ulcers, increased skin sloughing, lethargy and loss of appetite. Bd and Bsal have been implicated in significant declines and extinctions of amphibian populations worldwide [2,79]. (iii) Ranavirosis: caused by double-stranded DNA viruses belonging to the family Iridoviridae with various symptoms in amphibians, including haemorrhaging, skin ulcers, tissue necrosis and internal organ damage [80,81]. It can cause mass die-offs of infected amphibians and has been identified as a significant threat to populations in many parts of the world [82,83]. (iv) Parasitic infections: amphibians can be affected by a range of internal and external parasites, such as nematodes (roundworms), trematodes (flatworms), mites and ticks [84,85].

Pathogens/parasites may alter host physiology to enhance pathogen/parasite proliferation, survival and transmission [85]. Complex relationships between pathogen/parasite species and their interactions with each other and their hosts might impact infectious disease outcomes [86,87]. In this theme issue, [88] reviews the importance of tolerance and resistance against the fungi Bd and Bsal, the causative agents of chytridiomycosis. Tolerance measures the ability of an organism to limit the detrimental effects caused by a given infection, protecting the host without harming the pathogen [89]. While resistance refers to the ability to limit the intensity of that infection, protecting the host at the cost of the pathogen [90]. In accordance, [88] highlights that infection tolerance has important implications for pathogen spread and maintenance, drives some species to decline, and contributes to the dilution of natural selection for tolerance and resistance. Similarly, considering the importance of tolerance and resistance, [46] investigates how changes in host immunity through ontogeny can influence interactions among co-infecting parasite species in amphibians. The researchers exposed Cuban treefrogs (Osteopilus septentrionalis) to Bd fungus and a nematode (Aplectana hamatospicula) at different life stages: tadpole, metamorphic and post-metamorphic. The results showed ontogenetic differences in IgY levels and cellular immunity but no evidence of facilitative interactions between co-infecting parasites. However, the Bd fungus decreased immunity in metamorphic frogs, making them less resistant and tolerant to infection than other life stages. These findings suggest that immune changes through ontogeny can alter host responses to parasite exposure [46].

Another crucial factor impacting the outcome of host–pathogen interactions is the host microbial communities [91–93]. The adaptive microbiome hypothesis states that exposure to a pathogen alters an organism's microbiome to better cope with subsequent pathogen encounters [94–96]. To assess this hypothesis for chytridiomycosis, [54] investigates the effects of a Bd metabolite-based prophylactic inoculation on the host microbiome composition in larval P. regilla. Their results suggest a protective prophylactic-induced shift toward microbiome members antagonistic to Bd [54]. Furthermore, diet composition significantly impacts the diversity and function of host-associated gut microbial communities [97,98]. For example, high-quality diets can improve the host's nutrient uptake and alter the metabolites used and produced by the host and its microbial communities [99,100]. In this theme issue, [101] investigates how increasing salinization in freshwaters by road de-icing salt runoff affects gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). The authors found that elevating salinity and supplementing a basic larval diet with algae increased larval growth but also increased ranavirus loads. However, larvae given algae did not exhibit elevated corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. The authors suggest that algal supplementation may reduce stress responses to infection by regulating host metabolism and endocrine function [101].

In addition, the emergence of novel pathogens in naive multi-host communities can have differential impacts on species, with some acting as reservoirs and others amplifying transmission [102,103]. However, characterizing the roles of different species in wildlife communities during infectious disease emergence is challenging as these events are often unpredictable. How species-specific attributes influence exposure, probability of infection, and pathogen intensity during the fungal pathogen (Bd) emergence was the central question of [104] in this issue. The authors used field-collected data and found that some hosts disproportionately contributed to transmission dynamics [104]. This information is particularly important when considering reintroducing amphibians back into their original communities. If supersensitive hosts are reintroduced and are unable to overcome infections, this can have a detrimental impact on the success of conservation programmes. By identifying key species responsible for disease transmission, conservationists can better understand and mitigate the risk of disease transmission, thereby improving the chances of successful reintroduction programs.

5. Ecoimmunology

Ecoimmunology is a field of study that investigates the interactions between an organism's immune system and its environment. It is an interdisciplinary field integrating concepts and methods from ecology, evolution and immunology [105,106]. Ecoimmunology seeks to understand how the immune system of an organism adapts to environmental conditions and stressors, such as temperature, food availability and exposure to pathogens [107,108]. It also examines how immune responses influence the fitness and survival of individuals, populations and species [109,110]. Research in ecoimmunology has revealed that environmental conditions and stressors can significantly impact immune function, including changes in the number and type of immune cells produced, the speed and strength of immune responses and the susceptibility of individuals to diseases [21,32,62,111].

In this theme issue, [112] investigates the links between changes in body mass of captive cane toads (Rhinella marina) and their performance in immune assays. Toads that lost weight over three months had a higher phagocytic ability of whole blood owing to increased circulating levels of phagocytic cells. Other measures of immune performance were not correlated with mass change. Considering the challenges invasive species face as they expand into novel environments with substantial seasonal changes in food availability, energy restrictions might shift their immune function toward more economical and general avenues of combating pathogens [112]. Another paper of this issue, [113] investigates a native Brazilian toad species (Rhinella icterica), to establish a positive correlation between testosterone, corticosterone and immune function in the field, and a positive correlation between testosterone and immune function in captive male toads, suggesting these steroids show an immunostimulatory effect during the reproduction in anurans [113].

Considering that amphibians face many threats, including habitat degradation, introduced species, pollutants, emerging diseases, unpredictable temperature changes and rainfall, [72] reviews amphibians' response to some natural stressors. The authors demonstrate that in addition to possibly suppressing some innate and lymphocyte-mediated responses, elevated temperatures can alter amphibian skin and gut microbial communities, resulting in possible dysbiosis that fosters reduced resistance to pathogens. Thus, extreme heat and drought stresses due to climate change might increase the vulnerability of amphibians to diseases such as chytridiomycosis and ranavirus outbreaks [72]. The authors emphasize the importance of understanding host–pathogen physiology for determining how amphibian populations deal with stressful situations and cope with diseases in a changing world.

6. Conservation biology

The conservation physiology field comprises diverse physiological topics, such as immune and endocrine systems and nutritional traits, to understand organismal and population responses to environmental change, stressors and disease resistance [21,114]. Among vertebrates, amphibians are the most threatened and declining group, with more than 40% of amphibian species being recognized as endangered [115].

Amphibians have a complex life cycle and are predicted to be among the vertebrate taxa most affected by climate change, pathogens and other stressors [7,115,116]. In particular, climate change is expected to promote changes in wetland temperatures, hydroperiod or drought regimes, among others, with amphibians being a great model in the context of monitoring and predicting the health, persistence, and distribution of free-living populations in the face of environmental changes [7,117]. Conservation efforts often focus on preventing and managing the spread of diseases and addressing other factors contributing to amphibian declines. Pathogens like Bd and Bsal are relatively new; however, even for those known for longer, such as bacteria and ranaviruses, much remains unknown about their distribution and impacts on amphibian populations.

The papers in this theme issue provide knowledge, approaches and techniques that can address the impacts of changing landscapes, experimental manipulation and infectious disease across amphibian individual and populational scales. This theme issue brings also an array of ecoimmunological tools for amphibians, such as immune measures (e.g. white blood cell profile, plasma bacterial killing ability and phagocytosis assay), stress-induced hormonal and immune modulation, microbiome composition and function, and disease resistance. These tools can be used in wildlife and experimental conditions to manage emerging pathogens, and monitor and predict disease susceptibility in amphibian populations, helping to understand amphibian survival in a changing world. Moreover, the papers in this issue bring insights into the importance of understanding the amphibian immune response (at larval and adult stages) to the diseases to understand better the mechanisms that drive differences among populations and species [36,46,52,54,88,104], which could help to predict amphibian conservation strategies.

Preventing the spread of diseases will require careful management from conservation organizations and government agencies, including increased surveillance of wild populations, restrictions on the import and trade of amphibians, assessment of water and environmental quality, and effective quarantine measures for infected individuals. Understanding the relationship between immune function and the environment is essential for conservation efforts, as environmental stressors and habitat degradation can negatively impact immune function, leaving populations more vulnerable to diseases and other threats. By studying ecoimmunology, researchers can gain insight into how to promote the health and resilience of individuals and populations in changing and challenging environments. In addition, by understanding and managing the factors that threaten biodiversity, conservation biologists can help ensure a sustainable future for both human society and the natural world.

7. Conclusions, future directions, and limitations

Despite the fact that amphibians have been used as a model system for studying the climate change effects on the immune system and disease outcomes, our capacity to establish expectations across different environmental conditions is limited. Moreover, attempts to understand the integrated immune and endocrine responses and disease susceptibility following environmental changes are scarce. The papers in this theme issue underscore that amphibians have several cellular and non-cellular immune aspects that can be used to investigate wild and captive populations in many biotic and abiotic conditions. They also underline the importance of ontogeny, emphasizing the urge to understand the biotic and abiotic effects on immunity in amphibians at different life stages. The present papers suggest that stress-driven immunomodulation in distinct amphibian species is complex and bring essential insights into understanding amphibians' physiology, ecology, evolution and conservation. There is also research covering several aspects of the amphibian immune system associated with infectious diseases, which will advance comparative investigations on amphibian immunity in the proposed topics. Since investigated variables were tested and validated for multiple species, standardizing the methods for analysing amphibian immunity across species will help to achieve a more integrative approach for future studies. However, several knowledge gaps remain, and exploring future directions while considering the limitations is essential for advancing our understanding of these intricate relationships. Below we highlight the potential future directions and acknowledge the limitations that researchers might encounter when investigating amphibian ecoimmunology, stress modulation, and disease ecology.

(a) . Future directions

(i) . Investigating the role of the microbiome

The microbiome influences immune system function and stress responses in various organisms. Understanding the composition and function of the amphibian microbiome and its interactions with the host's immune system could provide valuable insights into disease susceptibility and stress modulation.

(ii) . Integrating multi-omics approaches

Combining genomic, transcriptomic, proteomic and metabolomic data can enhance our understanding of the underlying mechanisms involved in stress responses, immune function, and disease susceptibility in amphibians. Integrating multi-omics approaches will enable a comprehensive assessment of the complex interactions between genes, environment and pathogens.

(iii) . Functional and longitudinal studies

An important aspect of stress and immune responses against pathogens is their sequential development across the different organs within an organism and over time. While multi-omics approaches are powerful, they are fully adequate to capture the dynamic response of immune cells in space and time, their expansion, contraction, migration, homing, and effector function (cell killing, phagocytosis, etc.). While functional studies are challenging in non-model species (e.g. lack of reagents such as antibodies), new technology such as single-cell transcriptomics provides a powerful new opportunity to advance these types of studies.

(iv) . Assessing the effects of anthropogenic stressors

Amphibians face numerous anthropogenic stressors, such as habitat loss, pollution, and climate change. Future research should focus on examining how these environmental stressors impact amphibian immune function, stress responses, and disease dynamics. Identifying the mechanisms by which anthropogenic stressors compromise amphibian health will aid in developing effective conservation strategies.

(v) . Examining trade-offs in immune investment

Investigating the trade-offs between immune function and other life-history traits, such as reproductive investment and growth, is crucial. Understanding how amphibians allocate limited resources to different physiological processes will provide insights into the costs and benefits of immune responses and their ecological implications.

(b) . Limitations

(i) . Sample size and statistical power

Conducting studies with large sample sizes is challenging in the field of amphibian ecoimmunology owing to logistical constraints, limited funding, and the decline of amphibian populations and risk of disturbance of small populations (e.g. reproduction). Small sample sizes limit the statistical power and generalizability of research findings.

(ii) . Environmental variability

Amphibians inhabit very diverse environments with a wide variety of ecological conditions. The dynamic nature of these environments/microenvironments can introduce confounding factors and make it difficult to discern the specific effects of stressors and diseases on amphibian immune function.

(iii) . Pathogen diversity and interactions

Amphibians are susceptible to numerous pathogens, including viruses, fungi, bacteria and parasites. The interactions between multiple pathogens and their impacts on amphibian health are complex and not yet fully understood. Investigating the interactions and co-infections among pathogens will be crucial to unravel their specific and combined effects on amphibian immune responses.

(iv) . Long-term monitoring

Long-term studies that track individual amphibians across their lifespan are needed to understand the cumulative effects of stress, disease, and environmental changes. However, such studies require substantial resources and long-term commitment, making them challenging to conduct.

Data accessibility

This article has no additional data.

Authors' contributions

V.R.A.: conceptualization, project administration, writing—original draft; J.R.: project administration, writing—review and editing; S.C.M.T.: conceptualization, project administration, writing—original draft.

All authors gave final approval for publication and agreed to be held accountable for the work performed herein.

Conflict of interest declaration

We declare we have no competing interests.

Funding

We received no funding for this project.