Amphibian Immunity Research Articles

Screening and computational characterization of novel antimicrobial cathelicidins from amphibian transcriptomic data

As cold-blooded organisms living in damp and dark environments, amphibians have evolved robust defense mechanisms to protect themselves from predators and infections. Among the wide repertoire of bioactive compounds they produce are antimicrobial peptides (AMPs), which are required as part of innate immunity. One important class of AMPs is cathelicidins, known for their broad-spectrum activity against pathogens and their immunoregulatory roles. However, despite their promising biomedical potential and the increasing availability of omics data, few cathelicidins have been studied in amphibians, mostly through conventional experimental techniques. Here, we present 210 novel cathelicidin sequences from amphibian transcriptomes, identified through a comprehensive computational pipeline, which employed HMMER and BLAST tools to screen cathelicidin domains. These sequences reveal a typical tripartite domain architecture that was confirmed by SignalP and InterProScan analysis. Phylogenetic inference with IQ-TREE classified the sequences into six categories based on evolutionary relationships. Compared to cathelicidins from other vertebrates, amphibian mature peptides exhibit longer average lengths (around 50 amino acids), fewer aromatic and hydrophobic residues, and reduced thermal stability. Furthermore, these amphibian cathelicidins were characterized for their physicochemical and biological properties, revealing significant antimicrobial potential with lower hemolytic capability, especially in anurans, which suggests a balance between their antimicrobial and hemolytic activities predicted through AMPlify, ampir, AmpGram, and HemoPI. Secondary structure estimations, including three-dimensional modeling using AlphaFold2, indicate that amphibian cathelicidins predominantly feature α-helices and coils. Some representative models also display a high α-helix composition with amphipathic topology, facilitating interactions with simulated bacterial membranes as assessed by the PPM approach. Thus, these findings highlight the functional role of cathelicidins in amphibian immunity and their promising biomedical applicability, emphasizing the importance of applying computational methods to expand the scope and reveal the diverse landscape of cathelicidins across vertebrates.

Dietary carotenoid supplementation has long-term and community-wide effects on the amphibian skin microbiome.

The amphibian skin microbiome plays a crucial role in host immunity and pathogen defence, yet we know little about the environmental drivers of skin microbial variation across host individuals. Inter-individual variation in the availability of micro-nutrients such as dietary carotenoids, which are involved in amphibian immunity, may be one factor that influences skin microbial assembly across different life history stages. We compared the effect of four carotenoid supplementation regimes during different life stages on the adult skin microbiome using a captive population of the critically endangered southern corroboree frog, Pseudophryne corroboree. We applied 16S rRNA sequencing paired with joint-species distribution models to examine the effect of supplementation on taxon abundances. We found that carotenoid supplementation had subtle yet taxonomically widespread effects on the skin microbiome, even 4.5 years post supplementation. Supplementation during any life-history stage tended to have a positive effect on the number of bacterial taxa detected, although explanatory power was low. Some genera were sensitive to supplementation pre-metamorphosis, but most demonstrated either additive or dominant effects, whereby supplementation during one life history stage had intermediate or similar effects, respectively, to supplementation across life. Carotenoid supplementation increased abundances of taxa belonging to lactic acid bacteria, including Lactococcus and Enterococcus, a group of bacteria that have previously been linked to protection against the amphibian fungal pathogen Batrachochytrium dendrobatidis (Bd). While the fitness benefits of these microbial shifts require further study, these results suggest a fundamental relationship between nutrition and the amphibian skin microbiome which may be critical to amphibian health and the development of novel conservation strategies.

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

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'.

Do changes in body mass alter white blood cell profiles and immune function in Australian cane toads (Rhinella marina)?

Variation in food resources can result in dramatic fluctuations in the body condition of animals dependent on those resources. Decreases in body mass can disrupt patterns of energy allocation and impose stress, thereby altering immune function. In this study, we investigated links between changes in body mass of captive cane toads (Rhinella marina), their circulating white blood cell populations, and their performance in immune assays. Captive toads that lost weight over a three-month period had increased levels of monocytes and heterophils and reduced levels of eosinophils. Basophil and lymphocyte levels were unrelated to changes in mass. Because individuals that lost mass had higher heterophil levels but stable lymphocyte levels, the ratio of these cell types was also higher, partially consistent with a stress response. Phagocytic ability of whole blood was higher in toads that lost mass, owing to increased circulating levels of phagocytic cells. Other measures of immune performance were unrelated to mass change. These results highlight the challenges faced by invasive species as they expand their range into novel environments which may impose substantial seasonal changes in food availability that were not present in the native range. Individuals facing energy restrictions may shift their immune function towards more economical and general avenues of combating pathogens. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Selection of an anti-pathogen skin microbiome following prophylaxis treatment in an amphibian model system.

With emerging diseases on the rise, there is an urgent need to identify and understand novel mechanisms of prophylactic protection in vertebrate hosts. Inducing resistance against emerging pathogens through prophylaxis is an ideal management strategy that may impact pathogens and their host-associated microbiome. The host microbiome is recognized as a critical component of immunity, but the effects of prophylactic inoculation on the microbiome are unknown. In this study, we investigate the effects of prophylaxis on host microbiome composition, focusing on the selection of anti-pathogenic microbes contributing to host acquired immunity in a model host-fungal disease system, amphibian chytridiomycosis. We inoculated larval Pseudacris regilla against the fungal pathogen Batrachochytrium dendrobatidis (Bd) with a Bd metabolite-based prophylactic. Increased prophylactic concentration and exposure duration were associated with significant increases in proportions of putatively Bd-inhibitory host-associated bacterial taxa, indicating a protective prophylactic-induced shift towards microbiome members that are antagonistic to Bd. Our findings are in accordance with the adaptive microbiome hypothesis, where exposure to a pathogen alters the microbiome to better cope with subsequent pathogen encounters. Our study advances research on the temporal dynamics of microbiome memory and the role of prophylaxis-induced shifts in microbiomes contributing to prophylaxis effectiveness. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Diet- and salinity-induced modifications of the gut microbiota are associated with differential physiological responses to ranavirus infection in Rana sylvatica.

Greater knowledge of how host-microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae-a pattern consistent with increased growth and fat deposition in mammals-that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host-pathogen system. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Effects of exogenous elevation of corticosterone on immunity and the skin microbiome of eastern newts (Notophthalmus viridescens).

The amphibian chytrid fungus, Batrachochytrium salamandrivorans (Bsal) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

The amphibian immune system.

Amphibians are at the forefront of bridging the evolutionary gap between mammals and more ancient, jawed vertebrates. Currently, several diseases have targeted amphibians and understanding their immune system has importance beyond their use as a research model. The immune system of the African clawed frog, Xenopus laevis, and that of mammals is well conserved. We know that several features of the adaptive and innate immune system are very similar for both, including the existence of B cells, T cells and innate-like T cells. In particular, the study of the immune system at early stages of development is benefitted by studying X. laevis tadpoles. The tadpoles mainly rely on innate immune mechanisms including pre-set or innate-like T cells until after metamorphosis. In this review we lay out what is known about the innate and adaptive immune system of X. laevis including the lymphoid organs as well as how other amphibian immune systems are similar or different. Furthermore, we will describe how the amphibian immune system responds to some viral, bacterial and fungal insults. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Evolutionary ecology of host competence after a chytrid outbreak in a naive amphibian community.

Naive multi-host communities include species that may differentially maintain, transmit and amplify novel pathogens; therefore, we expect species to fill distinct roles during infectious disease emergence. Characterizing these roles in wildlife communities is challenging because most disease emergence events are unpredictable. Here, we used field-collected data to investigate how species-specific attributes influenced the degree of exposure, probability of infection, and pathogen intensity, during the emergence of the fungal pathogen Batrachochytrium dendrobatidis (Bd) in a highly diverse tropical amphibian community. Our findings confirmed that ecological traits commonly evaluated as correlates of decline were positively associated with infection prevalence and intensity at the species level during the outbreak. We identified key hosts that disproportionally contributed to transmission dynamics in this community and found a signature of phylogenetic history in disease responses associated with increased pathogen exposure via shared life-history traits. Our findings establish a framework that could be applied in conservation efforts to identify key species driving disease dynamics under enzootics before reintroducing amphibians back into their original communities. Reintroductions of supersensitive hosts that are unable to overcome infections will limit the success of conservation programmes by amplifying the disease at the community level. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Amphibian infection tolerance to chytridiomycosis.

Animal defences against infection involve two distinct but complementary mechanisms: tolerance and resistance. Tolerance measures the animal's ability to limit detrimental effects from a given infection, whereas resistance is the ability to limit the intensity of that infection. Tolerance is a valuable defence for highly prevalent, persistent or endemic infections where mitigation strategies based on traditional resistance mechanisms are less effective or evolutionarily stable. Selective breeding of amphibians for enhanced tolerance to Batrachochytrium spp. has been suggested as a strategy for mitigating the impacts of the fungal disease, chytridiomycosis. Here, we define infection tolerance and resistance in the context of chytridiomycosis, present evidence for variation in tolerance to chytridiomycosis, and explore epidemiological, ecological and evolutionary implications of tolerance to chytridiomycosis. We found that exposure risk and environmental moderation of infection burdens are major confounders of resistance and tolerance, chytridiomycosis is primarily characterized by variation in constitutive rather than adaptive resistance, tolerance is epidemiologically important in driving pathogen spread and maintenance, heterogeneity of tolerance leads to ecological trade-offs, and natural selection for resistance and tolerance is likely to be dilute. Improving our understanding of infection tolerance broadens our capacity for mitigating the ongoing impacts of emerging infectious diseases such as chytridiomycosis. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

A perspective into the relationships between amphibian (Xenopus laevis) myeloid cell subsets.

Macrophage (Mϕ)-lineage cells are integral to the immune defences of all vertebrates, including amphibians. Across vertebrates, Mϕ differentiation and functionality depend on activation of the colony stimulating factor-1 (CSF1) receptor by CSF1 and interluekin-34 (IL34) cytokines. Our findings to date indicate that amphibian (Xenopus laevis) Mϕs differentiated with CSF1 and IL34 are morphologically, transcriptionally and functionally distinct. Notably, mammalian Mϕs share common progenitor population(s) with dendritic cells (DCs), which rely on fms-like tyrosine kinase 3 ligand (FLT3L) for differentiation while X. laevis IL34-Mϕs exhibit many features attributed to mammalian DCs. Presently, we compared X. laevis CSF1- and IL34-Mϕs with FLT3L-derived X. laevis DCs. Our transcriptional and functional analyses indicated that indeed the frog IL34-Mϕs and FLT3L-DCs possessed many commonalities over CSF1-Mϕs, including transcriptional profiles and functional capacities. Compared to X. laevis CSF1-Mϕs, the IL34-Mϕs and FLT3L-DCs possess greater surface major histocompatibility complex (MHC) class I, but not MHC class II expression, were better at eliciting mixed leucocyte responses in vitro and generating in vivo re-exposure immune responses against Mycobacterium marinum. Further analyses of non-mammalian myelopoiesis akin to those described here, will grant unique perspectives into the evolutionarily retained and diverged pathways of Mϕ and DC functional differentiation. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Ontogeny of immunity and potential implications for co-infection.

Immunity changes through ontogeny and can mediate facilitative and inhibitory interactions among co-infecting parasite species. In amphibians, most immune memory is not carried through metamorphosis, leading to variation in the complexity of immune responses across life stages. To test if the ontogeny of host immunity might drive interactions among co-infecting parasites, we simultaneously exposed Cuban treefrogs (Osteopilus septentrionalis) to a fungus (Batrachochytrium dendrobaditis, Bd) and a nematode (Aplectana hamatospicula) at tadpole, metamorphic and post-metamorphic life stages. We measured metrics of host immunity, host health and parasite abundance. We predicted facilitative interactions between co-infecting parasites as the different immune responses hosts mount to combat these infectious are energetically challenging to mount simultaneously. We found ontogenetic differences in IgY levels and cellular immunity but no evidence that metamorphic frogs were more immunosuppressed than tadpoles. There was also little evidence that these parasites facilitated one another and no evidence that A. hamatospicula infection altered host immunity or health. However, Bd, which is known to be immunosuppressive, decreased immunity in metamorphic frogs. This made metamorphic frogs both less resistant and less tolerant of Bd infection than the other life stages. These findings indicate that changes in immunity altered host responses to parasite exposures throughout ontogeny. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Testosterone immunomodulation in free-living and captive Rhinella icterica male toads.

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'.

Contrasting effects of transdermal and implant corticosterone treatments in the American bullfrog wound healing.

Glucocorticoid (GC) release is triggered by adverse stimuli that activate the hypothalamus-pituitary-adrenal/interrenal axis. Glucocorticoids may enhance or suppress immune functions depending on the level of elevation. In this study, we investigated the effects of transient and chronic increase of corticosterone (CORT) on the wound healing of the American bullfrog. Frogs were submitted to a daily transdermal hormonal application that acutely elevated CORT plasma levels, or vehicle as a control. Other frogs were surgically implanted with a silastic tube filled with CORT that resulted in chronic elevation of CORT plasma levels or received empty implants as a control. A dermal biopsy was performed to create a wound and was photographed every 3 days. Individuals treated with transdermal CORT started healing faster than their control 32 days after the biopsy. Frogs that received CORT implants tended to heal slower than control subjects. Plasma bacterial killing ability was not affected by treatment, which reinforces the constitutive nature of this innate immune trait. By the end of the experiment, frogs from the acute CORT treatment had smaller wounds compared with those receiving the CORT-filled implants, highlighting the differential effects of acute (immunoenhancing) and chronic (immunosuppressive) elevation of CORT plasma levels. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Towards the generation of gnotobiotic larvae as a tool to investigate the influence of the microbiome on the development of the amphibian immune system.

The immune equilibrium model suggests that exposure to microbes during early life primes immune responses for pathogen exposure later in life. While recent studies using a range of gnotobiotic (germ-free) model organisms offer support for this theory, we currently lack a tractable model system for investigating the influence of the microbiome on immune system development. Here, we used an amphibian species (Xenopus laevis) to investigate the importance of the microbiome in larval development and susceptibility to infectious disease later in life. We found that experimental reductions of the microbiome during embryonic and larval stages effectively reduced microbial richness, diversity and altered community composition in tadpoles prior to metamorphosis. In addition, our antimicrobial treatments resulted in few negative effects on larval development, body condition, or survival to metamorphosis. However, contrary to our predictions, our antimicrobial treatments did not alter susceptibility to the lethal fungal pathogen Batrachochytrium dendrobatidis (Bd) in the adult life stage. While our treatments to reduce the microbiome during early development did not play a critical role in determining susceptibility to disease caused by Bd in X. laevis, they nevertheless indicate that developing a gnotobiotic amphibian model system may be highly useful for future immunological investigations. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

The amphibian invitrome: Past, present, and future contributions to our understanding of amphibian immunity

Many amphibian populations are declining worldwide, and infectious diseases are a leading cause. Given the eminent threat infectious diseases pose to amphibian populations, there is a need to understand the host-pathogen-environment interactions that govern amphibian susceptibility to disease and mortality events. However, using animals in research raises an ethical dilemma, which is magnified by the alarming rates at which many amphibian populations are declining. Thus, in vitro study systems such as cell lines represent valuable tools for furthering our understanding of amphibian immune systems. In this review, we curate a list of the amphibian cell lines established to date (the amphibian invitrome), highlight how research using amphibian cell lines has advanced our understanding of the amphibian immune system, anti-ranaviral defence mechanisms, and Batrachochytrium dendrobatidis replication in host cells, and offer our perspective on how future use of amphibian cell lines can advance the field of amphibian immunology.

Critical review of the phytohemagglutinin assay for assessing amphibian immunity.

Infectious diseases are a major driver of the global amphibian decline. In addition, many factors, including genetics, stress, pollution, and climate change can influence the response to pathogens. Therefore, it is important to be able to evaluate amphibian immunity in the laboratory and in the field. The phytohemagglutinin (PHA) assay is an inexpensive and relatively non-invasive tool that has been used extensively to assess immunocompetence, especially in birds, and more recently in amphibians. However, there is substantial variation in experimental methodology among amphibian PHA studies in terms of species and life stages, PHA doses and injection sites, and use of experimental controls. Here, we compile and compare all known PHA studies in amphibians to identify knowledge gaps and develop best practices for future work. We found that research has only been conducted on a limited number of species, which may not reflect the diversity of amphibians. There is also a lack of validation studies in most species, so that doses and timing of PHA injection and subsequent swelling measurements may not effectively evaluate immunocompetence. Based on these and other findings, we put forward a set of recommendations to make future PHA studies more consistent and improve the ability to utilize this assay in wild populations, where immune surveillance is greatly needed.

Environmental endocrine disruptors and amphibian immunity: A bridge between the thyroid hormone axis and T cell development

Immunity is susceptible to reprogramming by environmental chemical and endocrine signals. Notably, numerous thyroid disrupting chemicals (TDCs) have the potential to perturb immune endpoints, but data are lacking on the mechanisms by which TDCs can influence the development of the immune system. T cell immunity is particularly vulnerable to modulation by TDCs during thymic education, differentiation, and selection. The following review discusses the ways in which thyroid hormones may influence T cell development, as well as emerging TDCs with potential to impact both thyroid hormone physiology and immune outcomes. To overcome the challenges of studying TDC impacts on immune toxicological endpoints, a comparative approach using the amphibian Xenopus laevis is recommended. X. laevis are ideally suited to studying TDC impacts on immunity due to the importance of thyroid hormones for metamorphosis, and the wealth of immunological models to measure immune endpoints in both tadpoles and adult frogs.

When Defenses Fail: Atelopus zeteki Skin Secretions Increase Growth of the Pathogen Batrachochytrium dendrobatidis.

To combat the threat of emerging infectious diseases in wildlife, ecoimmunologists seek to understand the complex interactions among pathogens, their hosts, and their shared environments. The cutaneous fungal pathogen Batrachochytrium dendrobatidis (Bd), has led to the decline of innumerable amphibian species, including the Panamanian golden frog (Atelopus zeteki). Given that Bd can evade or dampen the acquired immune responses of some amphibians, nonspecific immune defenses are thought to be especially important for amphibian defenses against Bd. In particular, skin secretions constitute a vital component of amphibian innate immunity against skin infections, but their role in protecting A. zeteki from Bd is unknown. We investigated the importance of this innate immune component by reducing the skin secretions from A. zeteki and evaluating their effectiveness against Bd in vitro and in vivo. Following exposure to Bd in a controlled inoculation experiment, we compared key disease characteristics (e.g., changes in body condition, prevalence, pathogen loads, and survival) among groups of frogs that had their skin secretions reduced and control frogs that maintained their skin secretions. Surprisingly, we found that the skin secretions collected from A. zeteki increased Bd growth in vitro. This finding was further supported by infection and survival patterns in the in vivo experiment where frogs with reduced skin secretions tended to have lower pathogen loads and survive longer compared to frogs that maintained their secretions. These results suggest that the skin secretions of A. zeteki are not only ineffective at inhibiting Bd but may enhance Bd growth, possibly leading to greater severity of disease and higher mortality in this highly vulnerable species. These results differ from those of previous studies in other amphibian host species that suggest that skin secretions are a key defense in protecting amphibians from developing severe chytridiomycosis. Therefore, we suggest that the importance of immune components cannot be generalized across all amphibian species or over time. Moreover, the finding that skin secretions may be enhancing Bd growth emphasizes the importance of investigating these immune components in detail, especially for species that are a conservation priority.

The amphibian complement system and chytridiomycosis.

Understanding host immune function and ecoimmunology is increasingly important at a time when emerging infectious diseases (EIDs) threaten wildlife. One EID that has emerged and spread widely in recent years is chytridiomycosis, caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), which is implicated unprecedented amphibian declines around the world. The impacts of Bd have been severe for many amphibian species, but some populations have exhibited signs of persistence, and even recovery, in some regions. Many mechanisms may underpin this pattern and amphibian immune responses are likely one key component. Although we have made great strides in understanding amphibian immunity, the complement system remains poorly understood. The complement system is a nonspecific, innate immune defense that is known to enhance other immune responses. Complement activation can occur by three different biochemical pathways and result in protective mechanisms, such as inflammation, opsonization, and pathogen lysis, thereby providing protection to the host. We currently lack an understanding of complement pathway activation for chytridiomycosis, but several studies have suggested that it may be a key part of an early and robust immune response that confers host resistance. Here, we review the available research on the complement system in general as well as amphibian complement responses to Bd infection. Additionally, we propose future research directions that will increase our understanding of the amphibian complement system and other immune responses to Bd. Finally, we suggest how a deeper understanding of amphibian immunity could enhance the conservation and management of amphibian species that are threatened by chytridiomycosis.