Introduction

Global amphibian population declines first became apparent about 30 years ago, as herpetologists from around the world compared notes about the difficulty they were experiencing finding their study organisms (Wake 1991). Fears that they were witnessing early signs of an impending sixth mass extinction event (Wake and Vredenburg 2008) gave rise to a flurry of research to ascertain why amphibians were disappearing from so many localities. Amphibian population declines can result from numerous factors including habitat modification and fragmentation, exotic predators and competitors, over-exploitation, climate change, and emerging infectious diseases (reviewed in Berger et al. 2016; Collins, 2010; Kolby and Daszak 2016; Rollins-Smith 2017).

In the 1990s, the most dramatic, precipitous declines were observed in pristine habitat in Central America and Australia, which could not easily be explained by anthropogenic factors. Although researchers had previously described the spread of amphibian population extinctions occurring in waves, suggestive of pathogen spread (Laurance et al. 1996), the hypothesis was met with skepticism owing to the lack of knowledge of the causative agent (Alford and Richards 1997; Hero and Gillespie 1997). Subsequently, a new fungal pathogen, initially misidentified as a Perkinsus-like protist, and later recognized to be a chytrid fungus, Batrachochytrium dendrobatidis (hereafter denoted Bd), was identified in victims of epizootics in Australia and Central America (Berger et al. 1998; Waldman and Tocher 1998).

Bd was first considered a recently emerged clone that spread globally (Morehouse et al. 2003) to cause population declines. Later, highly diversified endemic Bd strains were identified in many localities around the world (Bataille et al. 2013; Farrer et al. 2011; Rodriguez et al. 2014). Although Bd was thought to be a novel pathogen of amphibians, the fungus now has been identified in amphibians collected over the past 100 years or longer (Fong et al. 2015; Goka et al. 2009; Rodriguez et al. 2014; Talley et al. 2015). Historical population declines appear to coincide with the incursion of new hypervirulent Bd strains or the emergence of virulence in endemic Bd strains (Carvalho et al. 2017). Recent population crashes in European salamanders have been linked to another chytrid pathogen, Batrachochytrium salamandrivorans (denoted Bsal) (Martel et al. 2013, 2014).

How does Bd damage hosts?

Bd infects the keratinized mouthparts of larvae, which may interfere with their feeding behavior, as well as the skin of adults, which serves as an important respiratory organ for amphibians. Infection might cause hyperkeratosis, thereby impeding respiration or water balance, or hosts might be poisoned by a fungal toxin (Berger et al. 1998; Pessier et al. 1999). Adult amphibians showing clinical signs of chytridiomycosis may experience lower plasma concentrations of sodium and potassium electrolytes, which may induce asystolic cardiac arrest (Voyles et al. 2009). However, individuals bearing low infection loads, although potentially suffering some deficits from aclinical chytridiomycosis, show no significant impairment in metabolic measures such as rehydration rates (Carver et al. 2010). These results suggest that tolerance of—as well as resistance to—the pathogen may be selected in amphibians living in habitats with enzootic Bd strains (Savage and Zamudio 2016).

As Bd invades epidermal tissue (Van Rooij et al. 2012), infection might rapidly induce innate immune responses against it. Indeed, amphibian skin contains many mucous and granular glands, which secrete functional mucins and antimicrobial peptides (AMPs) (Rollins-Smith and Conlon 2005). These AMPs inhibit Bd growth in vitro and serve as a key component of the innate immune response to Bd (Ramsey et al. 2010; Rollins-Smith et al. 2011). AMP repertoires vary among species, perhaps correlated with differential susceptibility to Bd (Rollins-Smith 2009; Woodhams et al. 2007). Symbiotic bacteria also may play a role in innate immunity, and their metabolites can exert synergistic effects with AMPs to inhibit Bd growth (Myers et al. 2012).

In the susceptible species Rana muscosa, Rana sierra, and Atelopus zeteki, Bd-infected individuals show reduced gene expression levels of keratin and collagen, key components of structural skin integrity (Ellison et al. 2014a; Rosenblum et al. 2012). By contrast, in several resistant species, genes needed to maintain skin integrity, such as collagen, are up-regulated after subjects are infected with Bd (Ellison et al. 2014b; Poorten and Rosenblum 2016). Decreased expression of ion channel genes and increased expression of potassium/chloride genes in Bd-susceptible species are consistent with the observation of electrolyte imbalance directly preceding death (Voyles et al. 2009), attributable perhaps to physical disruption of the epidermis (Rosenblum et al. 2012).

Soluble factors released by Bd, possibly derived from the cell wall, can actively interfere with adaptive immunity and prevent clearance of the pathogen. Bd inhibits the proliferation of splenocytes and induces lymphocyte apoptosis in vitro (Fites et al. 2013). Immunodeficiency thus occurs as insufficient T lymphocytes and B lymphocytes are present to respond to and eliminate Bd. Some lymphocyte genes are down-regulated in spleen of susceptible but not resistant or tolerant species in vivo (Ellison et al. 2014b). Effectors associated with Bd, such as CRN13, can induce necrosis in infected tissues (Ramirez-Garces et al. 2016). Patterns of transcriptional changes upon Bd infection vary widely among anurans (Ellison et al. 2014a, b; Poorten and Rosenblum 2016; Price et al. 2015; Rosenblum et al. 2012), but species resistant to Bd generally show evidence of stronger innate and adaptive immune system responses.

Adaptive immune responses appear to supplement innate immunity in conferring resistance to Bd. In African clawed frogs (Xenopus laevis), a species highly resistant to chytridiomycosis, AMPs of frogs subject to X-irradiation remain stable, yet Bd infection loads rise as spleen leukocyte numbers decrease (Ramsey et al. 2010). The antibodies IgM, IgY, and IgX bind to Bd, further suggesting a role for adaptive immune responses in conferring resistance (Ramsey et al. 2010). Bd-infected Litoria caerulea, a highly susceptible species, become immunocompromised as evidenced in lower total white blood cell and serum protein counts together with reduced splenic lymphocyte and immunoglobulin responses (Young et al. 2014).

Adult anurans are more susceptible to chytridiomycosis than tadpoles, perhaps because larvae lack keratinized tissues, other than their mouthparts, that can be infected by Bd. Nonetheless, larvae of some species suffer reduced growth and even mortality when infected by or exposed to Bd (Blaustein et al. 2005; Garner et al. 2009; Luquet et al. 2012). Ontogenetic changes in Bd resistance or tolerance correlate with aspects of immune system maturation, but the relationship, if any, remains to be studied. Metamorphosis entails radical reorganization of many tissue and organ systems including those involved in immune function, with a period of immunosuppression at metamorphic climax (Robert and Ohta 2009; Rollins-Smith 1998) when infected individuals are most vulnerable to the disease.

How does Bd infection alter MHC expression?

If Bd triggers an adaptive immune response, one might expect to find changes in transcriptional expression of MHC genes following infection. In most susceptible species that have been studied, including R. muscosa, R. sierra, and A. zeteki, MHC genes appear to be up-regulated in the skin after infection (Ellison et al. 2014a; Rosenblum et al. 2012). By contrast, in most resistant species, such as Xenopus (Silurana) tropicalis, Craugastor fitzingeri, Agalychnis callidryas, and Rana temporaria, no changes in MHC transcriptional expression have been found (Ellison et al. 2014b; Price et al. 2015; Rosenblum et al. 2009) (Table 1). However, down-regulation of MHC in spleen and liver of some susceptible species may be symptomatic of more general suppression of adaptive immune function as previously discussed.

Table 1 MHC transcriptional regulation in susceptible and resistant amphibians after Bd infection
Full size table

In these studies, although up- and down-regulation of MHC transcriptional expression was measured, translational levels were not determined and possibly remained unchanged. Moreover, discordant results among studies may reflect other variables, including temperature effects on pathogen infectivity or host immune function, ages and background of hosts, harvest timing for RNA isolation, and even sex or individual differences which might be especially important given the small sample sizes tested (e.g., see Ramsey et al. 2010; Zhu et al. 2014).

MHC genetic diversity and susceptibility to Bd infection

Susceptibility to chytridiomycosis varies among regions of the world. In localities where Bd has a history of endemism (e.g., parts of Asia, South America, North America), many species appear resistant to or tolerant of Bd, showing no clinical signs of disease. Epizootics typically occur in areas soon after initial incursions of Bd, but among the ensuing carnage, some species may thrive as others around them perish. Even in susceptible species, however, variation in resistance often is apparent. Natural selection presumably favors resistant variants, and populations of some species, thought to be extinct, now appear to be slowly recovering (Knapp et al. 2016; Scheele et al. 2017).

Previous studies showed that MHC variants confer resistance on amphibians to virulent pathogens that cause epizootics and mass mortality events (Barribeau et al. 2008; Teacher et al. 2009). Recent studies have found that differential survival of amphibians infected by Bd maps to genetic variation in the MHC class II peptide-binding region (PBR) (Bataille et al. 2015; Kosch et al. 2016; Savage and Zamudio 2011, 2016). The demonstration of adaptive immune responses to Bd was not necessarily expected given that infections often are limited to superficial epidermal cells (Richmond et al. 2009).

Acquired resistance to Bd is associated with particular amino acid residue positions situated in exon 2 of the MHC class IIB gene, which encodes the β1 segment of the antigen-binding groove that presents epitopes to T cells. The stability of the antigen-MHC complex depends mainly on deep pockets within the binding groove that interact directly with antigen residues. Nine pockets (P1–P9) are revealed by crystallography, of which four (P1, P4, P6, and P9) are polymorphic in the PBR and differ in how they bind to aliphatic, hydrophobic, and hydrophilic amino acids (Brown et al. 1993; Dai et al. 2008; Stern et al. 1994). Alleles that encode particular conformations of the P9 pocket appear key to Bd resistance among amphibians (Fig. 1) (Bataille et al. 2015). Specific properties of the P4 and P6 pockets also correlate with Bd resistance widely, but not universally, perhaps because of immune system differences among species, variation among Bd strains, or differences in environmental factors (Bataille et al. 2015).

Fig. 1
figure 1

Homology modeling of Bufo gargarizans MHC class II β1 domain allele Buga-1. Display of the typical three-dimensional structure of the vertebrate MHC class II β1 domain, with α-helices represented as red helical ribbons and β-sheets as thick purple arrows. The peptide-binding residues of the P9 pocket are indicated, with atomic structure for residues 56β and 57β. See Bataille et al. (2015) for further details

Full size image

The MHC of lowland leopard frogs (Rana yavapaiensis) is highly polymorphic, with 84 unique MHC class II PBR alleles identified (Savage and Zamudio 2016). In laboratory infection studies, individuals bearing “allele Q” were resistant to Bd. Population genetic analyses detected a significant signal of positive selection acting on codon 46 in pocket 9 (Savage and Zamudio 2011). Sampling in the field across several localities similarly found that this allele was positively associated with survival rates, but unlike in the laboratory experiments, heterozygosity conferred no advantage in natural conditions (Savage and Zamudio 2016).

Alpine tree frogs (Litoria verreauxii alpina) from populations with a history of Bd endemism appear more resistant to experimental infection with Bd than those from populations that are naïve to Bd. Comparing disease progression among three populations, infection loads were lower and survivorship higher in subjects from one locality in which Bd had been endemic for many decades. Subjects from a locality in which Bd has been historically absent had higher infection loads and succumbed more quickly to chytridiomycosis, but so too did subjects from another long-term Bd-infected locality. Twenty-two MHC IIβ alleles were recovered among the populations. Of 84 infected frogs, six survivors bore eight alleles containing identical residues at five codon positions, all present within pocket 9. Only subjects homozygous for alleles encoding this pocket conformation survived infection (Bataille et al. 2015). Similar conformations of the P9 pocket in the MHC II β1 domain appear to be fixed in Bd-resistant Bufo species and Bombina orientalis in Korea (Bataille et al. 2015).

In Panama, highland and lowland frog populations have evolved different immunogenetic responses to the pathogen. Bd has been enzootic in the highlands since the late 1990s but has only recently reached lowland populations. Highland populations of the túngara frog (Physalaemus pustulosus) live in amphibian communities that suffered declines when Bd initially spread to them. Surviving individuals tend to have MHC class II alleles with the resistant P9 conformation, and most individuals are homozygous for these alleles. By contrast, temperature regimens in the lowlands are not favorable for the transmission of Bd, population declines have not been observed, and individuals tend to bear these same alleles but in the heterozygous state (Kosch et al. 2016). Selection for resistance alleles thus appears to fluctuate based on environmental conditions that affect pathogen infectivity and virulence.

MHC class II molecules present antigens to CD4+ helper T cells from extracellular pathogens such as Bd, so most amphibian disease studies have focused on class II genes. MHC class I molecules are expressed on all nucleated somatic cells and mainly present antigens from intracellular pathogens to CD8+ cytotoxic T cells. Recent studies describe how Bd sometimes penetrates into cells (Van Rooji et al. 2012). In these cases, MHC class I molecules potentially may bind to intracellular Bd peptides for presentation to CD8+ T cells. In addition, peptides derived from extracellular Bd, rather than being presented by MHC class II, can be displayed to MHC class I molecules through the process of cross-presentation (Cresswell et al. 2005). Endangered southern corroboree frogs, Pseudophryne corroboree, which are facing steep population declines in the wild attributable to Bd, show high MHC class IA diversity. Genetic evidence points to strong positive and purifying selection at class IA sites that are associated with PBR pockets in resistant species (Kosch et al. 2017). Comparably high levels of genetic diversity in MHC class I in Japanese Rana species similarly raise the possibility of selection for Bd resistance in those species (Lau et al. 2016). Although a role for MHC class I genes in responding to Bd infection has yet to be demonstrated, class I  and II genes show similar expression patterns following Bd infection (Table 1). Further studies on MHC class I involvement in the adaptive immune response thus are warranted.

Bd-induced immunological memory

If amphibians demonstrate an adaptive immune response to Bd, immunization protocols might be developed to enhance their resistance to the pathogen. Results to date on the inducement of robust acquired immunity have been mixed. Exposure to formalin-killed Bd, by injection, did not reduce susceptibility of juvenile yellow-legged frogs (R. muscosa) to subsequent infection (Stice and Briggs 2010). Boreal toads (Anaxyrus boreas) injected with heat-killed Bd also survived no better than control subjects when infected with Bd (Rollins-Smith et al. 2009). Nonetheless, inoculation with Bd induced a systemic response in X. laevis, as evidenced by circulating antibodies specific to Bd (Ramsey et al. 2010).

As Bd infects the skin, inoculation by injection might not be appropriate for gauging the development of an adaptive immune response. Partial immersion of boorolong frogs (Litoria booroolongensis) in solution containing Bd zoospores readily caused subjects to become infected. Frogs then were cleared of infection by treatment with the fungicide itraconazole and subsequently reinfected using the same procedure. However, these individuals survived in no higher numbers than Bd-naïve individuals infected for the first time (Cashins et al. 2013). Control for immunosuppressive properties of itraconazole was lacking, though, and a reanalysis of the same data revealed that previously inoculated frogs demonstrated increased resistance upon reinfection (McMahon et al. 2014).

As Bd is viable only within a limited temperature range, hosts can be cleared of the disease by potentially less invasive heat treatments. Using such procedures, Cuban tree frogs (Osteopilus septentrionalis) inoculated with Bd presented decreased infection loads in each successive cycle of infection and clearance. This acquired immunity is attributable to increased abundance and proliferation of splenic lymphocytes rather than changes in skin peptide abundance (McMahon et al. 2014). Furthermore, prior exposure to Bd not only activated a strong immune system response but also induced a behavioral response, as Bd-exposed oak toads (Anaxyrus quercicus) showed an aversion to substrate contaminated by Bd zoospores that was lacking in Bd-naïve subjects (McMahon et al. 2014).

MHC resistance alleles incur life-history costs

MHC loci in natural populations often are highly polymorphic, presumably because multiple alleles encode many PBR conformations that bind to a wide array of epitopes. This variation can be favored by heterozygote advantage or negative fluctuating selection (Piertney and Oliver 2006). As the MHC is co-dominantly expressed, in the former case, heterozygotes potentially can present twice as many PBR conformations as homozygotes. In the latter case, hosts can present PBR conformations to which pathogens have had no opportunity to adapt. Adaptive immune responses are thought to be enhanced with increased levels of immunogenetic variation, although this may not be important in all circumstances (Slade 1992). In some systems, maximum resistance is achieved by having an optimal number of MHC class IIB alleles, less than the maximum possible, especially when hosts are infected by multiple parasites (Wegner et al. 2003). This might reflect deletion of self-reactive T cells, which restricts the pool of available T cells (Nowak et al. 1992).

Studies on the dynamics of immunogenetic responses to Bd are consistent with the hypothesis that when infected by a new, virulent pathogen, strong directional selection over the short term may favor particular alleles that confer resistance to it. However, the advantages of effective immune response to particular pathogens such as Bd may be offset by reduced immune capacity to respond to other pathogens. In boreal toads (A. boreas), a species highly susceptible to chytridiomycosis (Carey et al. 2006), more heterozygous than homozygous individuals were found to be infected by Bd in surveys of natural populations (Addis et al. 2015). Although the MHC was not characterized, this result is consistent with the expectation that Bd exerts strong selection for homozygosity of resistance alleles (also see Tracy et al. 2015). Selection for particular resistance alleles may vary among populations according to environmental factors that affect pathogen virulence and host resistance. Shifts in selection regimens may explain the variable results found in field and laboratory studies, and among populations living in different habitats, even within species.

In evolutionary terms, immune responses can incur fitness costs. Alleles that confer resistance not only to Bd but also to other pathogens, including ranavirus (Teacher et al. 2009) and the bacterial pathogen Aeromonas hydrophila (Barribeau et al. 2008), have been identified in amphibians. Bacterial pathogens, especially A. hydrophila, frequently have been linked to epizootics of amphibian populations but now are thought to represent secondary infections of individuals already suffering from chytridiomycosis or other diseases (Taylor et al. 1999). In experimental studies, X. laevis tadpoles bearing MHC alleles that confer resistance to A. hydrophila grew slower than those with susceptible MHC alleles (Barribeau et al. 2008). Slower growth and smaller size at metamorphosis typically reduce survivorship and the likelihood of successful reproduction in amphibians, so resistance alleles may incur a life-history cost. Similarly, resistance to chytridiomycosis may impose costs on growth and other life-history traits (Garner et al. 2009; An and Waldman 2016; Woodhams et al. 2016). These results point to a possible trade-off between immune function and other determinants of fitness. Thus, while certain MHC alleles confer disease resistance, they may not be selected when disease poses little or no threat. In these conditions, balancing selection is likely to supplant directional selection to enhance MHC variation.

Toll-like receptors

Toll-like receptors (TLRs) can recognize pathogen-associated molecular patterns (PAMPs), structures shared by many pathogens (Hayashi et al. 2001; Rollins-Smith et al. 2009). TLRs play a critical role in early pathogen recognition and defense pathways (Beutler and Rietschel 2003; Janeway and Medzhitov 2002). Activation of TLR-mediated cell signaling pathways can induce the up-regulation of genes that orchestrate immune responses, leading to cytokine production, proliferation, and survival (Misch and Hawn 2008; Rollins-Smith et al. 2009). In turn, this can activate cellular pathways culminating in the production of transcription factors that target MHC promoters regulating gene expression (Mak et al. 2014).

In Bd-infected A. zeteki, several TLR genes, such as TLR2 type-1-like and TLR5, were up-regulated in the skin, spleen, and intestine (Ellison et al. 2014a). TLR2 recognizes fungal-derived ligands, while TLR5 is known to recognize bacterial flagella (Akira and Takeda 2004). Among the up-regulated TLRs, TLR5 was most up-regulated upon Bd infection, suggesting that individuals experienced secondary bacterial infections. The possibility that secondary pathogens contribute to mortality in Bd-infected amphibians remains untested. Studies on other susceptible amphibians found no evidence for TLR activation following Bd infection (Rosenblum et al. 2009, 2012).

Conclusions

Chytridiomycosis, caused by B. dendrobatidis, threatens amphibian populations in many parts of the world. Variation exists within and among species in susceptibility to the pathogen, and this appears related to genetic variation in the MHC class II locus. Selection for resistance appears ongoing in the wild, as evidenced by slow population recoveries of individuals that may bear resistance alleles.

Comparison of transcriptomes of subjects before and after Bd infection reveals a diversity of responses among species. MHC class I and class II genes of some susceptible species are up-regulated following Bd infection, but translational levels are unknown. Most resistant species show little change in MHC gene expression after Bd infection.

Particular conformations of the MHC class II PBR appear to confer resistance to the pathogen and are shared among resistant species around the world. Laboratory and field experiments both demonstrate particular conformations of the P9 pocket, as well as specific properties of the P4 and P6 pockets, conferring resistance on hosts. Some studies suggest that not only MHC class II but also class I genes may be under selection for Bd resistance.

Repeated exposure to killed Bd zoospores fails to elicit an enhanced immune response in hosts. However, cycles of exposure to live Bd zoospores followed by treatment to clear hosts of the pathogen appear to produce a robust immune response that may serve as the basis for the development of immunization strategies.

Epizootics may result in strong directional selection for particular MHC PBR conformations, and amphibians with homozygous resistance alleles may benefit from enhanced resistance in some conditions. However, reduction in MHC diversity may incur life-history costs. Further research is needed to evaluate these costs with respect to Bd infection.

TLRs can be up-regulated in Bd-infected hosts. Their role in coordinating innate and adaptive immune responses to the pathogen requires further study.