Getting under—and through—the skin: ecological genomics of chytridiomycosis infection in frogs



    1. Sainte-Justine Hospital Research Centre, Department of Pediatrics, University of Montreal, 3175 Chemin de la Cote Sainte-Catherine, Montreal, Quebec H3T 1C5, Canada
    Search for more papers by this author
  • J. TUNG

    1. Department of Evolutionary Anthropology, Duke University, PO Box 90383, Durham, NC, USA
    2. Duke Population Research Institute, Duke University, PO Box 90420, Durham, NC, USA
    Search for more papers by this author


Jenny Tung, Fax: 001 919 660 7348; E-mail:


Amphibian species around the world are currently becoming endangered or lost at a rate that outstrips other vertebrates—victims of a combination of habitat loss, climate change and susceptibility to emerging infectious disease (Stuart et al. 2004). One of the most devastating such diseases is caused by the chytrid fungus Batrachochytrium dendrobatidis (Bd), which infects hundreds of amphibian species on multiple continents. While Bd itself has been characterized for some time, we still know little about the mechanisms that make it so deadly. In this issue of Molecular Ecology, Rosenblum et al. describe a genomic approach to this question, reporting the results of a genome-wide analysis of the transcriptional response to Bd in the liver, skin and spleen of mountain yellow-legged frogs (Rana mucosa and R. sierrae: Fig. 1) (Rosenblum et al. 2012). Their results indicate that the skin is not only the first, but likely the most important, line of defence in these animals. Strikingly, they describe a surprisingly modest immune response to infection in Rana, a result that may help explain variable Bd susceptibility across populations and species.

Figure 1.

 The frog and the fungus. Left, the mountain yellow-legged frog, one of hundreds of worldwide amphibian species in decline. Right, the chytrid fungus Batrachochytrium dendrobatidis (Bd), in part responsible for loss of these frogs. In Rana mucosa and R. sierrae, infection by Bd leads to a massive loss of skin integrity and frequently death. Photo credit: (left panel) Roland A. Knapp, Sierra Nevada Aquatic Research Lab; (right panel) Erica B. Rosenblum, University of Idaho.

To achieve these results, Rosenblum et al. adopted a strategy that combined development of new genomic resources, whole transcriptome profiling and laboratory-based experimental infection. They first characterized the transcriptomes of Rana mucosa and Rana sierrae across multiple individuals, tissues and infection statuses via 454 pyrosequencing. Using a custom NimbleGen microarray developed from the results of their 454 runs, they then profiled gene expression in the skin, liver and spleen of experimentally infected and control frogs of both species, at two time points. This procedure resulted in a large set of genes that were significantly differentially expressed between control and Bd-infected frogs. The study authors highlight strong similarities in the response to infection across the two Rana species (both of which are highly susceptible in the wild), as well as a discernible level of shared response between these two species and a third species, Silurana (Xenopus) tropicalis, profiled in an earlier paper (Rosenblum et al. 2009).

In particular, categorical enrichment analysis of the set of Bd-responsive genes in the skin revealed strong evidence for changes in keratin, collagen, elastin and fibrinogen pathways related to skin integrity. In contrast, liver- and spleen-expressed genes exhibited relatively little response to Bd infection; similarly, genes involved in the innate or adaptive immune response were not strongly enriched among the Bd-responsive set. Rosenblum et al. therefore concluded that the skin is the major focus for Bd invasion and disease and that the immune response to Bd infection is essentially absent—or at least strikingly muted—in these Bd-susceptible species. However, perhaps paradoxically, lack of a clear immune response in these susceptible species helps highlight the importance of the immune system for the Bd response in general, especially in combination with evidence from other species.

For instance, Rosenblum et al. show that R. muscosa and R. sierrae, which are especially susceptible to Bd, are not able to induce high levels of antimicrobial gene expression upon infection with Bd (at least as assessed by the set of antimicrobial genes included on their expression array). Indeed, the vast majority of differentially expressed genes with putative antimicrobial activity were actually downregulated after Bd infection, as were other important innate immune signalling pathways (for example, induction of NF-kb activity, which plays a central role in sustaining the pro-inflammatory immune response). Interestingly, antimicrobial peptides (AMPs) secreted at the skin surface are thought to be particularly important players in protection from Bd infection in other species. Depletion of AMPs in the model species Silurana (Xenopus) laevis, which is less susceptible to Bd, results in increased vulnerability to infection (Ramsey et al. 2010). Additionally, individual purified AMPs were shown to inhibit the growth of Bd in vitro (Rollins-Smith et al. 2009), and survival rate among different frog species appears to correlate with the effectiveness of the skin AMP mixture (Woodhams et al. 2007). Species-specific differences in the ability to induce an adequate innate immune response, particularly via potent AMP release, may therefore contribute to species-specific differences in resistance to chytridiomycosis. The results of Rosenblum et al. provide substantial, albeit circumstantial, evidence in support of this hypothesis. More importantly, however, the approach they have pioneered provides a natural framework for extending the characterization of immunogenetic responses in the skin to a broader set of resistant and susceptible species.

A significant advantage of their approach lies in its ability to interrogate a large set of genes simultaneously. Indeed, Rosenblum et al. not only were able to show a weak innate immune response to Bd infection in R. mucosa and R. sierrae, but also showed that these species do not mount an effective adaptive immune response to Bd either. Neither the liver nor the spleen showed elevated levels of CD4 expression postinfection, suggesting lack of systemic T-cell recruitment and a consequent failure to activate downstream adaptive immune defences. In contrast, the less susceptible species S. laevis does exhibit an adaptive immune response to infection: after Bd inoculation, S. laevis produces elevated levels of Bd-specific IgM and IgY serum antibodies (Ramsey et al. 2010) [an effect absent in R. mucosa: (Stice & Briggs 2010)]. A role for the adaptive immune response is also supported by an association between MHC genotype and survival after Bd infection across different frog populations (Savage & Zamudio 2011). Interestingly, some MHC genes actually were upregulated in the postinfection frogs studied in Rosenblum et al. A detectable response at the level of MHC expression, yet the absence of a strong T-lymphocyte response in R. mucosa and R. sierrae, suggests that at least one component of Bd susceptibility in these species may therefore relate to the intersection between MHC signalling and T-cell proliferation. I intriguingly, Bd is apparently able to induce soluble factors that inhibit the proliferation of T and B lymphocytes, at least in vitro (Rollins-Smith et al. 2011).

Like many genome profiling studies, the work presented by Rosenblum et al. raises as many questions as it answers. In a system like these frogs, which is amenable to laboratory-based manipulations, conducting a controlled experiment in relatively sterile laboratory conditions is a natural starting place. However, previous work also suggests that bacteria found on the skin of R. mucosa are able to release antifungal metabolites that increase protection from Bd (Woodhams et al. 2007; Walke et al. 2011), suggesting that the skin microbial community may play an important role in mediating the immune response to Bd. Future work could extend the framework utilized by Rosenblum et al., in which the frogs were largely sheltered against non-Bd pathogens, to replicate more natural settings in which other pathogens and mutualists might also be present. In particular, it would be very interesting to investigate whether frogs exhibit different gene regulatory responses to infection based on the microbiome present in their skin and whether susceptible versus resistant individuals differ in this respect. Similarly, given recent findings of population structure among Bd isolates (Farrer et al. 2011), combinatorial studies that test for interactions between host and pathogen genotypes using gene expression profiling could be leveraged to help explain geographic and population differences in Bd susceptibility.

More broadly, the work reported by Rosenblum et al. represents an important proof of principle for how genomic approaches can be integrated into, and shed new light on, questions of ecological and evolutionary importance. Indeed, one of the great promises of the ‘genome revolution’ has lain in its ability to break down disciplinary walls and to extend a genetic perspective to species that would otherwise have a sparse or absent genetic toolkit (Mitchell-Olds et al. 2008). As exemplified by this work, some of those species are, nevertheless, the most compelling subjects of study in ecology and conservation (as well as in evolution and behaviour). At the same time, this study also illustrates that work in this vein remains challenging: although Rosenblum et al. quite rightly emphasize the biological results of their work, they also overcame not-inconsequential methodological barriers, including the lack of a characterized transcriptome for their species of interest and the need to design a custom array to measure genome-wide gene expression. Some of these difficulties may be more readily overcome in the future with a switch to high throughput sequencing approaches (e.g. RNA-seq) rather than array-based technologies, which would permit simultaneous species-specific transcriptome characterization and gene expression profiling. As such data continue to proliferate natural populations will become increasingly tractable for ecological genomics research. In turn, genomic data will, we hope, play an increasingly important role in providing novel solutions to serious problems in conservation and ecological research, including the decline in worldwide amphibian populations.

J.T. is interested in the evolutionary genomics of natural populations, particularly the interplay between ecology, behavior, and genetics. L.B.B. studies the genetic and genomic basis of natural variation in immune response and susceptibility to disease.