Molecular Ecology (2012) 21, 3095–3097

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

L. B . BARREIRO* and J . TUNG†‡*Sainte-Justine Hospital Research Centre, Department of

Pediatrics, University of Montreal, 3175 Chemin de la Cote

Sainte-Catherine, Montreal, Quebec H3T 1C5, Canada,

†Department of Evolutionary Anthropology, Duke University,

PO Box 90383, Durham, NC, USA, ‡Duke Population Research

Institute, Duke University, PO Box 90420, Durham, NC, USA

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, cli-

mate change and susceptibility to emerging infectious

disease (Stuart et al. 2004). One of the most devastating

such diseases is caused by the chytrid fungus Batracho-

chytrium 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 transcrip-

tional response to Bd in the liver, skin and spleen of

mountain yellow-legged frogs (Rana mucosa and R. sier-

rae: 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.

Keywords: amphibians, conservation genetics, ecological

genetics, fungi, transcriptomics

Received 20 March 2012; revision received 2 April 2012;

accepted 12 April 2012

To achieve these results, Rosenblum et al. adopted a strat-

egy that combined development of new genomic resources,

whole transcriptome profiling and laboratory-based experi-

mental infection. They first characterized the transcripto-

mes of Rana mucosa and Rana sierrae across multiple

individuals, tissues and infection statuses via 454 pyrose-

quencing. Using a custom NimbleGen microarray devel-

Correspondence: Jenny Tung, Fax: 001 919 660 7348;

E-mail: jt5@duke.edu

� 2012 Blackwell Publishing Ltd

oped from the results of their 454 runs, they then profiled

gene expression in the skin, liver and spleen of experimen-

tally 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 combi-

nation 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 downreg-

ulated 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, antimi-

crobial 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 infec-

tion (Ramsey et al. 2010). Additionally, individual purified

AMPs were shown to inhibit the growth of Bd in vitro (Rol-

lins-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

Fig. 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.

3096 N E W S A N D VI E WS: PE R SPE C TIV E

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 abil-

ity 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 upreg-

ulated in the postinfection frogs studied in Rosenblum

et al. A detectable response at the level of MHC expres-

sion, yet the absence of a strong T-lymphocyte response in

R. mucosa and R. sierrae, suggests that at least one compo-

nent 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 labora-

tory-based manipulations, conducting a controlled experi-

ment 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 popula-

tion structure among Bd isolates (Farrer et al. 2011), combi-

natorial 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 geno-

mic 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 revolu-

tion’ 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 sub-

jects 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 bio-

logical results of their work, they also overcame not-incon-

sequential 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 simul-

� 2012 Blackwell Publishing Ltd

NEWS AND VIEWS: P ERSPECTIVE 3097

taneous species-specific transcriptome characterization and

gene expression profiling. As such data continue to prolif-

erate natural populations will become increasingly tractable

for ecological genomics research. In turn, genomic data

will, we hope, play an increasingly important role in pro-

viding novel solutions to serious problems in conservation

and ecological research, including the decline in worldwide

amphibian populations.

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� 2012 Blackwell Publishing Ltd

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J.T. is interested in the evolutionary genomics of natural popu-

lations, 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 dis-

ease.

doi: 10.1111/j.1365-294X.2012.05624.x