SYMPOSIUM

Genomic Studies of Disease-Outcome in Host–Pathogen DynamicsAna V. Longo,1,* Patricia A. Burrowes† and Kelly R. Zamudio*

*Department of Ecology and Evolutionary Biology, Cornell University, E145 Corson Hall, Ithaca, NY 14853, USA;†Department of Biology, University of Puerto Rico, PO Box 23360, San Juan, PR 00931-3360, USA

From the symposium ‘‘Methods and Mechanisms in Ecoimmunology’’ presented at the annual meeting of the Society for

Integrative and Comparative Biology, January 3–7, 2014 at Austin, Texas.

1E-mail: avl7@cornell.edu

Synopsis Pathogens act as agents of evolutionary change in host populations, altering the host’s allele frequencies and

phenotypes through selection. The mechanisms underlying these adaptive changes depend on which defense strategy the

host adopts upon infection. With increased anthropogenic change and loss of biodiversity, ecological impacts on adaptive

processes may reduce the ability of hosts to evolve resistance, or to persist within their tolerance limits, thus increasing

the capacity of pathogens to cause disease and mortality. In this review, we use amphibians and a pathogenic chytrid

fungus (Batrachochytrium dendrobatidis, Bd) to illustrate how integrating genomic approaches into current research, both

for hosts and pathogens, will improve our understanding of factors promoting the outcome of disease. As new emerging

pathogens continue threatening amphibian populations worldwide, we recommend that researchers focus on individuals

that survive after natural epizootics or experimental challenges. These survivors represent an underutilized and under-

exploited genetic resource for characterizing adaptive traits involved in the clearance of pathogens or in their tolerance.

We highlight two target areas that will benefit from focused research: (1) Identification of the genetic basis of the hosts’

defense strategies (resistance and tolerance) and of Bd’s pathogenicity traits and (2) genomic characterization of shifts in

fitness that drive seasonal and/or temporal patterns in host–pathogen interactions. To provide insights into hosts’ sur-

vival, we review recent literature—including experimental Bd challenges and longitudinal studies—that underscore the

complexity of Bd infections as determined by a combination of genetic and environmental factors. Given the heteroge-

neity of disease-outcomes and broad diversity of host species, amphibians provide a unique opportunity to identify novel

genetic determinants of resistance to a recently emerged fungal pathogen. Developing additional genetic resources (e.g.,

genomic profiles, resistance mapping, and dual RNA-seq) will advance our understanding of the components of the

innate and adaptive immune system acting on infected hosts in varying environments. These ecoimmunomic applica-

tions, which link host–pathogen eco-evolutionary processes with applied conservation efforts, will specifically benefit

threatened amphibians that remain safeguarded in captive colonies.

Introduction

Understanding and predicting the emergence and

dynamics of infectious diseases in wildlife is critical

to the health of humans and ecosystems (Daszak

et al. 2000; Beldomenico and Begon 2010). The det-

rimental effects of pathogens on hosts’ fitness have

downstream effects on population dynamics

(Anderson and May 1981) that in turn impacts com-

munity structure and ecosystem function (Wiles

et al. 2006; Crawford et al. 2010; Keesing et al.

2010). Pathogens act as agents of evolutionary

change, thereby altering allele frequencies through

selection (May and Anderson 1983; Altizer et al.

2003), but they can also modify the morphology

and behavior of hosts (Lefevre et al. 2009). The

mechanisms underlying these adaptive changes

depend upon which defense strategy the host

adopts after infection: Resistance involves hosts lim-

iting their infection load by inhibiting the reproduc-

tive potential of the pathogen. In contrast, hosts

employ tolerance to reduce negative fitness conse-

quences and damage caused by pathogens (Roy and

Kirchner 2000; Raberg et al. 2009). Global environ-

mental change has the potential to impact adaptive

processes and reduce the ability of hosts to evolve

resistance or to persist within their tolerance limits

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(Harvell et al. 2002; Keesing et al. 2010; Franks and

Hoffmann 2012). Therefore, we must understand the

interplay between ecological and evolutionary pro-

cesses that lead to an increase in the capacity of

pathogens to cause disease and mortality (Fig. 1).

Natural systems with seasonal cycles of epidemic

and endemic pathogen conditions provide the op-

portunity to characterize how changes in defense

strategies prompt shifts in the fitness advantage

from host to pathogen, and vice versa. Seasonal var-

iation may affect how well genotypes of hosts and

pathogens perform under a given set of environmen-

tal conditions, ultimately influencing their survival

and reproduction (Orr 2009). In this review, we

illustrate the importance of identifying the genetic ba-

sis of defensive strategies in amphibians, a vertebrate

class that is severely threatened by chytrid fungal

pathogens in the genus Batrachochytrium (Berger

et al. 1999, 2009; Lips et al. 2006; Heatwole 2013;

Martel et al. 2013). With more than 500 host species

infected (www.bd-maps.net), Batrachochytrium den-

drobatidis (hereafter Bd) is among the wildlife path-

ogens with the highest species diversity of hosts

(Fisher et al. 2012; Lips 2014). Indeed, host general-

ism enhances the survival of Bd by providing more

routes for transmission; therefore, hosts’ shared traits

(e.g., genetics, immunity, physiology, or behavior)

may hold the key to decipher the source and mech-

anisms promoting different outcomes of the disease.

Although several species have already lost the battle

against Bd (Lips et al. 2006; Pounds et al. 2006), the

ones persisting are fighting infection under enzootic

conditions that fluctuate over time (Briggs et al.

2010; Longo et al. 2010; Savage et al. 2011). Thus,

to answer the question ‘‘which factors predict

disease-outcome?’’, our field needs to move forward

from reporting patterns to more mechanistic frame-

works addressing the adaptive potential in host–

pathogen interactions. Mounting evidence from

almost two decades of research indicates that suscep-

tibility to Bd is a complex phenotype generated by a

combination of genetic and environmental factors

(Ribas et al. 2009; Savage and Zamudio 2011;

Becker et al. 2012), of which the genetic responses,

both of the host and the pathogen, have been under-

explored. Integrating genomic approaches will allow

us to characterize the functional and mechanistic di-

versity of host–pathogen responses, and develop new

methods to compare their genetic basis under differ-

ent environmental pressures.

Here, we highlight new target areas for research

necessary to integrate genomic approaches into dis-

ease dynamics. These approaches can significantly

strengthen our understanding of hosts’ survival

under epizootic and enzootic host–pathogen interac-

tions, especially for generalist multi-host pathogens

such as Bd. We outline genomic studies that re-

searchers should consider to develop effective man-

agement strategies while maximizing available

resources. Our proposed genomic-target areas focus

particularly on the dichotomy between individuals

that survive after natural epizootics or as part of

experimental challenges and individuals that die,

because survivors represent an underutilized and

underexploited genetic resource for characterizing

adaptive traits. Once a pathogen is established in a

population, the first target is to identify resistant and

tolerant genotypes of the host, as well as pathogens’

traits correlated with virulence. We know that all

hosts are not equal (Johnson and Hartson 2009;

Gervasi et al. 2013a); thus, detecting the genetic

basis of hosts’ defenses under controlled conditions

will help us uncover the evolutionary potential of

populations when threatened by emerging diseases.

Our second target is to elucidate the environmental

controls of hosts’ defenses by investigating shifts in

fitness advantage that drive host–pathogen dynamics.

Given that environmental factors affect multiple

aspects of the biology of both organisms—including

physiological processes, immune responses, and

behavior—we need to distinguish how these changes

affect adaptive responses and influence the temporal

dynamics of disease. We conclude by recommending

the integration of ecoimmunomics into amphibian

conservation programs. Combining ecoimmune

parameters (i.e., parasite density, host fitness, and

immune responses, sensu Graham et al. 2011) with

Fig. 1 Conceptual diagram illustrating how the environment

shapes host–pathogen relationships and interactions with other

species. The outcome of disease (survival or mortality) is a

consequence of evolutionary and ecological processes that

alter allele-frequencies and population dynamics.

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the newest genomic technologies will create a bridge

between host–pathogen eco-evolutionary processes

and applied conservation efforts.

Target I: Identify resistant/tolerant genotypes of the

host and the pathogenicity traits of Bd

Host–pathogen interactions provide three classical

conditions that allow us to examine the influence

of natural selection on populations: (1) Variation

in hosts’ defenses and in pathogenicity traits, (2)

heritability of traits, and (3) differential survival as

a function of these traits (Anderson and May 1981;

Antonovics et al. 2013). In the amphibian-Bd system,

field and experimental studies support variability

in hosts’ defensive traits conferring survival

(Woodhams et al. 2007a; Savage and Zamudio

2011; Gervasi et al. 2013a). Although complex envi-

ronmental interactions can modulate this variation

(see discussion in Target II), we are still early in

the process of identifying polymorphisms directly re-

lated to hosts’ defenses, the initial step in comparing

heterogeneity in interspecific responses among hosts

(Woodhams et al. 2007a; Gervasi et al. 2013a). In

contrast, we have a better idea of the evolutionary

changes promoting virulence (Rosenblum et al.

2009a; Joneson et al. 2011), yet the mechanisms by

which Bd bypasses or adapts to the host’s immunity

need to be fully characterized (Fites et al. 2013).

In Target I, we first review the amphibian-Bd lit-

erature for insights into survival, both of host and of

pathogen, that could be attributed to unidentified

genetic factors. Then, to elucidate the genetic basis

of host–pathogen interactions, we propose two po-

tential genomic studies that will identify adaptive

traits involved in the hosts’ survival and in the vir-

ulence of the pathogen.

Insights from experimental challenges with Bd

Experimental challenges are typically one of the first

steps used to investigate differences in survival

among host-groups of interest (e.g., species, age,

sex, treatment, and pathogens’ genotypes). One ad-

vantage of working with non-model organisms is

that often amphibians used in experiments come

from natural populations, thereby providing a large

pool of variable traits of hosts upon which selection

can act. On the other hand, experimenting with

wild-caught individuals precludes control of infec-

tion histories, genetic relatedness, presence of micro-

bial symbionts, and reproductive fitness. Of these

variables, absence of information on previous infec-

tion merits the development of new assays to deter-

mine whether wild-caught hosts are naıve, or have

been exposed and developed defenses against Bd.

Despite these shortcomings, studies of wild-caught

hosts can still demonstrate amphibians’ susceptibility

to infection and consequent mortality by clearing

individuals with fungicide, raising amphibians from

eggs to metamorphs, or by randomizing hosts in

treatments.

Instead of highlighting the mortality effects of Bd

on individuals that are part of experimental chal-

lenges, we argue that survivors deserve special atten-

tion because they potentially carry the adaptive traits

involved in survival of the disease. Here, we examine

variability in hosts’ survival by collecting survival

probabilities from several Bd experimental exposures

published in 2013 (Supplementary Table S1). Our

review is far from exhaustive, but sufficiently com-

prehensive to underscore how factors related to the

pathogen’s genotype (Gervasi et al. 2013b;

Langhammer et al. 2013), dosage of inoculum

(Doddington et al. 2013; Gervasi et al. 2013a,

2013b; Paetow et al. 2013), and identity and age of

the host species (Gervasi et al. 2013a; Ortiz-

Santaliestra et al. 2013) significantly influence hosts’

survival. We found that, with the exception of one

trial, the probability of surviving infections ranged

from 6% to 97% (Supplementary Table S1). We es-

timated median survival, defined as the number of

days in which 50% of the individuals inoculated with

Bd are still alive, and showed that median survival of

the host is negatively related to the dosage of Bd

inoculum (Fig. 2). Bd dosages of 104–105 zoospores

caused a sharp decline in median survival 1 month

after inoculation (Fig. 2), whereas lower dosages took

a longer time to achieve the 50% survival threshold.

Fig. 2 Relationship between median survival of the host

and the dosage of inoculum used in different challenges

of Batrachochytrium dendrobatidis published in 2013. See

Supplementary Table S1 for complete records and references.

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This slight survival advantage for the host was ob-

served only at lower pathogen densities but might be

extremely important in explaining persistence of

populations in the wild.

Assuming that all inoculations from this sub-

sample of experiments were 100% effective, surviving

amphibians had to employ either some resistance

strategy to clear or reduce Bd infection (Savage and

Zamudio 2011; Woodhams et al. 2011), or some tol-

erance strategy to control damage caused by the

pathogen. Thus, we can infer that observed differ-

ences in survival might be driven by the host’s

genetic factors (that yet remain to be identified),

considering that researchers ignored their genetic

background upon collection. If so, the expectation

is that genetically homogenous populations should

be more susceptible to pathogens, because functional

variation at resistance loci is low and transmissibility

between genetically similar hosts occurs more rapidly

(King and Lively 2012).

Variation in a host’s survival can arise from dif-

ferent defense strategies, including innate and ac-

quired immune responses. Cutaneous antimicrobial

peptides (AMPs), which are part of the innate

immune response, have antifungal activity against

Bd (Woodhams et al. 2007b; Ramsey et al. 2010;

Rollins-Smith et al. 2011; Daum et al. 2012), but

not all hosts produce the diversity and quantities

of AMPs required to overcome infections

(Woodhams et al. 2006). On the other hand, studies

of acquired immune responses indicate that higher

heterozygosities in alleles of the major histocompat-

ibility complex (MHC) are positively associated with

resistance to Bd (May et al. 2011; Savage and

Zamudio 2011). Further, specific MHC alleles and

coordinated expression of acquired immune path-

ways are associated with resistance in some species

(Savage and Zamudio 2011; Ellison et al. forthcom-

ing 2014). Thus, studies both of innate and acquired

immunity underscore the potential of the host’s ge-

notype as a predictor of the risk of disease. Clearly,

further genomic characterization of hosts’ immune

function will likely reveal additional networks of

genes linked to resistance phenotypes or genetic fac-

tors underlying interspecific interactions if particular

loci are involved in selecting host-associated micro-

biota more generally (Benson et al. 2010). To fully

understand susceptibility to disease, we must con-

sider immune responses as a complex phenotypic

trait (Knight 2012) that requires an integrated ap-

proach combining experimental manipulations with

studies of natural populations experiencing different

selective regimes through time.

Genomic studies to characterize hosts’ genotypes

from surviving and diseased individuals

Genomic methods for high-throughput marker dis-

covery, such as restriction-site-associated DNA se-

quencing (RAD-seq), genotyping by sequencing,

and functional assays (RNA-seq), allow us to geno-

type multiple individuals at thousands of random

and functionally expressed loci, and explore associa-

tions between genes and disease phenotypes (Davey

et al. 2011). Although most non-model species, by

definition, lack the genomic resources (e.g., popula-

tion genetic studies; sequenced and annotated ge-

nomes) necessary to identify particular variants of

the host, these genomic approaches can help target

genomic regions of interest, providing an initial step

in uncovering potential associations of genotype and

phenotype. For instance, by comparing single-nucle-

otide polymorphisms from dead and surviving indi-

viduals, we can identify alleles potentially associated

with the hosts’ defenses, regardless of the source of

the response (innate or acquired immunity). An ideal

study would genotype individuals to reveal neutral

and adaptive loci (Allendorf et al. 2010) in a fully

factorial experiment with standardized dosages of

pathogens and multiple strains of Bd to identify

thresholds of susceptibility, recovery time of the dis-

ease, and general and specific resistance. This would

allow us to develop the host’s susceptibility panels

and test for functional changes in gene expression

through transcriptome profiling (RNA-seq). This de-

tailed experimental approach has the power to quan-

tify temporal changes in hosts’ immune responses

(Ribas et al. 2009; Decaestecker et al. 2011;

Rosenblum et al. 2012b), by sub-sampling from

each treatment for temporal quantification of the

intensity of infection, body condition, and clinical

signs of chytridiomycosis (Rosenblum et al. 2012b).

Thus far, very few species have been part of studies

using this type of controlled exposure (Ribas et al.

2009; Rosenblum et al. 2009b, 2012b; Savage et al.

2014; Ellison et al. forthcoming 2014), and we are

still far from a solid comparative understanding

of how these underlie interspecific differences. We

expect more studies of this sort in the future as

the comparative framework for amphibian immunity

becomes available.

Genomic studies to characterize and quantify

variation in pathogenicity

In contrast to studies of amphibians’ immunity, for

which genomic studies are still relatively scarce, we

have made considerable progress toward understand-

ing the evolutionary history of Bd as an organism

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that is pathogenic to amphibians. Genomic studies

point to the enrichment of genes related to extracel-

lular and enzymatic activity as an important evolu-

tionary step in the transition of Bd from its closest

non-pathogenic relative Homolaphlyctis polyrhiza

(Joneson et al. 2011). In addition, the recent discov-

ery of Bd’s sister species (Batrachochytrium salaman-

drivorans) suggests that pathogenicity is conserved

between the members of this genus (Martel et al.

2013). Bd’s pathogenicity mechanism involves the

production of unidentified toxic factors that inhibit

the proliferation of T and B lymphocytes in the host

(Fites et al. 2013), thus precluding effective acquired

immunity. Continued proliferation of the pathogen

disrupts the integrity and osmoregulatory function of

the host’s skin (Voyles et al. 2009). However, strains

of Bd display high variation in genotypic, pheno-

typic, and proteomic traits (Fisher et al. 2009;

Voyles 2011), perhaps caused by novel adaptations

following jumps to new hosts. Therefore, further

characterization of these pathogen adaptations

should aim to quantify the interaction between the

pathogenicity of Bd and its hosts’ immunogenomics.

Now that we are accumulating genomic detail

on particular strains, we can implement focused

approaches to target and measure the expression of

putative pathogenic factors, for example, serines and

aspartic peptidases (Joneson et al. 2011; Rosenblum

et al. 2013), in time-series or dose-dependent chal-

lenges, via quantitative polymerase chain reaction.

Given that strains might become more pathogenic

under suitable conditions (Woodhams et al. 2008),

quantifying these factors will allow us to accurately

predict when the symptoms of disease develop and

the host’s fitness deteriorates.

One potential exciting possibility is to perform

dual RNA-seq of Bd and its amphibian host

(Westermann et al. 2012). Dual RNA-seq will simul-

taneously detect time-dependent changes in gene

expression from early phases of infection through

either mortality or survival. In particular, we

should look for genes promoting colonization and

adherence of the fungus (those will be correlated

with pathogen density (Rosenblum et al. 2012a)),

or for genes involved in inflammatory responses

such as mycotoxins or proteases (Joneson et al.

2011; Rosenblum et al. 2012a; Fites et al. 2013). As

new emerging technologies improve procedures for

isolating cells and for amplification, we expect that

dual RNA-seq of single epithelial cells will deliver

real-time measurements of hosts’ immune responses

and subsequent strategies for evading the pathogen.

Comparing dual RNA-seq results from in vitro and

in vivo experiments will allow us to decipher specific

responses affected by the physiological conditions

encountered in the host’s skin (Westermann et al.

2012).

Finally, genomic approaches will also allow us to

quantify and characterize variation in pathogenicity

from a phylogenetic perspective, through compara-

tive analyses of major Bd lineages (Farrer et al. 2011;

Schloegel et al. 2012; Rosenblum et al. 2013).

Putative pathogenic factors occur in genomic regions

characterized by loss of heterozygosity and variation

in number of copies, and show signatures of adap-

tation in the pathogen (Rosenblum et al. 2013).

Thus, understanding in more detail the function of

these genomic regions will likely reveal how strains

of Bd attenuate through consecutive passages

(Langhammer et al. 2013), but can be reactivated

by re-isolation from hosts (Brem et al. 2013). The

results of these lineage-based comparative studies will

elucidate mechanisms of pathogenicity, and also

potentially allow us to reconstruct the transmission

of Bd and its jumps to new hosts.

Target II: Elucidate the environmental controls

of hosts’ defenses

Our genomic studies need to transcend from the

laboratory to the field to account for the diversity

of environmental conditions under which host–

pathogen relationships occur. Certainly, considering

natural environmental variation brings additional

challenges, in part promoted by our limited knowl-

edge of the underlying short-term ecological and

long-term evolutionary processes that have shaped

interactions of hosts, pathogen, and the environment

(Fig. 1). In the amphibian-Bd system, traditionally

we have assumed seasonal variability in prevalence

and infection intensity to be due to abiotic factors

(Retallick et al. 2004; Kriger and Hero 2007), despite

the fact that it can also result from biotic changes

inherent in the host. For instance, the environment

can interact with both organisms by maintaining

polymorphisms at adaptive loci (Lazzaro and Little

2009), thus causing fluctuations in the frequency of

hosts’ genotypes carrying resistance alleles (Fig. 3), or

alternatively, Bd genotypes with high pathogenicity.

We know that genetic variation and identity of the

host contribute to the relative abundance of certain

microbial taxa in the hosts’ skin (Srinivas et al.

2013). Therefore, the consequences of these changes

in frequency of alleles can extend to other microbes

associated with hosts, which interact (either posi-

tively or negatively) with Bd. Understanding how

selection favors genetically-based polymorphisms in

the resistance of the host or in the pathogen’s

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virulence, under specific environmental conditions,

will be the next step to explain the widespread het-

erogeneity in survival of hosts across species and

across populations.

In Target II, we review the environmental compo-

nents of disease-outcomes by focusing on longitudi-

nal studies in wild amphibian populations. To show

how biotic factors inherent in the host modulate

host–pathogen dynamics, we propose two genomic

studies that will identify genetic and environmental

sources for these shifts in fitness-advantage, thus

improving our ability to predict factors leading to

epizootics in the future.

Insights from longitudinal studies

One of the most important goals for the conserva-

tion and management of amphibians infected with

Bd is to determine how disease affects the size of

the host’s population under natural conditions. In

contrast to controlled experimental settings, natural

populations often exhibit high variation in rates of

exposure to the pathogen, and in, abiotic factors and

food resources, all of which may directly affect dis-

ease-outcome and survival of the host. Longitudinal

surveys of disease combined with mark-recapture

studies provide the only reliable estimates of hosts’

survival and detectability in the wild, parameters that

are essential for evaluating population trends and

patterns affecting the spread of the pathogen (Lips

et al. 2006; Ryan et al. 2008; Briggs et al. 2010).

However, such studies are not particularly common

in the literature, perhaps reflecting logistic or eco-

nomic constraints in our ability to access remote

areas multiple times throughout the year.

Amphibian population trends observed at the time

of emergence of Bd have provided indisputable evi-

dence for a rapid decline in hosts’ population size

and survival as a consequence of chytridiomycosis

(Lips et al. 2006; Vredenburg et al. 2010). For sus-

ceptible species, emergence of a pathogen can cause

extirpation if defense traits are absent, or lead to

extreme population bottlenecks even if alleles for re-

sistance or tolerance are present. Under epizootic

conditions, the incidence of disease builds up rapidly

over a few months and mortality is high (Lips et al.

2006; Brem and Lips 2008; Vredenburg et al. 2010),

providing limited opportunity for selection for de-

fensive traits that could promote their spread

through populations of the host. Harlequin frogs

(Atelopus spp.) constitute one such example of a

Bd-susceptible genus that lacks sufficiently robust

defensive traits to persist post-epizootic. Atelopus

were once common throughout Central and South

America, and many species are now endangered or

extinct due to chytridiomycosis (Pounds et al. 2006;

Lips et al. 2008). The majority of emergences of Bd

were unnoticed in amphibian assemblages across the

world, and now we are left with species that persist

with enzootic infections (Retallick et al. 2004; Briggs

et al. 2010; Longo et al. 2010; Savage et al. 2011).

Some of these species show signs of demographic

recovery (Tobler et al. 2012; Newell et al. 2013),

making them ideal candidates for studying the evo-

lution of strategies for resistance or tolerance.

However, the most common phenomenon, both in

temperate and tropical regions, is to persist but still

carry seasonal infections that affect population sizes

(Murray et al. 2009; Longo et al. 2010; Tobler and

Schmidt 2010; Savage et al. 2011). This underscores

the idea that either the environment: (1) maintains

polymorphisms at loci for defensive traits or (2) pro-

vides possibilities for variable immune responses

across seasons or life-history stages. We must exam-

ine each of these hypotheses because they have dif-

ferent implications for determining the timing of

new epizootics.

Frequent environmental fluctuations could lead to

the maintenance of polymorphisms in defensive

traits in natural populations of the host under enzo-

otic pathogen conditions. Evolutionary theory pre-

dicts that resistance-traits that are costly will be

more polymorphic than tolerance-traits (Roy and

Kirchner 2000). Beneficial alleles for resistance

cannot fix in populations if hosts lose their fitness

advantage as disease declines (Roy and Kirchner

2000). The fact that Bd has an environmental reser-

voir, and is not necessarily limited to amphibian

hosts (McMahon et al. 2013), may increase the

Fig. 3 Hypothesized seasonal dynamics in the host’s resistance

(dashed line). Active population of the host in time A differs from

that at time B in that more individuals carry resistance-alleles

(R-alleles) at adaptive loci. Pathogen-load (solid line) modified

from multiple studies of seasonal variation in the prevalence of Bd

and its intensity of infection (Retallick et al. 2004; Longo et al.

2010; Savage et al. 2011).

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benefits of fixing alleles conferring resistance in some

populations (Savage and Zamudio 2011) and out-

weighs the costs of maintaining those traits. In situ-

ations in which selective pressures change over time,

within-population seasonal variation in the effective-

ness of immunity-related genes might alter the le-

thality of the disease (Savage and Zamudio 2011).

In other words, if hosts’ immune responses are mod-

ulated by changes in temperature or, alternatively,

pathogen density, we can expect additional shifts in

the fitness advantage from the host to the pathogen

(Fig. 3). At lower temperatures, Bd produces more

zoospores that remain active for longer periods

(Woodhams et al. 2008). At the same time, amphib-

ians decrease immune responses when environmental

conditions extend beyond their optimal values

(Raffel et al. 2006), which in turn leads to increased

rates of infection (Longo et al. 2010). Therefore,

at higher temperatures, hosts should have the fit-

ness advantage and rely on resistance strategies.

Identifying whether these temporal cycles are part

of host–pathogen coevolutionary processes will

allow a better interpretation of the genetic basis of

susceptibility to disease.

Amphibian species often have distinct phenologies,

with life-history stages dependent upon specific envi-

ronmental cues. Therefore, quantifying hosts’ sur-

vival at different stages can point us to specific

life-cycle events that may increase susceptibility or

resistance to disease. For instance, in a 7-year

mark-recapture study of Fleay’s barred frog

(Mixophyes fleayi) in Australia, survival of adults

promoted an increase in density of individuals in

the population (Newell et al. 2013). In contrast,

other species exhibit increased recruitment of juve-

niles, which allow populations to stabilize under low

abundance and high mortality of adults (Muths et al.

2011; Tobler et al. 2012). In anuran hosts co-existing

with enzootic pathogen infections, we expect to find

different mechanisms driving demographic cycles of

recovery and crash, depending on which life-stage

carries the fitness advantage against Bd (Fig. 4).

The maturation of immunity with age will give the

fitness advantage to adults because hosts will have

higher capacity in their pathogen-recognition reper-

toire. However, if hosts overcome trade-offs between

immunity and development, or alternatively, immu-

nity and reproduction, we expect a shift in the fitness

Fig. 4 Age-specific strategies for survival of the host following infection with Bd.

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advantage to younger life stages (Fig. 4). These cases

exemplify that to halt declining population trends,

hosts’ defensive strategies must switch mortality-

effects to compensatory rather than additive

(Tobler et al. 2012), an area of mitigation that has

been poorly explored thus far.

Genomic studies to investigate shifts in fitness

advantage that drive seasonal host–pathogen

dynamics

Host populations experiencing seasonal variation in

the prevalence and intensity of pathogens provide

one of the best opportunities for comparing genomic

variation and detecting selection using temporal data

on allele frequencies within populations (Fig. 3).

Using neutral loci, we can quantify effective popula-

tion size through time in the host and in the path-

ogen, which will help us elucidate the type of

evolutionary interactions between the two species.

If effective population size does not change, we can

assume that populations of host and pathogen have

both reached equilibrium. Thus, we expect no net

detrimental effects of disease favoring the evolution

of tolerance, if this defensive strategy is constant and

not modulated by any environmental factor.

However, if we do find temporal fluctuations in

host/pathogen effective population sizes, then we

can infer that seasonal changes in defensive capacity

and/or pathogenicity are driving the dynamics of the

host’s population size. We propose that examining

the origin, integration, and maintenance of adaptive

(resistance) alleles in the population’s genotype pool

over time, both for the host and the pathogen, will

uncover how pervasive these antagonistic interactions

are in natural populations. For example, environ-

mental changes in temperature inducing mortality

in the host may lead to a reduction in the frequency

of individuals carrying alleles for resistance, thus

promoting higher loads of pathogen (Fig. 3).

Understanding the timing of these dynamics of

allele frequencies will elucidate how different loci

contribute to the high variability of the host’s re-

sponses to disease. In addition, we should character-

ize how adaptive loci are expressed under different

environmental conditions or ages of hosts. We al-

ready have evidence for particular alleles significantly

reducing the risk of mortality from chytridiomycosis

(Savage and Zamudio 2011), but we do not know

how expression is affected in natural environmental

or ontogenetic contexts. A temporal analysis of dy-

namics in adaptive loci will offer important insights

into the contribution of genetic variation in different

disease-phenotypes of the host.

With respect to the pathogen, demographic pro-

cesses such as migration, population size, mutation

rates, and generation time can interact to introduce

genetic variation. Typically, pathogens are expected

to adapt faster than their hosts. Recent studies sup-

port this observation in Bd by showing high Internal

Transcribed Spacer (ITS) haplotype diversity and

long-term persistence in some localities such as

Brazil and Asia (Goka et al. 2009; Bai et al. 2012;

Bataille et al. 2013; Rodriguez et al. 2014). Therefore,

we need to assess whether these molecular signatures

in the pathogen are concordant with the generation

of counter-responses in the host. Factorial studies

comparing pathogens’ responses under four settings

(with and without host; laboratory versus field) can

separate the effects of the hosts’ defenses from the

effects of environmental controls affecting the

pathogen.

Genomic studies to characterize indirect mechanisms

of defense: the role of microbial assemblages in the skin

The immune system plays an essential role in shap-

ing the structure of microbial assemblages in frog

skin, including the likelihood of infection by Bd.

Although Bd shares the cutaneous niche with other

microorganisms, the microbiota have the potential to

be Bd-antagonistic, and thus contribute an indirect

mechanism of defense for the host (Harris et al.

2006). Using skin metagenomics, we can identify

phylotypes negatively correlated with the intensity

of infection by Bd, as well as testing their potential

use as probiotics (Bletz et al. 2013). By combining

skin metagenomics with scans of the host’s genome,

we can elucidate how the host’s genetic factors con-

tribute the control of the core microbiota, that is,

taxa present in most individuals (Loudon et al.

2013), and how environmental effects change mi-

crobes’ relative abundance. With these associations,

we might expect to find loci controlling the presence

and/or abundance of individual microbial taxa, or

groups of related taxa, as well as pleiotropic effects

if immune responses are nonspecific (Benson et al.

2010). In addition, functional analyses of Bd-antag-

onistic taxa can reflect whether their contribution

falls into a resistance strategy that limits Bd growth

or provides tolerance by aiding the host in physio-

logical processes such as osmoregulation.

Conclusions

In this review, we emphasize that survival of hosts

in the amphibian-Bd system is the norm rather than

the exception, and that many populations persist

with enzootic infections. Bd was first discovered

8 A. V. Longo et al.

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nloaded from

and recognized as an infectious pathogen causing

chytridiomycosis and population declines in amphib-

ians in 1996 (Berger et al. 1998, 1999; Skerratt 2009)

and was subsequently formally described as a new

species by Longcore et al. (1999). Since then, our

methods for quantifying survival have relied primar-

ily on ecological processes shaping host–pathogen

dynamics than on the evolutionary consequences

for the long-term persistence of amphibians. In

fact, molecular dating suggests that Bd lineages are

tens of thousands of years older than previously

thought (Rosenblum et al. 2013), indicating that

many amphibians overcame pathogen-pressure by

engaging different defensive strategies. We can hy-

pothesize that high host-diversity should favor pat-

terns of coevolutionary alternation (Davies and

Brooke 1989), in which pathogens can shift among

species over many generations, thereby evolving a

preference for hosts with higher competence. Why

Bd increased pathogenicity over recent years

(Phillips and Puschendorf 2013), or what factors

will drive its evolutionary potential under changing

climates, are important questions that are still pend-

ing answers.

Here, we highlighted several genomic approaches

to understand the disease-outcome that will espe-

cially benefit amphibians currently threatened by

Bd. In cases in which the emergence of the disease

was predictable (Lips et al. 2006), many amphibian

conservationists took preemptive actions to protect

susceptible species from disease-induced extinctions.

Now, many species remain safeguarded in captive

colonies with the goal of reintroducing them back

into natural habitats. Once the genetic determinants

of defense traits are identified for these species, se-

lective breeding for tolerance may offer protection

against multiple lineages of Bd, thereby improving

the likelihood of success in future reintroductions.

To do this, we have recommended rigorous tests

that incorporate the triad of ecoimmune parameters

(i.e., the hosts’ fitness, density of the pathogen within

the host, and changes in immune responses, sensu

Graham et al. 2011) with new genomic approaches

to characterizing responses of hosts and pathogens

to variable environments. This new subfield of

ecoimmunology—ecoimmunomics—will link host–

pathogen eco-evolutionary processes to efforts in

applied conservation.

Acknowledgments

The authors thank C. Downs, J. Adelman, and

G. Demas for inviting us to participate in the sym-

posium. A. Ellison and two anonymous reviewers

provided constructive comments on previous drafts

of this article. They gratefully acknowledge our

editor, H. Heatwole, for his thorough editorial feed-

back, which greatly improved our article.

Funding

This work was supported by Population Evolutionary

Processes [DEB-1120249 to K.R.Z.] and Doctoral

Dissertation Improvement [DEB-1310036 to K.R.Z.

and A.V.L.] grants funded by the National Science

Foundation, as well as by fellowships and grants to

A.V.L. from the Ford Foundation’s Predoctoral

Fellowship, Cornell University’s (CU) SUNY/Sage

Diversity Fellowships, CU Department of Ecology

and Evolutionary Biology, CU Graduate School,

Andrew W. Mellon Graduate Student Grants, and

Atkinson’s Center for Sustainable Biodiversity

Fund. The Research Coordination Network for

Ecoimmunology [IOS-0947177 to L. Martin,

D. Ardia, and D. Hawley] covered travel costs to

attend the symposium.

Supplementary data

Supplementary data available at ICB online.

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