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: [email protected]
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
Integrative and Comparative BiologyIntegrative and Comparative Biology, pp. 1–12
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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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