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Function al Ecology. 20 18; 1–13. wileyonlinelibrar y.com/journal/fec
© 2018 The Aut hors. Functional Ecolog y
© 2018 British Ecological Society
Carotenoid- based displays have become textbook examples of
honest signals of individual quality (Dugatkin, 2013). Although
some studies have failed to find consistent evidence that
carotenoid- based traits are related to physiological performance
(Dale, 2000; Dowling & Mulder, 2006; Smith, Raberg, Ohlsson,
Granbom, & Hasselquist, 2007), behavioural ecologist s have gener-
ally embraced the idea that carotenoid- based coloration can serve
as a condition- dependent signal of quality (Alonso- Alvarez et al.,
2004; Hill, 1991; Kemp, Herberstein, & Grether, 2012; McGraw &
Ardia, 2003; Peters, Denk, Delhey, & Kempenaers, 2004; Velando,
Beamonte- Barrientos, & Torres, 2006). Accordingly, recent re-
search on carotenoid- based ornamentation has focused on the
mechanisms by which coloration can be a reliable signal of funda-
mental aspects of performance, such as immune system function or
avoidance of oxidative stress.
Current discussions of the mechanisms that might create a
link between carotenoid coloration and performance have been
dominated by the resource trade- off hypothesis, which proposes
that carotenoid coloration is an h onest signal of individual quality be-
cause only high- quality individuals can allocate sufficient carotenoid
resources away from critical physiological processes to achieve full-
colour ornamentation (Figure 1). The resource trade- off hypothesis
is founded on three key assumptions: (1) carotenoid pigments are
limited resources such that most individuals do not possess suffi-
cient carotenoids to maximize per formance in all avenues simultane-
ously; (2) carotenoid pigments play impor tant roles in physiological
processes, particularly in immune response and/or antioxidant de-
fence; and hence, (3) carotenoid- based ornaments reflect quality
because only high- quality individuals can allocate to both sides of
the trade- off associated with carotenoid pigments.
The main purpose of this synthesis was to provide a critical
assessment of these three major assumptions of the resource
trade- off hypothesis for explaining honesty in carotenoid-
based coloration. We limit the scope of our discussion to stud-
ies of birds, which are the group of animals that have been
most extensively studied with regard to carotenoid signalling.
DOI : 10.1111 /1365 -2435 .1312 2
1Depar tment of Biologic al Sciences, Auburn
University, Auburn, Alabama
2School of Biological Sciences, Monash
University, Clayton, Vic., Australia
Rebecc a E. Koch, School of Biological
Science s, Monas h University, Clayton, Vic .,
Handling Editor: Brett Sandercock
1. Within the past several decades, resource trade-offs have emerged as the com-
monly accepted explanation for how carotenoid-based coloration links to indi-
vidual per formance. However, the literatur e on carotenoid signalling is i nconsistent
in how carotenoid resource trade-offs are defined, assessed and interpreted.
2. We provide a clear statement of the resource trade-off hypothesis for explaining
the honesty of carotenoid-based ornaments, its key assumptions and evidence for
(or against) each assumption.
3. Focusing on class Aves, we perform a critical assessment of theoretical and em-
pirical evidence for carotenoid resource limitation and for direct physiological
benefits of carotenoid pigments to immune and antioxidant performance.
4. We identify important inconsistencies in how data related to physiological func-
tion and carotenoid coloration have been interpreted in the light of the resource
trade-off hypothesis, and we suggest directions for future research.
antioxidants, carotenoid pigments, coloration, ornamentation
KOCH and HILL
Comparisons among classes of vertebrates or between verte-
brates and invertebrates can provide important insights, but
tackling physiological differences among major taxonomic
groups further complicate an already complex topic. By focus-
ing on birds in this review, we hope to highlight patterns and
questions that will have important implications for other taxa,
and we encourage application of the questions raised here to
In this review, we assess trade- off mechanisms proposed to
explain honest signalling in carotenoid- coloured ornaments while
also considering evidence for the direct physiological benefits of
carotenoids to the individual. In the process of evaluating the cur-
rent state of understanding of the main assumptions of the carot-
enoid resource trade- off hypothesis, we examined 179 empirical
studies of carotenoid- based traits in birds published between
1992 and 2017; we provide a list of these studies for reference
(Suppor ting Information Table S1, Appendix S1, Figures S1 and S2
for more detail). While debates regarding the physiological bene-
fits of carotenoids or whether carotenoids are limited resources
are not new, here, we focus on a specific, unifying framework
and its main predictions. We highlight where assessment can be
made based on currently available data and highlight where more
information is needed to adequately test the carotenoid resource
trade- off hypothesis.
Without carotenoid limitation, carotenoid resource trade- offs can-
not be the basis for honest signalling in carotenoid- based traits.
Thus, the question of whether carotenoids are limiting resources for
birds is a central question related to the resource trade- off hypoth-
esis in avian species (Hadfield & Owens, 2006; Hill, 1994; Hudon,
1994; Olson & Owens, 1998; Simons, Maia, Leenknegt, & Verhulst,
2014). An important distinction is that carotenoid limitation can be
interpreted from two distinct perspectives: individuals may be lim-
ited in the quantity of carotenoids they can acquire from the envi-
ronment, or individuals may be limited in the size of their internal
“pool” of carotenoid resources available for physiological use. While
these two interpretations of carotenoid limitation are related—an
individual cannot acquire internal carotenoid resources without
consuming c arotenoids from the environment—it is important to
consider that internal carotenoid limitation can exist without envi-
ronmental constraints (e.g., due to limit ations in quantities that can
be assimilated from the diet), and vice versa (e.g., finite quantities in
the environment may still be more than sufficient for all processes).
Environmental carotenoid limitation was among the earliest ex-
planations for variation in the expression of carotenoid- based color-
ation in both fish (Endler, 1980; Grether, Hudon, & Millie, 1999) and
FIGURE1 Simple vs. complex interpretations of the fundamental carotenoid resource trade- off hypothesis. Many studies work under
a simplified framework (grey boxes, thick black arrows) in which dietary carotenoids are absorbed from the diet, circulated, then deposited
either as external colorants or as internal immune/antioxidant boosters. However, the true pathways through which ingested carotenoids
travel are far more complex (white boxes, thin dashed arrows). (a) While not all carotenoids ingested are absorbed, many are ac tively
absorbed through the activity of SCARB1 (Toomey et al., 2017), and some small levels of passive absorption occur; passive absorption
may play a greater role when ingested carotenoid quantities are very large, such as in experimental supplementation manipulations. Active
absorption may be selective for certain carotenoids or vitamins, and trade- offs may occur bet ween which molecules are absorbed at a given
time (Fitze et al., 2007; García- de Blas et al., 2016; McGraw, Hill, Navara, & Parker, 2004). (b) The bloodstream is a major site of internal
carotenoid storage and movement, but carotenoids are also stored in the fat, liver and other tissues (McGraw, 20 06); metabolically active
tissues, more than the bloodstream, are the likely locations of any physiological activities of carotenoids beyond serving as colorants (c).
Biotransformation of carotenoids into dif ferent forms, including cleavage of retinol precursors to form beta- carotene (such as by BCMO1;
Hill & Johnson, 2012) or conversion of dietary yellow carotenoids into red ketocarotenoids (such as by CYP2J19; Lopes et al., 2016; Mundy
et al., 2016), may drastically alter the concentration and types of carotenoids present internally. Excess carotenoids may also be cleaved
(such as by BCO2; Hill & Johnson, 2012) and excreted, and measuring such carotenoids may provide important information about whether or
not an individual faces internal carotenoid limitation
KOCH and HILL
birds (Hill, 1990; Hill, Inouye, & Montgomerie, 20 02). The necessity
that animals acquire carotenoids through their diet is sometimes
taken as clear evidence that carotenoid acquisition is limiting, but
carotenoid resources can be finite without being limiting. It is pos-
sible that essentially all wild birds ingest sufficient carotenoids to
meet their physiological needs (Hudon, 1994). Unfortunately, ques-
tions related to environmental carotenoid limitation are difficult
to answer under natural conditions wherein a population of birds
may consume a wide variet y of food items, individuals may dif fer
in which and how much of those various items they consume, and
the internal processes of carotenoid assimilation (which may differ
among individuals, species, season and even specific carotenoid pig-
ments; McGraw, 2005; Tella et al., 200 4) are largely unknown in any
given study system. Studies testing for preferences for carotenoid-
rich food in birds with carotenoid- based ornamentation have found
only mixed support (Bascuñán, Tourville, Toomey, & McGraw, 2009;
Behbahaninia, Butler, Toomey, & McGraw, 2012; Catoni, Metzger,
Schaefer, & Bairlein, 2011; Senar et al., 2010), indicating that many
colourful birds do not need to preferentially forage for carotenoid-
rich food in order to produce colour ful ornaments (but see Walker
et al., 2014). Currently, environmental carotenoid limitation is not
widely considered to be a major determinant of carotenoid- based
colour signalling (Hadfield & Owens, 2006; Møller et al., 2000;
Svensson & Wong, 2011).
While there is general acceptance that most populations of birds
are not limited by the quantity of carotenoids they can acquire from
the environment, experimental studies show that even wild popu-
lations of birds can respond to dietary carotenoid supplementation
(Ewen, Thorogood, Karadas, & Cassey, 2008; Sternalski, Mougeot,
Perez- Rodriguez, & Bretagnolle, 2012). If natural environments are
not carotenoid- limited, then how can supplementation affect the
coloration or physiology of wild birds? That supplementation has
sometimes been found to increase coloration, immune response or
other measurements in birds with access to their normal diets raises
questions about our assumptions of environmental carotenoid lim-
itation—and about supplementation itself.
It is difficult to validate the biological meaning of response to
artificial carotenoid supplementation. Dietary supplementation in-
herently provides carotenoids in quantities and frequencies that dif-
fer from levels that would normally be consumed in the wild. Even
if such supplementation does not lead to abnormally high plasma
carotenoid levels, supplemented birds may be rapidly transpor ting
excess carotenoids to other tissues. Carotenoids provided in sup-
plementation can also enable birds to bypass metabolic pathways.
For instance, when the red carotenoid canthaxanthin is provided in
diet (Hill, 1992), birds can bypass the oxidation of yellow pigments
to red (Lopes et al., 2016). More subtle changes in the ratio of ca-
rotenoids in supplemented diets—such as the ratio of lutein to zea-
xanthin—can further alter the effects of supplementation (Fit ze,
Tschirren, Gasparini, & Richner, 2007; García- de Blas, Mateo, &
Alonso- Alvarez, 2016). Experimental supplementation may there-
fore provide different quantities of various carotenoids at artificially
high relative abundances and for different periods of time than what
any bird would experience in the wild (Koch, Wilson, & Hill, 2015),
which makes it difficult to assess the relevance of results of supple-
mentation to variation observed in wild populations.
We can reconcile conflict between general acceptance that wild
birds are not environmentally limited by carotenoids with findings
that supplementation can modify bird phenotype if we accept that
carotenoid supplementation provides birds with unnatural quanti-
ties or types of carotenoids with uncertain consequences for phys-
iology and ornamental trait expression. Future research into honest
carotenoid signalling may benefit from a shif t away from using ca-
rotenoid supplementation in experiments. Given the number of
variables already interacting to affect how supplemental carotenoid
regimens alter avian physiology (dose, duration, type, life- history
stage of subjects and season), it may be most productive for future
studies of wild birds to avoid dietary manipulation and instead focus
on physiological challenges or measurements of standing variation.
In studies of birds held in captivity, it may be most useful to provide
subjects with a seed, insec t or fruit diet that more closely replicates
natural carotenoid availability, without the potentially confounding
effects of dosing with purified synthesized carotenoids. Over 40%
of the studies we examined performed carotenoid supplementation
(Suppor ting Information), and such experiments have made clear
contributions to our understanding of avian physiology and color-
ation; however, we argue that future studies would be better ser ved
by focusing on internal carotenoid use rather than external carot-
Even if acquiring carotenoids from the environment poses no
limitation on carotenoid use, the challenges of distributing carot-
enoids internally could still create trade- offs in how carotenoids are
allocated. This concept of internal carotenoid limitation assumes
that the quantities of carotenoids absorbed from the diet, trans-
ported through the body and stored for future use are finite and
small enough that birds may not be able to allocate sufficient ca-
rotenoids to both external ornamentation and internal processes.
Importantly, it is well known that internal carotenoid resources and
their allocation may differ widely thro ugh seasons based on breeding
(particularly for females depositing carotenoids in yolk) or moult (for
species with plumage coloration), and studies must consider that lim-
itation may be present in some seasons but absent in others—though
changes in dietary consumption may also compensate for changing
need (Hill, 1995; Isaksson, Von Post, & Andersson, 2007; McGraw,
Nolan, & Crino, 2011; Sassani, Sevy, Strasser, Anderson, & Heath,
2016). Internal carotenoid limitation is most commonly tested by
assessing correlations among different physiological processes that
may require carotenoids, such as immune system function and orna-
mental colour production. We discuss these types of studies in detail
in the following sections.
However, carotenoid limitation is, at its heart, a quantitative
topic that depends on the amount s of carotenoids that are available
to a given physiological process at the time they are needed. There
are now sufficient data to estimate the quantities of carotenoids that
an average individual in a well- studied species like the house finch
(Haemorhous mexicanus) may require to properly colour its feathers
KOCH and HILL
(Suppor ting Information Appendix S2); we estimated that the aver-
age moulting male house finch requires about 41 μg of carotenoids
in total to colour his ornamental plumage, while he possesses about
58 μg in his liver, blood and fat at any given time during moult. That
carotenoids may be limiting is conceivable if a male house finch pos-
sesses only 17 μg of excess carotenoids in his body during moult,
stored throughout various tissues. However, our calculations do not
estimate the quantities of carotenoids that are entering each bird’s
body ever y day through its diet, offsetting the quantities of carot-
enoids it deposits in its feathers. These quantities of carotenoids
should be validated by tracking of carotenoids through an animal’s
body. Ultimately, questions of internal c arotenoid limitation should
be answered by measuring the size of the internal resource “pool” in
a bird’s tissues with the quantities of carotenoids required to fully
colour an ornament and/or participate in an important physiological
It is widely stated that carotenoids play an important role in immune
system function in birds, but their direct involvement as antioxidants
in avian systems is uncertain and contentious (Costantini & Møller,
2008; Hartley & Kennedy, 200 4; Perez- Rodriguez, 2009; Simons,
Cohen, & Verhulst, 2012). Studies aimed at testing physiological
functions of carotenoids have been conducted with such diverse ap-
proaches that a quantitative meta- analysis, while useful for assess-
ing general patterns (Simons et al., 2012; Weaver, Santos, Tucker,
Wilson, & Hill, 2018), misses important relationships between par-
ticular experimental designs, study systems and result s. Thus, we
assessed the evidence that carotenoids play a key role in immune
defence and in the avoidance of oxidative stress using a qualitative
assessment of published studies. Moreover, while we provide a brief
description of a wide variety of studies related to the carotenoid re-
source trade- off hypothesis in Supporting information Table S1, we
aim to avoid overgeneralization of studies by “‘vote-counting’ posi-
tive or negative relationships” (Koricheva, Gurevitch, & Mengersen,
2013), and instead encourage readers to assess results in- context
within publications themselves. In the following sections, we high-
light some important sources of variation and outlying questions
regarding tests of the physiological benefits of carotenoids. The
question of whether or not carotenoids play important physiological
roles besides ser ving as pigments for coloration is fundamental to
determining whether carotenoids are valuable resources that must
be differentially allocated among functions at all.
Carotenoids are so widely accepted as enhancers of vertebrate
immune function that some recent studies state the role of carot-
enoids as beneficial to immunocompetence as a well- established
truth (Benito, Gonzalez- Solis, & Becker, 2011; Giraudeau, Chavez,
Toomey, & McGraw, 2015; Merrill, Naylor, & Grindstaf f, 2016).
Originally founded on a small number of empirical papers examining
the potential benefits of a carotenoid- rich diet in humans (Krinsky,
1989; Stahl & Sies, 2005) and then expanded to behavioural ecology
by foundational theoretical papers such as Lozano (1994), studies
have since examined correlations between carotenoid- based col-
oration, circulating carotenoid levels, carotenoid consumption and
measurements of immune response to test whether carotenoids en-
The specific means by which carotenoids may improve immune
system performance are rarely articulated and remain largely un-
resolved. Studies investigating carotenoids and immune benefits
often cite studies of mammalian species (Bendich, 1989; Jyonouchi,
Zhang, Gross, & Tomita, 1994; Kim et al., 200 0), or a review by
Chew and Park (2004) that also discusses experiments performed
almost entirely on mammalian subjects. Briefly, carotenoids have
been implicated in lymphocyte proliferation and activity, though
their biochemical participation in such processes remains uncertain
(Chew & Park, 2004). It is possible that carotenoids may be local-
ized to specific organelles, such as the mitochondria, where they
may prevent oxidative damage and therefore facilitate proper cellu-
lar function in immune cells (Chew & Park, 20 04; Hill, 2014; Koch,
Josefson, & Hill, 2017)—but this hypothesis remains to be tested.
Carotenoids have also been cited as important to preventing dam-
age to self during the innate immune process of oxidative burst in
which immune cells target pathogens for oxidative damage by rap-
idly releasing pro- oxidants (Chew & Park, 20 04), though the process
functions differently in mammalian vs. avian cells (Harmon, 1998),
and thus far there is little evidence to suggest a role of carotenoids
in oxidative burst in birds (Koch et al., 2018; Sild, Sepp, Manniste, &
Horak, 2011). While research into the potential benefit of dietary
carotenoid supplements in poultry has been fairly extensive, such
studies are not focused on providing biologically relevant doses and
are largely not useful tests of how carotenoid function may contrib-
ute to honest signalling (Koutsos, Lopez, & Klasing, 2007; Koutsos,
López, & Klasing, 20 06; Meriwether, Humphrey, Peterson, Klasing,
& Koutsos, 2010; Shanmugasundaram & Selvaraj, 2011; Surai, 2002,
The first step towards understanding the role of carotenoids in
immune response within an ecologically relevant context is to per-
form studies of how, where and when carotenoids are absorbed,
transported throughout different tissues in the body and deposited
in ornaments. Carotenoid trade- of fs can be convincingly estab-
lished only if the endpoints of carotenoid mobilization are deter-
mined. Using techniques to label carotenoid pigments and track
their absorption, transport and conversion throughout the body (see
Conclusions) will be imperative for elucidating carotenoid movement
among and storage within tissues and how these patterns may differ
among individuals, sexes and species. By combining such methods
with various immune challenges, conclusive tests of allocation trade-
offs are possible.
In conjunction with studies that track the allocation of carot-
enoids, we encourage the development and use of experimental
KOCH and HILL
manipulations that more closely simulate the kinds of pathogenic or
parasitic challenges a bird may be expected to encounter in nature.
The carotenoid trade- off literature is dominated by results based on
quantification of parasites (such as coccidia) or assessment of im-
mune function based on swelling response to injection of phytohem-
agglutinin (PHA; Supporting information Table S1). A primary reason
that these measures are so widespread is that they have no appar-
ent long- term effects on a bird and can be performed even in the
field with no obvious side effects. While methods like PHA injection
are convenient and pose relatively low risk of harm to experimen-
tal subjects, the results can be difficult to interpret (Adamo, 2004;
Biard, Hardy, Motreuil, & Moreau, 20 09). Challenging birds with a
live pathogen or parasite is arguably the most biologically relevant
means to test whether carotenoids function in immune response
(Brawner, Hill, & Sundermann, 2000; Lindstrom & Lundstrom, 200 0),
particularly when we do not yet know the specific immune processes
that may involve carotenoids. However, the experimental benefits of
using live pathogens or parasites must be weighed against the ethics
of inducing suffering in vertebrate animals.
Better methods for measuring immune response is a focus of
the growing field of eco- immunology (Demas, Zysling, Beechler,
Muehlenbein, & French, 2011; Graham et al., 2011; Mar tin, Weil,
& Nelson, 2006), and we urge ecologists, animal behaviourists
and evolutionary biologists alike that study carotenoid coloration
to adopt new and better techniques for assessing immune system
function. We also suggest that information in the avian and poul-
try disease literature (Boseret, Losson, Mainil, Thiry, & Saegerman,
2013; Dorrestein, 2009; Joseph, 2003; Lister & Houghton- Wallace,
2012; Pattison, McMullin, Bradbury, & Alexander, 2008) is an im-
portant resource for finding pathogens that can be dosed to sub-
jects in a controlled setting. Information from songbird veterinary
studies may be particularly applicable to studies that keep captive
populations of passerines commonly used in tests of carotenoid-
based signalling, like zebra finches (Taeniopygia guttata), European
greenfinches (Carduelis chloris) or house finches. A wide variety of
viral, bacterial and fungal diseases have been described in passerine
species (Dorrestein, 2009; Joseph, 2003), each of which may be a
potentially useful challenge for a study of wild birds held in captivity.
Using diseases known to veterinary medicine may help researchers
reduce harm to animals by t aking advantage of known treatments
and also may provide a useful resource for how to detec t and mea-
In sum, that carotenoids are essential to proper immune func-
tion is not yet justified by consistent empirical evidence. There is
persistent variation in the results of studies testing for relationships
between carotenoids and immunocompetence such that a straight-
forward, universally beneficial ef fect of carotenoids seems unlikely.
We urge future research on these problems to: (1) perform in- depth
analyses of where and in what quantities carotenoids move through
the body to develop specific hypotheses for mechanisms by which
carotenoids may interact with the immune system; then, (2) carry
out targeted immune challenges and/or measurements that hone in
one these specific hypothesized processes.
Before the former has been accomplished (developing specific
hypothesis for where carotenoids participate in immune defence),
studies interested in testing for a benefit of carotenoids should
avoid using specific immune test s with questionable relevance to
overall disease resistance (e.g., PHA). We instead suggest studies
consider using challenges and measurement s that best gauge bi-
ologically relevant immune defence, such as pathogen infection
and measurement of clearance (Lindstrom & Lundstrom, 200 0),
tolerance (Adelman, Kirkpatrick, Grodio, & Hawley, 2013), survival
(Hanssen, Hasselquist, Folstad, & Erikstad, 2004) or a comprehen-
sive suite of measures integrating among branches of the immune
system (Adamo, 20 04; Demas et al., 2011; Millet, Bennett, Lee, Hau,
& Klasing, 2007; Salvante, 2006). Injecting birds with bacterial lipo-
polysaccharide (LPS) may be a useful option for inducing systemic
immune response without a live pathogen, but similarly, we encour-
age use of this technique to be combined with overall measures of
performance (such as body temperature, cytokine levels; Adamo,
2004; Demas et al., 2011; Millet et al., 2007). With any challenge,
however, we emphasize that a decrease in coloration during immune
response does not alone signify that carotenoid pigments are being
used directly in the response. Isolating the movement of carotenoids
through the animal body, particularly during states of challenge, is a
key to advancing the field.
Whether or not carotenoids function as important antioxidants in
the bodies of birds is contentious (Costantini & Møller, 2008; Perez-
Rodriguez, 2009; Svensson & Wong, 2011). Two meta- analyses
have examined relationships between circulating carotenoids and/
or carotenoid- based coloration and oxidative stress parameters
and both reported small and generally non- significant effect sizes
(Costantini & Møller, 2008; Simons et al., 2012). Others have sug-
gested that carotenoids may indic ate oxidative state without di-
rectly participating as antioxidant s, such as by becoming “bleached”
during oxidative stress (Hartley & Kennedy, 2004) or because only
particular oxidative states (García- de Blas et al., 2016) or high lev-
els of cellular functionality (Johnson & Hill, 2013) facilitate the con-
version of dietary carotenoids into ornamental carotenoids. While
carotenoids are, by their chemical nature, effective electron receiv-
ers under most conditions and hence are potential antioxidants (El-
Agamey et al., 2004; Krinsky & Yeum, 2003; Stahl & Sies, 20 03), it
is uncertain whether the physiological systems of birds are adapted
to deploy carotenoids as important antioxidants to maintain redox
balance (Perez- Rodriguez, 2009). The key question is whether the
right forms of carotenoids are present in sufficient quantities in the
necessary organ- level, cellular or even subcellular locations to have
a biologically significant effect on an individual’s ability to maintain
healthy oxidative balance.
A challenge central to any studies examining oxidative stress
parameters is that the interplay between pro- oxidants and antiox-
idants causes complex and variable fluctuation in levels of antioxi-
dants, pro- oxidants and oxidative damage (Costantini, 2014; Hõrak
KOCH and HILL
& Cohen, 2010; Monaghan, Metcalfe, & Torres, 2009), and such
levels may var y between different locations in an individual’s body.
As in many tests of immune function, studies on live animals are re-
stricted to assessing oxidative stress from blood samples to avoid
terminal experimentation (Cohen, de Magalhães, & Gohil, 2010), al-
though levels of oxidative damage markers or antioxidant capacity
in the blood may have limited relevance to oxidative stress in other
organs (Santos, 2017; but see Stier et al., 2017). Nonetheless, given
that blood is a main location of carotenoid storage and transpor t,
measuring blood- based oxidative stress parameters may be consid-
ered relevant to gauging carotenoid antioxidant activity. However,
centralized and metabolically active organs like the liver are likely
to play a more direct role in maintaining the oxidative state of birds,
so estimates of carotenoids and oxidative stress in blood will likely
be only an indirect measure of functions occurring elsewhere in the
It is impor tant to note that experiments would ideally examine
the relationships between antioxidants, pro- oxidants and damage
in the specific tissues most pertinent to the research objectives.
For example, measuring oxidative parameters in flight muscle may
be a good target for testing activity- related oxidative stress, while
sites of infec tion or immune cell congregation may be better suited
for testing immune- related oxidative stress. In addition, the chem-
ical paraquat is sometimes administered in oral doses as a gener-
alized oxidative challenge, but it can have localized effec ts where
it accumulates within the digestive tract (Koch & Hill, 2017). A s a
result, measuring oxidative damage in the blood may only indirectly
correspond to any responses to oxidative stress occurring due to
paraquat—and a paraquat challenge may not be expec ted to alter
oxidative stress in tissues important to the production of coloured
ornaments (Isaksson & Andersson, 2008). A whole- body challenge
like low- dose ionizing radiation may be more effective than more
localized challenges like diquat or paraquat (Koch & Hill, 2017) in
inducing oxidative stress that c an be meaningfully assessed in blood-
A recent study by Tomášek et al. (2016) has proposed that an
important explanation for inconsistency in relationships between
carotenoids and oxidative stress is that the current commonly used
methods for measuring antioxidant defences fail to capture the ef-
fects of carotenoids because they focus on hydrophilic rather than
lipophilic reactions (given that carotenoids themselves are lipophilic).
García- de Blas et al. (2016) also found that hydrophilic antioxidant
capacity in red- legged partridges was largely af fected by hydrophilic
antioxidants rather than lipophilic carotenoids. The implication that
the most commonly used methods for estimating antioxidant de-
fences in studies of carotenoid- based coloration in birds are likely
to miss effects of carotenoid activit y is intriguing and may provide
some explanation for the inconsistency of previous studies, although
measurement of antioxidant capacity is only one aspect of oxidative
stress that has been tested.
Overall, studies of the relationships between carotenoids and ox-
idative stress have yielded complex result s that are hard to interpret
and do not clearly support the hypothesis that carotenoids ser ve as
key antioxidants in birds. As with tests of the immune benefits of
carotenoids, a critical next step is performing detailed experiments
to try to isolate where and in what quantities carotenoids are pres-
ent in the avian body and how, specifically, these carotenoids may or
may not interact with pro- oxidants. Labelling carotenoid molecules
(see Conclusions) will also allow for new tests of where carotenoids
are present, and—in combination with new oxidative challenge tech-
niques (Koch & Hill, 2017)—how and where they may be transformed
in response to oxidative stress. A better understanding of the sub-
cellular locations of carotenoids and the role that they play in anti-
oxidant defences is essential to drawing conclusions about whether
the antioxidant potential of carotenoids in vitro does in fact reflect
an important and significant role of carotenoids as antioxidants in
vivo—and how the deposition of carotenoids in coloured ornaments
may or may not alter the ability of an individual to maintain a healthy
oxidative balance (von Schantz, Bensch, Grahn, Hasselquist , &
Wittzell, 1999). Given ongoing ambiguity in the antioxidant function
of carotenoids in the avian body, we encourage researchers to avoid
making general assumptions that carotenoid antioxidant activity
may explain patterns in results.
The literature associated with the resource trade- off hypothesis
is founded on the prediction that, in populations of animals with
carotenoid- based colour displays, carotenoid coloration enables as-
sessment of individual quality. The various ways in which quality can
be defin ed and assessed wit h respect to condi tion- depend ent signals
have been considered in detail in several recent papers (Hill, 2011,
2014; Lailvaux & Kasumovic, 2011; Wilson & Nussey, 2010). Here,
it is impor tant to consider how concepts of quality can influence
the interpretation of physiological data with regard to carotenoid
resource trade- offs. There is a general lack of consistency within
studies that test carotenoid resource trade- offs in how individual
quality is defined, resulting in added confusion in the literature (Hill,
2011). Studies of the honesty of carotenoid- based ornaments tend
to invoke individual quality from one of three broad perspectives:
(1) oxidative or immune health state, (2) magnitude of internal ca-
rotenoid resource pools or (3) some aspect of inherent, underlying
functionality (Table 1). These three aspec ts of quality are not mutu-
ally exclusive, but instead tend to represent the way that “high” or
“low” quality is interpreted and tested within a given study.
In studies of carotenoid signalling that are focused on health
state, it is commonly observed that birds presented with an immune
challenge, such as a pathogen or parasite infection, develop paler
carotenoid coloration than control individuals (Brawner et al., 2000;
Faivre, Gregoire, Preault, Cezilly, & Sorci, 2003; Rosenthal, Murphy,
Darling, & Tarvin, 2012). In these cases, carotenoid- based traits may
be interpreted as honest signals of quality because increasing phys-
iological challenge inhibits fullcolour expression (Table 1). In other
KOCH and HILL
TABLE1 Distinguishing features of the main different axes along which individuals are considered to vary in quality in studies of carotenoid trade- offs
Health state Carotenoid coloration
reflects current health
state or a similar
Higher quality individuals
have been subjected to
health state (e.g.,
Challenging an individual
will decrease his
How we predict internal carotenoid levels (and external coloration)
to vary in response to challenge differs depending on how we
expect carotenoids to interact wit h physiological responses.
Carotenoids may either be “use d up” in boosting response
(causing a negative correlation between internal carotenoid levels
and physiological response) or may be beneficial simply when
present in larger quantities (causing a positive correlation
between internal carotenoid levels and physiological response).
Higher quality individuals
have larger pools of
Individuals with larger
carotenoid resource pools
will have increased
The biological relevance of artificially increasing or decreasing
dietar y carotenoid levels is almost impossible to verif y. We do not
know how a given dietary programme may actually affect internal
carotenoid levels, given variation in carotenoid absorption and
transportation that is difficult to measure.
health state and
reflects the combined
effec ts of health state
and internal carot-
Healthy, carotenoid- rich
individuals may be
considered high quality,
both health state
Uncertain Without clear knowledge of t he relative quantities of carotenoids
“used up” (or not) by a health challenge, it is difficult to predict in
any given system how dietary carotenoid supplementation or
deprivation may affect coloration and/or response.
reflects some aspect
of intrinsic physiologi-
Higher quality individuals
are those that per form
best within one treatment
group (e.g., the individuals
with bes t coloration
despite an immune
Under identical conditions,
individuals will vary in
oxidative stress measures,
some individuals will
exhibit superior perfor-
mance across multiple
metrics compared with
Without isolating the genetic and/or physiological for under lying
variation in individual performance, it is difficult to disentangle
underlying differences in performance from external differences
in environment and other external factors (even within individuals
held under the same conditions).
KOCH and HILL
experiments, studies report positive relationships between supple-
mentation of dietary carotenoids, amount of circulating carotenoid
pigments (a proxy for the size of internal carotenoid resource pools)
and the expression of colourful traits (Hõrak, Sild, Soomets, Sepp, &
Kilk, 2010; McGraw & Ardia, 2003). Such observations suggest that
individuals with more pigment s (larger resource pools) have higher
quality ornaments (Table 1) and that coloration is affected by chang-
ing access to dietary pigments. When these two general patterns
are considered in combination with the hypothesis that carotenoids
may directly boost physiological function, predictions regarding the
relationships between internal carotenoid pigments, physiological
performance and external carotenoid coloration become extremely
challenging to formulate (Table 1).
It is difficult to articulate a hypothesis that incorporates the neg-
ative effects of physiological challenges on carotenoids, positive
effects of carotenoids on coloration and positive effects of carot-
enoids on physiological function. The main challenge is that we do
not know the specifics of how or where carotenoids are used by im-
mune or antioxidant systems, as described in the previous section.
Thus, it is not possible to make realistic predictions in experiments
that investigate the interactions among these variables. For exam-
ple, one might propose that internal carotenoid pigments are “used
up” while boosting physiological function, in which case the predic-
tion would be a negative relationship between strength of physiolog-
ical response and carotenoid levels. Alternatively, carotenoids might
boost immune func tion without being consumed, in which case the
prediction would be a positive relationship between physiological re-
sponse and carotenoid levels. These two predictions both assume
that carotenoids serve a direct beneficial role in individual perfor-
mance, but they yield opposite predictions regarding the direction
of the relationship between internal carotenoid levels and measures
of that performance.
One method to attempt to test whether carotenoids are con-
sumed in the process of boosting response (and whether they are
involved in the response at all) may be to induce a systemic physio-
logical challenge while manipulating dietary carotenoid access and
measuring carotenoids or ornamental coloration. Hypothetically, if
carotenoids are consumed during response and are present in phys-
iologically limiting amounts, then only supplemented individuals
should be able to maintain coloration during a challenge. However,
results of studies testing for these interactions are often difficult to
interpret. For example, both Sild et al. (2011) in greenfinches and
Alonso- Alvarez et al. (2004) in zebra finches found no significant
interaction between immune challenge (LPS injec tion) and carot-
enoid supplementation on plasma carotenoid levels or ornamental
coloration, respectively. In contrast to predictions, they found that
immune challenge decreased circulating carotenoids or coloration
by a constant amount, regardless of dietary carotenoid manipula-
tion—though supplemented individuals always had greater circulat-
ing carotenoids or coloration when compared to control individuals.
The question then becomes, what caused the decrease in carot-
enoid circulation or ornamental expression during the challenge?
Detailed measurement of the strength of individual response and
the mobilization of internal carotenoid resources pools would be
necessary to distinguish whether these challenged individuals paid
a set “price” in carotenoid resources to boost response to the chal-
lenge, or whether they simply decreased carotenoid absorption by a
set amount while mounting a response.
The study by Alonso- Alvarez et al. (2004) also demonstrates an
interesting case of how examining the same results from a different
perspective can alter interpretations. The authors found no direct,
categorical effect of carotenoid supplementation on the ability of
zebra finch red blood cells to resist oxidative attack; however, they
did find that individuals with the greatest increase in plasma carot-
enoid content had the strongest performance on the oxidative at-
tack test (Figure 2). This latter result could be seen as supportive
of the resource trade- off hypothesis: perhaps individuals with the
greatest quantity of carotenoids in internal tissues gained the larg-
est benefit in resistance to oxidative damage. Without knowing how
and where c arotenoids may perform such antioxidant function, how-
ever, we cannot separate this resource trade- off interpretation from
alternative hypotheses. It is also possible, for example, that some
individuals (i.e., high- quality individuals) had fundamentally superior
performance across multiple physiological arenas such that they had
stronger defence against free radical attack as well as a greater ca-
pacity to absorb carotenoids from the diet (Hill, 2011). This lat ter
hypothesis may be a better fit in that it predicts the same pattern
of association between increase in plasma carotenoid levels and in-
crease in resistance to oxidative stress, and it also explains a lack
of direct effect of supplementation on oxidative damage resist ance
(Figure 2), as carotenoids were no longer required to directly partic-
ipate in antioxidant reactions. These alternative interpretations em-
phasize that it is important to consider the complexity of the system
under study and to be explicit regarding the assumptions that lead
to a conclusion that carotenoid trade- offs are involved. The use of
this additional analysis demonstrates how examining variation at the
individual level rather than among discrete treatments may be par-
ticularly fruitful for interpreting results, given that variation within
treatment groups or problems with the treatments themselves (e.g.,
supplementation dose) may obscure meaningful patterns.
To summarize, widespread uncertainty exists in the carotenoid
literature about whether we define high- quality individuals as those
that are (1) currently healthy, (2) currently possessing large quan-
tities of carotenoids, some combination of both or (3) inherently
superior in one or more physiological metrics. The result of this un-
certainty is that a wide range of observations can be used to support
resource trade- off hypothesis even if alternative explanations exist,
such as index hypotheses that propose trait quality as an indicator
of internal conditions rather than as a direct product of costly trade-
offs (Biernaskie, Grafen, & Perry, 2014; Hill, 2011; Weaver, Koch &
Hill, 2017). While it is not erroneous to discuss how results fit (or
fail to fit) particular frameworks, the danger is in suggesting most
every observation to be supportive of a favoured hypothesis. A lack
of exclusive predictions hinders our ability to draw accurate conclu-
sions about support for or against the resource trade- off hypothesis
and discourages consideration of alternative hypotheses as stated
KOCH and HILL
evidence for the current trade- off paradigm continues to build.
We encourage future studies to consider approaching experiments
with the perspective of quality as the “underlying functionalit y” of
individuals (Table 1), as it sidesteps many potential methodological
issues (e.g., comparing supplemented to unsupplemented individu-
als when the supplemental dose has unknown biological relevance)
and hones in on the physiological differences among individuals that
allow some to express higher quality coloration than others.
Several common threads emerge from our consideration of the ca-
rotenoid resource trade- off hypothesis. There is a critical need for
better understanding of the specific biochemical activity of carote-
noids in the animal body, for more detailed observations of the quan-
tities and types of carotenoids present in various organs and cellular
locations (and their movements between these locations), and for
the articulation and testing of specific mechanisms that link produc-
tion of carotenoid- based colour displays to individual qualit y. For no
species have we yet quantified and characterized the full journey
of carotenoids through an individual’s body including the quanti-
ties of carotenoids absorbed, the internal locations to which those
carotenoids are transported, where they are stored, how and when
stored carotenoids are used, the quantities and loc ations of carot-
enoids needed to achieve full coloration of an ornament, and the ef-
fects of experimental supplementation or physiological challenge on
processes like carotenoid absorption and distribution. While these
processes will vary among species and among individuals within a
species, establishing the general patterns in these processes even in
model avian species will significantly advance our ability to under-
stand the role of carotenoids in physiological performance as well
It is already possible to estimate the quantities of carotenoids
present in the body or deposited in the feathers in species where d e-
tailed carotenoid analyses of many tissues have been reported, such
as the house finch (Supporting Information Appendix S2). However,
it will be more informative to experimentally label specific carot-
enoid molecules and follow them directly as they travel through the
body and undergo transformations. Carotenoid radiolabelling has
been used sparingly in animals, but radiolabelled canthaxanthin has
been used in two studies that tracked carotenoid absorption and/or
metabolism in chickens (Schiedt , 1989) and trout (Hardy, Torrissen,
& Scott, 1990); developing labelled dietar y carotenoids for use in
modern studies will open up a rich new resource for discovering
patterns of carotenoid movement and use in the body (Jansen &
Lugtenburg, 1996). Characterizing carotenoid absorption, transport,
storage and conversion in both healthy and immune- challenged
birds, for example, will provide clear answers to long- standing ques-
tions about how immune activation modifies carotenoid coloration,
and whether the carotenoids appear to directly participate in re-
sponse (e.g., if carotenoids are found to be transported to immune
cells or tissues).
In addition, break throughs in deducing the enzymes that con-
trol carotenoid coloration in birds (Lopes et al., 2016; Mundy et al.,
2016; Toews, Hofmeister, & Taylor, 2017; Toomey et al., 2017) pro-
vide exciting oppor tunities to measure gene expression and to con-
duct knock- down or knockout experiments to further parse how
FIGURE2 An example of the challenges of interpreting experiments manipulating carotenoid dose and environmental conditions.
Highlighted is a subset of results from a carefully executed study by Alonso- Alvarez et al. (2004). These authors supplemented an
experimental group of zebra finches with carotenoids in the drinking water (a) and compared their antioxidant defence (red blood cell
resistance to oxidative challenge) to that of control birds not supplemented with carotenoids. They found that carotenoid- supplemented
birds had increased carotenoid- based bill coloration and plasma carotenoid content, but no difference in antioxidant defences relative to
control birds (b). However, within both treatment groups, birds with higher plasma carotenoid content had higher antioxidant defences
(c). There are t wo divergent explanations for these patterns: (1) plasma carotenoids boosted antioxidant defences directly, or (2) birds
that were better at absorbing and transporting carotenoids also had inherently better defences against oxidative challenge. Given that
the supplementation treatment itself had no effect on antioxidant defences, the former explanation is not well supported. The challenge
for researchers is separating causative (explanation 1, above) from correlative (explanation 2) relationships between carotenoids and
KOCH and HILL
and why individuals may differ in their internally sequestered and
externally displayed levels of carotenoids. A knock- down in a main
carotenoid absorption gene, SCARB1, has already been character-
ized in the domestic canary, a species with carotenoid- based plum-
age coloration both in domestic and in wild populations (Toomey
et al., 2017). Studying SCARB1 knock- downs like that of the canary
provides researchers the unique opportunit y to test the effects
of severe physiological carotenoid depletion without relying on
dietary manipulations (Koch et al., 2018). As technology contin-
ues to advance and allow for manipulation of target genes within
vertebrate systems, studying knock- outs in carotenoid absorption
(Toomey et al., 2017) or transformation (Lopes et al., 2016; Mundy
et al., 2016) genes will yield transformative information regarding
the costs or benefits of specific types of carotenoids and the pro-
cesses involving them. Simultaneously, examining the expression of
these genes under different environmental, seasonal or develop-
mental stages offers a new opportunity to advance our understand-
ing of the mechanisms underlying colour variation and to make
inferences about the role of such mechanisms in signal honesty.
We note several of these gene products and their functions in the
caption of Figure 1.
Until we have established a better fundamental understand-
ing of carotenoid physiology, we urge researchers studying carot-
enoid coloration to maintain an open perspective with regard to
whether or not carotenoids ser ve key physiological functions, such
as immune enhancement or free radical scavenging, and whether
resource trade- offs are the bases for honest carotenoid coloration.
Correlations between carotenoid levels, coloration and physiologi-
cal per formance can only yield so much information without a bet-
ter understanding of underlying mechanisms. The goal of this study
was not to review and evaluate alternative hypotheses for the re-
source trade- off hypothesis (Hill, 2011; Weaver, Koch, & Hill, 2017),
but it is important to keep alternatives in mind when interpreting
results that are non- significant or do not match the predictions of
carotenoid resource trade- offs. Tackling old questions with new
approaches—be they new genetic techniques or new perspectives
on the role of carotenoids in physiological function—will be key to
substantiating or questioning the carotenoid resource trade- off
hypothesis across systems.
The auth ors would like to than k J. Johnson, W. Hood an d M. Toomey fo r
contributing to the quantitative carotenoid analysis in the Supporting
Informat ion. We also thank the Hil l and Hood labs at Aubu rn University
for feedback on early drafts of this manuscript.
R.E.K. wrote the first draft, devised the figure and tables, worked
with G.E.H. on revisions and performed the literature review; G.E.H.
led major manuscript revisions.
This article does not contain data.
Rebecca E. Koch http://orcid.org/0000-0002-9068-6231
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