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Testing the resource tradeoff hypothesis for carotenoid-based signal honesty using genetic
variants of the domestic canary
Rebecca E. Koch1,2*, Molly Staley1,3,4, Andreas N. Kavazis5, Dennis Hasselquist6, Matthew B.
Toomey7,8, and Geoffrey E. Hill1
1 Department of Biological Sciences, Auburn University, Auburn, AL, 36849, U.S.A.
2 School of Biological Science, Monash University, Clayton, VIC, 3168, Australia
3 Center for the Science of Animal Welfare, Brookfield Zoo, Chicago, IL, 60513, U.S.A.
4 Department of Biology, Loyola University Chicago, Chicago, IL, 60660, U.S.A.
5 School of Kinesiology, Auburn University, Auburn, AL, 36849, U.S.A.
6 Department of Biology, Lund University, Ekologihuset, Sölvegatan 37, 223 62 Lund, Sweden
7 Department of Pathology and Immunology, Washington University School of Medicine, St.
Louis, MO, 63110, U.S.A.
8 Department of Biological Science, University of Tulsa, Tulsa, OK, 74104, U.S.A.
* Correspondence and requests for materials should be addressed to R.E.K. (email:
rebecca.adrian@monash.edu).
Key words: condition-dependent trait, immunocompetence, antioxidant, ornament
Journal of Experimental Biology • Accepted manuscript
http://jeb.biologists.org/lookup/doi/10.1242/jeb.188102Access the most recent version at
First posted online on 15 March 2019 as 10.1242/jeb.188102
SUMMARY STATEMENT
Contrary to resource tradeoff predictions, white canaries with mutations that disable the
absorption or deposition of carotenoids show no significant increase in oxidative stress nor any
loss of immune system function.
Journal of Experimental Biology • Accepted manuscript
ABSTRACT
Carotenoid-based coloration in birds is widely considered an honest signal of individual
condition, but the mechanisms responsible for condition dependency in such ornaments remain
debated. Currently, the most common explanation for how carotenoid coloration serves as a
reliable signal of condition is the resource tradeoff hypothesis, which proposes that use of
carotenoids for ornaments reduces their availability for use by the immune system or for
protection from oxidative damage. However, two main assumptions of the hypothesis remain in
question: whether carotenoids boost the performance of internal processes like immune and
antioxidant defenses, and whether allocating carotenoids to ornaments imposes a trade-off with
such benefits. In this study, we tested these two fundamental assumptions using types of
domestic canary (Serinus canaria) that enable experiments in which carotenoid availability and
allocation can be tightly controlled. Specifically, we assessed metrics of immune and antioxidant
performance in three genetic variants of the color-bred canary that differ only in carotenoid
phenotype: ornamented, carotenoid-rich yellow canaries; unornamented, carotenoid-rich “white
dominant” canaries; and unornamented, carotenoid-deficient “white recessive” canaries. The
resource tradeoff hypothesis predicts that carotenoid-rich individuals should outperform
carotenoid-deficient individuals and that birds that allocate carotenoids to feathers should pay a
cost in the form of reduced immune function or greater oxidative stress compared to
unornamented birds. We found no evidence to support either prediction; all three canary types
performed equally across measures. We suggest that testing alternate mechanisms for the
honesty of carotenoid-based coloration should be a key focus of future studies of carotenoid-
based signaling in birds.
Journal of Experimental Biology • Accepted manuscript
INTRODUCTION
An outstanding challenge in behavioral and evolutionary ecology is understanding how
ornamental traits can serve as honest signals of individual condition (Higham, 2014). Numerous
studies have now documented that a range of ornamental traits are positively associated with
aspects of individual health and vitality (Hill, 2014), but the mechanism by which honest signals
resist cheating remains contentious (Hill, 2011; Weaver et al., 2017).
One of the most commonly studied classes of ornamentation in animals is carotenoid-
based coloration, which includes most of the red, orange, and yellow coloration of birds. Many
studies have presented evidence that carotenoid-based coloration serves as a reliable signal of
condition (reviewed in Svensson and Wong, 2011). The resource tradeoff hypothesis, which is
currently the most widely accepted hypothesis for how carotenoid-based signals of condition
remain honest, hinges on the assumptions that 1) internal carotenoid pigments provide benefits to
immune and/or antioxidant defenses within the body, and 2) animals are limited in the quantity
of carotenoids physiologically available (Koch and Hill, 2018). By this idea, only the highest
quality individuals can afford to allocate carotenoids to ornaments (Figure 1, top).
The resource tradeoff hypothesis offers an intuitive explanation for why sick, weak, or
otherwise low-quality individuals may be constrained from producing high-quality, richly
colored carotenoid signals. However, tests of the two central assumptions of the hypothesis—that
carotenoids offer physiological benefits, and that they are limited in internal availability—have
yielded inconsistent evidence (reviewd in Koch and Hill, 2018). The first assumption has faced
particularly strong criticism: while carotenoids are potent antioxidants under a wide range of
conditions in vitro, it remains uncertain whether they play a significant role in antioxidant
defenses in vivo in vertebrates (Hartley and Kennedy, 2004; Costantini and Møller, 2008; Perez-
Journal of Experimental Biology • Accepted manuscript
Rodriguez, 2009; Koch et al., 2018). Moreover, while carotenoids are often described as
beneficial to immune system function, evidence that carotenoids actually play a positive role in
immune defense remains scant and is generally restricted to a handful of studies in mammalian
systems (Chew and Park, 2004; Koch and Hill, 2018; Svensson and Wong, 2011). Likewise,
there is little empirical evidence to support the assumption that carotenoids are limiting in the
diets of wild animals. Much of the evidence for access to carotenoids affecting ornamentation
comes from studies of animals supplemented with large doses of carotenoids. These methods
have their own complications (Koch et al., 2016a), and while supplementation sometimes
appears to alleviate an apparent resource limitation (McGraw, Nolan and Crino, 2011), other
studies have found no such effects (Navara and Hill, 2003).
A key challenge to studies of the resource tradeoff hypothesis is that while researchers
can manipulate the size of internal carotenoid resource pools through diet or the potential
physiological need for carotenoids through immune or oxidative challenges, it has not been
possible to directly manipulate the availability or the allocation of carotenoids within the bodies
of animals. Accordingly, there is a clear need for tests in which the costs and benefits of
carotenoid allocation can be assessed more directly with fewer confounding mechanisms for
observed effects. Different types of the domestic canary (Serinus canaria) with mutations
affecting key carotenoid-related pathways provide an opportunity for such direct tests.
In this study, we consider three types of canaries with different levels of carotenoid
availability and usage. First, the standard yellow lipochrome canary (Y) has feathers that are
bright yellow due to pigmentation with carotenoids that are absorbed from the diet, converted
into different forms, and deposited in growing feathers (Koch et al., 2016b). White recessive
(WR) canaries have a mutation that almost completely eliminates their ability to absorb
Journal of Experimental Biology • Accepted manuscript
carotenoids from their diet (Toomey et al., 2017). Because vertebrates cannot synthesize
carotenoid pigments de novo, this mutation results in extreme carotenoid deficiency as well as
white plumage (Wolf et al., 2000). Lastly, white dominant (WD) canaries have a mutation that
prevents the deposition of carotenoid pigments into feathers during molt. While this mutation has
not yet been traced to a specific gene, its phenotypic effects are well known: WD canaries absorb
carotenoids and circulate them at the same levels as yellow canaries, but they allocate no
carotenoids to ornamentation and have white plumage (Figure 2). This canary system therefore
presents three levels of carotenoid usage: ornamented Y canaries absorb and potentially allocate
carotenoids to both feathers and physiological needs; WD canaries absorb and circulate
carotenoids but do not allocate them to ornamental feather coloration, potentially leaving more
carotenoids for physiological needs; and, WR canaries absorb essentially no carotenoids and thus
have no carotenoids to allocate to either physiological needs or ornamentation (Figure 1). By
comparing birds with and without internal carotenoid resources (WR vs. WD), we can test for
the physiological benefit of carotenoids (assumption 1), and by comparing birds with and
without carotenoid-based ornamentation (WD vs. Y), we can test for a physiological cost to
“spending” carotenoids (assumption 2) as colorants during molt (the period of ornamental
carotenoid deposition). Koch et al. (2018) previously tested assumption 1 in WR and Y canaries;
here, we take advantage of the WD system to perform a test of assumption 2, that there is a cost
of carotenoid allocation.
Journal of Experimental Biology • Accepted manuscript
We compared the performance of WR, WD, and Y canaries on several measures of
immunocompetence and antioxidant capacity. Based on the framework of the resource tradeoff
hypothesis, we predicted that during molt, carotenoid-rich, ornament-free WD birds should
outperform both carotenoid-deficient WR birds and carotenoid-spending Y birds; however,
outside of molt, carotenoid rich WD and Y birds should perform equally well, compared to
carotenoid-deficient WR birds.
MATERIALS AND METHODS
Study system and husbandry
We performed our study using a research colony of after-hatch-year canaries held at the Auburn
University Avian Research Laboratory 1 in Auburn, AL. All procedures were approved by the
Auburn University Animal Care and Use Committee (PRNs 2014-2465, 2014-2499, 2015-2724,
and 2015-2789). We performed several experimental procedures and analyses from January
through August 2016, as described in Figure 3.
The three canary color types that we studied, WR, WD, and Y, are all of the same breed
(“color-bred” canaries) and differ only in their carotenoid phenotype. The WD and WR
phenotypes are the product of Mendelian dominant or recessive alleles, respectively. While the
mutation responsible for the WR phenotype has been isolated to SCARB1, which functions in
carotenoid absorption (Toomey et al., 2017), the mutation in WD canaries has not yet been
identified. However, decades of observation and careful breeding by aviculturists as well as our
own observations and carotenoid analyses demonstrate clearly that WD birds differ from Y birds
only in their lack of ornamental carotenoid deposition. Throughout the study, we held all
canaries on a carotenoid-controlled diet of mixed canary seed (All Natural Canary Blend, Jones
Journal of Experimental Biology • Accepted manuscript
Seed Company; Lawton, OK, USA) coated with a carotenoid-free vitamin powder (AviVita Plus,
Avitech Bird Supplies; Frazier Park, CA, USA), which provides adequate dietary carotenoids for
yellow birds to fully color their feathers (Koch et al., 2018). The vitamin supplement prevented
any symptoms of retinol deficiency in the WR canaries, which cannot absorb retinoid precursor
carotenoids from their diet (Wolf et al., 2000).
Carotenoid analyses
Prior to the commencement of the main experiments, we performed carotenoid content analyses
on plasma samples taken outside of molt from four WR, four WD, and four Y canaries in our
colony; these samples were stored for less than 6 months at -80C prior to analysis. We also
collected skin and feather samples from four birds of each color type that died prior to
experimentation; these samples were stored for less than 12 months at -80C prior to analysis.
The carotenoid content of all samples was analyzed using high performance liquid
chromatography according to the methods described in Toomey et al. (2017) and Koch et al.
(2018).
Vaccination and antibody response
Because there is little conclusive evidence to suggest a direct role of carotenoids in any one
immune mechanism in birds (Koch and Hill, 2018; Svensson and Wong, 2011), we selected
broad measures of immune system function that inform on multiple aspects of
immunocompetence and that have biological relevance to immune defense without causing
lasting harm to the birds. First, we tested the canaries’ ability to mount an adaptive immune
response both after first exposure (primary response) and later exposure (secondary response) to
Journal of Experimental Biology • Accepted manuscript
an antigen that stimulates antibody production (through vaccination). Antibody production in a
primary response represents the ability of the innate immune system to recognize a novel antigen
and induce an adaptive immune response against that antigen, while the secondary response is
largely contingent on immunological memory but will also be influenced by the functional state
of the innate immune system (Hoebe et al., 2004; Iwasaki and Medzhitov, 2015). Carotenoids
have been implicated in boosting lymphocyte proliferation and performance in mammals
(reviewed in Chew and Park, 2004), so antibody production is one possible target for finding a
physiological benefit to internal carotenoids.
We vaccinated and measured the circulating antibodies of experimental birds twice, both
outside (January 2016) and within (late August 2016) the molt period (Figure 3). Experimental
procedures were identical for both periods, though the measurements taken outside of molt
represent the first exposure of the birds to the antigen (primary response) and the measurements
from molt represent the second exposure (secondary response). Briefly, we first drew a baseline
sample of blood (75 L) from each bird, then injected them intramuscularly with 100 L of
pharmaceutical-grade tetanus vaccine (2 lf of tetanus toxoid; also contained 2.7 lf of diphtheria
toxoid; TENIVAC, Sanofi Pasteur, Lyon, France), dosing 50 L each into the breast muscles on
the left and right sides of the sternum. After 10 days, we drew a second blood sample (75 L)
from each bird. Both blood samples were centrifuged immediately, and plasma was stored at -
80C until further analysis. Samples of plasma from before and after vaccination were shipped to
Lund University (Lund, Sweden) for anti-tetanus antibody analysis. Anti-tetanus antibody levels
were quantified from plasma using previously described enzyme linked immunosorbent assay
(ELISA) methods developed for songbirds (Hasselquist et al., 1999; Ilmonen et al., 2000). The
net “antibody response” to tetanus for each individual was calculated as the difference between
Journal of Experimental Biology • Accepted manuscript
pre- and post-vaccination measurements, which are reported in units of milli-optical-density per
minute (milliOD/min), as described in Koch et al. (2018).
Total antioxidant capacity
To test for any detectable effect of carotenoid presence, absence, or ornamental deposition on
antioxidant defenses, we assessed a measure of hydrophilic antioxidant capacity. While
carotenoids are lipophilic and therefore may contribute only indirectly to this measure of
antioxidant capacity (Tomášek et al., 2016), similar tests of hydrophilic antioxidant capacity
have been common in studies of the resource tradeoff hypothesis, yielding inconsistent results
(Alonso-Alvarez et al., 2008; García-de Blas et al., 2016; Hõrak et al., 2010; Morales et al.,
2009). Resolving whether or not carotenoids contribute significantly to antioxidant capacity is a
priority for testing the resource tradeoff hypothesis (Koch and Hill, 2018). During both molt
(July-August 2016) and non-molt (June 2016) periods, we measured total antioxidant capacity in
plasma samples using the TAC kit (OxiSelect Total Antioxidant Capacity (TAC) Assay Kit, Cell
BioLabs; San Diego, CA, USA; Figure 3). We diluted 5 L of plasma in 15 L of phosphate-
buffered saline (PBS) in duplicate for each individual, and report results in units of M Copper
Reduction Equivalents (CREs; Koch et al., 2018). For the measurements during molt, we used
plasma collected after the immune challenge described below so that we captured TAC during a
state of potential immune-induced oxidative stress (Costantini and Møller, 2009).
We also attempted several methods of quantifying oxidative damage in our canary
plasma (d-ROMs and ELISAs for protein carbonyls or 4-hydroxynonenal). However, none of
these assays was sensitive enough for accurate measurement in the small quantities of plasma
that we were able to collect from our birds.
Journal of Experimental Biology • Accepted manuscript
LPS challenge
To expand on our measure of adaptive immunity, we tested two different aspects of innate
immunity; specifically, we tested the response of live birds to bacterial lipopolysaccharide (LPS),
a potent innate immune stimulant, and non-specific innate immune defenses against bacteria
associated with soluble immune proteins in plasma (see next section). First, during molt (July-
August 2016), we challenged birds with bacterial lipopolysaccharide (LPS). LPS is commonly
used to invoke an acute innate immune response in animals without causing lasting disease. In
songbirds, LPS injection has generally been found to affect body temperature, food consumption,
and body mass (Owen-Ashley and Wingfield, 2006), as well as cause an increase in oxidative
stress (Costantini and Møller, 2009). We quantified response to LPS using these broad measures
of sickness response to capture any effect of carotenoid presence or absence (or allocation of
carotenoids to ornaments) on overall acute innate immune response symptoms. Carotenoid-based
coloration (Rosenthal et al., 2012) and circulating carotenoid levels (Alonso-Alvarez et al., 2004;
Sild et al., 2011) have been found to decrease in response to LPS challenge in songbirds, though
the mechanism causing that decrease remains uncertain.
We monitored the molt stage of each bird from July through the end of August and
challenged birds when they had emerging pinfeathers, were beginning to show loose feather
plumes across the majority of their ventral and/or dorsal sides, and displayed evidence of molted
wing and tail feathers. Full details of the LPS challenge procedure are described in Koch et al.
(2018). Briefly, we isolated a subset of birds in experimental cages the day before LPS injection,
then dosed them the following morning with an intra-abdominal injection of 1 mg/mL LPS from
E. coli (O55:B5; List Biological Laboratories, Inc.; Campbell, CA, USA) dissolved in PBS. We
recorded the mass and body temperature (using a Leaton Digital Thermocouple Thermometer
Journal of Experimental Biology • Accepted manuscript
inserted ~1 cm into the vent; Shenzhen DeXi Electronics Co.; Shenzhen, China) of each bird
immediately before and 8 hours after injection. Also 8 hours after injection, we collected 150 L
of blood in two heparinized capillary tubes, and immediately spread one drop on a microscope
slide for cell counts; the remaining blood was centrifuged to extract plasma and red blood cell
samples, then stored at -80C until further analysis (TAC measurement, described above).
Finally, we provided birds with a known quantity of seed for 24 hours before and then 24 hours
following injection, which we weighed to calculate food consumption before and after the
challenge.
From the blood smear collected after LPS injection, we also measured the
ratio of heterophils (the avian analog to mammalian neutrophils) to lymphocytes. This measure,
called H:L ratio, has previously been found to be a general indicator of immune activation in
birds such that a higher value indicates greater activation (Al-Murrani et al., 2006; Davis et al.,
2004; Gross and Siegel, 1983), and has been related to carotenoid-based color expression in
songbirds (Maney et al., 2008). To collect this measure, we first fixed and stained (Fisher
Healthcare PROTOCOL Hema 3 Fixative and Solutions; Fisher Scientific, Pittsburgh, PA, USA)
the slides. We then used standard techniques for avian blood cell counting and type identification
to determine the number of heterophils and lymphocytes present in 10,000 total cells per
individual (estimated based on total slides viewed and average cell counts per three
representative slide views with a single layer of cells). We divided total number of heterophils by
total number of lymphocytes to calculate the H:L ratio.
Journal of Experimental Biology • Accepted manuscript
Bacterial killing assay
To follow up our results from the LPS challenge, we performed an additional innate immune
response measure, isolating the complement-dependent antibacterial defenses of the plasma
(Demas et al., 2011; Merle et al., 2015). Again, while the mechanisms by which carotenoids may
be directly involved in plasma bacterial killing ability are not known, this measure has
previously been found to increase in birds supplemented with increased dietary carotenoids
(Leclaire et al., 2011; McGraw et al., 2011) and therefore is a promising target for detecting
possible benefits of carotenoids. On a subset of plasma extracted from the pre-vaccination blood
sample during molt in August 2016 (Figure 3), we performed a modified microplate-based
plasma bacterial killing assay (French and Neuman-Lee, 2012) as described in Koch et al.
(2018). Briefly, we tested bacterial killing of the canary plasma against a bacterial solution (E.
coli, ATCC 8739, reconstituted from pellet in sterile PBS; Microbiologics Epower; St. Cloud,
MN, USA) diluted to 1 x 105 CFUs. We mixed 80 L of diluted canary plasma (diluted 1:4 in
sterile PBS) with 8 L of the bacterial solution, then incubated the mixture at 37.4C for 30
minutes. After incubation, we plated 20 L of each sample in triplicate on a 96-well microplate,
added 125 L of sterile tryptic soy broth to each well, then read the plate at a wavelength of 600
nm. After 12 additional hours of incubation at 37.4C, we took a second reading at 600 nm. On
each plate, we also tested positive controls (bacteria but no plasma), negative controls (plasma
but no bacteria, to test for contamination), and “FBS controls” (containing sterile fetal bovine
serum instead of canary plasma, to control for non-immune interactions between bacteria and
plasma components, which appeared to boost bacterial growth).
Journal of Experimental Biology • Accepted manuscript
To assess the results, we first calculated an average net absorbance for each individual
and control by subtracting baseline values from 12-hour values. We then eliminated any
absorbance readings that differed more than 10% from the other two values for an individual and
averaged the remaining net absorbance values for each individual. We divided this final net
absorbance for each individual by the final net absorbance of the FBS controls on the same
microplate to obtain a value for percent difference in bacterial growth between samples and
positive control. This percent-bacteria-killed value for each individual was used in further
analyses. However, we found that individuals tended to consistently either completely kill (<10%
bacterial growth compared to FBS positive controls) or completely fail to kill (>90% bacterial
growth compared to FBS positive controls) their bacterial challenge, so we also performed a
binomial regression analysis to assess statistical patterns in the data. For this analysis, we
excluded data points with percent-bacteria-killed values between 10 and 90% so that all
remaining individuals could be categorized as having fully killed or fully failed to kill their
bacterial challenge.
Statistics
For all measurements examined, we used ANOVA to test for significant differences in response
measurements based on color type, sex, or the interaction of sex and color type. We tested for
differences between the sexes because measurements used in our study, such as those of immune
system function, have previously been found to differ between the sexes in songbirds with
carotenoid-based coloration (Love et al., 2008; McGraw et al., 2011). However, given that sex
and the interaction of sex and color type played little role in our results (Tables S1-4), we focus
on differences in performance among the three color types. We used the Tukey post-hoc method
Journal of Experimental Biology • Accepted manuscript
to test pairwise comparisons between WR vs. WD and WD vs. Y results for each measure. For
measures collected outside of molt only (TAC and primary antibody response to tetanus), we
also ran identical analyses in which we pooled the measurements of WD and Y color types and
compared them against WR (“carotenoid-rich” vs. “carotenoid-poor”); because allocation of
carotenoids to feathers only occurs during molt, WD and Y birds should not differ outside of
molt. For LPS injection results only, we also performed two-tailed paired t-tests to assess
whether injection significantly affected mass, food consumption, or temperature across all
canaries. Preliminary results indicated that baseline values for food consumption, body
temperature, and mass may differ between sexes or colors, so our ANOVAs of these LPS effects
also included a term for initial, pre-injection values. Finally, for bacterial killing assay analyses,
we tested for significant differences using binomial regression (on categorical data coded as
“fully killed” or “fully failed to kill”) as well as ANOVA (on continuous data of percent bacteria
killed). All statistical analyses were performed in R (version 3.5.0; R Core Team, 2018). All
reported error values are standard errors.
RESULTS
Carotenoid analyses
As expected, we found WR canaries to lack significant carotenoids in plasma (0.75 ± 0.18
µg/mL), skin (0.0003 ± 0.0003 µg/mg), and feathers (no carotenoids detected). WD canaries
similarly lack carotenoids in their skin (0.00009 ± 0.0007 µg/mg) and feathers (0.0001 ± 0.00006
µg/mg), but have carotenoid-rich plasma (23.6 ± 7.1 µg/mL). Y canaries possess relatively
carotenoid-rich feathers (0.12 ± 0.04 µg/mg), skin (0.15 ± 0.07 µg/mg), and plasma (20.3 ± 10.6
µg/mL).
Journal of Experimental Biology • Accepted manuscript
Vaccination and antibody response
Outside of molt, in the primary response to vaccination with tetanus, we found no significant
differences in performance among birds based on color type (Figure 4, Table 1), sex, or their
interaction (all P > 0.11). Comparing “carotenoid-rich” (WD and Y) canaries to “carotenoid-
poor” (WR) for these results collected outside of molt revealed that carotenoid-rich canaries had
lower antibody responses than carotenoid-poor canaries (F1,28 = 4.75; P = 0.04). During molt, in
the secondary response, there was a significant effect of sex (P = 0.046): males, on average, had
a stronger antibody response (1.84 ± 0.11 milliOD/min) than did females (1.48 ± 0.22). Color
type or the interaction of color type and sex had no significant effects on secondary antibody
response (both P > 0.23).
Total antioxidant capacity
Both within and outside of molt, WD, WR, and Y canaries did not differ significantly in total
antioxidant capacity (both P > 0.41; Table 1; Figure 4). There was a significant effect of sex on
TAC, but only within molt (P = 0.04; P = 0.47 outside of molt); molting males had greater
average TAC (2001 ± 266 CRE) than females (1208 ± 143 CRE). The interaction of sex and
color type had no significant effect on TAC either during or outside of molt (P>0.17). An
analysis pooling WD and Y performance outside of molt (see above) also revealed no significant
differences between carotenoid-rich and carotenoid-poor canaries (F1,51 = 1.36, P = 0.72).
Journal of Experimental Biology • Accepted manuscript
Response to LPS challenge
The initial values of each individuals’ mass, temperature, or food consumption always influenced
the magnitude by which its values changed after LPS injection (all P < 0.001), and WR and WD
(but not Y and WD) canaries also differed significantly in their initial body temperature (P =
0.02). Color type, sex, or their interaction had no other significant effects on baseline body
temperature, food consumption, or mass, though there was a trend toward a difference between
the sexes in initial food consumption (P = 0.087) and between the color types? in initial mass (P
= 0.078; all other P > 0.1).
Among all canaries, LPS injection induced a loss of mass (average decrease: 1.20 g; t =
17.18, df = 56, P < 0 .001) within 24 hours, and a body temperature increase (average increase:
0.46ºC; t = 6.67, df = 34, P < 0.001) within 8 hours, but no change to food consumption over 24
hours (average increase: 0.11 mg consumed / g body mass / hour; t = 0.56, df = 60, P = 0.58).
We found that color type of the canary significantly affected the magnitude of LPS-induced loss
of mass (P = 0.02; Table 1). This is driven by differences between WR and WD birds: WR
canaries tended to lose more mass than WD canaries, though there was no such pattern between
Y and WD birds (Table 1). Sex had no significant effect on change in mass, but there was a trend
toward a significant difference in change in body temperature between males and females
(average temperature increase in females: 0.58 ± 0.09ºC; in males: 0.33 ± 0.10ºC; P = 0.052),
and there was a significant difference in change in food consumption between males and females
(average change in females: -0.51 ± 0.36 mg consumed / hour / g body mass; in males: 0.16 ±
0.24 mg consumed / hour / g body mass; P=0.027). Neither temperature increase nor change in
food consumption differed among color types or with the interaction of sex and color type (Table
1, all P>0.17).
Journal of Experimental Biology • Accepted manuscript
The ratio of heterophils to lymphocytes present in blood smears prepared eight hours
after LPS injection also did not differ between color types, sexes, or their interaction (all P >
0.11; Table 1).
Bacterial killing ability
We found no significant differences in bacterial killing ability between the sexes, among the
colors, or with their interaction, either in a binomial regression analysis where individuals were
coded as having either killed (>90% bacterial clearance) or failed to kill (<10% clearance) their
challenge (all P>0.53), or in an ANOVA of percent clearance (P>0.10; Table 1).
DISCUSSION
In this study, we used strains of canaries with mutations in key carotenoid pathways to test two
of the central assumptions of the carotenoid resource tradeoff hypothesis: 1) carotenoids provide
physiological benefits, and 2) animals tradeoff subtraction of physiological benefits with
production of ornamentation as carotenoids are allocated to ornaments (Koch and Hill, 2018).
After comparing performance across several measures of immunocompetence and one measure
of antioxidant capacity in WD vs. Y (ornament-free vs. ornamented) and WD vs. WR
(carotenoid-rich vs. carotenoid-free) canaries, we found no empirical support for either
assumption.
These comparisons offer what are perhaps the most direct tests to date of whether internal
carotenoid resources provide significant physiological benefits to birds and whether allocating
these carotenoid resources to ornamental plumage coloration decreases these benefits. While
most previous tests of the resource tradeoff hypothesis have relied on experimental
Journal of Experimental Biology • Accepted manuscript
manipulations, such as modified dietary carotenoid intake and/or physiological challenge, our
experimental groups were fed identical diets and held under identical conditions. Indeed, Y, WR,
and WD canaries appear functionally identical in all aspects of phenotype except carotenoid
usage because they are variants of the same breed of canary, differing only in mutations that
affect carotenoid coloration.
The patterns in our data do not conform to the predictions of the resource tradeoff
hypothesis: there were no consistent trends toward WD canaries performing better than Y
canaries during molt or better than WR canaries outside of molt. Despite uneven sample sizes
due to the difficulty of locating WD canaries, our data did not exhibit consistent trends in the
direction predicted by the resource tradeoff hypothesis, making it unlikely that a greater sample
size would significantly alter our conclusions. The only measurements in which we detected a
significant effect of color type or carotenoid phenotype on response were: 1) carotenoid-rich
types (WD and Y) exhibited lower primary antibody response to tetanus outside of molt than did
carotenoid-poor WR canaries; 2) WR canaries had higher baseline body temperature prior to
LPS injection; and, 3) WR canaries lost more mass during LPS challenge than did WD canaries,
though WR also tended to have higher baseline mass and the magnitude of their mass loss is
similar to that of Y canaries. None of these relationships suggest a major role of internal
carotenoids in boosting physiological response or of a cost to depositing carotenoids as
ornamental colorants during molt. We nevertheless encourage future studies to explore other
potential avenues of physiological benefit of carotenoids, such as lipophilic antioxidant capacity,
which has recently been proposed to be the best method for detecting the potential antioxidant
effects of carotenoids (Tomášek et al., 2016a). Moreover, isolating the genetic mutation(s)
responsible for the WD phenotype would be extremely valuable both for describing the
Journal of Experimental Biology • Accepted manuscript
mechanistic origin of the “ornament-free” canary and for advancing our understanding the
genetic basis of variation in plumage ornaments.
Our observations corroborate patterns that we found in our previous analysis comparing
only WR and Y birds (Koch et al., 2018). In that study, we were also able to assay the levels of
one endogenous antioxidant (glutathione) in WR and Y red blood cells, and we found no
difference between the birds; however, without a more comprehensive panel of measurements,
we cannot rule out that WR birds may have compensatory mechanisms to circumvent their lack
of carotenoids. Indeed, the trend toward higher average body mass among WR birds (compared
to WD and Y birds) that we recorded at the start of our immune challenge experiment in this
study suggests some underlying differences may exist between the color types. Previous genetic
exploration of the WR canaries compared them to yellow canaries of vastly different genetic
lines (e.g. lines bred for varying body conformation) in order to isolate the genetic regions
responsible for the WR phenotype (Toomey et al., 2017). Further genetic analyses of closely
related WR and WD/Y canaries, such as those tested in our experiments, will be important to
rule out whether WR birds have been selected for compensatory adaptations to their
physiological lack of carotenoids. However, given that we found no consistent differences in
performance among the three color types of canaries we examined here or between WR and Y
canaries in our previous study (Koch et al., 2018), we consider it unlikely that we have
overlooked any substantial sources of variation among these birds.
Our results provide an important new piece of evidence in the discussion surrounding the
honesty of carotenoid-based signals, but a definitive explanation for condition-dependent
coloration remains elusive. One possibility is that retinol (Vitamin A) may play a greater role
than previously assumed in the physiological benefits that have been attributed to carotenoids
Journal of Experimental Biology • Accepted manuscript
(Hill and Johnson, 2012; Koch et al., 2018). By providing all our birds with a supplement that
includes retinol, we prevented retinoid deficiency in the WR birds, which cannot absorb the
carotenoid precursors to retinol (Wolf et al., 2000). However, in wild birds, individuals low in
carotenoids are unlikely to have access to other sources of retinol, so low-carotenoid birds may
also be low in retinol (and vice versa; Simons et al., 2015). Given that retinol has a wide range of
well accepted health benefits in vertebrates (Hill and Johnson, 2012), it is plausible that some of
the health benefits that have been credited to carotenoids themselves arise because carotenoids
are a valuable source of retinoids.
Another possibility is that the basic premise of the resource tradeoff hypothesis is
incorrect and carotenoids play no important functions in vertebrates other than social signaling.
A physiological cost to absorbing or converting carotenoid pigments from the diet could
maintain condition-dependence in carotenoid-based signals without requiring carotenoids to play
a direct role in boosting physiological processes (Weaver et al., 2018); however, we found no
consistent evidence of such physiological costs to carotenoid uptake or conversion in our WD or
Y canaries. An alternative hypothesis for the basis for carotenoids as honest signals that invokes
no physiological functions for carotenoids is the shared pathway hypothesis (Hill, 2011). This
hypothesis proposes that the quality of external coloration can be linked to the quality of vital
cellular processes if both coloration and physiological well-being are dependent on the
functionality of shared mechanistic pathways. By this idea, individual “condition” is defined as
the performance of cellular processes that drive variation both in honest signals and in the
qualities they signal, and carotenoids themselves are not required to have any physiological
function besides being colorants (Weaver et al., 2017). However, testing the shared pathway
hypothesis requires isolating and testing the performance of that shared pathway; while a
Journal of Experimental Biology • Accepted manuscript
potential shared cellular process has been proposed—mitochondrial function (Hill, 2014; Koch,
Josefson and Hill, 2017)—it awaits empirical testing. We encourage future research to critically
test the resource tradeoff hypothesis as an explanation for condition-dependence in carotenoid-
based signals in birds and to devise new means of more directly testing the functions of
carotenoids within the animal body. Until such time as techniques for manipulating specific
genetic pathways in vertebrates are widely accessible, domestic birds with existing genetic
mutations in key carotenoid pathways offer a rich and accessible system for such studies.
ACKNOWLEDGEMENTS
We thank J. Corbo for assistance with carotenoid analyses and A. Hegemann and C. Birberg for
assistance with tetanus antibody assessment. Members of the Hill and Hood labs and Auburn
University undergraduates assisted with live animal procedures.
COMPETING INTERESTS
No competing interests declared.
FUNDING
This work was supported by grants from the National Science Foundation (Doctoral Dissertation
Improvement Grant 1501560 to R.K.) and the Swedish Research Council (VR; 621-2013-4357
and 2016-04391 to D.H.).
DATA AVAILABILITY
Additional figures and tables depicting the results of this study are available at Monash
University figshare (https://doi.org/10.26180/5c5bb4f82d869).
Journal of Experimental Biology • Accepted manuscript
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Journal of Experimental Biology • Accepted manuscript
Figures
Figure 1. Diagram illustrating the hypothetical allocation of carotenoids from diet to tissue
to physiological process or coloration. The carotenoid resource tradeoff hypothesis proposes
that carotenoid pigments absorbed from the diet into the bloodstream must be differentially
allocated to either boosting internal processes or coloring ornaments. WD canaries (shown here
with yellow wings to distinguish them from WR canaries) do not allocate carotenoids to plumage
coloration and therefore can, in theory, allocate all ingested carotenoids to boosting function.
WR canaries cannot absorb carotenoids from their diet, so they have no carotenoids to allocate to
either process. If we assume that carotenoids are beneficial to internal processes and that
depositing them in coloration imposes an allocation trade-off, then immune and antioxidant
Journal of Experimental Biology • Accepted manuscript
function will be predicted by the size of the carotenoid allocation arrow pointing to it (or lack
thereof): ornament-free WD canaries will outperform ornamented Y canaries, and carotenoid-
rich Y canaries will in turn outperform carotenoid-deficient WR canaries.
Journal of Experimental Biology • Accepted manuscript
Figure 2. The white dominant canary possesses internal carotenoids but does not deposit
them to color its feathers. White dominant canaries feature yellow subcutaneous fat (A), yellow
plasma (B), and white plumage (C).
Journal of Experimental Biology • Accepted manuscript
Figure 3. Timeline of experiments. The molt period of the canaries lasts about two months,
from late June to late August. We performed physiological tests of WD, Y, and WR canaries
during both the molt and non-molt periods in 2016. The exact start date of molt measurements
varied according to the molt stage of individual birds, and subsequent measures were staggered
to allow adequate time between blood sampling. TAC = total antioxidant capacity; LPS =
bacterial lipopolysaccharide; H:L = heterophil to lymphocyte ratio; BKA = bacterial killing
assay.
Journal of Experimental Biology • Accepted manuscript
Figure 4. Hypothesized and actual (mean SE) results of two physiological measures taken
inside and outside of molt. Based on the carotenoid resource tradeoff hypothesis, the predicted
patterns in the data are clear: WD and Y birds should feature strong physiological performance
outside of molt (A), but WD birds should outperform Y birds within molt (B); WR birds should
always perform poorly. We found none of these expected patterns in measures of total
antioxidant capacity (C, D; ANOVA P > 0.4 for color type) and antibody response to tetanus
(primary response outside of molt, E; secondary response during molt, F; ANOVA P > 0.1 for
color type). Sample sizes are printed in the bars of C-F.
Journal of Experimental Biology • Accepted manuscript
Table 1. Results of statistical analyses of the effects of canary color type on immune or
antioxidant performance
Overall model
(Color type)
Post-hoc: WR vs.
WD
Post-hoc: Y vs. WD
Stage
Measurement
F (df)
P
Difference
P
Difference
P
NON-
MOLT
Primary
antibody
response
1.255
(2,26)
0.273
0.51
0.17
0.16
0.88
TAC (CRE)
0.93
(2,23)
0.41
-59.2
0.97
-330.5
0.41
MOLT
Secondary
antibody
response
1.58
(2,46)
0.24
-0.48
0.34
-0.48
0.34
LPS-mediated
mass change
(g)
4.42
(2,50)
0.017
0.52
0.034
0.29
0.35
LPS-mediated
temperature
change (ºC)
0.73
(2,28)
0.49
-0.26
0.73
-0.34
0.60
LPS-mediated
food
consumption
change
(g/hour/g)
0.51
(2,54)
0.60
-0.53
0.66
-0.51
0.69
TAC (CRE)
0.52
(2,20)
0.60
-300.1
0.74
-418.6
0.58
H:L
2.354
(2,45)
0.11
0.17
0.11
0.16
0.15
BKA (%
killed)
1.85
(2,47)
0.17
24.8
0.34
33.7
0.14
The overall model refers to the main ANOVA results, while post-hoc results refer to pairwise
comparisons between focal color types using the Tukey method. In the post-hoc results, a
negative difference indicates that WD birds had lower values than WR or Y birds. Degrees of
freedom (df) values indicate the degrees of freedom of the model and the error, respectively.
Journal of Experimental Biology • Accepted manuscript
Table S1. Average anti-tetanus antibody responses for the primary response (outside of the
molt season) and secondary response (during molt) for WD, WR, and Y canaries.
Descriptive results
ANOVA results
Color
type
Sample
size
Average
response
± SE
(milliOD/min)
Variable
F (df)
P
Primary response
(non-molt)
WD
3 F, 1 M
0.51 ± 0.04
Color type
2.358 (2,26)
0.114
WR
14 F, 9 M
1.02 ± 0.12
Sex
1.255 (1,26)
0.273
Y
3 F, 2 M
0.67 ± 0.12
Interaction
0.783 (2,26)
0.467
Secondary response
(molt)
WD
6 F, 2 M
2.08 ± 0.26
Color type
1.164 (2,46)
0.321
WR
10 F, 13 M
1.61 ± 0.17
Sex
4.190 (1,46)
0.046
Y
7 F, 14 M
1.60 ± 0.20
Interaction
1.476 (2,46)
0.239
F = female, M = male; df = degrees of freedom (numerator, denominator).
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Table S2. Average total antioxidant capacity of plasma samples from WD, WR, and Y
canaries, inside and outside of molt.
Descriptive results
ANOVA results
Color type
Sample size
Average response
± SE (CRE)
Variable
F (df)
P
Non-molt
WD
3 F, 8 M
1595 ± 221
Color type
0.925 (2,23)
0.411
WR
2 F, 7 M
1536 ± 171
Sex
0.541 (1,23)
0.469
Y
2 F, 7 M
1265 ± 133
Interaction
1.908 (2,23)
0.171
Molt
WD
2 F, 3 M
1889 ± 550
Color type
0.516 (2,20)
0.605
WR
7 F, 4 M
1589 ± 173
Sex
4.821 (1,20)
0.040
Y
6 F, 4 M
1471± 240
Interaction
0.346 (2,20)
0.711
CRE = copper reduction equivalents; F = female, M = male; df = degrees of freedom (numerator,
denominator).
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Table S3. Baseline physiological metrics and their LPS-mediated changes in WD, WR, and
Y canaries during molt.
Descriptive results
ANOVA results
Color
type
Sample size
Average response
± SE
Variable
F (df)
P
Initial mass
(g)
WD
6 F, 2 M
22.73 ±1.26
Color type
2.691 (2,5)
0.078
WR
10 F, 16 M
25.40 ± 0.72
Sex
1.008 (1,51)
0.320
Y
7 F, 16 M
23.60 ± 0.59
Interaction
0.526 (2,51)
0.594
Change in
mass (g)
WD
6 F, 2 M
-0.85 ± 0.19
Color type
4.421 (2,50)
0.017
WR
10 F, 16 M
-1.37 ± 0.01
Sex
0.242 (1,50)
0.625
Y
7 F, 16 M
-1.14 ± 0.10
Interaction
2.258 (2,50)
0.115
Initial value
12.536 (1,50)
<0.001
Initial body
temperature
(°C)
WD
4 F, 3 M
40.67 ± 0.20
Color type
4.552 (2,29)
0.019
WR
8 F, 7 M
41.22 ± 0.12
Sex
0.007 (1,29)
0.932
Y
6 F, 7 M
40.97 ± 0.09
Interaction
0.099 (2,29)
0.906
Change in
body
temperature
(°C)
WD
4 F, 3 M
0.60 ± 0.11
Color type
0.733 (2,28)
0.490
WR
8 F, 7 M
0.45 ± 0.11
Sex
4.109 (1,28)
0.052
Y
6 F, 7 M
0.41 ± 0.12
Interaction
0.371 (2,28)
0.693
Initial value
16.262 (1,28)
<0.001
Initial food
consumption
(mg
consumed /
hour / g
body mass)
WD
6 F, 2 M
2.79 ± 0.43
Color type
0.376 (2,55)
0.688
WR
11 F, 17 M
3.08 ± 0.26
Sex
3.032 (1,55)
0.087
Y
8 F, 17 M
2.76 ± 0.34
Interaction
2.347 (2,55)
0.105
Change in
food
consumption
(g
consumed /
hour / g
body mass)
WD
6 F, 2 M
0.34 ± 0.51
Color type
0.510 (2,54)
0.603
WR
11 F, 17 M
-0.19 ± 0.23
Sex
5.185 (1,54)
0.027
Y
8 F, 17 M
-0.17 ± 0.40
Interaction
1.811 (2,54)
0.173
Initial value
19.724 (1,54)
<0.001
Heterophil to
lymphocyte
ratio (post-
LPS only)
WD
6 F, 3 M
0.17 ± 0.039
Color type
2.354 (2,45)
0.107
WR
9 F, 15 M
0.34 ± 0.039
Sex
0.556 (1,45)
0.460
Y
6 F, 12 M
0.33 ± 0.058
Interaction
0.244 (2,45)
0.785
Negative values for change in mass, temperature, or food consumption indicate that
measurements decreased in value after LPS injection. F = female, M = male; df = degrees of
freedom (numerator, denominator).
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Table S4. Bacterial killing ability of WD, WR, and Y canaries during molt.
Descriptive results
ANOVA results
Binomial GLM results
Color
type
Sample
size
Average response
± SE (Percent bacterial killing),
fraction of individuals who fully-
killed their challenge
Variable
F (df)
P
Variable
Z
P
WD
5 F, 3 M
39.05 ±17.35, 3/8
Color type
1.845 (2,47)
0.169
Intercept
-0.444
0.657
Color type
(WR vs.
WD)
0.365
0.715
Color type
(Y vs. WD)
0.011
0.991
WR
10 F, 10
M
58.22 ±10.56, 12/20
Sex
0.395 (1,47)
0.533
Sex
-0.188
0.851
Y
6 F, 15
M
65.49 ± 9.77, 14/21
Interaction
2.472 (2,47)
0.095
Interaction
(WR vs. WD
by Sex)
0.634
0.526
Interaction
(Y vs. WD
by Sex)
-0.011
0.992
Individuals were considered to have “fully-killed their challenge” if they had a percentage of
bacteria killed greater than 90%. Results are presented both for an ANOVA performed on
continuous data of percent bacterial killing, and a binomial generalized linear model (GLM) on
categorical data indicating whether or not an individual fully-killed their challenge. F = female,
M = male; df = degrees of freedom (numerator, denominator).
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S1. Mean ± SE anti-tetanus antibody responses of the three color types (left panel) and
the two sexes (right panel). Small points represent individual raw data; numbers at the base of
each panel represent sample sizes.
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S2. Mean ± SE total antibody capacity (TAC) of the three color types (left panels) and
the two sexes (right panels). Small points represent individual raw data; numbers at the base of
each panel represent sample sizes.
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S3. Mean ± SE measurements taken prior to or after bacterial lipopolysaccharide (LPS)
injection.
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S4. Mean ± SE heterophil to lymphocyte ratio of the three color types (left panels) and
the two sexes (right panels). Small points represent individual raw data; numbers at the base of
each panel represent sample sizes.
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
Figure S5. Mean ± SE bacterial killing capacity (percent bacteria killed relative to positive
controls). Small points represent individual raw data; numbers at the base of each panel
represent sample sizes.
23 30
0
25
50
75
100
FM
Sex
Percent Bacteria Killed
9 22 22
0
25
50
75
100
WD WR Y
Color Type
Percent Bacteria Killed
Journal of Experimental Biology: doi:10.1242/jeb.188102: Supplementary information
Journal of Experimental Biology • Supplementary information
