Red fish, blue fish: Trade-offs between pigmentation and immunity in Betta splendens

Abstract

Carotenoid pigments are responsible for many examples of sexually attractive red, orange, and yellow coloration in animals and play an important role in antioxidant and immune defenses. Because vertebrates cannot synthesize carotenoids, limited dietary availability may impose a trade-off between maintaining ornamental coloration and health. We used an experimental approach to test the carotenoid trade-off hypothesis in the fighting fish Betta splendens, by examining whether carotenoid allocation strategies differ among conspecifics that exhibit a gradient of body coloration from blue to red. We found that male redness is underlain by carotenoids and that females preferred to associate with red males over blue males, suggesting a sexually-selected advantage to being red. Moreover, we found strong experimental support for the carotenoid trade-off hypothesis, as individuals that varied in color did not appear to allocate carotenoids equally to both immune response and coloration. Redder fish given supplemental carotenoids increased in both immune response (to a phytohemagglutination challenge) and redness compared with controls. In contrast, bluer fish given supplemental carotenoids did not become more red but instead benefited immunologically more so than either control or redder supplemented fish. These results enhance our understanding of the evolution and plasticity of carotenoid mobilization and utilization pathways in animals. Copyright 2007, Oxford University Press.

Full text

Red fish, blue fish: trade-offs between
pigmentation and immunity in Betta splendens
Ethan D. Clotfelter,
a
*Daniel R. Ardia,
b
*and Kevin J. McGraw
c
a
Department of Biology, Amherst College, Amherst, MA 01002, USA,
b
Department of Biology,
Franklin and Marshall College, Lancaster, PA 17604, USA, and
c
School of Life Sciences, Arizona State
University, Tempe, AZ 85287, USA
Carotenoid pigments are responsible for many examples of sexually attractive red, orange, and yellow coloration in animals and
play an important role in antioxidant and immune defenses. Because vertebrates cannot synthesize carotenoids, limited dietary
availability may impose a trade-off between maintaining ornamental coloration and health. We used an experimental approach to
test the carotenoid trade-off hypothesis in the fighting fish Betta splendens, by examining whether carotenoid allocation strategies
differ among conspecifics that exhibit a gradient of body coloration from blue to red. We found that male redness is underlain
by carotenoids and that females preferred to associate with red males over blue males, suggesting a sexually-selected advantage to
being red. Moreover, we found strong experimental support for the carotenoid trade-off hypothesis, as individuals that varied in
color did not appear to allocate carotenoids equally to both immune response and coloration. Redder fish given supplemental
carotenoids increased in both immune response (to a phytohemagglutination challenge) and redness compared with controls.
In contrast, bluer fish given supplemental carotenoids did not become more red but instead benefited immunologically more so
than either control or redder supplemented fish. These results enhance our understanding of the evolution and plasticity of
carotenoid mobilization and utilization pathways in animals. Key words: carotenoids, coloration, immune response, pteridines,
sexual selection. [Behav Ecol 18:1139–1145 (2007)]
Pigment-based colors are common visual signals in the an-
imal kingdom (Needham 1974). Carotenoid pigments are
widely used to produce red, orange, and yellow coloration,
especially in fishes, lizards, and birds (Evans and Norris
1996; Macedonia et al. 2000; Hill and McGraw 2006a). In
many instances, these carotenoid-based colors are sexually
attractive to prospective mates (Kodric-Brown 1993; Blount
et al. 2003; Maan et al. 2006). Carotenoid pigments also serve
a variety of physiological roles (Vershinin 1999), with one
major function as an immunostimulant and antioxidant
(Bendich 1989; Chew 1993; McGraw and Ardia 2003). Because
carotenoid pigments are derived from dietary sources and
cannot be synthesized de novo, their availability is under nu-
tritional control in a variety of taxa (Grether et al. 1999;
Alonso-Alvarez et al. 2004; Hill and McGraw 2006b). A conse-
quence of this scarcity is that carotenoid allocation is condi-
tion dependent (Hill and Montgomerie 1994; von Schantz
et al. 1999), which leads to a presumed trade-off between alloca-
tion to functions such as coloration and immunity (Lozano 1994;
von Schantz et al. 1999; Faivre et al. 2003; Alonso-Alvarez et al.
2004; Peters et al. 2004). Individuals with the brightest colors
are presumed to be those that have sufficient carotenoids for
meeting both immunological and coloration functions and
hence are the healthiest and most desirable mates.
Several types of studies have attempted to elucidate such
a carotenoid trade-off. At a very basic level, the fact that di-
etary supplementation with carotenoids enhances both immu-
nity and coloration (Blount et al. 2003; McGraw and Ardia
2003; Alonso-Alvarez et al. 2004) suggests that carotenoid-
limited animals must dedicate carotenoids more to one or
another function or suffer both somatically and sexually. Sec-
ond, experimental manipulations of health status in animals
that deposit carotenoids in bare parts (e.g., beaks, legs, and
flesh) have shown that immunocompromised animals fade in
color (Faivre et al. 2003; Peters et al. 2004), suggesting that
carotenoids are retrieved from colorful tissues to fight patho-
genic or parasitic challenges. These studies have not tested
the alternative that carotenoid deposition or metabolism was
instead impaired by the immune challenge and thus whether
trade-offs cause these color changes remains unknown. Third,
in perhaps the best test of carotenoid trade-offs to date (Fitze
et al. 2007), it was recently found that when 2 types of caro-
tenoids (xanthophylls and carotenes) were provided to nes-
tling great tits (Parus major), the carotenoids used in plumage
coloration (xanthophylls) were not the carotenoids that influ-
enced immunocompetence (carotenes). Through all this, how-
ever, we still await a rigorous, experimental test of the
carotenoid trade-off hypothesis in adult animals that display
sexually attractive, pigment-based coloration.
In contrast to this prior work, an ideal system for testing
carotenoid trade-offs would be in species that show distinct
color morphs that vary in their carotenoid dependency or
their ability to mobilize carotenoids (Sinervo and Lively
1996; Craig and Foote 2001; Craig et al. 2005; Pryke and
Griffith 2006) and thus may employ different carotenoid allo-
cation strategies. In such a system, a key prediction for the
carotenoid trade-off hypothesis would be that individuals
that lack or have reduced carotenoid coloration should allo-
cate relatively more dietary carotenoids to their immune
system. Moreover, animals with extensive carotenoid coloration
should suffer decreased immunocompetence compared with
less carotenoid-colored animals given the same level of carot-
enoid uptake, due to increased allocation to coloration.
Therefore, the goal of this study was to test whether geneti-
cally based intraspecific differences in body coloration affect
relative allocation of carotenoids to coloration versus immune
system. We did so by examining the effect of dietary carotenoid
*These authors contributed equally to this work.
Address correspondence to E.D. Clotfelter. E-mail: edclotfelter@
amherst.edu.
Received 12 February 2007; revised 30 August 2007; accepted 5
September 2007.
Behavioral Ecology
doi:10.1093/beheco/arm090
Advance Access publication 10 October 2007
The Author 2007. Published by Oxford University Press on behalf of
the International Society for Behavioral Ecology. All rights reserved.
For permissions, please e-mail: journals.permissions@oxfordjournals.org

supplementation on a range of color phenotypes in the
Siamese fighting fish (Betta splendens). Artificial selection on
B. splendens has produced a range of color morphs, particu-
larly reds and blues, which provides a powerful tool for exam-
ining intraspecific carotenoid allocation strategies.
Our initial objectives were to determine the pigment basis
for redness in B. splendens and the extent to which red body
color in males is attractive to females. We used traditional
biochemical methods to measure tissue concentrations of
carotenoids and pteridines (drosopterins), another class of
pigments that can contribute to red and orange coloration
in fishes (Dupont 1958; Henze et al. 1977). Pteridines can be
synthesized de novo (Hurst 1980); thus, it is important to con-
sider the possibility that fish compensate for carotenoid scar-
city by using pteridines as skin colorants (Grether et al. 2001).
We predicted that both carotenoids and drosopterins would
contribute to red coloration. With respect to female mate
choice, we predicted that female B. splendens would favor
red males over blue males in a dichotomous choice test. Little
is known about female preferences for male redness in either
wild-type or domestic stocks of this species, but female prefer-
ences for carotenoid-dependent coloration in males have been
found in many other fishes (Kodric-Brown 1993; Candolin
1999; Maan et al. 2006).
Our main objective, however, was to test the trade-off hy-
pothesis by comparing carotenoid allocation strategies as
a function of a fish’s initial body coloration. We supplemented
dietary carotenoids to individuals over a range of body colors
from blue to red (measured with UV–Vis spectrophotometry)
in order to determine how the need to devote pigments to
skin color affected the ability to allocate carotenoids to color-
ation at the expense of the immune system. First, we tested
whether carotenoid supplementation increased redness and
enhanced the inflammatory response to phytohemagglutinin
(PHA). Similar work has been done on guppies (Poecilia retic-
ulata) (Grether et al. 2004) and salmonids such as rainbow
trout (Oncorhynchus mykiss) and sockeye salmon (Oncorhynchus
nerka) (Amar et al. 2000; Craig and Foote 2001; Amar et al.
2004). Based on these studies and those cited above, we pre-
dicted that B. splendens given supplemental carotenoids would
become redder as well as elevate their immune response.
After establishing that carotenoids boost both immune re-
sponse and coloration (see Results), we tested a central pre-
diction of the trade-off hypothesis: that initial body coloration
affects allocation of supplemented carotenoids. Because fish
could allocate additional carotenoids to either immune activ-
ity or coloration, we predicted that redder individuals would
use supplemented carotenoids to augment both coloration
and immunity, whereas less- or nonred individuals would use
carotenoids to improve immunity and not coloration and thus
show greater increases in immune response and smaller
changes in coloration compared with redder fish.
MATERIALS AND METHODS
Fish were housed in individual, visually isolated 1-l beakers
filled with municipal tap water that had been subjected to
reverse osmosis and reconstituted to a conductivity of 100–
150 lS. Water was changed at a rate of 25% every other day.
Fish were maintained at 27 C and a 12:12 h light:dark cycle.
Female preferences for male color
To establish whether female B. splendens have a mating pref-
erence for red males, we conducted a dichotomous mate
choice test in the laboratory. We obtained sexually mature,
male B. splendens from a commercial supplier. We measured
standard length (SL) 60.01 mm with digital calipers. Five
uniformly red males and 5 uniformly blue males were selected
and matched for size (SL red ¼37.61 60.56 mm, SL blue ¼
37.68 60.52 mm; t
8
¼0.10, P¼0.92). They were also
qualitatively matched for temperament, as measured by ago-
nistic responsiveness to their mirror image (Clotfelter et al.
2006). Female mate choice was assessed by placing focal fe-
males in a 15 315 330-cm tank; test males were housed in
two 15 315 315-cm tanks placed perpendicularly to the
female choice tank such that they could not see each other.
Females acclimated to the choice tank for 5 min, after which
time they were allowed to view males for a 5-min prechoice
period to ensure that they visited both males before data col-
lection began. We then measured the time spent by females
(in seconds) in each third of the tank during a 5-min choice
period. The positions of red and blue males were alternated
between the left and right tanks to eliminate the effects of
potential side biases by females.
Carotenoid supplementation
To the human eye, predominant body coloration ranged from
blue to purple to red. Fish were paired with respect to these
categories of body coloration (see below for spectral analyses
of skin color), and then one member of each pair was
randomly assigned to the experimental (carotenoid supple-
mented) and control treatment groups. The carotenoid-
supplemented diet (in flake form) contained the following
(percent by mass): spray-dried white fish meal (20%), wheat
flour (20.2%), vegetable oil (0.9%), vitamins including vita-
min A in palmitate form (0.5%), water (58.1%), and b-carotene
(0.3%); a similar diet has been used for guppies (Grether et al.
2004). The control diet was identical with the exception that
a similar quantity of water was substituted for b-carotene.
Both diets were prepared by Ocean Star International, Inc.
(Snowville, UT) Sixty-one male B. splendens (28 controls and
33 carotenoid supplemented) were used in our diet experi-
ment. Fish were fed a ration equivalent to 5% of their body mass
twice daily for 8 weeks. Post hoc comparisons showed that ex-
perimental groups did not differ in either initial coloration or
body size (see Results).
Color measurements
Before and after the 8-week diet treatment, we measured the
reflectance of each fish using an Ocean Optics USB2000 spec-
trometer connected to a PX-2 pulsed xenon light. Fish were
removed from their home beakers and immobilized against
a moist sponge. We measured reflectance from a 1-cm diam-
eter region of the left side of the caudal peduncle for 10 s. We
assessed reflectance at 5-nm intervals over the wavelength
range of 300–700 nm using a 400-lm reflection probe (Ocean
Optics R400-7) held at a 45angle 5 mm from the sample
(Lahti 2006). Integration time was set at 100 ms, and reflec-
tance was averaged over 100 scans; boxcar smoothing was set
to 5. We standardized measurements with a diffuse tile made
of polytetrafluoroethylene that reflects .98% of light over all
sampled wavelengths (Ocean Optics WS-1).
We summarized reflectance data using principal compo-
nents (PCs) analysis (Jolliffe 1986), thought to be the most
appropriate means of reducing spectrophotometric data for
analysis (Cuthill et al. 1999). We reduced color data to 3 PCs,
which explained 96.2% of the variance in the sample, thus
creating 3 independent measures of color. PC1 (hereafter
‘‘brightness’’) explained 71.8% of the variance in the sample,
loaded negatively across the entire (300–700 nm) range of
wavelengths, and corresponded to differences in brightness
(Endler 1990). Individuals with high values of PC1 were those
with low brightness (i.e., low reflectance). PC2 (hereafter
1140 Behavioral Ecology

‘‘redness’’) explained 17.2% of the variation and loaded neg-
atively between 320 and 520 nm and highly positively between
600 and 700 nm, thus making PC2 an assessment of red versus
blue coloration. Higher values of PC2 were redder individuals.
Plots of PC loadings versus wavelength are shown in Figure 1.
We validated PC2 as an index of redness by regressing the
final PC2 value for each fish on the wavelength at peak re-
flectance from our reflectance curves, which yielded a signifi-
cantly positive relationship (R
2
¼0.10, F
1,58
¼6.71, P¼
0.012). The reflectance curves we obtained from red fish were
qualitatively similar to those for the red ventral coloration of
threespine sticklebacks (Gasterosteus aculeatus) (Rush et al.
2003; Rick et al. 2004).
Immune response
We assessed generalized cell-mediated immunity using expo-
sure to PHA, measured as an inflammatory response (Martin
et al. 2006). We previously validated this technique for use in
B. splendens (Ardia and Clotfelter 2006) by comparing the in-
flammatory response of fish injected with PHA with those
injected with saline. Each individual was anesthetized in tri-
caine methanesulfonate and placed on a wet sponge under
a 6.33dissecting microscope. On the right side of the caudal
peduncle, 3–5 scales were removed to mark the injection site
for consistent measurements. Prior to injection, the thickness
of the caudal peduncle at the location of scale removal was
measured with a digital micrometer (60.001 mm accuracy)
3 times (F
98,196
¼34.3, P,0.0001, repeatability ¼0.94). After
measurements, each individual was injected at the location of
scale removal with 4 lg of PHA (L-8751, Sigma–Aldrich, St.
Louis, MO) in 2 ll of phosphate-buffered saline. After 24 h,
each fish was anesthetized again and the thickness of the
tissue at the location of injection and scale removal was re-
measured (repeatability F
60,120
¼22.1, P,0.0001, repeatabil-
ity ¼0.84). The response of each individual was recorded as
the difference (in millimeters) between postinjection thick-
ness and preinjection thickness (Smits et al. 1999).
Pigment analysis
At the end of the carotenoid supplementation period, we
euthanized fish and immediately removed a 0.5 30.5-cm sec-
tion of dermis and epidermis from the caudal peduncle; tissue
samples were stored at 80 C until analysis. Thawed tissue
was then ground in 2 ml methyl tertiary-butyl ether (MTBE)
for 2 min in a mixer mill (McGraw et al. 2003). The jar was
then rinsed with 1 ml MTBE to remove any residual pigment
and combined with the 2 ml extract in a 9 ml screw-cap glass
tube. We then added 2 ml of 1% NH
4
OH to the tube, vor-
texed it for 1 min, and then centrifuged it for 5 min at 3000
rpm. This method partitioned the carotenoids into the top
(MTBE) layer and the pteridines into the bottom (NH
4
OH)
layer. We used absorbance spectrophotometry separately on
the 2 fractions to determine carotenoid and pteridine concen-
trations based on standard calculations (McGraw et al. 2002).
Carotenoids in the MTBE fraction absorbed light maximally at
447 nm and presumably were yellow xanthophylls; pteridines
in the ammonium hydroxide fraction absorbed maximally at
490 nm and were presumably drosopterins (Grether et al.
2001). Thus, in our calculations, we used 2550 as the extinction
coefficent for xanthophyll carotenoids (Bauernfeind 1981)
and 10 000 as the extinction coefficient for drosopterins
(Wilson and Jacobsen 1977).
Statistical analysis
All variables met the assumptions of parametric statistics. We
used SAS 9.1.3 to conduct statistical analyses. We tested for the
effect of color on mate choice using a mixed model analysis of
variance (ANOVA) and compared initial allocation of fish to
treatments by color using an unpaired t-test. The effect of
supplementation on immune response, coloration, and tissue
pigment concentrations was tested using analysis of covari-
ance, with initial fish coloration and SL included as covariates.
Thus, we report least square means that account for the ef-
fects of these covariates. We included interaction terms in the
models that corresponded to our a priori predictions. Tests
were 2 tailed, and differences were considered significant at
P,0.05. Means are shown with 6standard error (SE).
RESULTS
Pigment basis of coloration
To understand the basic relationship between pigments and
skin color in this species, we analyzed data for control fish
only. Tissue pigment concentrations were highly significant
predictors of fish brightness (PC1) (F
2,22
¼18.5, P,
0.0001). Drosopterins were positively correlated with PC1
(F
1,22
¼15.8, b¼0.67, P¼0.001), indicating that high levels
of drosopterins were found in fish with low brightness. Caro-
tenoids had no relationship to final brightness (F
1,22
¼1.2,
b¼0.18, P¼0.29). Skin pigment concentrations were also
significant predictors of fish redness (F
2,22
¼4.1, P¼0.03),
which varied with a continuous distribution defined by PC2.
Wavelength (nm)
200 300 400 500 600 700 800
0.05
0.06
0.07
0.08
0.09
0.10
0.11
0.12
0.13
Principal Component 1 (“brightness”)
A
200 300 400 500 600 700 800
-0.1
0.0
0.1
0.2
0.3
Principal Component 2 (“redness”)
B
Figure 1
Loadings of 2 PCs from a PCs
analysis of light reflected from
the caudal peduncle of male
Betta splendens, plotted against
light wavelength (nanometers).
(A) PC1 is a measure of skin
brightness, in which greater
values indicate lower bright-
ness (i.e., reflectance) and (B)
PC2 is a measure of redness.
Clotfelter et al. •Carotenoid trade-offs in Betta splendens 1141

Redder fish had more carotenoids (F
1,22
¼5.4, b¼0.54,
P¼0.03) but fewer drosopterins (F
1,22
¼7.5, b¼0.64,
P¼0.01).
Supplementation of B. splendens with dietary carotenoids
had weak effects on the concentrations of skin carotenoids
(controls ¼5.16 60.45 lg/g, N¼27; supplemented ¼5.96 6
0.33 lg/g, N¼32; t
57
¼1.46, P¼0.15) and drosopterins
(controls ¼3.95 60.49 mg/g, N¼26; supplemented ¼5.15 6
0.52 mg/g, N¼33; t
57
¼1.65, P¼0.10), tending to in-
crease both carotenoid and pteridine levels. However, dietary
carotenoid supplementation increased skin carotenoid con-
centrations in fish that were red at the start of the experiment
(initial PC2 score) (effect of supplementation: F
1,55
¼7.6, P¼
0.008; initial PC2 score: F
1,55
¼4.4, P¼0.04; initial PC2 3
supplementation: F
1,55
¼6.8, P¼0.01). These fish also had
more drosopterins in their skin, but their drosopterin concen-
trations increased only marginally due to carotenoid supple-
mentation (effect of supplementation: F
1,53
¼2.7, P¼0.10;
initial PC2 score: F
1,53
¼4.3, P¼0.04); there was also no
interaction between initial PC2 score and the effect of supple-
mentation (F
1,53
¼0.08, P¼0.78). When we tested only fish
with initial positive redness (PC2) scores, we found a signifi-
cant effect of carotenoid supplementation on skin carotenoid
concentrations (t
28
¼2.7, P¼0.04; controls ¼5.17 10.41,
N¼13; supplemented ¼6.79 10.37, N¼18) but no differ-
ence in skin drosopterin concentrations (t
28
¼0.6, P¼0.54;
controls ¼4.75 10.7, N¼13; supplemented ¼5.34 10.6,
N¼18).
Female preference for red coloration in males
Female B. splendens (N¼23) spent significantly more time in
the side of the tank nearest the red male (149.09 612.12 s) as
with the blue male (96.48 612.21 s) (Figure 2; mixed model
ANOVA F
1,44
¼9.36, P¼0.004). Female preference was un-
affected by which pair of male stimulus fish we used (removal
of term led to no change in 2 log likelihood ratio). Note that
this was the only experiment in which fish were categorized
dichotomously (red vs. blue) rather than continuously (PC2).
Carotenoid supplementation boosts red coloration
Post hoc comparisons showed that we allocated fish to treat-
ment groups irrespective of their initial brightness (control ¼
0.90 61.34, N¼28; supplemented ¼0.27 61.53, N¼33;
t
59
¼0.31, P¼0.76), redness (control ¼0.59 60.78,
N¼28; supplemented ¼0.19 60.67, N¼33; t
59
¼0.77,
P¼0.45), or SL (control ¼37.56 61.45 mm; supplemented ¼
39.06 60.34 mm; t
59
¼1.09, P¼0.28).
We were able to measure initial (before carotenoid supple-
mentation) and final brightness and redness in 60 (27 con-
trols and 33 supplemented) of the 61 fish (erroneous final
color measurement on one fish resulted in a significant out-
lier; Cook’s distance ¼0.76). The carotenoid trade-off hypoth-
esis assumes that the delivery of supplemental carotenoids will
enhance coloration, and consistent with this, we found that
fish supplemented with carotenoids became significantly
more red (PC2) (least square mean 6SE change in redness;
controls ¼2.13 60.51, N¼27; supplemented ¼0.33 6
0.46, N¼33; F
1,54
¼43.76, P,0.0001). Carotenoid supple-
mentation did not, however, induce a significant change in
skin brightness (PC1) (control ¼0.43 615.04, N¼27;
supplemented ¼1.58 615.24, N¼33; t
59
¼0.30, P¼0.77).
Carotenoid supplementation boosts immune response
A second key assumption of the trade-off hypothesis is that
carotenoid supplementation boosts immune activity. Also con-
sistent with this, we found that carotenoid supplementation
significantly increased the immune response of male B. splendens,
as measured by the swelling of the caudal peduncle in re-
sponse to PHA injection (Figure 3; mean postinjection swell-
ing in mm 6SE: control ¼0.087 60.01 mm, N¼28;
supplemented ¼0.17 60.02 mm, N¼33; carotenoid supple-
mentation group: F
1,58
¼12.68, P¼0.001). We obtained sim-
ilar results when we used the ratio of preinjection swelling to
postinjection swelling as our dependent variable (data not
shown).
Initial coloration affects trade-offs between immune
response and coloration
In support of a key prediction for the carotenoid trade-off
hypothesis—namely, that animals with less carotenoid-dependent
coloration should devote comparatively more carotenoids to
an immune response than should animals with more such
coloration—we found that initial body coloration affected al-
location strategies of carotenoids between coloration and im-
munity. Fish with initially low PC2 values (more blue) showed
a larger increase in immune activity (PHA response) than did
redder fish when supplemented with carotenoids (Figure 4;
0
20
40
60
80
100
120
140
160
180
200
Blue male Neutral zone Red male
Female choice
Time (seconds)
Figure 2
Female Betta splendens (N¼23) spent more time (F
1,44
¼9.36, P¼
0.004) associating with red males than they did with blue males.
0.00
0.05
0.10
0.15
0.20
Contro
lC
arotenoid
supplemented
Immune response to PHA (mm)
Figure 3
Fish supplemented with dietary carotenoids (N¼33) were able to
mount a significantly greater immune response to the PHA injection
than were the fish on the control diet (N¼28; F
1,58
¼12.68, P¼
0.001).
1142 Behavioral Ecology

overall model: F
4,56
¼7.83, P,0.0001; initial redness: F
1,56
¼
6.78, P¼0.01; initial redness 3supplementation: F
1,56
¼7.59,
P,0.01; supplementation: F
1,56
¼15.5, P¼0.0002). Carot-
enoid supplementation increased redness (least square mean 6
SE change in redness; controls ¼2.13 60.51, N¼27; sup-
plemented ¼0.33 60.46, N¼33; F
1,54
¼43.76, P,0.0001)
but only in fish that were initially more red (Figure 5; initial
redness: F
1,54
¼116.6, P,0.0001; initial redness 3supple-
mentation: F
1,54
¼85.3, P,0.0001). Supplementation de-
creased fish brightness, as indicated by higher PC1 values
(least square mean 6SE change in brightness; controls ¼
1.12 60.82, N¼27; supplemented ¼2.58 60.73, N¼
33), such that reflectance in less-bright fish decreased even
further in response to supplementation than in fish with high-
er initial brightness (initial brightness: F
1,54
¼166.6, P,
0.0001; initial brightness 3supplementation: F
1,54
¼10.8,
P¼0.001). Neither immune responses nor SLs were signifi-
cantly related to changes in redness or brightness when they
were included as covariates (F1.2, P0.17).
DISCUSSION
Organisms are predicted to make trade-offs when resources
are scarce and serve multiple functions. Carotenoids are
thought to be an example of a scarce resource; they must be
acquired through the diet, and they provide benefits to both
health and sexually selected coloration. We report experimen-
tal evidence demonstrating carotenoid trade-offs in B. splendens;
male B. splendens displayed different carotenoid allocation
strategies based on their initial coloration. Unlike other spe-
cies in which this trade-off has been examined, where the
ability to maintain carotenoid-based coloration is condition
dependent and results in a range of red and less-red pheno-
types, male B. splendens have genetically determined color
morphs. Redder individuals (positive PC2 values) provided
with supplemental carotenoids showed an increased inflam-
matory response to PHA and greater redness, whereas bluer
individuals (negative PC2 values) showed no change in color-
ation and instead mounted an even greater immune response.
In other words, because bluer fish were faced with an inher-
ently more relaxed carotenoid trade-off for health versus col-
oration (fewer carotenoids devoted to color), they apparently
diverted their accumulated pool of carotenoids more to one
function (immune response) than to the other (color).
Role of carotenoids in coloration, mate choice,
and immunity
We first found support for 3 key assumptions of the caroten-
oid trade-off hypothesis in B. splendens: that carotenoid pig-
ments 1) are used for coloration, 2) are a predictor of female
preference, and 3) boost both immunity and coloration when
in abundance. We found that coloration in B. splendens is un-
derlain by both carotenoids and pteridines, as is true for many
other red or orange color patches in fish (Henze et al. 1977)
and lizards (Macedonia et al. 2000). Carotenoid-supplemented
fish became redder in color, and naturally redder fish had
higher carotenoid concentrations in their skin as a result of
the supplementation. Furthermore, carotenoid supplementa-
tion decreased fish brightness, providing further evidence
that dietary carotenoids were allocated to skin coloration.
There was also evidence that redder fish had greater drosop-
terin concentrations in their skin. Grether et al. (2001) re-
ported that skin pteridine concentrations in Trinidadian
guppies covaried positively with natural carotenoid availability
due to population genetic differences in drosopterin content.
Grether et al. (2005) also found that experimental manipula-
tions of dietary carotenoids can marginally and inversely im-
pact skin drosopterin concentrations. Clearly, more work is
needed to better understand the complementarities and com-
petitions between these 2 classes of integumentary colorants.
Second, we report a role of red coloration in sexual selec-
tion in this species. Female B. splendens display a preference
for associating with red males. Such female preferences for
carotenoid-dependent signals in males have been reported in
other fishes (Kodric-Brown 1993; Candolin 1999; Maan et al.
-10 -5 0 5 10
Initial PC2
-0.1
0.0
0.1
0.2
0.3
0.4
Immune response to PHA (mm)
Supplemented
Control
RedBlue
Figure 4
Fish that were initially bluer (negative PC2 values; see text for
explanation) and received the carotenoid-supplemented diet
showed a larger boost in immune activity than did redder fish
(positive PC2 values) or than did fish on the control diet (overall
model: F
4,56
¼7.83, P,0.0001; initial redness: F
1,56
¼6.78, P¼
0.01; initial redness 3supplementation: F
1,56
¼7.59, P,0.01;
supplementation: F
1,56
¼15.5, P¼0.0002).
-10 -5 0 5 10
Initial Redness
-20
-10
0
10
20
Change in Redness
Supplemented
Control
RedBlue
Less red
More red
Figure 5
Carotenoid supplementation increased redness (PC2; see text for
explanation), and the change in redness varied depending on initial
redness (initial redness: F
1,54
¼116.6, P,0.0001; initial redness 3
supplementation: F
1,54
¼85.3, P,0.0001).
Clotfelter et al. •Carotenoid trade-offs in Betta splendens 1143

2006) but have not been previously reported for B. splendens
or any member of its perciform family (Osphronemidae).
Finally, we demonstrated that dietary supplementation with
carotenoids significantly increased the ability of male B. splen-
dens to mount an inflammatory response to PHA injection.
Our results provide additional evidence for the immunoen-
hancing role of carotenoids in vertebrates and that environ-
mental scarcity of carotenoids may lead to the evolution of
allocation strategies. Moreover, our use of the generalized
swelling response to PHA as a metric of immune response
(Ardia and Clotfelter 2006) complements the humoral meas-
ures (Amar et al. 2004) or allografting outcomes (Grether
et al. 2004) used in other fish studies.
Experimental support for the trade-off hypothesis: the
effect of initial coloration
In our key test of the carotenoid trade-off hypothesis, we pro-
vided evidence that individuals vary in their carotenoid allo-
cation strategy depending on their degree of carotenoid-
based skin coloration. We found that redder fish appeared
to allocate their supplemental carotenoids to both immune
response and color, as they increased in redness over the sup-
plementation period and increased their inflammatory re-
sponse to PHA relative to control fish. In contrast, bluer fish
(individuals with negative PC2 scores) given supplemental
carotenoids did not change color but instead mounted
a greater inflammatory response to the PHA challenge than
observed in either control or redder supplemented fish.
This study is the first to demonstrate that, within members
of the same sex, individuals whose coloration is less caroten-
oid based have a qualitatively different carotenoid allocation
strategy than do redder conspecifics. Grether et al. (2004)
have previously shown that carotenoid enhancement of im-
munity in guppies is sex specific because males—and not
females—have carotenoid-based coloration. Most studies that
have attempted to shed light on carotenoid trade-offs in ani-
mals have examined species whose yellow-to-red coloration is
purely carotenoid based and is environmentally (condition)
dependent (Blount et al. 2003; McGraw and Ardia 2003;
Alonso-Alvarez et al. 2004; Peters et al. 2004). In such systems,
carotenoid trade-offs have been more difficult to evaluate be-
cause there is no obvious group of animals that is constrained
in carotenoid allocation (i.e., even drab animals can divert
supplemental pigments to both immunity and coloration).
While we realize that this genetic color polymorphism has
been derived through artificial selection, we believe that these
results provide a model for examining intra- and interspecific
differences in carotenoid allocation strategies, as artificially
selected fish represent extremes of a naturally occurring color
gradient. Wild B. splendens possess both blues and reds to
varying degrees, and many of the domestic fish we used in
this study fell along a similar continuum. Furthermore, we
showed that even the bluest fish had carotenoids in their
dermis, demonstrating their ability to develop carotenoid-
based pigmentation. Thus, selection for uniformly blue and
red coloration in domestic strains led to associated changes in
carotenoid allocation strategies, leading to changes in both
coloration and carotenoid usage. Applying this approach to
examining differences among populations or closely related
species that differ in the extent of carotenoid-based coloration
may help elucidate the rate and extent of change in the costs
and benefits of allocating carotenoids to coloration versus
immune response.
Overall, our results indicate that body coloration in poly-
chromatic species can have a strong effect on carotenoid
allocation strategies. Research investigating the role of evolu-
tionary trade-offs between sexual ornaments and immunity
should examine underlying differences in carotenoid alloca-
tion strategies that may be caused by differences in coloration.
FUNDING
Dean of Faculty’s office at Amherst College (E.D.C.); School
of Life Sciences and College of Liberal Arts and Sciences at
Arizona State University (K.J.M.).
We thank Greg Grether for advice on fish diets and Mark Lamon and
Nanette Bunker at Ocean Star International, Inc. for producing our
experimental and control diets. The Jeff Podos laboratory, particularly
David Lahti, at the University of Massachusetts kindly loaned us their
spectrometer and their expertise. Maureen Manning provided out-
standing logistical support through many stages of this project. Addi-
tional thanks to Alexandria Brown, Katie Moravec, and Neron
Thomas for laboratory and animal care assistance. This research was
conducted with the approval of the Institutional Animal Care and
Use Committee of Amherst College. Anne Houde and 2 anonymous
referees provided helpful comments on earlier versions of the
manuscript.
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