Seasonal changes in parasite load and a cellular immune response in a colour polymorphic lizard.

Abstract

Permanent colour polymorphisms may be maintained by complex interactions between physiological traits (e.g. immunity) and environmental pressures. In this study we investigate morph specific variation in parasite load and cellular immune response (induced by a Phytohaemagglutinin, PHA injection) in a colour polymorphic population of the Dalmatian wall lizard (Podarcis melisellensis), where adult males have bright white, yellow or orange throats and ventral sides. Orange males have larger heads and can bite harder than the others. To examine seasonal effects, analyses were performed at an early and late stage in the reproductive season (May and September). Infection with mites and ticks did not differ among morphs, but was more severe at the end of the reproductive season. Fewer orange individuals were infected with haemogregarines at the end of the season, but white males were always more infected (higher number of haemogregarines in their blood) than other morphs. White and yellow males showed an increased PHA response towards the end of the season, but PHA response decreased in the orange morph. Finally, across all morphs, a relationship was found between ectoparasite load and PHA response. Our study provides indications of alternative life-history strategies among colour morphs and evidence for an up-regulation of the immune function at the end of the reproductive season.

Full text

Page 1

BEHAVIORAL ECOLOGY - ORIGINAL PAPER
Seasonal changes in parasite load and a cellular immune response
in a colour polymorphic lizard
Katleen Huyghe•Annette Van Oystaeyen•
Frank Pasmans•Zoran Tadic ´ •Bieke Vanhooydonck•
Raoul Van Damme
Received: 28 October 2009/Accepted: 15 April 2010/Published online: 9 May 2010
? Springer-Verlag 2010
Abstract
maintained by complex interactions between physiological
traits (e.g. immunity) and environmental pressures. In this
study we investigate morph specific variation in parasite
load and cellular immune response (induced by a Phyto-
haemagglutinin, PHA injection) in a colour polymorphic
population of the Dalmatian wall lizard (Podarcis meli-
sellensis), where adult males have bright white, yellow or
orange throats and ventral sides. Orange males have larger
heads and can bite harder than the others. To examine
seasonal effects, analyses were performed at an early and
late stage in the reproductive season (May and September).
Infection with mites and ticks did not differ among morphs,
but was more severe at the end of the reproductive
season. Fewer orange individuals were infected with
Permanent colour polymorphisms may be
haemogregarines at the end of the season, but white males
were always more infected (higher number of haemogre-
garines in their blood) than other morphs. White and yel-
low males showed an increased PHA response towards the
end of the season, but PHA response decreased in the
orange morph. Finally, across all morphs, a relationship
was found between ectoparasite load and PHA response.
Our study provides indications of alternative life-history
strategies among colour morphs and evidence for an
up-regulation of the immune function at the end of the
reproductive season.
Keywords
Immune defence ? Seasonal differences ? Lacertidae
Parasites ? Colour polymorphism ?
Introduction
The vertebrate immune system constitutes an effective
defence against parasites and pathogens, but it is also
expensive to develop and maintain. Therefore, investment
in immune defence must be traded off with other fitness
components (Schmid-Hempel and Ebert 2003): animals
that invest heavily in traits such as parental care (Moreno
et al. 2001), mating behaviour (McKean and Nunney 2001)
and sexual ornamentation (Verhulst et al. 1999) may opt to
tolerate an infection rather than to fight it (Sheldon and
Verhulst 1996). Compounding to the complexity, the ver-
tebrate immune system itself consists of several compo-
nents that may act in synergy but may also compete for
available resources (Braude et al. 1999; Zuk and Stoehr
2002; Buchanan et al. 2003; Gasparini et al. 2009). Such
trade-offs may help to explain the ecologically and evo-
lutionary important intraspecific variation in immune
function that is being observed in a growing number or taxa
Communicated by Anssi Laurila.
K. Huyghe (&)
Department of Biodiversity and Evolutionary Biology, Museo
Nacional de Ciencias Naturales (MNCN-CSIC), Calle Jose ´
Gutie ´rrez Abascal 2, 28006 Madrid, Spain
e-mail: katleen.huyghe@ua.ac.be
K. Huyghe ? A. Van Oystaeyen ? B. Vanhooydonck ?
R. Van Damme
Department of Biology, University of Antwerp,
Universiteitsplein 1, 2610 Antwerp, Belgium
F. Pasmans
Department of Pathology, Bacteriology and Avian Diseases,
Faculty of Veterinary Medicine, Ghent University, Salisburylaan
133, 9820 Merelbeke, Belgium
Z. Tadic ´
Department of Animal Physiology, University of Zagreb,
Rooseveltov trg 6, 10000 Zagreb, Croatia
123
Oecologia (2010) 163:867–874
DOI 10.1007/s00442-010-1646-9

Page 2

(e.g. fish: Clotfelter et al. 2007; frogs: McCallum and
Trauth 2007; lizards: Svensson et al. 2001; Calsbeek et al.
2008; birds: Pryke et al. 2007; mammals: Cutrera et al.
2010).
Polymorphic species provide excellent opportunities to
study the interplay between immune defence and other
life history traits. Morphs are thought to represent dif-
ferent fitness optima, produced by correlational selection
on alternative suits of life-history traits. In many cases of
male polymorphism, one morph pursues an aggressive,
dominant reproductive strategy, often by physically
defending a territory. The other morph then takes a more
tranquil approach, behaves less aggressively towards
conspecifics, and employs alternative mating tactics such
as sneaking to obtain copulations. The dominant morph
must invest in morphological, physiological and behav-
ioural traits that help to maintain its superior rank but are
also likely to trade-off with its immune function. The
immunocompetence handicap hypothesis proposes that the
association between a dominant mating strategy and
reduced immunocompetence arises from the dual effect of
testosterone, which promotes secondary sexual traits such
as ornaments, bright colours and aggressive behaviour,
but is simultaneously immunosuppressive (Folstad and
Karter 1992, but see Roberts et al. 2004). Alternatively,
the trade-off may simply reflect differential energy allo-
cation: if pursuing a dominant reproductive strategy is
energetically demanding, dominant morphs may not have
sufficient reserves to mount the immune response, espe-
cially towards the end of the reproductive season
(Duckworth et al. 2001; Alonso-Alvarez et al. 2007;
Roberts and Peters 2009).
Femalepolymorphismis
(Svensson et al. 2009), but in the few cases studied, female
morphs tend to differ in reproductive traits such as clutch
size and egg mass, and the success of either morph depends
strongly on the social environment (Svensson et al. 2001;
Vercken et al. 2007). Here too, differences in immune
function between morphs may arise from direct energetic
trade-offs, or via the adverse effects of hormones (e.g.
elevated levels of corticosterone may minimize the effects
of stressors but at the same time reduce immunocompe-
tence: Comendant et al. 2003).
Several recent studies have found empirical support for
the prediction that morphs should exhibit differential
investment in immune function. For instance, in the
Gouldian finch (Erythrura gouldiae) aggressive red-headed
males exhibit immunosuppression in socially competitive
environments, while the non-aggressive black heads do not
(Pryke and Griffith 2006). In the side-blotched lizard (Uta
stansburiana), orange-throated females experience a higher
immunosuppressive effect from social crowding than yel-
low-throated conspecifics (Svensson et al. 2001; see also
much lessdocumented
Vercken et al. 2007; Calsbeek et al. 2008). In the tawny
owl (Strix aluco) the interaction between the innate and
humoral branches of the immune system is synergistic in
dark nestlings, but antagonistic in pale nestlings (Gasparini
et al. 2009). These theoretical and empirical consider-
ations corroborate the idea that immune function may
play an important role in the maintenance of colour
polymorphisms.
The Dalmatian wall lizard, Podarcis melisellensis, also
shows colour polymorphism. Male P. melisellensis differ in
throat and ventral colouration, which can be bright white,
yellow or orange. The colour morphs also differ in several
other traits that seem relevant for their respective repro-
ductive strategy. Orange males are on average larger, they
have relatively larger heads, they can bite harder and
behave more aggressively than the other morphs (Huyghe
et al. 2009a, b). As a result, they enjoy a higher dominance
status (Huyghe et al. unpublished data). We here test the
hypothesis that this advantage is balanced by a decreased
immunocompetence. We expect that high testosterone
levels and/or high investment in territorial defence will
exhaust orange males as the breeding season advances,
resulting in lowered immune responses and increased
susceptibility to infectious disease.
Our main objectives are (1) to investigate the relation-
ships between immune defence function (cellular immune
response), parasite loads and colour, (2) to detect morph-
specific variation in immune function, which could provide
insights into the physiological mechanisms maintaining
colour polymorphism within this species, and (3) to test
whether there is a seasonal effect on immune function for
the different morphs. Therefore, we measured immune
function both early (May) and late (September) in the
reproductive season in a polymorphic population of
P. melisellensis.
Materials and methods
Study species
Podarcis melisellensis is a small lacertid lizard, endemic
to the Adriatic coastline and islands in the Adriatic Sea.
In 2008, sexually mature males (snout-vent length,
SVL[55 mm) were captured by noose on the island
Lastovo (Croatia) early during the reproductive season
(May, N = 74) and at the end (September, N = 38). Liz-
ards were kept individually in cloth bags and transported to
a field based laboratory, where the following measurements
were taken. Within 48 h following capture, lizards were
returned to the field in seemingly good health. Individuals
were categorized in morphs by visual assessment of their
colour (white, yellow or orange).
868Oecologia (2010) 163:867–874
123

Page 3

Morphometrics
Each individual’s SVL was measured using digital calli-
pers (Mitutoyo, accuracy: 0.01 mm) and each was weighed
with a Pro Scout balance (to the nearest 0.1 g). As a con-
dition index residual values of a linear regression analysis
of mass (dependent) over SVL (independent) were used
(both log10transformed). Tail type was scored as intact,
broken or regenerated.
Parasites
The degree of ectoparasite infestation was assessed by
brushing each lizard for 5 min with a soft paint brush over
a white paper, so that ticks (fam. Ixodidae) and mites (fam.
Trombiculidae) could be collected in a tube filled with 70%
aqueous methanol. This resulted in 100% removal of vis-
ible ectoparasites. Later, ectoparasites were counted using
a binocular loupe at 1009 magnification.
To assess infestation by haemoparasites, a small amount
of blood was collected with a heparinized capillary tube by
puncturing the postorbital sinus. Thin blood smears were
made on glass microscope slides. Smears were air dried
and fixed for 15 min in absolute methanol and later these
were Hemacolour-stained (Merck, Darmstadt, Germany)
and sealed to facilitate long-term storage. Blood smears
showed similar concentrations of red blood cells and were
surveyed for 15 min, counting the number of haemogre-
garines (Karyolysis sp.) at 5009 magnification using a light
microscope.
Cellular immune response
We quantified the delayed cutaneous hypersensitivity
response (Belliure et al. 2004; Oppliger et al. 2004) as an
indicator of one aspect of immunity, at the cellular level.
This response was assessed by injecting one foot of every
individual with a 20 lL solution containing 50 mg of
phytohaemagluttinin (PHA; Sigma–Aldrich, L-8754) in
10 mL phosphate buffered saline (PBS). PHA influences a
variety of cell types and, therefore the response to PHA
injection is complex, but can serve as an index for
heightened immune cell activity (Kennedy and Nager
2006; Martin et al. 2006). Thickness of the foot was
measured before injection and 24 h later, using digital
callipers (Mitutuyo, accuracy: 0.01 mm). The other foot
was treated in the same way, but injected with 20 lL of
PBS serving as a control. The immune response was cal-
culated as the change in thickness of the PHA injected foot
minus the change in the control foot. Larger localized
swelling indicated an increased immune activity at the
cellular level. Measurements were made in triplicate and
the median was used in the analyses.
Statistical analyses
We investigated parasite prevalence (proportion of indi-
viduals infected) using hierarchical loglinear analyses,
testing for possible differences between times in the
breeding season (early vs. late), among colour morphs, and
the interaction effect of season 9 morph. Generalized
linear models were used to test for effects of season and
colour, and their interaction on the degree of parasite
infection (the number of parasites per individual) and on
tail type (intact, broken or regenerated). The variation in
PHA response and body condition among morphs, season
and their interaction was tested using analyses of variance.
Chi square (v2) and significance (P) values are only
reported for the significant effects. To test the predictability
of infection with parasites, logistic regression analyses
were used (infected vs. uninfected). Relationships among
traits were investigated using correlation analyses.
Results
Differences among morphs across the reproductive
season
A greater proportion of the population was infected with
ectoparasites at the end of the season than at the beginning:
63% in May versus 100% in September (v2= 26.61,
df = 1, P\0.001), but prevalence was equal for all
morphs (v2= 0.26, P = 0.88, Fig. 1a). Accordingly,
ectoparasite load (mean number of parasites per lizard) was
more severe late in the reproductive season (v2= 41.08,
df = 1, P\0.001), but it did not differ among morphs
(v2= 0.34, df = 2, P = 0.85; Fig. 2a).
The proportion of individuals infected with haemogre-
garines differed significantly among morphs across the
season (v2= 7.98,df = 2,
increased for the white and yellow morphs during the
season, but decreased for the orange morph (Fig. 1b). The
degree of infection (mean number of haemogregarines per
lizard) changed across the season (v2= 31.41, df = 1,
P\0.001) and differed among morphs (v2= 8.74,
df = 2, P = 0.02): infection was more severe at the
beginning of the reproductive season and white males had a
higher number of haemogregarines in their blood (Fig. 2b).
The response to injection with PHA differed signifi-
cantly among morphs across the season (interaction effect
season 9 colour: F2,84= 4.79, P = 0.01). White and yel-
low males showed an increased response later in the sea-
son, while the immune response of orange males remained
the same (Fig. 3).
Body condition did not differ between colour morphs
(F2,107= 0.20,
P = 0.82) or season
P = 0.02): infestation
(F1,107= 0.029,
Oecologia (2010) 163:867–874 869
123

Page 4

P = 0.87). Tail break frequencies were not different among
morphs (v2= 0.070, df = 2, P = 0.97) and were the same
early and late in the season (v2= 0.019, df = 1, P = 0.89).
Relationships among health factors
We documented a significant correlation between presence
and absence of ectoparasites with PHA response (logistic
regression analysis, v2= 16.72, df = 3, P = 0.001). PHA
response was the sole significant predictor variable in the
model (B = 21.16, v2= 8.73, df = 1, P = 0.003); SVL
and mass did not explain a significant proportion of the
variance of infection (both v2\2.79, both P[0.10).
Moreover, when taking only the infected individuals into
account, a correlation was found between the numbers of
ectoparasites and PHA response (Spearman’s r = 0.34,
P = 0.001, Fig. 4a), but not with SVL or mass (both
r\0.15, both P[0.17). Lizards carrying more ectopar-
asites seemed to have a higher PHA response, but when the
percentage of infectedindividuals
(ectoparasites)
0
20
40
60
80
100
white
yellow
orange
a
September
May
percentage of infected individuals
(haemoparasites)
0
20
40
60
80
100
b
Fig. 1 Percentage of individuals infected with parasites (infestation)
for the different morphs early (May, N = 74) and late (September,
N = 38) in the reproductive season. a Ectoparasites: more individ-
uals were infected in September, no differences among morphs,
b Haemogregarines: more white and yellow individuals infected in
September, more orange in May
percentage of infectedindividuals
(ectoparasites)
0
20
40
60
80
100
white
yellow
orange
a
September
May
percentage of infected individuals
(haemoparasites)
0
20
40
60
80
100
b
Fig. 2 Mean and standard error of number of parasites for the
different morphs early (May) and late (September) in the reproductive
season. a Ectoparasites: more severe infection in September, no
differences among morphs b Haemogregarines: more severe infection
in May, a higher number of haemogregarines was found in white
males
September
May
PHA response (mm)
0.0
0.2
0.4
0.6
0.8
white
yellow
orange
Fig. 3 Mean and standard errors of the response to PHA injection,
corrected for injection with PBS (in mm) for the different morphs
early (May) and late (September) in the reproductive season. White
and yellow males showed an increased response later in the season,
while the immune response of orange males remained the same
870Oecologia (2010) 163:867–874
123

Page 5

number of ectoparasites increases, the capacity to respond
to a PHA injection appears to drop again (Fig. 4a: qua-
dratic fit line, r2= 0.24 vs. linear fit line, r2= 0.087). A
quadratic regression produced a better fit than the linear
regression (extra sum-of-squares F test, F1,83= 24.02,
P\0.0001). This correlation did not differ among morphs
across the season (three-way interaction colour 9 sea-
son 9 PHA, dependent = # ectoparasites not significant:
v2= 0.11, P = 0.95).
There was a marginally significant relationship between
infestation by ectoparasites and haemogregarines (Hierar-
chical loglinear analysis: v2= 3.96, P = 0.06).
The presence or absence of haemogregarines in the
blood could be predicted by mass (B = -16.11, v2= 4.52,
P = 0.03), but not SVL or PHA (both v2\3.66, both
P[0.06); a significant model was generated in a logistic
regression analysis (model v2= 8.36, P = 0.04), showing
that individuals with a low mass have a higher probability
of carrying haemogregarines in their blood. The number
of haemogregarines was not correlated with SVL (r =
-0.15, P = 0.33), mass (r = -0.14, P = 0.34) or PHA
response (r = -0.25, P = 0.14, Fig. 4b).
There was no correlation between body condition and
PHA response (r = 0.037, P = 0.74).
Discussion
At the onset of this study, we hypothesized that male P.
melisellensis morphs might differ in their ability to with-
stand challenges of their immune system, and that this
difference may become apparent near the end of the
reproductive season. We found that seasonal changes in
different aspects of the immune function were not similar
for all morphs.
All morphs showed an increased ectoparasite prevalence
(proportion of individuals infected) and infection rate (the
number of ectoparasites) as the season progressed (Figs. 1a
and 2a). The finding that ectoparasite load was equal for
the different morphs is in line with earlier findings that
morphs do not show different activity patterns (Huyghe
et al. 2007). Susceptibility to ectoparasite infection is partly
related to activity level in several species (e.g. Bouma et al.
2007). Morphs may show similar so-called avoidance
behaviour, a non-specific immune defence technique where
infection with pathogens is ‘avoided’. Parasite load
increased for all morphs during the breeding season, which
can be related to an increased presence of parasites and/or
increased activity towards the end of the season, and thus a
higher probability of becoming infected. Alternatively, the
increase in parasite load could be an indirect effect of e.g.
decreased survival of uninfected individuals.
The number of individuals with haemogregarines in
their blood was lower at the end of the season than at the
beginning for orange, but higher for white and yellow
males (Fig. 1b). Within infected lizards, the number of
haemogregarines found in the blood was higher at the
beginning of the season, and white males were always
infected to a greater extent (Fig. 2b). The finding that fewer
orange individuals suffered from haemoparasite infection
in September could indicate several things. In the lizard
Lacerta vivipara for example, haemoparasite load was
positively correlated with reproductive effort (Sorci et al.
1996), and two different pathways were suggested that
could lead to this relation. When haemoparasites have a
severe impact on the host’s survival, a selective benefit
should exist when maximizing investment in current
reproduction. Alternatively, a reduced anti-parasite defence
could be the consequence of higher reproductive invest-
ment. Reproductive investment is lower near the end of the
breeding season, explaining the lower levels of haemo-
parasite load (number of haemogregarines per lizard)
within infected individuals. P. melisellensis morphs could
also differ in timing of their reproductive effort and con-
sequently in parasite infestation (number of individuals
infected) and parasite load (number of parasites). On the
other hand, lizards become infected with blood parasites by
taking up the vectors for haemogregarines as food items
(i.e. ticks and mites) (Smallridge and Bull 1999), and as the
numbers of these vectors increase in the environment as the
# ectoparasites
1 10 2030 40
PHA response (mm)
0.2
0.4
0.6
0.8
1
a
# haemogregarines
11050 100 300
0.2
0.4
0.6
0.8
1
b
Fig. 4 Relationship between
the response to a PHA injection
(in mm), corrected for injection
with PBS, and the number of
parasites. Open circles early
season (May) and closed circles
late season (September).
a Ectoparasites.
b Haemogregarines in blood
Oecologia (2010) 163:867–874 871
123

Page 6

season progresses, we would expect a concordant increase
in haemoparasite infection (number of parasites per infec-
ted individual). Possibly, the infected individuals with high
haemoparasite loads did not survive and were, therefore,
not found in September.
A correlation between tail regeneration and blood
parasitism was found in Lacerta vivipara (Oppliger and
Clobert 1997): tailless infected lizards had a slower rate of
tail regeneration than parasite-free lizards. The period of
tail regeneration is crucial for lizards, as predation may
increase, and the longer the time required for regeneration,
the greater the risk of predation and other costs associated
with tail loss. In the present study, we found no differences
in tail break frequency among morphs and between times
in the reproductive season. The variation in blood parasite
infestation and load were thus not accompanied with var-
iation in tail break frequencies. Although we have no data
on tail regeneration itself in P. melisellensis, our data
suggest do not support the correlation between parasites
and tail regeneration.
The elevated PHA-response of orange males at the
beginning of the season (Fig. 3) may partly be a conse-
quence of a higher incidence of injuries (acquired
immunity). Orange males are more aggressive than the
others and, therefore, may be more involved in competi-
tive interactions, possibly resulting in injuries (Huyghe
et al. 2007), especially during the breeding season. During
these interactions, males tend to bite each other fiercely
on the head and neck region. The ability to mount a cell-
mediated immune response to a mitogenic stimulus such
as PHA may have important fitness consequences,
because it constitutes a generalized short-term response to
allergens and wounds (Zuk and Johnsen 1998). Therefore,
individuals with a higher response have an advantage and
a higher survival probability (Gonzalez et al. 1999).
Differences in PHA response have been reported for other
polymorphic species, such as Podarcis muralis, where
yellow morphs have a lower response (Sacchi et al. 2007),
and the female morphs of Anolis sagrei (Calsbeek et al.
2008). White and yellow male P. melisellensis showed an
increased PHA response near the end of the breeding
season, which could mean that they, as the season pro-
gresses, become increasingly involved in aggressive
interactions. In this case, morphs would ‘divide’ the
breeding season, with orange males being more aggres-
sive and more successful in the beginning, and the other
morphs at the end. Currently no information is available
on the possible fluctuations in aggressive behaviour, male-
male competition, and success during the breeding season.
We only know that, early in the season, orange males are
more aggressive and have a higher probability of winning
inter-male competitive events (Huyghe et al. unpublished
data).
In Podarcis muralis lizards, the immune response to
PHA-injection was negatively correlated with the inten-
sity of haemogregarine infection (Amo et al. 2005), but
this relation was not found in P. melisellensis. In birds,
PHA swelling is traded off with other physiological
functions, indicated by the fact that PHA swellings are
weaker when concurrent with other costly activities
(Martin et al. 2006). The amount of swelling in response
to a PHA injection showed in P. melisellensis however a
quadratic relation with ectoparasite load (Fig. 4a): indi-
viduals carrying more ectoparasites seemed to have a
higher PHA response, but when ectoparasite infection
becomes more severe, the capacity to respond to a PHA
injection seems to drop again. Individuals which were not
carrying any ectoparasite had a lower PHA response than
the ones which were. Seemingly, infection by ectopara-
sites is related to the ability to mount a cellular immune
response to a mitogen injection, but in an unexpected
way. PHA swelling in our study could perhaps be an
indication for high quality individuals which are able to
survive high levels of ectoparasite infection and which are
capable of responding more effectively to PHA, compared
to individuals with lower ectoparasite loads and lower
swelling responses. Alternatively, there may be a priming
effect, in which those with high loads of ectoparasites
may already be primed for immunological responses
(PHA swelling) because their immune system is con-
stantly challenged, and those with decreased ectoparasite
loads may exhibit a lowered swelling response because
their immune systems have not been primed in the same
way. When ectoparasite load was very high, PHA
responses dropped again, indicating a threshold beyond
which animals cannot bear the costs of severe parasite
infection and mounting a swelling response.
Our study provides two strong indications of alternative
life-history strategies among different colour morphs of the
lizard species Podarcis melisellensis. Firstly, we found a
differential haemogregarine parasitemia infection, with
white males having more severe infections than males of
other colours. Secondly, the response to PHA injection
differed among morphs and seasons, with yellow and white
males showing an increase in response as the season pro-
gressed. It remains unclear whether these differences are
caused by immunological factors, tolerance or behavioural
differences. Furthermore, the results of our study indicate
an up-regulation of the immune function at the end of the
breeding season, when lower numbers of haemogregarines
were found and the PHA response increased in two mor-
phs. This may be an important strategy to survive stressful
conditions during winter. As this phenomenon is poorly
studied in reptile species, more research is necessary to
gain insights in this mechanism and its consequences on
survival.
872Oecologia (2010) 163:867–874
123

Page 7

Acknowledgments
Husak for help in the field, and J. Meaney for improving the English.
All data was collected in accordance with University of Antwerp
animal welfare standards and protocol, and field permit no. 532-08-
01-01/3-08-03, provided by the Croatian Ministry of Culture. B.
Vanhooydonck is a postdoctoral fellow of the Fund for Scientific
Research, Flanders (FWO-Vl).
We would like to thank A. Herrel and J.F.
References
Alonso-Alvarez C, Bertrand S, Sorci G (2007) Energetic reserves,
leptin and testosterone: a refinement of the immunocompetence
handicap hypothesis. Biol Lett 3:271–274. doi:10.1098/rsbl.
2007.0020
Amo L, Lo ´pez P, Martı ´n J (2005) Prevalence and intensity of
haemogregarine blood parasites and their mite vectors in the
common wall lizard, Podarcis muralis. Parasitol Res 96:378–
381. doi:10.1007/s00436-005-1354-2
Belliure J, Smith L, Sorci G (2004) Effect of testosterone on T cell-
mediated immunity in two species of Mediterranean Lacertid
lizards. J Exp Zool 301A:411–418. doi:10.1002/jez.a.20068
Bouma MJ, Smallridge CJ, Bull MJ, Komdeur J (2007) Susceptibility
to infection by a haemogregarine parasite and the impact of
infection in the Australian sleepy lizard Tiliqua rugosa. Parasitol
Res 100:949–954. doi:10.1007/s00436-006-0379-5
Braude S, Tang-Martinez Z, Taylor GT (1999) Stress, testosterone,
and the immunoredistribution hypothesis. Behav Ecol 10:333–
350
Buchanan KL, Evans MR, Goldsmith AR (2003) Testosterone,
dominance signaling and immunosuppression in the house
sparrow, Passer domesticus. Behav Ecol Sociobiol 55:50–59.
doi:10.1007/s00265-003-0682-4
Calsbeek R, Bonneaud C, Smith TB (2008) Differential fitness effects
of immunocompetence and neighbourhood density in alternative
femalelizardmorphs.J
10.1111/j.1365-2656.2007.01320.x
Clotfelter ED, Ardia DR, McGraw KJ (2007) Red fish, blue fish:
trade-offs between pigmentation and immunity in Betta splen-
dens. Behav Ecol 18:1139–1145. doi:10.1093/beheco/arm090
Comendant T, Sinervo B, Svensson EI, Wingfield J (2003) Social
competition, corticosterone and survival in female lizard mor-
phs. J Evol Biol 16:948–955
Cutrera AP, Zenuto RR, Luna F, Antenucci CD (2010) Mounting a
specific immune response increases energy expenditure of the
subterranean rodent Ctenomys talarum (tuco-tuco): implications
for intraspecific and interspecific variation in immunological
traits. J Exp Biol 213:715–724. doi:10.1242/jeb.037887
Duckworth RA, Mendonc ¸a MT, Hill GE (2001) A condition
dependent link between testosterone and disease resistance in
the house finch. Proc R Soc Lond B 268:2467–2472. doi:
10.1098/rspb.2001.1827
Folstad I, Karter AJ (1992) Parasites, bright males, and the
immunocompetence handicap. Am Nat 139:603–622
Gasparini J, Piault R, Bize P, Roulin A (2009) Synergistic and
antagonistic interaction between different branches of the
immune system is related to melanin-based coloration in nestling
tawny owls.J Evol Biol
j.1420-9101.2009.01831.x
Gonzalez G, Sorci G, Møller AP, Ninni P, Haussy C, De Lope F
(1999) Immunocompetence and condition-dependent sexual
advertisement in male house sparrows (Passer domesticus).
J Anim Ecol 68:1225–1234
Huyghe K, Vanhooydonck B, Herrel A, Tadic ´ Z, Van Damme R
(2007) Morphology, performance, behavior and ecology of three
AnimEcol77:103–109.doi:
22:2348–2353. doi:10.1111/
color morphs in males of the lizard Podarcis melisellensis. Integr
Comp Biol 47:211–220. doi:10.1093/icb/icm043
Huyghe K, Herrel A, Adriaens D, Tadic ´ Z, Van Damme R (2009a) It
is all in the head: morphological basis for differences in bite
force among colour morphs of the Dalmatian wall lizard. Biol J
Linn Soc 96:13–22
Huyghe K, Husak JF, Herrel A, Tadic ´ Z, Moore IT, Van Damme R,
Vanhooydonck B (2009b) Relationships between hormones,
physiological performance and immunocompetence in a color-
polymorphic lizard species, Podarcis melisellensis. Horm Behav
55:488–493. doi:10.1016/j.yhbeh.2009.02.005
Kennedy MW, Nager RG (2006) The perils and prospects of using
phytohaemagglutinin in evolutionary ecology. Trends Ecol Evol
21:653–655. doi:10.1016/j.tree.2006.09.017
Martin LB II, Han P, Lewittes J, Kuhlman JR, Klasing KC, Wikelski
M (2006) Phytohemagglutinin-induced skin swelling in birds:
histological support for a classic immunoecological technique.
Funct Ecol 20:290–299. doi:10.1111/j.1365-2435.2006.01094.x
McCallum ML, Trauth SE (2007) Physiological trade-offs between
immunity and reproduction in the northern cricket frog (Acris
crepitans). Herpetologica 63:269–273
McKean KA, Nunney L (2001) Increased sexual activity reduces
male immune function in Drosophila melanogaster. Proc Natl
Acad Sci USA 98:7904–7909. doi:10.1073/pnas.131216398
Moreno J, Sanz JJ, Merino S, Arriero E (2001) Daily energy
expenditure and cell-mediated immunity in pied fly catchers
while feeding nestlings: interaction with moult. Oecologia
129:492–497. doi:10.1007/s004420100767
Oppliger A, Clobert J (1997) Reduced tail regeneration in the
common lizard, Lacerta vivipara, parasitized by blood parasites.
Funct Ecol 11:652–655
Oppliger A, Giorgi MS, Conelli A, Nembrini M, John-Alder HB
(2004) Effect of testosterone on immunocompetence, parasite
load, and metabolism in the common wall lizard (Podarcis
muralis). Can J Zool 82:1713–1719. doi:10.1139/Z04-152
Pryke SR, Griffith SC (2006) Red dominates black: agonistic
signaling among head morphs in the colour polymorphic
Gouldian finch. Proc R Soc Lond B 273:949–957. doi:10.1098/
rspb.2005.3362
Pryke SR, Astheimer LB, Buttemer WA, Griffith SC (2007)
Frequency-dependent physiological trade-offs between compet-
ing colour morphs. Biol Lett 3:494–497. doi:10.1098/rsbl.
2007.0213
Roberts M, Peters A (2009) Is testosterone immunosuppressive in a
condition-dependent manner? An experimental test in blue tits.
J Exp Biol 212:1811–1818. doi:10.1242/jeb.031047
Roberts ML, Buchanan KL, Evans MR (2004) Testing the immuno-
competence handicap hypothesis: a review of the evidence.
Anim Behav 68:227–239. doi:10.1016/j.anbehav.2004.05.001
Sacchi R, Rubolini D, Gentilli A, Pupin F, Razzetti E, Scali S,
Galeotti P, Fasola M (2007) Morph-specific immunity in male
Podarcis muralis. Amphib-Reptil 28:408–412
Schmid-Hempel P, Ebert D (2003) On the evolutionary ecology of
specific immune defence. Trends Ecol Evol 18:27–37
Sheldon BC, Verhulst S (1996) Ecological immunology: costly
parasite defenses and trade-offs in evolutionary ecology. Trends
Ecol Evol 11:317–321
Smallridge CJ, Bull MC (1999) Transmission of the blood parasite
Hemolivia mariae between its lizard and tick host. Parasitol Res
85:858–863
Sorci G, Clobert J, Michalakis Y (1996) Cost of reproduction and cost
of parasitism in the common lizard, Lacerta vivipara. Oikos
76:121–130
Svensson E, Sinervo B, Comendant T (2001) Condition, genotype-by-
environment interaction, and correlational selection in lizard
life-history morphs. Evolution 55:2053–2069
Oecologia (2010) 163:867–874 873
123

Page 8

Svensson EI, Abbott JK, Gosden TP, Coreau A (2009) Female
polymorphisms, sexual conflict and limits to speciation pro-
cesses in animals. Evol Ecol 23:93–108. doi:10.1007/s10682-
007-9208-2
Vercken E, Massot M, Sinervo B, Clobert J (2007) Colour variation
and alternative reproductive strategies in females of the common
lizard Lacerta vivipara. J Evol Biol 20:221–232. doi:10.1111/
j.1420-9101.2006.01208.x
Verhulst S, Dieleman SJ, Parmentier HK (1999) A tradeoff between
immunocompetence and sexual ornamentation in domestic fowl.
Proc Natl Acad Sci USA 96:4478–4481
Zuk M, Johnsen TR (1998) Seasonal changes in the relationship
between ornamentation and immune response in red jungle fowl.
Proc R Soc Lond B 265:1631–1635
Zuk M, Stoehr AM (2002) Immune defense and host life history. Am
Nat 160:S9–S22
874Oecologia (2010) 163:867–874
123