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2002).
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pecific
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Proc. R. Soc. B
Evolutionary Studies, University of Groningen, PO Box 14, 9750 AA
Haren, The Netherlands.shape the pace-of-life syndrome requires insights into
evolved responses, which reflect historical constraints and
adaptation by natural selection, and individual responses,
which reveal both the potential for selection and
physiological constraints on the response to selection.
Comparative studies of physiological correlates of life
histories are largely restricted to metabolism: low meta-
bolic rates are thought to be associated with low
reproductive investment and high survival rate (Pearl
1928; Daan et al. 1990; Zera & Harshman 2001;
conditions, such as risk of disease, and suggests that birds
in relatively disease-free environments, such as marine and
arctic habitats, have evolved less robust immune defenses
(Piersma 1997). In both scenarios, immune function is an
important physiological attribute in the evolution of life
histories. Within species, variation in immune function
might indicate fitness-related individual ?quality? differ-
ences. In particular, individuals having better body
condition might support a higher level of metabolism
and a more robust immune system (Norris & Evans 2000).
Comparative ecological and evolutionary immunology
faces the problem of defining a functional measure of
overall immunity to infectious challenges that allows for*Author for correspondence (b.i.tieleman@rug.nl).? Present address: Animal Ecology Group, Centre for Ecological andReceived
Acceptedost fundamental trade-off in life-history evolution is
verse relationship between reproductive success
ity) and adult survival rate (Cody 1966; Charnov
icklefs 2000). Attempts to explain this relationship
acillated between emphasis on ecological factors
s risk of predation, food availability and density
ence, and physiological parameters, notably energy
iture (Lack 1947; Skutch 1949; Ashmole 1963;
& Daan 1980; Martin 2004; Speakman et al. 2004;
a et al. 2004). Recently, interest has focused on
ting demographic parameters related to survival
production, and various physiological attributes,
ng metabolism and immune function, all subsumed
the ?pace-of-life syndrome? (Ricklefs & Wikelski
Understanding the evolutionary processes that
investigations into the trade-off between surviva
reproduction have focused on the costs of increased
loads, especially during reproduction, elucidating
ging effects of oxidative stress and compromised im
function in individual animals (Deerenberg et al.
Wiersma et al. 2004).
Development, maintenance and use of the im
system incur costs in terms of both time and nu
(Klasing & Leshchinsky 1999). Accordingly, inters
variation in immune function might be related to lon
in such a way that higher levels or a better qua
immune response evolved in longer-lived species
increased investment in self-maintenance over rep
tion. An alternative, though not mutually exc
hypothesis relates immunocompetence to environm1. INTRODUCTION
The m
Ricklefs & Wikelski 2002). In comparisons within species,
l andConstitutive innate im
of the pace-of-life syn
B. Irene Tieleman1,*,?, Joseph B
and Kirk
1Department of Biology, University of Missouri-St Louis
2Department of Evolution, Ecology and Organismal
Columbus, O
3Department of Animal Science, Univer
We studied the relationship between one componen
an indicator of the ?pace-of-life syndrome?, among
tropical house wren (Troglodytes aedon), to gain ins
and physiology. To assess constitutive innate imm
ecological and evolutionary immunology that qua
in vitro assay utilises a single blood sample to provide
immunity. We found that the bactericidal activity o
among individuals within a species. This variation w
BMR. However, among species, bacteria killing a
BMR, suggesting that species with a slower pace-o
immune capability. Among individuals of a single
positively correlated with mass-adjusted BMR, poi
on which natural selection potentially could act.
Keywords: life history; immune function7 January 2005
11 May 2005
1unity is a component
rome in tropical birds
Williams2, Robert E. Ricklefs1
Klasing3
001 Natural Bridge Road, St Louis, MO 63121, USA
logy, Ohio State University, 318 W 12th Avenue,
3210, USA
of California, Davis, CA 95616, USA
immune function and basal metabolic rate (BMR),
tropical bird species and among individuals of the
ts into functional connections between life history
ty we introduced a new technique in the field of
es the bactericidal activity of whole blood. This
unctional, integrated measure of constitutive innate
hole blood varied considerably among species and
not correlated with body mass or whole-organism
ity was negatively correlated with mass-adjusted
fe have evolved a more robust constitutive innate
cies, the house wren, bacteria killing activity was
g to physiological differences in individual quality
etabolic rate; tropics; bactericidal activity
doi:10.1098/rspb.2005.3155
Published onlineq 2005 The Royal Society
related quality differences among individuals within a
species, and to compare inter- and intraspecific patterns,
bac
e 1a
dy m
.2
.7
.4
.0
.4
.0
.9
.3
.7
.8
.5
.9
2 B. Irene Tieleman and others Immunity and metabolic rate in birdscomparisons across a range of species, and laborious
protocols that are not practical in field studies. Additional
complications arise from the multifaceted nature of the
immune system which has led to the development of an
array of techniques that highlight single, often non-
comparable components of the immune system (Norris
& Evans 2000; Adamo 2004). Finally, some tests of
immune function are difficult to interpret because
excessive responses may indicate immunopathology,
making it possibly difficult to infer quality of response
from quantity of response.
We introduce a technique novel to physiological
ecology that presents a functional, integrated and
comparative measure of constitutive innate immunity
by quantifying the bactericidal activity of whole blood.
Constitutive innate immunity, a mixture of humoral (e.g.
natural antibodies, complement, acute phase proteins)
and cellular components (e.g. macrophages, heterophils,
thrombocytes), establishes the first line of defence
against invading pathogens. Its functioning is ostensibly
unaffected by previous challenges and the development
Table 1. Body mass, mass-corrected BMR (averageGs.e.) and
units, CFU, killedGs.e.) for 12 species of tropical birds.
(NB. Values reported for the house wren in this table and figur
different dilutions of blood and media (see ?2).)
species
bo
(g)
bicoloured antbird Gymnopithys leucaspis 27
blue-crowned motmot Momotus momota 99
blue?gray tanager Thraupis episcopus 30
clay-coloured robin Turdus grayi 72
crimson-backed tanager Ramphoceles dimidiatus 26
dusky antbird Cercomacra tyrannina 14
grey-headed tanager Eucometis penicillatus 30
house wren Troglodytes aedon 13
red-capped manakin Pipra mentalis 14
ruddy ground dove Columbina talpacoti 44
slaty antshrike Thamnophilus punctatus 20
spotted antbird Hylophylax naevioides 16of pathogen-specific antibodies, making it ideal for
studies across species or individuals and across environ-
ments with differing abundances and diversities of
pathogens. In addition, components of the innate
immune system, including natural antibodies, are
encoded in the germ-line genome, thus providing genetic
variation for evolution, and they play an essential role in
activating the acquired arm of the immune system for
specific defences against infection (Carroll & Prodeus
1998; Ochsenbein & Zinkernagel 2000). The bacteri-
cidal activity of whole blood is an excellent predictor of
the susceptibility of humans to a variety of bacterial
infections (Keusch et al. 1975). Keusch et al. (1975)
measured intracellular bactericidal activity of leukocytes
in whole blood to diagnose patients with an inherited
disorder of phagocytic cells, characterized by the
inability of phagocytes to kill certain types of bacteria
and fungi, and resulting in recurrent life-threatening
bacterial and fungal infection.
To test the hypothesis that the immune response is an
integral part of the evolved life history, we described the
Proc. R. Soc. Bwe explored the relationship between bacteria killing
activity of blood and BMR among individual tropical
house wrens (Troglodytes aedon).
2. METHODS
(a) Animals
We mist-netted individuals of 12 bird species during March?
July 2004 in the tropical humid lowland rainforest of
Soberania National Park and around Gamboa, Panama
(table 1). Upon capture, we immediately took a blood sample
for the immune assay, and transported blood and birds to ourrelationship between constitutive innate immunity and
basal metabolic rate (BMR), an indicator of the pace-of-
life, in an interspecific comparison of tropical birds. We
predicted that species with low BMR would have higher
levels of investment in immunity, including the constitu-
tive innate arm. In addition, to determine whether
constitutive innate immunity might indicate fitness-
tericidal activity of whole blood (average % of colony forming
cannot be compared with values in figure 1b due to the use of
ass BMR
(kJ dK1 gK0.638) n
bactericidal
activity
(% CFU killed) n
3.4G0.11 8 99.9G0.04 5
2.2G0.15 4 99.1G0.38 8
4.1G0.11 8 11.2G6.66 15
3.8G0.16 10 82.9G4.20 30
4.0G0.12 8 49.0G9.53 11
3.0G0.25 3 93.3G6.50 2
3.7G0.23 3 90.9G4.90 7
3.5G0.07 28 89.0G3.06 27
4.1G0.17 7 K3.4G8.92 6
3.1G0.56 7 K3.2G5.60 18
3.5G0.41 3 99.6G0.27 3
3.6G0.16 6 94.6G2.56 10laboratory. All birds were housed in cages at an ambient
temperature of about 25 8C, and provided with fruit,
mealworms, re-hydrated crickets and/or seeds, depending
on a species? diet. Usually we measured metabolism the night
after capture, but occasionally we kept birds for up to 3 days.
All house wrens for the intraspecific comparisons were
feeding 12 day old nestlings when we captured them for
blood sampling and BMR-measurements. After metabolism
measurements, we released them before sunrise at their nest
sites; in all cases birds resumed feeding their offspring. By
restricting our study to one geographic location, and by taking
advantage of the wide variety of life histories among tropical
birds, we minimized the effect of environment as confounding
factor in our analysis.
(b) Bacteria killing assay
We assessed the bactericidal activity of fresh whole blood
in vitro. We collected blood samples of birds in the field within
5 min of capture, a period previously shown to minimize the
effect of capture stress on our bacteria killing assay (Matson
et al., unpublished work). We sterilized the area around the
wing vein with 70% ETOH, allowed the ETOH to evaporate,
influx via the syringe pump with calculated influx showed a
Immunity and metabolic rate in birds B. Irene Tieleman and others 3then punctured the wing vein with a sterile needle, and
collected 50?100 ml of blood in sterile heparinized haemato-
crit tubes (Fisher Scientific). Haematocrit tubes were
transported in sterilized plastic boxes to the laboratory,
where the blood was processed within half an hour after
obtaining the sample.
A laminar flow hood (AC600, AirClean Systems,
Raleigh, NC) provided a sterile working environment in
the laboratory. For interspecific comparisons, we diluted
20 ml of blood with 180 ml CO2-independent media
(#18045, Gibco-Invitrogen, Carlsbad, CA) enriched with
5% complement-inactivated foetal calf serum and 4 mM
L-glutamine. To increase the resolution of our antibacterial
assay for house wrens, a species with a relatively high killing
activity, we diluted 10 ml of blood in 190 ml media. To each
dilution we added 20 ml of a suspension of living Escherichia
coli (ATCC #8739). The E. coli were reconstituted from
lyophilized pellets (Epower Microorganisms #0483E7,
MicroBioLogics, St Cloud, MN) in phosphate buffered
saline (Sigma, St Louis, MO) following company instruc-
tions and adjusted to a concentration of 150?200 colonies
per 75 ml of diluted blood?bacteria mixture. The mixture of
diluted blood and E. coli was incubated for 30 min at 41 8C,
and then 75 ml was spread onto agar plates (D-ifco soybean-
casein digest agar, USP, Fisher catalog #DF0369176).
Each sample was plated in duplicate. The number of
bacteria in the added bacteria suspension was quantified
with control dilutions consisting of 200 ml of media mixed
with 20 ml of bacteria-suspension. We made 1?5 control
dilutions per session, and used the average when nO1.
Plates were incubated upside down at ambient temperature
(25?30 8C) and the number of colony-forming units was
counted the following day.
We tested for variation among control plates by making
8 control dilutions from the same bacteria suspension
(20 ml of suspension in 200 ml of media) and plating each
dilution in duplicate. Following our normal procedure, we
calculated the average of each pair of control plates. We
found that the coefficient of variation among controls was
3.8% (average number of coloniesGs.d.: 120.8G4.60,
nZ8). Our normal control dilutions were plated directly,
without incubating them. When we compared 27 pairs of
unincubated control dilutions with dilutions that had been
incubated for 30 min at 41 8C, we found a 7.8% increase
in number of colonies after incubation (unincubatedGs.d.:
148G43.2; incubatedGs.d.: 159G51.5; paired-tZ2.66,
pZ0.013, nZ27). For calculations of bactericidal activity,
we used the number of colonies from unincubated control
plates because they reflect the initial situation at the
instant that the blood starts acting on the bacteria.
Components within blood can prevent bacteria growth or
kill bacterial cells, in either of which case bacteria will not
grow. However, bacterial cells may divide in blood as
indicated by negative killing values. The bactericidal
activity of blood was expressed as the number of colony-
forming units remaining after incubation of the blood?
bacteria mixture relative to the number inoculated (control
plates).
(c) Oxygen consumption
We measured rates of oxygen consumption for post-
absorptive birds during their nocturnal phase by standard
flow-through respirometry methods. Birds were placed in
stainless steel metabolism chambers that had an air-tightProc. R. Soc. Bmean difference of G2% for our 10 trials.
After a 3 h equilibration period, we recorded O2-
concentration, the dewpoints of inlet and outlet air, the
temperature of the dewpoint hygrometer, and Ta in the
chamber, using a data logger (Campbell Scientific, model
CR23X). When, during the fourth hour of measurements,
the traces for O2-consumption were stable for at least 10 min,
we noted these times and used these data for calculations.
After completing the metabolism measurements, we immedi-
ately measured the body temperature of birds with a
Physitemp thermometer (Model Batt-12) and a 36 gauge
copper?constantan thermocouple. The relative humidity of
outlet air was always between 10 and 40%. Oxygen
consumption was calculated with eqn (4) of Hill (1972).
We used 20.08 J mlK1 O2 to convert oxygen consumption to
heat production (Schmidt-Nielsen 1997).
(d) Statistics
Bactericidal activity was expressed in percentages that
varied from below zero, when bacteria proliferated during
the incubation, to 100%, when all of the bacteria were
killed. The negative values complicated data transform-
ation, and together with the nonlinear relationships between
variables, prompted us to use non-parametric Spearman
rank correlation analysis as implemented in SPSS 12.0.
Metabolism data were analysed using linear regression
analysis.
Interspecific correlations may be statistically biased if
closely related species resemble each other as a result of
common ancestry (Felsenstein 1985; Harvey & Pagel 1991).
We made a phylogeny based on the DNA-hybridization
relationships determined by Sibley & Ahlquist (1990) and
used Abouheif ?s (Abouheif 1999; Rheindt et al. 2004) Test
for Serial Independence to determine whether a phylogenetic
effect was present in our data for mass, BMR and bactericidal
activity. None of these variables was significantly autocorre-
lated (BMR pZ0.33, mass pZ0.12, bactericidal activity
pZ0.33), and therefore we did not include phylogeny in our
analyses.Lexan lid, formed by a thick rubber gasket. Birds perched on
a wire-mesh platform over a layer of mineral oil that trapped
feces. The chambers sat in a large cool-box with a Peltier
device (Sable Systems, Pelt-4) to control ambient tempera-
ture (Ta)G0.1 8C. We measured birds at 31?32 8C, which is in
their thermoneutral zone, as determined by measurements
over a range of Tas for each species.
Compressed air coursed through columns of Drierite to
remove water, through previously calibrated (Levy 1964)
Mykrolis mass flow controllers (FC-2900; 2 SLPM) set
between 500 and 700 ml minK1 (STP), depending on
species, and then into the chamber. Exiting air passed
through an Edgetech dewpoint hygrometer (Dewprime II,
factory recalibration August 2003). A subsample was then
routed through silica gel, Ascarite, and silica gel to remove
water and CO2 before measuring the fractional concentration
of O2 with an Applied Electrochemistry oxygen analyser
(S3A-II). We calibrated the entire system for measuring
O2-consumption by infusing pure O2 into our chamber using
a syringe pump with a previously determined flow rate while
simultaneously pushing room air through another port into
the chamber at a known rate. Comparisons of known O2
4 B. Irene Tieleman and others Immunity and metabolic rate in birds
% 
CF
U 
kil
led
)
60
80
100
mass-corrected BMR (kJ d?1 g? 0.638)
2.0 3.0 3.5 4.0
ba
ct
er
ic
id
al
 a
ct
iv
ity
 (%
 C
FU
 ki
lle
d)
0
20
40
60
80
100
all birds
rs= ?0.59
p= 0.042
passerines only
rs= ? 0.87
p= 0.001 RUGD
BCMM
(b)
(a)
2.53. RESULTS
(a) Interspecific relationships of bactericidal
activity, body mass and BMR
Average bactericidal activity varied among species
between K3% and C100% (table 1). We found no
significant relationship between bactericidal activity and
body mass ( rsZK0.007, nZ12, pZ0.98) or between
bactericidal activity and whole-animal BMR
( rsZK0.19, nZ12, pZ0.56). We adjusted BMR for
body mass by dividing BMR by mass0.638, the exponent
taken from an allometric equation relating BMR to body
mass in birds (Tieleman & Williams 2000). Relating
mass-adjusted BMR and bactericidal activity for all
species we found a significant negative correlation
( rsZK0.59, pZ0.042, figure 1a). Because our data
set included only two non-passerines (blue-crowned
motmot and ruddy ground dove), we verified that
restricting the analysis to passerines only did not alter
the results; the correlation between mass-adjusted BMR
and bactericidal activity was maintained ( rsZK0.87,
nZ10, pZ0.001). These data suggest that species with a
BMR higher than predicted for their body mass have a
these variables. We hypothesize that Columbiformes
residual BMR (kJ d?1)
?3 ?2 ?1 0 1 2 3 4 5
ba
ct
er
ic
id
al
 ac
tiv
ity
 (
?20
0
20
40
r s= 0.47
p= 0.031
Figure 1. (a) Relationship between mass-adjusted basal
metabolic rate (BMR) and bactericidal activity (% colony
forming units, CFU, killed) of whole blood among 12 species
of tropical birds. The two non-passerine species are marked
BCMM (blue-crowned motmot) and RUGD (ruddy ground
dove). (b) Relationship between residual BMR and bacteri-
cidal activity of whole blood among individuals of the house
wren.
Proc. R. Soc. Bmight have evolved a different evolutionary strategy to
resist or control infections, with apparent small invest-
ments in constitutive innate immunity, but a strong
systemic inflammatory response to infections as indicated
by high levels of acute phase proteins after inoculation
with heat-killed E. coli (K. C. Klasing & M. Wikelski,
unpublished data). In agreement with this idea, Matson
et al. (2004) recently reported that the mourning dove
(Zenaida macroura), another member of the Columbi-
formes, had the lowest constitutive complement and
natural antibody levels in a comparative study on
constitutive innate humoral immunity of 11 avian species.
Comparing pigeons and doves with other bird species may
therefore provide interesting evolutionary insights into
trade-offs between the constitutive innate arm, the acute
phase response, and the adaptive arm of the immune
system (Norris & Evans 2000; Lee & Klasing 2004). We
wonder if these immunological trade-offs might be relatedlower bactericidal activity of their blood than species
with a relatively low BMR.
(b) Intraspecific relationships of bactericidal
activity, body mass and BMR
In house wrens, BMR was positively related with body
mass according to the regression equation BMR
(kJ dK1)Z1.215 (s.e. 0.403) mass (g)C2.227 (s.e.
5.376; r2Z0.26, d.f.Z27, pslopeZ0.006, nZ13 males, 15
females). Bactericidal activity (measured for 21 birds) was
not significantly correlated with body mass ( rsZK0.14,
nZ21, pZ0.53) or whole-animal BMR ( rsZ0.29, nZ21,
pZ0.21). To correct for body mass we calculated residual
BMR (ZmeasuredKpredicted by the regression equation
relating BMR and mass), and found a significant positive
correlation between residual BMR and bactericidal
activity ( rsZ0.47, nZ21, pZ0.031, figure 1b).
4. DISCUSSION
Constitutive innate immunity was assessed as the ability of
whole blood to kill bacteria. Interpretation of bactericidal
activity is unambiguous because the principal job of the
immune system is to kill potential pathogens and our assay
is a direct measurement of this crucial function. Bacteri-
cidal activity varied substantially among 12 species of
tropical birds and among individuals within one species,
the house wren, and covaried with a physiological measure
of the ?intensity of living?. Bactericidal activity of blood
was not related to mass or to whole-animal BMR, but
correlated with mass-corrected BMR. If BMR were
inversely related to lifespan, then the negative interspecific
correlation between immune function and BMR would
support our hypothesis that the level of investment in
constitutive innate immunity can be understood in the
context of life-history evolution. In this case, our data are
consistent with the idea that species with longer average
lifespans invest more into immune function to protect
future opportunities to reproduce. In any case, the
association of high immune competency with low
metabolic rate might reflect a basic trade-off between
rate of activity and self-maintenance functions.
The ruddy ground dove, with low mass-corrected BMR
and low bactericidal activity of its blood, deviated
markedly from the overall negative correlation between
have a relatively high BMR that allows them to work hard
during the nestling period while maintaining high body
Immunity and metabolic rate in birds B. Irene Tieleman and others 5condition, reflected in high immunocompetence. Alter-
natively, low quality individuals might have relatively high
BMR and increased bactericidal activity because of active
infections. The acute phase response to pathogens results
in increased BMR and increased levels of bactericidal
acute phase proteins in the blood (Gabay & Kushner
1999). Future experimental and/or mechanistic work on
the link between metabolism and constitutive innate
immunity should yield insights into the validity of these
ideas.
The bactericidal activity of whole blood represents a
functional, integrated measure of constitutive innate
immunity previously missing from the tools available to
ecological and evolutionary immunology (Norris & Evans
2000; Merchant et al. 2003). While the current study
presents results on the ability of whole blood to kill one
strain of E. coli, this assay can be extended to other
bacteria and yeast species or strains with different
characteristics, to increase the robustness of the overall
measure of innate immunity. Additional potential
improvements include modifying the blood dilution or
the incubation time of the blood?bacteria mixture in order
to increase the resolution of the results at the extremes of
the range. Nonetheless, our results show that this measure
has relevance in both comparative and intraspecific
studies. In vitro bactericidal activity of whole blood
provides a functional and field compatible measure of
constitutive innate immune function.
We thank the Smithsonian Tropical Research Institute for
logistic help throughout the study, Jennie Bennett for
developing the bacteria killing assay for use in wild birds,
Thomas Dijkstra for practical help in the field, and Kevin
Matson for practical help and comments on a previous draft.
This research was funded by National Science Foundation
grant IBN-0212587.
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