ECOPHYSIOLOGY
Lynn B. Martin II ? Dennis Hasselquist
Martin Wikelski
Investment in immune defense is linked to pace of life in house sparrows
Received: 9 June 2005 / Accepted: 8 November 2005 / Published online:1 February 2006

 Springer-Verlag 2006
Abstract The evidence for a relationship between life
history and immune defense is equivocal, although the
basic premise is intuitively appealing: animals that live
short lives and reproduce early and rapidly should not
waste resources on defenses they might never use. One
possible reason for a lack of strong support for this
hypothesis could be the inherent complexity of the ver-
tebrate immune system. Indeed, di?erent components of
the vertebrate immune system vary in their relative costs
and bene?ts, and therefore only some defenses may
complement variation in species? life history. To address
this hypothesis, we compared multiple types of immune
activity between two populations of house sparrows
(Passer domesticus) with distinct life histories, one from
Colon, Panama, which lay small clutches over an ex-
tended breeding season (i.e., slow-living) and the other
from Princeton, New Jersey, which lay larger clutches in
a smaller window of time (i.e., fast-living). We expected
(a) that more costly types of immune defenses would be
stronger in the slow-living sparrows and (2) that the
slow-living sparrows would show a greater increase in
whole-body energy expenditure after immune challenge
compared to their fast-living counterparts. We found
that secondary antibody response to a novel antigen was
more rapid and energetic investment in immune activity
was greater in slow-living sparrows. However, cell-
mediated immune activity was more robust in fast-living
sparrows, and other measures of defense were not dif-
ferent between populations. These results provide partial
support for a relationship between life history and im-
mune defense in this species, but they also indicate that
this relationship is not clear-cut. Further study is nec-
essary to identify the in?uence of other factors, partic-
ular pathogen environment during development, on the
architecture of the immune system of wild animals.
Keywords Humoral ? Immunocompetence ? Innate ?
Passerine ? RMR ? Trade-o?
Introduction
The concept of immunocompetence, or the capacity of an
organism to prevent or control infection (Owens and
Wilson 1999), became popular in the ecological literature
when it was ?rst characterized as a commodity that was
diminished when animals elevated circulating testoster-
one to augment or express their sexual ornaments
(Folstad and Karter 1992). To date, evidence corrobo-
rating this ??immunocompetence handicap hypothesis?? is
equivocal (Grossman 1985; Hasselquist et al. 1999;
Owen-Ashley et al. 2004; Greenman et al. 2005). Still, the
conceptualization of immune defense as a malleable
investment commodity attracted the interest of ecologists
and evolutionary biologists (Sheldon and Verhulst 1996;
Lochmiller and Deerenberg 2000; Norris and Evans
2000) and led to many new discoveries. One of the most
striking discoveries is that immune defenses are quite
variable among and within species (Nelson and Demas
1996; Martin et al. 2001; Tella et al. 2002). Historically, it
was predicted that the random nature of generating
lymphocyte diversity would produce similar levels of
defense (per unit body size) among taxa (Cohn and
Langman 1990). What remains unclear now is why var-
iability in immune defense exists; presumably all animals
would bene?t from defense against pathogens at all times
of their lives, so why is this not seen in nature?
Communicated by Carol Vleck
L. B. Martin II (&) ? M. Wikelski
Department of Ecology and Evolutionary Biology,
Princeton University, Princeton, NJ, USA
E-mail: lmartin@mail.psy.ohio-state.edu
Tel.: +1-614-5389540
Fax: +1-614-4513116
D. Hasselquist
Department of Animal Ecology, Lund University, Lund, Sweden
E-mail: dennis.hasselquist@zooekol.lu.se
L. B. Martin II
Departments of Psychology and Neuroscience,
The Ohio State University, Townshend Hall,
Columbus, OH 43210, USA
Oecologia (2006) 147: 565?575
DOI 10.1007/s00442-005-0314-y
Mounting evidence indicates that the energetic (De-
mas 2004), nutritional (Lochmiller and Deerenberg
2000), and immunopathological (Ra?berg et al. 1998)
costs of immune defense may be important. Trade-o?s
between immune defense and other costly activities ap-
pear common in wild species. In passerine birds,
induction of immune activity often negatively a?ects
reproductive success (Ilmonen et al. 2000; Ra?berg et al.
2000; Bonneaud et al. 2003), tissue growth (Martin
2005), and survival (Hanssen et al. 2004). Similarly, the
large developmental costs of certain immune defenses,
particularly the generation of a diverse T and B cell
repertoire, have been proposed to explain why many
songbird species maintain long incubation periods in
nest-predator rich environments (Ricklefs 1992).
We expect that if such cost-bene?t counterbalances
explain why organisms can mount robust immune re-
sponses at only certain times in their lives, a similar
perspective might explain how the immune systems of
animals are generally organized. For instance, animals
living in parasite-dense environments should invest
heavily in immune defense relative to those living in
parasite-poor habitats (Piersma 1997). Similarly, ani-
mals living relatively slow-paced lives (e.g., animals that
mature and breed late and modestly; Wikelski et al.
2003) should invest heavily in immune defense because
they are more likely to be exposed repeatedly to
pathogens than their fast-living relatives (Klasing and
Leshchinsky 1999). Recently, two studies found evi-
dence for relationships between immune defense and
pace of life in birds (Martin et al. 2001; Tella et al.
2002). In both cases, species exhibiting life history traits
representing a slow pace of life (e.g., long-develop-
mental period, small-clutch size, or large-body size)
showed greater levels of one type of immune defense,
phytohemagglutinin (PHA) induced wing-web swelling.
Recently, we found that the same measure of immune
defense varied between populations of house sparrows
(Passer domesticus) living their lives at di?erent paces.
First, we found that Panamanian sparrows, which lay
small clutches over a 10-month breeding season (slow-
living; Martin et al., submitted), maintained similar
levels of PHA responsiveness year-round. Sparrows
from New Jersey on the other hand, which lay larger
clutches over a shorter breeding season (fast-living;
Summers-Smith 1988), showed a reduction in PHA
swellings during the height of the breeding season
(Martin et al. 2004). Additionally, we found that cor-
ticosterone, an immunosuppressant in vertebrates when
maintained at high levels for extended periods, did not
a?ect PHA swelling in the slow-living sparrows. How-
ever, it did suppress PHA swelling in the fast-living
birds (Martin et al. 2005). We interpreted this di?erence
to indicate that slow-living sparrows do not decrease
immune investments even when under chronic stress.
Although the ?ndings of these studies are intriguing,
their reliance on a single immunological measure can
only indicate that life history?immunology relation-
ships probably exist. The related possibility, that the
pace of life of animals directly shapes their immuno-
logical portfolios, remains unresolved.
In this study, we attempted to address this possibility.
Speci?cally, we wanted to determine whether all or only
some types of immune activity varied between the same
two populations of house sparrows investigated earlier.
In other words, we wanted to determine if a slow pace of
life was related to greater overall immunocompetence.
We relied on the immune-defense component model of
Schmid-Hempel and Ebert (2003) to identify the
appropriate techniques to use to test this hypothesis.
This heuristic model recognizes that immune defenses
are complex and can be (a) maintained at a certain level
irrespective of the disease environment (constitutive), (b)
activated only in response to a disease challenge
(inducible), (c) targeted against a particular pathogens
(speci?c) and/or (d) generally responsive to a variety of
di?erent threats (non-speci?c). Further, the Schmid-
Hempel and Ebert model (2003) indicates that defenses
vary in terms of the resources necessary to develop, use,
and/or maintain them (Lochmiller and Deerenberg
2000; Klasing 2002), and each defense type di?ers in its
ability to control a given pathogen challenge (Kaufmann
et al. 2002). Finally, the model notes the hierarchical but
redundant organization of immune defenses. It recog-
nizes that some defenses may be obsolete if strong de-
fenses are in place upstream, but it also acknowledges
that the same endpoint can often be achieved by multiple
immunological strategies. In Table 1, we list the types of
defenses we measured in our study. In addition to
comparing these immune defenses outright, we mea-
sured energy expenditure in response to one type of
immune challenge in slow and fast-living sparrow pop-
ulations (Martin et al. 2003). This comparison allowed
us to directly test whether slow-living birds invest more
energy in defense than their fast-living counterparts over
the short term.
Materials and methods
Field sites and study species
The house sparrow is a small (
25 g), granivorous
passerine found on every continent but Antarctica, in
most cases because of human introduction (Summers-
Smith 1988). Although house sparrows are not native to
the western hemisphere, populations exhibit many of the
latitudinal life history clines of indigenous species,
including a decrease in clutch size (Summers-Smith
1988), an increase in the length of the breeding season
(Summers-Smith 1988), and a decrease in rate of energy
turnover (Kendeigh and Blem 1974; Kendeigh 1976)
towards the equator. Birds in this study were from a
North-temperate site (Princeton, New Jersey, USA:
4021?N, 7440?W), and a Neotropical site (Colon,
Panama: 91?N, 801?W; see Martin et al. 2004 for de-
tails). In 2003, we characterized the reproductive life
history of the Panamanian (slow-living) population and
566
found that these sparrows lay smaller clutches (3.3 eggs/
clutch; New Jersey: clutch size=4.6) and breed over
much of the year (10 months; Martin, unpublished
data), traits that are distinct from all temperate North
American populations studied to date (Summers-Smith
1988). Table 2 presents a more detailed characterization
of the reproductive life histories of the slow-living pop-
ulation (Martin et al., submitted) and a population at
the same latitude as Princeton, New Jersey (North 1973).
To date, we have not characterized the reproductive life
history of the fast-living population we have studied, but
we have no reason to suspect that it would di?er dra-
matically from other populations at the same latitude
(Summers-Smith 1988).
For the duration of all immune assays, birds were
held in captivity and receivedad libitum mixed seeds and
water every day and boiled, mashed chicken eggs and/or
live mealworms (Tenebrio molitor) every third day.
Photoperiod and temperature were held at ambient
levels of each latitude during the natural antibodies,
KLH, and RMR comparisons (see below); during E. coli
DTH challenges, all birds were held on long photope-
riods (14L:10D) at 25?2C and 40?5% R.H. (see
additional details below) in climate-controlled rooms in
Princeton, NJ. For all immune assays, individual spar-
rows were used only once unless otherwise noted. We
took this approach in case prior immune activation af-
fected subsequent immune responsiveness. All assays
were conducted between the months of February and
August, as both populations have been found to breed
during this period. Generally, we included similar
numbers of males and females in each comparison, but
because of low sample size, we did not have su?cient
statistical power to detect signi?cant e?ects of sex on our
results. Further, we used only birds that showed no signs
of molt, as feather growth can a?ect some types of im-
mune activity in passerines (Martin 2005) or vice versa
(Sanz et al. 2004).
We chose the house sparrow as a model species for
several reasons. First, by using a single species, we could
eliminate phylogenetic artifacts inherent to interspeci?c
studies, and we could standardize our immunological
methods (Kreukniet et al. 1994). Second, by using a
granivorous human commensal, we could reduce the
impact of uncontrollable factors, such as diet (Lochm-
iller and Deerenberg 2000), on our results. Last, by
conducting all experiments in a similar window of time,
we could be reasonably sure that immunological di?er-
ences identi?ed in this study were not due solely to time
of year or stress (Nelson 2004; Martin et al. 2004, 2005).
We were aware from the outset that our approach
(comparing two populations) would not enable us to be
sure that immunological di?erences detected were solely
due to life history variation (sensu Garland and Adolph
1994). Still, given (a) the extensive literature on physio-
logical and life history variation in North American
house sparrows and (b) the many insightful two-species/
two-population comparisons in the past (Weathers and
Greene 1998; Klein et al. 1999; Ghalambor and Martin
2000), we felt that our approach would be a useful ?rst
step to determining whether and how animals? life his-
tories shape their immune systems.
Table 1 Measures of immune function compared between fast and slow-living house sparrows
Immune defense Challenge used Response measured High response indicates
Constitutive
Natural antibodies/ Rabbit red blood cells
(RRBCs)
Agglutination of RRBCs High surveillance for extracellular
parasitesComplement Lysis of RRBCs
Induced, speci?c
T-cell memory Keyhole limpet hemocyanin
(KLH)
Swelling of wing patagium Strong capacity to respond to previously
encountered antigens
Intact, heat-killed E. coli
Antibody proliferation
(B cell)
Keyhole limpet hemocyanin
(KLH)
Primary antibody response Strong capacity to recognize and make
antibodies for a novel antigen
Secondary antibody response Strong capacity to respond to previously
encountered antigens
Energy investment
Cost of acute phase
response
Phytohemagglutinin (PHA) Change in resting metabolic
rate
Large investment in acute phase response
Table 2 Reproductive life history characters of two populations of house sparrows
Location Latitude Breeding
season (months)
Clutch
size (mean)
Clutches
year1
Eggs
female1 year1
Incubation
(days)
Nestling
(days)
Hatching
(%)
Fledging
(%)
Slow-living 9 10 3.4 3.2 10.8 10.5 16.2 0.84 0.86
Fast-livinga 43 4 5.0 1.5 7.4 11.7 15.4 0.51 0.61
aData from North 1973
567
Constitutive defenses
Natural antibody levels and lysis activity
We used a rabbit red blood cell (RRBC) agglutination/
lysis assay to characterize natural antibody (NAb) levels
(predominantly IgM) and complement/NAb-mediated
lysis capacity of plasma (Matson et al. 2005). In Feb-
ruary?April 2003, we captured wild birds and took 50 ll
blood samples from each animal; plasma was then
stored at 20C until assay (January 2004). In each
assay, we randomized samples among assay runs and
then added 25 ll of plasma from each sparrow and an
equal volume of saline to six columns of a ?at-bottom
96-well plate. To the remaining two columns, we added
saline and a positive control (RRBC-activated chicken
plasma collected on heparin: #ES1032P, Biomeda). We
then performed a serial dilution of the plasma and the
positive standards to the remaining wells on the plate.
To the ?lled plate, we then added 25 ll of a 1% RRBC
suspension (washed cells collected on citrate and
reconstituted in Alsevers: #wrb100, Hemostat) to all
wells except the last row; these wells contained only
saline and served as negative controls.
Once a plate was ?lled, we covered it with Para?lm,
placed it on a shaker for 2 min, incubated it in a warm
water bath (37C) for 90 min, then tilted the plate at a
45 angle for an additional 20 min to allow for potential
separation of the blood cell pellet. After this period, a
photograph of the plate was taken using a scanner
connected to a desktop computer. Seventy minutes later,
this procedure was repeated. Photographs were taken at
these time points in particular to enable to score both
agglutination and lysis in the same plasma sample
(Matson et al. 2005). Maximum agglutination was
scored as the concentration of plasma in which the blood
cell pellet remained completely intact after 20 min of
tilting. Lysis was scored (90 min post-incubation photo)
as the plasma dilution at which >75% RBCs had rup-
tured. This and all other assays (except KLH antibodies:
see below) were conducted blind to treatment by the
same person (L. Martin).
Inducible defenses
Cell-mediated immunity (delayed-type hypersensitivity
(DTH))
We used two substances to assess T-cell mediated
immunological memory, keyhole limpet hemocyanin
(KLH) and heat-killed Escherichia coli bacteria. Al-
though KLH is usually favored for measuring antibody
responses, many substances that possess a diverse com-
plement of epitopes (i.e., antigenic sites) can be used to
assay T-cell memory (Turk 1967). We chose these two
substances particularly because pilot studies in our lab
and previous work from other labs (Smith et al. 2005)
indicate that many substances can induce swelling
(i.e., T-cell mediated memory). For KLH, birds were
captured from the wild (Feb?May 2003) and held in
captivity in pairs in cages for 24 h prior to initial injec-
tion. To induce hypersensitivity to KLH, we injected
100 ll of a 1 mg ml1 KLH (Sigma H7017) in 0.9%
pyrogen-free saline solution into the wing web of each
bird 3? over the course of 2.5 months at equal intervals.
For the E. coli assay, we used the fast-living birds from
the preceding KLH assay (after a 3-week recovery per-
iod) and slow-living sparrows from a prior common
garden experiment (Martin et al. 2004). For E. coli, we
injected the wing web of each bird with approximately
10 k bacteria suspended in 50 ll saline (Lee et al. 2005).
Injections were prepared using dehydrated bacteria
(ATCC# 11303) that were re-suspended in broth, incu-
bated at 37C for 24 h, spun down, washed twice in
saline, diluted 1:10, and ?xed in 10% formalin over-
night. Just prior to all injections and 24 and 48 h after
the third injection, we measured wing-web swelling with
a Teclock pocket thickness gauge (model: SI-510) to the
nearest 0.1?? and later converted these measures to metric
units. To ensure that we were measuring T-cell mediated
immunological memory, we measured swelling 24 and
48 h after all three injections in the E. coli group. We
were unable to do the same for the KLH groups because
of time constraints (imposed by concurrent ?eld work).
For graphical clarity, we report only 24-h swellings;
di?erences in swelling at 48-h intervals were similar.
Humoral immunity (antibody responses)
We used KLH to induce primary and secondary anti-
body responses in the two sparrow populations; these
assays were conducted from May to June 2003. First, we
injected the wing web of all birds (KLH groups from
above experiment) with 100 ll of a 1 mg ml1 KLH in
pyrogen-free saline. This approach enabled us to mea-
sure cell-mediated and humoral immune activity in the
same bird simultaneously. To assess the primary anti-
body response to KLH, we took small blood samples
(50 ll) from the brachial vein of each bird just before the
?rst injection and 5, 10, and 15 days post-injection; to
assess the secondary antibody response, we injected
KLH into all birds again (12 days after last blood
sample) then took additional blood samples just prior to
and 3, 6, and 9 days after this second injection. After
collection, plasma was removed from all blood samples,
brie?y stored on ice, and frozen at 20C until anti-
bodies were measured (December 2003).
We used a previously developed ELISA assay to
measure KLH antibodies in our sparrows (Hasselquist
et al. 1999). Brie?y, 96-well plates were coated with
KLH, plasma from house sparrows was added to each
well (in duplicate), then incubated with an anti-red-
winged blackbird antibody (produced in rabbits), and a
goat?anti-rabbit antibody labeled with peroxidase. All
samples in these assays were coded and measured by a
person unaware of the aim of this study.
568
Energy invested in immune defense
Cost of an acute phase response
We measured the energetic cost of one type of immune
response to directly determine if investment in immune
defense varied between the two populations of sparrows.
In February 2003, we captured birds (n=7; three male
and four female birds) from the wild in Colon, Panama,
and that same night we placed them in 1 l respiratory
chambers and measured rates of oxygen consumption.
We used these data to calculate resting metabolic rate
(RMR, see Martin et al. 2003 for details), the rate of
energy expenditure of post-absorptive birds during this
quiescent phase (night) under thermoneutral conditions
(Ta: 28?30C; relative humidity: 80%; Ascho? and Pohl
1970). For the next three nights after capture, we mea-
sured energy expenditure of each bird to establish its
baseline RMR, as we have found that house sparrow
RMRs tend to decrease over the ?rst several nights in
captivity (Martin et al. 2003).
After this period of acclimation, we injected the wing
webs of all birds with 100 ll of 1 mg ml1 phytohe-
magglutinin (PHA-P: Sigma L9017) in pyrogen-free
saline and measured RMR for four additional nights
(Martin et al. 2003). We chose to use PHA to induce
systemic in?ammatory immune activity, as it increases
resting metabolic rate (Martin et al. 2003) and circulat-
ing levels of acute phase proteins (Matson et al. 2002) in
temperate house sparrows and induces local macro-
phages and basophils to secrete IL-1 and TNF-a, two
fever-inducing cytokines, in chickens (Stadecker et al.
1977). We used RMR the night prior to injection as the
baseline rate of energy expenditure of each bird. We did
not measure RMR of a control (saline-injected) group of
sparrows simultaneously (sensu Martin et al. 2003) be-
cause our metabolic system was committed to other
projects. Also, data for the third night post-PHA injec-
tion became corrupted for slow-living birds and could
not be used in population comparisons. For fast-living
house sparrows, we used data from a previous study on
Illinois house sparrow metabolic responses to PHA
challenges (February 2000; Martin et al. 2003). We
chose the particular measurement paradigm outlined
above because the similar methodologies allowed us to
compare data from this study to data collected from
fast-living birds. In that study, however, only adult
males were used.
Data analysis
Prior to performing statistical comparisons, all data
were tested for normality and equality of variances using
1-sample Kolmogorov?Smirnov and Levene?s tests, and
histograms of each variable were examined visually.
When data were parametrically distributed, we used ei-
ther independent sample T tests, ANOVA in conjunc-
tion with simultaneous Bonferroni post hoc tests, or
repeated-measures general linear models (GLM) for
statistical comparisons. When data were non-normally
distributed, we transformed them. If transformations
were unsuccessful, we used non-parametric Kruskal?
Wallis or Mann?Whitney U tests. When sample size was
low for statistical comparisons, we performed power
analyses and report observed power estimates (b). We
used SPSS v 10.0 (1999) for all statistical comparisons,
setting a=0.05.
Results
Constitutive defenses
Natural antibody levels and lysis activity
Neither NAb levels (t33=0.31, P=0.76) nor lysis activity
(U=127.5, P=0.40) was signi?cantly di?erent between
house sparrow populations (slow-living: n=20; fast-liv-
ing: n=15); sex of individuals also had no e?ect on ei-
ther measure. However, both measures were comparable
to those of a population from St. Louis, MO, USA
(Matson et al. 2005).
Inducible defenses
Cell-mediated immunity (DTH)
To ensure that we were measuring DTH and not non-
speci?c local in?ammation, we compared swelling in-
duced in birds after three di?erent injections of E. coli
bacteria spread over the course of several weeks (slow-
living: n=14, fast-living: n=11). Swelling induced soon
after (<48 h) a single injection would indicate a
non-speci?c in?ammatory response (i.e., predominantly
non-T-cell mediated); swelling induced after multiple
injections, however, would indicate cell-mediated mem-
ory orchestrated by T-cell clonotypes receptive to anti-
gens of E. coli (Turk 1967). Initial and second injections
induced very little swelling in either population, indi-
cating weak or no non-speci?c in?ammation (Fig. 1).
However, after the third injection, fast-living sparrows
exhibited signi?cantly larger swellings than they did
after being injected once or twice [Fig. 1: F2,23=12.4,
P<0.001; Bonferroni post hoc test, third injection ver-
sus ?rst (P=0.001) and second injection (P<0.001)].
This result did not hold in slow-living sparrows, how-
ever, as swellings did not increase signi?cantly over the
three injections (F2,23=1.5, P=0.23). Due to time con-
straints, we were unable to perform a similar test for
induction of DTH by KLH. However, we sensitized
animals to this antigen in the same manner as E. coli,
and thus assume that any swelling after three injections
represents (predominantly) cell-mediated hypersensitiv-
ity and not non-speci?c in?ammation.
To determine if cell-mediated immune activity dif-
fered between sparrow populations, we compared
569
swellings between populations 24 h after the third
injection of either E. coli or KLH. We found that
swelling induced by killed E. coli tended to be greater in
fast-living sparrows (Fig. 2a: F1,23=4.14, P=0.054;
b=0.494). We also included sex as a covariate in this
model, but found that it strongly reduced statistical
power but did not change the outcome of the prior
comparison (F1,23=2.04, P=0.155; sex: F1,23=0.010,
P=0.74, b=0.062). Swelling induced by KLH, however,
was distinctly greater in fast-living birds (Fig. 2b;
F1,25=26.0, P<0.001); sex of individuals, however, did
not a?ect this result (F1,25=14.991, P<0.001; sex:
F1,25=2.38, P=0.137, b=0.314).
Humoral immunity (antibody response)
To assess di?erences in the humoral arm of the immune
system between populations, we compared primary and
secondary antibody responses to KLH (slow-living:
n=13; fast-living: n=11). Neither sparrow population
showed a signi?cant primary antibody response to KLH
(Fig. 3a: F1,22=0.007, P=0.94). Therefore as expected,
there was no signi?cant di?erence in the primary re-
sponse between populations (F1,22=0.58, P=0.45) nor
was sex signi?cant (F1,22=0.001, P=0.976, b=0.050).
Both populations, however, showed signi?cant second-
ary antibody responses to KLH (Fig. 3b: F1,24=33.2,
P<0.001). Although there was no overall e?ect of
population on the secondary response (F1,24=0.26,
P=0.874), the time-course of the response varied with
latitude (F1,24=5.7, P=0.03). Inclusion of sex in the
model, however, only resulted in a weakening of the
Fig. 2 T-cell memory as expressed by wing-web swelling to a killed
E. coli bacteria (slow-living: n=13; fast-living: n=11) and b
keyhole limpet hemocyanin (KLH; slow-living: n=14; fast-living:
n=11) in slow versus fast-living house sparrows. P values from
independent samples T test. Bars are means + 1SE
Fig. 3 a Primary and b secondary antibody response to KLH
(mOD per min +1) for slow-living (n=14) and fast-living (n=11)
house sparrows (means ? 1SE). P values from repeated measures
ANOVA (see Materials and methods). Note the logarithmic scale
used
Fig. 1 T-cell memory as expressed by wing-web swelling in
response to repeated injections of killed E. coli bacteria in slow-
living Panamanian (open bars, n=13) and fast-living New Jersey
(?lled bars, n=11) house sparrows (Passer domesticus). Bars depict
means + 1SE. P value from repeated-measures ANOVA; letters
indicate group membership according to simultaneous Bonferroni
post hoc test. NS indicates no signi?cant change in amount of
swelling after multiple injections
570
latitudinal e?ect (F1,22=3.817, P=0.064; sex:
F1,22=0.040, P=0.843, b=0.054). Figure 3b suggests
that slow-living birds tended to produce antibodies more
rapidly but reach lower maximum titers; fast-living birds
tended to produce more total antibodies, but they took
longer to do so. When we compared antibody titers in
each population at the earliest time point of the sec-
ondary response (3d post-injection), we found that slow-
living sparrows indeed produced signi?cantly more
antibodies compared to fast-living sparrows (Fig. 3b;
t24=2.52, P=0.02). Contrastingly, when we compared
antibody levels after 9 days when fast-living sparrows
appeared to have higher titers, we found only a mar-
ginally signi?cant di?erence (t24 = 1.87, P=0.07).
Correlations between induced immune responses
To determine if birds that mounted strong primary
antibody responses also had strong secondary responses
to KLH, we performed Pearson correlation analysis
between the maximum primary antibodies and maxi-
mum secondary antibodies of each bird. We found no
signi?cant relationship, however, in either population
(slow-living: P=0.56; fast-living: P=0.34). To deter-
mine if a strong cell-mediated response (DTH) was re-
lated to a strong humoral response (antibody titer) in the
same bird, we performed Pearson correlation analysis on
maximum KLH-induced swelling and maximum levels
of KLH-speci?c antibodies during the secondary re-
sponse. Again, we found no signi?cant relationship
within individuals in either population (slow-living:
P=0.56; fast-living: P=0.80).
Energy invested in immune defense
At capture, slow-living house sparrows were signi?-
cantly lighter than fast-living sparrows (slow-living:
23.4?1.8 g, n=7; fast-living: 28.4?2.3 g, n=7;
t12=4.5, P=0.01). Because of this di?erence, we di-
vided the RMR of each bird by its mass the night prior
to metabolic measurement and compared these values
between groups. We found that both sparrow popula-
tions showed a decrease in RMR over the ?rst 3 days of
measurements (prior to immune challenge) followed by
a general increase after PHA challenge (cubic model:
F1,14=10.9, P=0.006). Slow-living sparrows, however,
showed a di?erent metabolic response to PHA challenge
than fast-living birds; RMR of slow-living birds tended
to increase and remain elevated for all 4 days post-
challenge, whereas RMR for fast-living birds did not
increase until 48 h after challenge and remained elevated
for only the following 24 h (Fig. 4a; see also Martin
et al. 2003). Overall, latitude of origin had a signi?cant
e?ect on RMR over the course of the experiment
(F1,14=8.58, P=0.01). To ensure that this result was not
a consequence of di?erent sexes being measured in slow-
living birds but only males being measured in fast-living
birds, we compared metabolic rate between male and
female slow-living house sparrows. We found no e?ect
of sex on RMR (F1,7=0.03, P=0.87; b=0.052).
To compare the relative proportion of the energy
budget that each population expended on immune
activity, we calculated the percent increase in RMR on
the 3 days post-immune challenge (using RMR of the
evening prior to the challenge as a baseline), and com-
pared the average of these values between populations.
We found that slow-living sparrows showed a greater
mean increase in energy expenditure to PHA challenge
compared to fast-living birds (Fig. 4b: t12=2.4,
P=0.037). Also in all birds, PHA induced signi?cant
wing-web swelling as it did in several studies from our
lab (Martin et al. 2004, 2005; Greenman et al. 2005);
data are not shown here for brevity.
Discussion
Poultry breeders have been attempting to engineer dis-
ease-resistant strains of chicken and turkeys for many
years without success (Kreukniet et al. 1994; Parmentier
Fig. 4 a Resting metabolic rate (RMR, ml O2 g
1 h1) before and
after phytohemagglutinin (PHA) injection into wing-web and b
cost of the PHA response, expressed as percentage of RMR in
slow-living (open symbols) and fast-living (solid symbols) house
sparrows (means ? 1SE). Diagonal lines (//) in (a) indicate missing
data due to failure of data recording during experiment. In b P
value is from independent samples T test; n=7 in each group
571
et al. 1996). They are beginning to recognize that their
di?culties may stem from the nature of the immune
system itself. Just like in the endocrine or nervous sys-
tems, di?erent components of the vertebrate immune
system have unique roles and subsequently impart dis-
tinct costs and bene?ts (Lochmiller and Deerenberg
2000; Ra?berg et al. 2002). In this study, we sought to
determine if the pace of life of house sparrows in?uenced
the organization of their immune systems. In order to do
this, we felt it was critical that the complexity of the
immune system be taken into account. Table 3 sum-
marizes the immunological di?erences we found in our
sparrow populations. Although our data show that im-
mune activity is variable between populations, they only
partly support our prediction for stronger immune de-
fenses in the slow-living house sparrows. The lack of
di?erences in NAb levels and lysis activity and the
unexpected result for stronger cell-mediated immunity in
the fast-living sparrows indicates that other factors be-
sides life history must shape the immune portfolios of
these birds.
The costs and bene?ts of immune defense
Inducible defenses
Speci?c, inducible defenses include primary and sec-
ondary antibody proliferation (mediated by B cells) and
cytotoxic and helper T-cell activity (Elgert 1996). The
advantages of these defenses lie in their speci?city and
low costs of use once they are generated (Ra?berg et al.
2002); disadvantages lie in their high developmental
costs (Klasing and Leshchinsky 1999), predilection for
autoimmune damage (Graham 2002), and long latency
prior to utility (Elgert 1996). In terms of B-cell mediated
immune function (humoral defense), secondary but not
primary antibody responses were di?erent between
populations. Speci?cally, slow-living birds produced
antibodies more rapidly than fast-living birds, but total
levels of antibodies tended to be greater at the height of
the response in fast-living birds. Rapid production of
antibodies is important for checking secondary infec-
tions (Elgert 1996)?the sooner antibodies target
(opsonize) pathogens, the less likely pathogens are to
take hold.
Such immunological di?erences may be complemen-
tary of life history variation. Speci?cally, one way in
which slow-living birds might generate antibodies
quicker than fast-living birds is to possess more lym-
phocytes receptive to the large number of epitopes of an
antigen such as KLH (Cohn and Langman 1990; Harris
and Markl 1999). This endpoint could be achieved
during ontogeny via generation of a diverse B (and/or T)
cell repertoire, which would promote responsiveness to a
greater number of components of the antigen in adults
(Ricklefs 1992; Klasing and Leshchinsky 1999). Such a
diversi?cation would be expensive, however, because of
the random nature of lymphocyte receptor generation
(Cohn and Langman 1990; Klasing and Leshchinsky
1999). Still, one might expect that if such di?erential B-
cell diversi?cation did take place, more rapid antibody
production would manifest during the primary, not
secondary, antibody response (Klasing and Leshchinsky
1999). Further, it is unclear how much a delay in the
production of antibodies would hinder the control of a
secondary infection in fast-living birds anyway. Perhaps
by generating high antibody titers late in an infection,
fast-living birds could compensate for delayed early
antibody production without dramatic consequences.
In terms of cell-mediated immunity, we found that
delayed-type hypersensitivity to two di?erent antigens
was generally stronger in fast-living birds. Alone, these
results would seem to indicate that fast-living sparrows
are better at eliminating intracellular infections or con-
trolling tumors than their slow-living brethren (Turk
1967). In conjunction with previous ?ndings though,
speci?cally that PHA-induced wing-web swelling is
weaker in fast versus slow-living sparrows in March?
April (Martin et al. 2004), such a generalization becomes
less viable. Work in other taxa has shown that DTH
responses often vary depending on the substances used
to induce the response. In these house sparrows, di?er-
ent substances (KLH, E. coli, and PHA) may have
activated di?erent immune cell populations leading to
di?erent amounts of swelling in each population (Turk
1967). In light of these inconsistencies among DTH re-
sponses, it is di?cult to say which population actually
possesses greater cell-mediated immunocompetence.
Greater E. coli and KLH-induced DTH responses in the
fast-living population may represent greater T-cell
mediated immune defense, or they may represent a
greater predisposition to allergic reaction in that popu-
lation (Turk 1967). This inconsistency suggests that the
typical ??more-is-better?? interpretation of some DTH
swelling responses may be unjusti?ed.
Constitutive defenses
Constitutive immune defenses are continuously mobi-
lized defenses that include physical barriers to infection
as well as circulating immune cells and cell products
(Elgert 1996). The main bene?t of these defenses is that
they are (usually) able to restrict the entry of pathogens,
but their costs have yet to be calculated. Neither NAb
levels nor lysis activity was di?erent between sparrow
Table 3 Comparisons of immune activity between two populations
of house sparrows
Immune defense Greater in Relative cost
Natural antibody levels Neither Low
Complement activity Neither Low
T-cell memory Fast-living Low
Antibody proliferation Slow-living High
Energy expenditure Slow-living High
572
populations in this study. This lack of a di?erence may
indicate that constitutive defenses in general are not
di?erent between populations. More likely though, this
lack of variation arises as a consequence of the short
period of time populations have been separated
(
150 years, Summers-Smith 1988; Ridgely and
Gwynne 1989). Some immune defenses, particularly
germ-line encoded ones such as natural antibodies
(Parmentier et al. 2004), may not have had su?cient
time to diverge.
Energy invested in defense
Slow-living birds showed a much greater increase in
resting metabolic rate (RMR) after injection with PHA
than did fast-living birds. Whole-body increases in en-
ergy expenditure are probably due to the induction of
acute phase responses by PHA in both populations.
Typically, PHA is used to induce only local immune
activity in birds (Stadecker et al. 1977; Greenman et al.
2005). However, some studies have used it to induce
acute phase responses (Matson et al. 2002; Martin et al.
2003). Although it is not clear what di?erences in energy
expenditure indicate in terms of the strength of acute
phase responses per se, it is apparent that slow-living
sparrows make larger energetic investments in this type
of immune activity than fast-living birds (sensu Lee et al.
2005).
Why does immune activity vary between populations?
It is apparent from the above data that relationships
between life history and immune defense in animals are
unlikely to be simple. In a life history context, immune
defense has been predicted to be greatest in animals: (a)
with a long lifespan (Klasing and Leshchinsky 1999), (b)
with long development times (Ricklefs 1992), (c) with a
slow pace of life (Ricklefs and Wikelski 2002; Martin
et al. 2004), or (d) of large body size (Tella et al. 2002;
Nunn 2002). None of these generalizations incorporate
speci?c predictions about components of the immune
system themselves, but our approach of looking for
immunological variation in two populations of house
sparrows with pre-existing di?erences in life history
characters represents a natural experiment for testing of
our ??pace of life?? hypothesis. We found some support
for our initial predictions: energy expended after
induction of an acute phase response was signi?cantly
greater in slow versus fast-living house sparrows, and
slow-living sparrows had more rapid antibody responses
than fast-living birds. Other immune measures, partic-
ularly cell-mediated immune activity, varied counter to
our expectations indicating that slow-living sparrows are
not generally ??more immunocompetent?? that their fast-
living relatives.
One important factor that may determine the nature
of populations? immune defenses is pathogen exposure
during development. In our house sparrow populations,
ecto-parasite and blood parasite levels are similar during
the early and late breeding season, but during the non-
breeding season, no blood parasites are detectable in
fast-living birds (Martin et al., unpublished manuscript).
If we assume that this pattern of parasite infections is
representative of other threats to house sparrows in both
places, we can interpret some of our immunological
data. For instance, more e?cient antibody-mediated
responses in slow-living birds may represent a strategy to
control persistent repeat infections using the most eco-
nomical and targeted means of defense (Klasing and
Leshchinsky 1999). However, if cost-minimization due
to high probability of infection was the sole driving
factor in?uencing defense portfolios, one would not
expect slow-living sparrows to show such strong acute
phase (energetic) responses because these are the most
costly and damaging types of defenses (Klasing and
Leshchinsky 1999). Slow-living animals (with presum-
ably long lifespan) could be jeopardizing their survival
probabilities combating infections in this way. This
inconsistency for strong acute phase responses in the
face of supposedly persistent disease threats may indi-
cate that other pathogens, including bacteria and viru-
ses, must follow di?erent seasonal trajectories than the
blood and ecto-parasites mentioned above.
Another possible in?uence on the immunological
patterns we found involves simple shortcomings of
methodology (Adamo 2004). Kreukniet et al. (1994)
discovered that chickens that had strong tissue swelling
responses to PHA had weak in vitro cellular prolifera-
tion to the same challenge. These contrary results show
that even when immunological methodologies are well-
developed, as they tend to be in domestic fowl, inter-
pretation can still be contingent upon the assay used.
Pre-existing di?erences between individuals or popula-
tions in comparisons like ours may also obscure immu-
nological uniqueness. For instance, there are two
approaches to conducting studies like ours. One option
is to solely study free-living animals. This practice allows
one to measure traits of interest in animals without
potential confounds of captivity stress. The shortcoming
of this approach is that one cannot usually take repeated
measurements of the same individuals over short time
scales, particularly because individuals can also be
stressed by repeated capture and because it may be dif-
?cult or impossible to recapture net-shy species like
house sparrows. More importantly, one cannot guar-
antee that wild animals do not also experience natural
stressors (e.g., temperature, predation, food limitation)
that may have confounding e?ects on physiological
traits themselves. Captivity allows one to make repeated
measures and standardize diet and other environmental
conditions (e.g., light, ambient temperature), but it may
also induce stress, potentially in di?erent manners or
degrees in di?erent populations of animals. Indeed,
stress can have large e?ects on the type and intensity of
immune responses (Sapolsky et al. 2000). In house
sparrows, we have already found that PHA-induced
573
wing-web swelling can be in?uenced by exogenously
administered corticosterone (Martin et al. 2005). Fur-
thermore, it is becoming clear that single measures of
immune activity are often state dependent. That is, if an
animal is growing feathers, going through maturation,
developing reproductive organs, or partaking in repro-
ductive behaviors, immune activity may change
(Greenman et al. 2005; Martin 2005). For this reason,
we did not include birds in heavy molt in this study, and
we conducted all of our comparisons during the breed-
ing seasons of both populations. Still, variation among
individuals in breeding state within each population may
have a?ected some immune measures. If this e?ect was
su?ciently large, however, we would not expect to ?nd
any di?erences in immune activity in a life history con-
text.
Conclusion
In a descriptive study like this one, it is impossible to
know whether immunological variation between two
populations is due to particular factors of interest
(Garland and Arnold 1994). Nevertheless, the extensive
variation seen in many physiological and life history
characteristics of North American house sparrows
(Kendeigh 1976; Summers-Smith et al. 1988) strongly
suggests that immunological di?erences detected in this
study are partly adaptive and perhaps genetic (Martin
et al. 2004). Moreover, because the immune system is
arguably one of the most complex phenomena in biol-
ogy, studies like this one can provide useful background
for future, more controlled work in understanding the
forces that determine the architecture of species? immune
systems. Although substantial molecular understanding
exists with regard to the form and function of the ver-
tebrate immune system, we have gained little integrative,
adaptive perspective on how it works. To continue to
make progress in this direction, we suggest keeping the
Schmid-Hempel and Ebert (2003) immune defense
component model in mind when designing future stud-
ies. Furthermore, researchers should try to directly
characterize disease-resistance in populations when
possible (Adamo 2004), appreciating that these studies,
too, may be limited in their scope. Indeed, it is unclear
whether the ability of a species to combat or prevent one
disease would be representative of its ability to ?ght all
diseases. Lastly, one should recognize that prophylactic
behavioral defenses, such as responsiveness to novel
foods, environments, and/or objects (Martin and Fitz-
gerald 2005), may be important ?rst lines of defense in
some cases. Ultimately, such a multi-tiered approach
will lead us to a robust, evolutionary understanding of
the vertebrate immune system.
Acknowledgements Many people helped with assays and bird care
and capture including Paula Capece, Lisa Fitzgerald, Peggy Han,
Kelly Lee, Lars Ra?berg, and Laura Spinney. We thank the man-
agement of the Belle Mead Farmer?s Co-op and the Princeton
Shopping Center for allowing us to work on their property in
New Jersey, and Jeanne Altmann, Michaela Hau, Henry Horn,
Kirk Klasing, Dustin Rubenstein, Alex Scheuerlein and Brian
Trainor for comments on earlier drafts. We thank Robert Ricklefs
for allowing us to conduct the NAb assay in his lab, and Kevin
Matson for assistance in performing the assay. Funding for this
work comes from grants to LBM from the American Museum of
Natural History, the American Ornithologist?s Union, the Prince-
ton University Program in Latin American Studies, the Pew
Charitable Trusts Training Program in Biocomplexity, and the US
Environmental Protection Agency STAR Fellowship, to DH from
the Swedish Research Council for Environment, Agriculture and
Spatial Planning (FORMAS), Crafoord Foundation, and Carl
Tryggers Foundation, and to MW from NSF-IRCEB #0212587.
All methods used in this study were approved by the Princeton
University Institutional Animal Care and Use Committee (protocol
number 1492), the National Environmental Authority of Panama
(ANAM), and the Smithsonian Tropical Research Institute
(STRI).
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