Content uploaded by Christian C Voigt
Author content
All content in this area was uploaded by Christian C Voigt
Content may be subject to copyright.
4514
INTRODUCTION
All aerobic organisms produce reactive oxygen species (ROS) as
by-products during energy production in mitochondria. In fact, it is
estimated that around 2–3% of oxygen consumed by cells is
diverted to generate superoxide and hydrogen peroxide, two of the
most important ROS (Chance et al., 1979). ROS are highly reactive
and thus can lead to oxidative damage by peroxidation of membrane
fatty acid chains, modification of DNA and loss of sulfhydryls and
carbonylation in proteins (Sohal et al., 1990; Sohal and Weindruch,
1996; Goyns, 2002). An animal can mitigate these negative effects
by raising an antioxidant barrier, which may consist of both
exogenous, diet-derived antioxidants such as vitamin E, and
endogenously produced antioxidants such as uric acid or antioxidant
enzymes (e.g. superoxide dismutases and peroxidases), converting
ROS into less reactive molecules. An imbalance between pro-
oxidants and antioxidants results in oxidative stress, which may
impair the metabolism of an organism by causing oxidative damage
as described above (Rose et al., 2002). Oxidative stress causes
senescence in cells and is therefore hypothesised to be an important
modulator of life-history trade-offs in vertebrates (Costantini, 2008;
Nussey et al., 2009); it has consequently been regarded as the main
cause of ageing in the literature of past decades (Harman, 1955).
However, the free-radical theory of ageing has recently been
challenged, as experimental and correlative studies do not always
support the hypothesis that high oxidative stress leads to shorter
lifespans (Speakman and Selman, 2011).
As the creation of ROS generally increases proportionately with
the amount of energy produced, i.e. with mass-specific metabolic
rate, animals with a high mass-specific metabolic rate should have
a shorter life span than species with a low mass-specific metabolic
rate (Pearl, 1928; Harman, 1955; Sacher, 1959). Evidence for this
free-radical theory of ageing suggested by Harman (Harman, 1955)
has been found in many empirical studies. In general, small
mammals with relatively high basal metabolic rates have lower life
expectancies than large mammals with low basal metabolic rates
(Hulbert et al., 2007). Variations in longevity among species have
also been shown to correlate negatively with the amount of
superoxide anion radicals produced in mitochondria (Tolmasoff et
al., 1980; Sohal and Weindruch, 1996) and oxidative damage of
mitochondrial DNA (Adelman et al., 1988; Barja and Herrero, 2000).
The negative correlation of mass-specific metabolic rate and
longevity among mammals comes with a few exceptions: for
example, small-sized bats may live about 3–4 times longer than
similar-sized terrestrial mammals (Austad and Fischer, 1991;
Wilkinson and South, 2002), but at the same time, their metabolic
rates are exceptionally high because of their ability of powered flight
(Munshi-South and Wilkinson, 2010). Thus, the question arises of
whether the high mass-specific metabolic rate of bats produces more
pro-oxidants than that of similar-sized terrestrial mammals, and if
so, whether bats show increased oxidative damage. If bats indeed
have to cope with high oxidative stress, what factor predisposes
them for long life expectancies? Initial studies on oxidative stress
SUMMARY
Oxidative stress – the imbalance between reactive oxygen species (ROS) and neutralising antioxidants – has been under debate
as the main cause of ageing in aerobial organisms. The level of ROS should increase during infection as part of the activation of
an immune response, leading to oxidative damage to proteins, lipids and DNA. Yet, it is unknown how long-lived organisms,
especially mammals, cope with oxidative stress. Bats are known to carry a variety of zoonotic pathogens and at the same time
are, despite their high mass-specific basal metabolic rate, unusually long lived, which may be partly the result of low oxidative
damage of organs. Here, we asked whether an immune challenge causes oxidative stress in free-ranging bats, measuring two
oxidative stress markers. We injected 20 short-tailed fruit bats (Carollia perspicillata) with bacterially derived lipopolysaccharide
(LPS) and 20 individuals with phosphate-buffered saline solution (PBS) as a control. Individuals injected with LPS showed an
immune reaction by increased white blood cell count after 24 h, whereas there was no significant change in leukocyte count in
control animals. The biological antioxidant potential (BAP) remained the same in both groups, but reactive oxygen metabolites
(ROMs) increased after treatment with LPS, indicating a significant increase in oxidative stress in animals when mounting an
immune reaction toward the inflammatory challenge. Control individuals did not show a change in oxidative stress markers. We
conclude that in a long-lived mammal, even high concentrations of antioxidants do not immediately neutralise free radicals
produced during a cellular immune response. Thus, fighting an infection may lead to oxidative stress in bats.
Key words: reactive oxygen metabolites, immune response, bat, antioxidants.
Received 8 May 2013; Accepted 28 August 2013
The Journal of Experimental Biology 216, 4514-4519
© 2013. Published by The Company of Biologists Ltd
doi:10.1242/jeb.090837
RESEARCH ARTICLE
Inflammatory challenge increases measures of oxidative stress in a free-ranging,
long-lived mammal
Karin Schneeberger
1,2,
*, Gábor Á. Czirják
1
and Christian C. Voigt
1,2
1
Leibniz Institute for Zoo and Wildlife Research, Alfred-Kowalke-Strasse 17, 10315 Berlin, Germany and
2
Department of
Animal Behaviour, Freie Universität Berlin, Takustrasse 6, 14195 Berlin, Germany
*Author for correspondence (schneeberger@izw-berlin.de)
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4515Oxidative stress and immune response in bats
in bats have shown that bats have lower levels of protein oxidation
than terrestrial mammals (Brunet-Rossinni, 2004). Potentially, bats
may have potent repair mechanisms for damage caused by oxidation.
It has recently been shown that genes regulating repair mechanisms
for DNA damage are positively selected for in bacteria (Sghaier et
al., 2008) and potentially also in vertebrates. However, low oxidative
damage in bats may also be explained by (1) low pro-oxidant
production, (2) high antioxidant levels, or (3) a combination of both.
Indeed, bats seem to produce lower levels of pro-oxidants than
terrestrial mammals (Brunet-Rossinni, 2004), and also have higher
levels of both enzymatic and non-enzymatic antioxidants in their
organs (Wilhelm Filho et al., 2007). Thus, low oxidative stress may
be causative for the exceptional longevity of bats. However, it
remains to be investigated why bats have such a low level of
oxidative stress and what factors influence the production of pro-
oxidants and antioxidants.
Besides their unusually long lifespan, bats are also outstanding
with respect to their pathogen load, particularly as a reservoir for
important zoonotic pathogens (Wibbelt et al., 2010; Wood et al.,
2012) such as lyssaviruses (Kuzmin et al., 2011), coronaviruses (Li
et al., 2005) and paramyxoviruses (Drexler et al., 2012). Although
it is crucial to understand how the immune system of bats works
and how they defend themselves against these pathogens,
surprisingly little is known about bat immunity and the factors
influencing it (Dobson, 2005). Recent studies have shown a
correlation between immune parameters and ecological factors such
as dietary niche and roost use in bats (Allen et al., 2009;
Schneeberger et al., 2013). Also, experiments on Mexican free-tailed
bats (Tadarida brasiliensis) demonstrated that they can mount a
considerable cellular immune response after injection of mitogens
such as phytohaemagglutinin (Allen et al., 2009). As in most other
animals, variations in immune responses can be linked to disease
susceptibility, such as the white-nose syndrome in temperate-zone
bats that eradicated millions of bats in North America during the
last decade (Lorch et al., 2011; Moore, 2011). However, mounting
an immune response is not only energetically costly (Lochmiller
and Deerenberg, 2000) but also associated with an increased
production of ROS. During an immune response, the host metabolic
rate is usually elevated (Sheldon and Verhulst, 1996), which leads
to higher mitochondrial activity and consequently to increased ROS
production (Finkel and Holbrook, 2000). Additionally, different
white blood cell subtypes involved in immune responses produce
ROS to directly kill pathogens (Dröge, 2002), and to enhance the
activation of T-lymphocytes (Dröge, 2002; Reth, 2002). Thus, ROS
have a signalling function during an immune response and a direct
negative effect on parasites and pathogens, but can at the same time
also damage the tissue of the host. Mounting an immune response
therefore should not only lead to an increase of ROS but also change
antioxidant levels to mitigate the negative effect. A meta-analysis
of avian studies has found a positive association between immune
responses and oxidative stress markers; however, findings on how
immune responses influence both pro-oxidants and antioxidants are
inconsistent (Costantini and Møller, 2009). Furthermore, some of
these studies only involve either pro-oxidants or antioxidants, yet
it is important to measure both in order to assess the level of oxidative
stress (Costantini and Verhulst, 2009). Also, carotenoids, which are
among the most frequently assessed antioxidants in birds, have
recently been shown to play a rather minor role in the antioxidant
defence of birds (Costantini and Møller, 2008).
As bats are special with respect to their longevity, high mass-
specific metabolic rate and disease susceptibility, but apparently
show low oxidative damage, our aim was to study whether an
immune response leads to an increase in oxidative stress in bats.
Most studies on birds show an increase in ROS and a decrease in
antioxidants after an immune challenge (Costantini and Møller,
2009). Because birds and bats have high metabolic rates, we would
expect a similar effect of immune activation on oxidative stress for
the two taxa. However, antioxidants used to counterbalance pro-
oxidants may differ between birds and bats: two essential
antioxidants, α-tocopherol and retinol, have been found in all
Neotropical bat species investigated so far (Müller et al., 2007),
while β-carotene and lutein, among the most important antioxidants
in birds, were missing in five out of six bat species. Furthermore,
in contrast to many birds (Chaudhuri and Chatterjee, 1969), bats –
just like haplorhine primates, including humans (Homo sapiens),
capybaras (Hydrochoerus hydrochaeris) and guinea pigs (Cavia
porcellus) – are unable to synthesise vitamin C because they lack
L-gulonolactone oxidase (Birney et al., 1976). Thus, it might not be
feasible to extrapolate findings from birds to bats and other
mammals.
Here, we conducted an immune challenge experiment in the short-
tailed fruit bat, Carollia perspicillata (Linnaeus 1758), an abundant,
frugivorous bat species commonly found in lowland regions of the
Neotropics. This species can be kept in captivity for short periods
and has been used before in immunological studies (Greiner et al.,
2010). We asked whether mimicking a bacterial infection via
injection of lipopolysaccharide (LPS) and the resulting immune
response leads to a change in reactive oxygen metabolites (ROMs),
representing total ROS produced, and antioxidant level. Our
expectation was that the concentration of ROMs would increase
and the level of antioxidants would decrease in the bats in order to
avoid oxidative stress.
MATERIALS AND METHODS
We captured 12 C. perspicillata (six males and six females) in
November and December 2011 and 28 C. perspicillata (14 males
and 14 females) in November and December 2012, respectively,
within the vicinity of ‘La Selva’ Biological Station (10°25′N,
84°00′W, Province Heredia, Costa Rica) using nylon mist nets (2.5 m
height; Ecotone, Gdynia, Poland) at ground level. The experiments
took place within 1 week of capture of the bats. We marked bats
individually and kept them in an outdoor flight cage (3.4×6.1×2.5 m)
where they were fed ad libitum with banana and papaya and provided
with water. The experiment started after all bats had been allowed
to habituate to the captive conditions for at least 3 days.
At the start of the experiment, we caught all animals in the flight
cage, put them in individual cotton bags and weighed them using
a spring balance (50 g capacity, Pesola, Baar, Switzerland). We took
an initial blood sample of ~60 μl from each individual by puncturing
the antibrachial vein with a sterile needle (no. C721.1, Carl Roth
GmbH, Karlsruhe, Germany) and transferring blood drops into
heparinised capillary tubes (no. 521-9100, VWR, Darmstadt,
Germany). In these samples, we measured basal antioxidant status
and immunity (see below). Then we assigned half of the individuals
of each sex randomly to the experimental group and the other half
to the control group. Animals from the experimental group were
injected subcutaneously with 50 μl of 1 mg ml
–1
LPS (Escherichia
coli, no. L2630, Sigma-Aldrich, Munich, Germany) in phosphate-
buffered saline solution (PBS; no. L1825, Biochrom AG, Berlin,
Germany) using a sterile disposable syringe (no. 0053.1, Carl Roth
GmbH). LPS is an endotoxin that induces an immune reaction in
treated animals, as well as sickness behaviour such as reduced
locomotion and feeding behaviour (Kozak et al., 1994). Furthermore,
endotoxins are known to generally increase oxidative stress markers
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4516 The Journal of Experimental Biology 216 (24)
(Victor et al., 2004), leading to oxidative damage (Skibska et al.,
2006). Individuals of the control group were injected with 50 μl PBS
without the antigen. We weighed each bat again 24 h post-injection
and took an additional blood sample. Because of the small sample
volume, we were restricted to measuring only part of the likely
complex immunological response to LPS. We therefore produced
blood smears and performed total white blood cell (WBC) counts
as a proxy for detecting an immunological reaction to LPS. The
remaining blood samples were centrifuged and the plasma was taken
and stored at −80°C until further analysis of oxidative stress
parameters. All bats were released at their site of capture after
collection of the final blood sample.
Blood smears were stained with May–Gruenwald’s solution (no.
T863.2, Carl Roth GmbH) and Giemsa (no. T862.1, Carl Roth
GmbH). We manually estimated total WBC count by counting the
cells in 10 visual fields with a microscope under 200× magnifications
(Schneeberger et al., 2013).
We measured markers of oxidative stress [dROM and biological
antioxidant potential (BAP)] using the Free Radical Analytical
System (FRAS4 evolvo; H&D srl, Parma, Italy), following the
instructions provided by the manufacturer. We measured the
concentration of ROMs using dROM-kits, which represents the total
level of hydroperoxide in plasma that is created during peroxidation
of amino acids, lipids and proteins, representing free radicals from
which ROMs are formed (Alberti et al., 2000; Buonocore et al., 2000).
We added 10 μl of plasma to a buffered chromogen, where the
derivates of ROMs form a coloured compound that can be measured
photometrically at a maximum absorbency peak of 505 nm after 5 min
of incubation at 37°C. According to Lambert–Beer’s law, the
absorbance is directly proportional to the concentration of ROMs and
is expressed as U Carr, where 1 U Carr is equivalent to 0.08 mg dl
–1
hydrogen peroxide. The antioxidant potential of the plasma was
measured using BAP-kits. We dissolved 10 μl of plasma into a
coloured solution containing ferric ions (FeCl
3
) and a chromogenic
substrate (a sulphur-derived compound). After 5 min of incubation at
37°C, we measured the degree of decolouration by the plasma
antioxidants by photometry with FRAS4 evolvo. The intensity of
decolouration is directly proportional to the ability of the plasma to
reduce ferric ions and thus to the concentration of non-enzymatic
antioxidants expressed as mmol l
–1
.
All statistical tests were run using R statistical software (R
Development Core Team, 2010). As individuals were not caught
and handled at the same time, we tested whether the delay between
capture and handling had an effect on measurements of cellular
immune response and oxidative stress. We did not find such an
influence on WBC count (Spearman rank correlation; ρ=–0.160;
P=0.157), dROM (ρ=0.046; P=0.684) or BAP (ρ=0.067; P=0.559),
excluding the potential of capture and handling stress to confound
our subsequent analysis.
To test whether potential changes in measures of oxidative stress
and WBC count could be accounted for by treatment with LPS or
PBS, we calculated mixed effects models using the package ‘lme4’
(Bates et al., 2011) with the interaction between treatment and time
of sampling (before or after injection) as well as sex as fixed factors,
and of dROM, BAP or WBC count as response variable. We log-
transformed the response variables in order to achieve normal
distribution of model residuals. Individual identity was included as a
random factor in all models to account for repeated measures of the
same individual. P-values were extracted using the ‘pvals.fnc’
function of the package ‘languageR’ (Baayen, 2011). To test whether
WBC count correlated with measures of oxidative stress, we
conducted Spearman rank correlation tests.
RESULTS
Body mass was significantly connected with treatment and day of
sampling (χ
2
=12.56; P=0.006), but not with sex (χ
2
=0.22; P=0.639).
Body mass decreased 24 h after LPS injection (t=–3.77; P<0.001;
Fig. 1A), but not after PBS injection (t=–0.51; P=0.612; Fig. 1B).
WBC count differed significantly between day of sampling and
treatment (χ
2
=11.34; P=0.010), but not between males and females
(χ
2
=1.11; P=0.737). WBC count increased significantly 24 h after
injection with LPS (t=2.99; P=0.006; Fig. 1C), but only tended to
increase after PBS injection (t=1.84; P=0.070; Fig. 1D).
Baseline dROM for C. perspicillata (N=40) averaged
100.33±29.75 U Carr (mean ± 1 s.d.) and BAP was
2294±0.48 μmol l
−1
before treatment. dROM was significantly
linked to treatment and day of sampling (χ
2
=12.52; P=0.006), but
not to sex (χ
2
=0.22; P=0.637). dROM increased significantly after
injection with LPS (t=3.34; P=0.001; Fig. 2A), but not after injection
with PBS (t=–1.13; P=0.261; Fig. 2B). BAP was not related to day
of sampling and treatment (χ
2
=3.38; P=0.336; Fig. 2C,D) or to sex
(χ
2
<0.001; P>0.999).
dROM was in general positively correlated to WBC count
(ρ=0.23; P=0.044), but BAP and WBC count were not significantly
connected (ρ=0.02; P=0.848).
DISCUSSION
Bats are exceptionally long-lived mammals (Austad and Fischer,
1991; Wilkinson and South, 2002) even though they have higher
mass-specific metabolic rates than similar-sized, terrestrial mammals
Body mass (g)
WBC/10 visual fields
31
29
27
25
23
21
19
17
15
250
200
150
100
50
0
Before After Before After
AB
CD
*
*
Fig. 1. Body mass decreases in individuals of the experimental group 24 h
post-treatment with lipopolysaccaride (LPS; A), but not in individuals of the
control group injected with phosphate-buffered saline solution (PBS; B).
White blood cell (WBC) count per 10 visual fields increases in animals of
the experimental group (C), but not in individuals of the control group (D).
Red lines indicate mean values before and after treatment and asterisks
indicate a significant difference at P<0.05.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4517Oxidative stress and immune response in bats
(Munshi-South and Wilkinson, 2010). Bats have a lower production
rate of ROS (Brunet-Rossinni, 2004) and also lower levels of
oxidative damage (Brunet-Rossinni, 2004). Here, we asked whether
mounting an immune response in bats leads to an increase in ROMs,
representing the total ROS created, and whether this changes the
concentration of antioxidants to mitigate oxidative stress. To the
best of our knowledge, this is the first study on the effect of mounting
an immune response on oxidative stress markers in a free-ranging
mammal.
Total WBC count increased significantly in the experimental
group 24 h after antigen treatment, but not in the control group. The
contrasting results between the control and experimental group
indicate that LPS caused a cellular immune reaction in individuals
of the experimental group. This is also supported by the observation
that bats of the experimental group lost body mass, while body mass
remained constant in individuals of the control group. The loss of
body mass and associated decrease in food ingestion might be the
result of LPS-induced sickness behaviour. In mice, it has previously
been shown that LPS results in reduced locomotion and decreased
food intake (Kozak et al., 1994).
dROM increased significantly in bats injected with LPS, and
WBC count correlated with dROM. Thus, the mounting of a cellular
immune response may lead to a higher production of ROS in free-
ranging bats, which is then represented by an increased concentration
of ROMs in plasma. This pattern is similar to what has been found
in various studies on birds (reviewed by Costantini and Møller,
2009). The high level of ROS during a natural infection is usually
a combination of oxidants released by the pathogen itself (Halliwell
et al., 1993) and the ROS released by the host during the mounting
of an immune response (Dröge, 2002; Reth, 2002). As we did not
inject bats with active pathogens but with the endotoxin only, we
can rule out the possibility of the pro-oxidants being synthesised
by the pathogen. Thus, the elevated dROM level in plasma of
animals injected with LPS is a consequence of physiological
processes involved in sickness behaviour, such as elevated metabolic
rate (Finkel and Holbrook, 2000) as well as ROS produced to
enhance the cellular immune response and to directly kill pathogens
(Dröge, 2002; Reth, 2002). This is supported by the finding that
dROM correlated positively with WBC count. Indeed, studies on
purified human monocytes showed that LPS directly stimulates the
production of superoxide, one of the most important pro-oxidants
in vertebrates (Landmann et al., 1995).
The negative effects of ROS on the organism can be mitigated
by the production or ingestion of antioxidants. Thus, with an increase
of ROS concentration represented by dROM levels in plasma, we
would also expect a change in antioxidant levels. However, in our
experiment, the concentration of antioxidants remained the same
before and after treatment in individuals injected with LPS or PBS.
Similar experiments in birds on how antioxidant levels change after
mounting an immune response are inconsistent. For example,
carotenoid levels but not total non-enzymatic antioxidant levels
increased after an immune challenge in wild kestrel nestlings [Falco
tinnunculus (Costantini and Dell’Omo, 2006)]. In red-legged
partridges (Alectoris rufa), the total antioxidant concentration
remained the same before and after injection with
phytohaemagglutinin (Perez-Rodriguez et al., 2008), but increased
in greenfinches [Carduelis chloris (Hõrak et al., 2007)]. The same
was true for chicken (Gallus gallus domesticus) injected with LPS
(Cohen et al., 2007): there was no change in the level of various
antioxidants before and 24 h after treatment. Also, an experimental
study on the effect of supplemental feeding of mice with antioxidant-
rich wine showed no effect of LPS on total antioxidant levels after
24 h (Percival and Sims, 2000). Similar to the findings of these
studies, we found that the antioxidant concentration did not change
after treatment with LPS in C. perspicillata. Potentially, an
antioxidant barrier needs longer than 24 h to be raised, as there may
be a time lag between an increase of free radicals and the
corresponding antioxidant response (Hõrak and Cohen, 2010;
Meitern et al., 2013). In their study on red-legged partridges, Perez-
Rodriguez and his colleagues (Perez-Rodriguez et al., 2008) argued
that, alternatively, pro-oxidants could have been buffered by
carotenoids, which they have measured separately and found to
decrease after the immune challenge. However, recently it has been
argued that although highly promoted, carotenoids have a rather
weak contribution to the avian antioxidant capacity (Costantini and
Møller, 2008). Also, carotenoids are not part of the antioxidant
barrier in most bats (Müller et al., 2007), which is why the absence
of an effect on antioxidants may have been caused by factors other
than an immediate buffering of ROS by carotenoids as suggested
in birds (Perez-Rodriguez et al., 2008). Potentially, the elevated
concentration of ROS may be partly compensated for by a short-
term increase of antioxidants ingested by food such as vitamin E
or by mobilising enzymatic antioxidants. The study of Percival and
Sims (Percival and Sims, 2000) implies that the high level of
antioxidants caused by supplemental feeding may not further
increase to cope with released ROS when the immune challenge is
short. However, as dROM increased after injection with LPS, ROS
were apparently not immediately neutralised by antioxidants.
Furthermore, the mean baseline BAP of 2294 mmol l
–1
in C.
perspicillata indicates a rather moderate concentration of
dROM (U Carr)
BAP (µmol l
–1
)
300
250
200
150
100
50
0
4500
4000
3500
3000
2500
1500
Before After Before After
AB
CD
2000
*
Fig. 2. The concentration of reactive oxygen metabolites (dROM) increases
in bats 24 h after injection with LPS (A), but not with PBS as a control (B).
Antioxidant concentration (BAP) remained the same in both the
experimental (C) and control group (D). Red lines indicate mean values
before and after treatment and asterisks indicate a significant difference at
P<0.05.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4518 The Journal of Experimental Biology 216 (24)
antioxidants, comparable with the level observed in rats
[1874 mmol l
–1
(Iwata et al., 2010)] and mice [2896 mmol l
–1
(Maruoka and Fujii, 2012)].
We conclude that an immune response can upregulate some
markers frequently associated with oxidative stress even in a long-
lived mammal that is known to have a reduced pro-oxidant production
compared with terrestrial mammals (Brunet-Rossinni, 2004; Wilhelm
Filho et al., 2007). Thus, bats may suffer from long-term consequences
of elevated oxidative stress after episodes of acute infection or during
chronic infection. The long lifespan of bats is therefore even more
puzzling, as bats carry a large variety of pathogens and our results
indicate that infections do increase oxidative stress. It remains to be
investigated whether natural infections with bat-borne and bat-
specific pathogens increase oxidative stress, and how bats cope with
the oxidative damage caused by ROS.
LIST OF ABBREVIATIONS
BAP biological antioxidant potential (mmol l
–1
)
FRAS free-radical analytic system
LPS lipopolysaccharide
ROM reactive oxygen metabolite (concentration given in U Carr)
ROS reactive oxygen species
WBC white blood cell
ACKNOWLEDGEMENTS
We are grateful to Daniel Lewanzik, Oliver Lindecke and Tobias Teige for help in
the field and Dr Alexandre Courtiol for statistical advice. We thank the
Organization for Tropical Studies (OTS) for allowing us to use their facilities and
for providing logistic support and the Costa Rican authorities for granting
permission to conduct this research [186-2012-SINAC]. We also thank two
anonymous referees who provided helpful comments on previous drafts of the
manuscript.
AUTHOR CONTRIBUTIONS
K.S. and C.C.V. conducted the fieldwork; K.S. counted the white blood cells,
measured pro-oxidants and antioxidants, and analysed the data; C.C.V.
supervised the study. All authors participated in the design of the study, discussed
the results and wrote the manuscript.
COMPETING INTERESTS
No competing interests declared.
FUNDING
This study was supported by funds of the Leibniz Institute for Zoo and Wildlife
Research and the German Science Foundation (Deutsche
Forschungsgemeinschaft) [DFG Vo 890/25].
REFERENCES
Adelman, R., Saul, R. L. and Ames, B. N. (1988). Oxidative damage to DNA: relation
to species metabolic rate and life span. Proc. Natl. Acad. Sci. USA 85, 2706-2708.
Alberti, A., Bolognini, L., Carratelli, M., Della Bona, M. A. and Macciantelli, D.
(2000). The radical cation of N,N-diethyl-para-phenylendiamine: a possible indicator
of oxidative stress in biological samples. Res. Chem. Intermediat. 26, 253-267.
Allen, L. C., Turmelle, A. S., Mendonça, M. T., Navara, K. J., Kunz, T. H. and
McCracken, G. F. (2009). Roosting ecology and variation in adaptive and innate
immune system function in the Brazilian free-tailed bat (Tadarida brasiliensis). J.
Comp. Physiol. B 179, 315-323.
Austad, S. N. and Fischer, K. E. (1991). Mammalian aging, metabolism, and ecology:
evidence from the bats and marsupials. J. Gerontol. 46, B47-B53.
Baayen, R. H. (2011). languageR: Data sets and functions with ‘Analyzing Linguistic
Data: A practical introduction to statistics’. R package version 1.4. http://CRAN.R-
project.org/package=languageR
Barja, G. and Herrero, A. (2000). Oxidative damage to mitochondrial DNA is inversely
related to maximum life span in the heart and brain of mammals. FASEB J. 14, 312-
318.
Bates, D., Maechler, M. and Bolker, B. (2011). lme4: Linear mixed-effects models
using S4 classes. R package version 0.999375-39. http://CRAN.R-
project.org/package=lme4
Birney, E. C., Jenness, R. and Ayaz, K. M. (1976). Inability of bats to synthesise
L-
ascorbic acid. Nature 260, 626-628.
Brunet-Rossinni, A. K. (2004). Reduced free-radical production and extreme longevity
in the little brown bat (Myotis lucifugus) versus two non-flying mammals. Mech.
Ageing Dev. 125, 11-20.
Buonocore, G., Perrone, S., Longini, M., Terzuoli, L. and Bracci, R. (2000). Total
hydroperoxide and advanced oxidation protein products in preterm hypoxic babies.
Pediatr. Res. 47, 221-224.
Chance, B., Sies, H. and Boveris, A. (1979). Hydroperoxide metabolism in
mammalian organs. Physiol. Rev. 59, 527-605.
Chaudhuri, C. R. and Chatterjee, I. B. (1969).
L-Ascorbic acid synthesis in birds:
phylogenetic trend. Science 164, 435-436.
Cohen, A., Klasing, K. and Ricklefs, R. (2007). Measuring circulating antioxidants in
wild birds. Comp. Biochem. Physiol. 147B, 110-121.
Costantini, D. (2008). Oxidative stress in ecology and evolution: lessons from avian
studies. Ecol. Lett. 11, 1238-1251.
Costantini, D. and Dell’Omo, G. (2006). Effects of T-cell-mediated immune response
on avian oxidative stress. Comp. Biochem. Physiol. 145A, 137-142.
Costantini, D. and Møller, A. P. (2008). Carotenoids are minor antioxidants for birds.
Funct. Ecol. 22, 367-370.
Costantini, D. and Møller, A. P. (2009). Does immune response cause oxidative
stress in birds? A meta-analysis. Comp. Biochem. Physiol. 153A, 339-344.
Costantini, D. and Verhulst, S. (2009). Does high antioxidant capacity indicate low
oxidative stress? Funct. Ecol. 23, 506-509.
Dobson, A. P. (2005). Virology. What links bats to emerging infectious diseases?
Science 310, 628-629.
Drexler, J. F., Corman, V. M., Müller, M. A., Maganga, G. D., Vallo, P., Binger, T.,
Gloza-Rausch, F., Rasche, A., Yordanov, S., Seebens, A. et al. (2012). Bats host
major mammalian paramyxoviruses. Nat. Commun. 3, 796.
Dröge, W. (2002). Free radicals in the physiological control of cell function. Physiol.
Rev. 82, 47-95.
Finkel, T. and Holbrook, N. J. (2000). Oxidants, oxidative stress and the biology of
ageing. Nature 408, 239-247.
Goyns, M. H. (2002). Genes, telomeres and mammalian ageing. Mech. Ageing Dev.
123, 791-799.
Greiner, S., Stefanski, V., Dehnhard, M. and Voigt, C. C. (2010). Plasma
testosterone levels decrease after activation of skin immune system in a free-ranging
mammal. Gen. Comp. Endocrinol. 168, 466-473.
Halliwell, B., Bomford, A. B., Stern, A., Golenser, J., Chevion, M., Callahan, H.
L., Aldunate, J., Morello, A. and Cross, C. E. Balasubramanian et al. (1993).
Free Radicals in Tropical Diseases. Chur, Switzerland: Harwood Academic
Publishers.
Harman, D. (1955). Aging: A Theory Based on Free Radical and Radiation Chemistry.
Berkeley, CA: University of California Radiation Laboratory.
Hõrak, P. and Cohen, A. (2010). How to measure oxidative stress in an ecological
context: methodological and statistical issues. Funct. Ecol. 24, 960-970.
Hõrak, P., Saks, L., Zilmer, M., Karu, U. and Zilmer, K. (2007). Do dietary
antioxidants alleviate the cost of immune activation? An experiment with
greenfinches. Am. Nat. 170, 625-635.
Hulbert, A. J., Pamplona, R., Buffenstein, R. and Buttemer, W. A. (2007). Life and
death: metabolic rate, membrane composition, and life span of animals.
Physiol.
Rev. 87, 1175-1213.
Iwata,
N., Okazaki, M., Kamiuchi, S. and Hibino, Y. (2010). Protective effects of oral
administrated ascorbic acid against oxidative stress and neuronal damage after
cerebral ischemia/reperfusion in diabetic rats. J. Health Sci. 56, 20-30.
Kozak, W. I. E. S., Conn, C. A. and Kluger, M. J. (1994). Lipopolysaccharide induces
fever and depresses locomotor activity in unrestrained mice. Am. J. Physiol. 266,
R125-R135.
Kuzmin, I. V., Bozick, B., Guagliardo, S. A., Kunkel, R., Shak, J. R., Tong, S. and
Rupprecht, C. E. (2011). Bats, emerging infectious diseases, and the rabies
paradigm revisited. Emerg. Health Threats J. 4, 7159.
Landmann, R., Scherer, F., Schumann, R., Link, S., Sansano, S. and Zimmerli, W.
(1995). LPS directly induces oxygen radical production in human monocytes via LPS
binding protein and CD14. J. Leukoc. Biol. 57, 440-449.
Li, W., Shi, Z., Yu, M., Ren, W., Smith, C., Epstein, J. H., Wang, H., Crameri, G.,
Hu, Z., Zhang, H. et al. (2005). Bats are natural reservoirs of SARS-like
coronaviruses. Science 310, 676-679.
Lochmiller, R. L. and Deerenberg, C. (2000). Trade-offs in evolutionary immunology:
just what is the cost of immunity? Oikos 88, 87-98.
Lorch, J. M., Meteyer, C. U., Behr, M. J., Boyles, J. G., Cryan, P. M., Hicks, A. C.,
Ballmann, A. E., Coleman, J. T. H., Redell, D. N., Reeder, D. M. et al. (2011).
Experimental infection of bats with Geomyces destructans causes white-nose
syndrome. Nature 480, 376-378.
Maruoka, H. and Fujii, K. (2012). Effects of exercise and food consumption on the
plasma oxidative stress. J. Phys. Ther. Sci. 24, 37-41.
Meitern, R., Sild, E., Kilk, K., Porosk, R. and Hõrak, P. (2013). On the
methodological limitations of detecting oxidative stress: effects of paraquat on
measures of oxidative status in greenfinches. J. Exp. Biol. 216, 2713-2721.
Moore, M. S. (2011). Ecological immunology in little brown myotis (Myotis lucifugus;
Chiroptera) affected by white-nose syndrome. PhD dissertation. Boston University,
Boston, MA, USA.
Müller, K., Voigt, C. C., Raila, J., Hurtienne, A., Vater, M., Brunnberg, L. and
Schweigert, F. J. (2007). Concentration of carotenoids, retinol and α-tocopherol in
plasma of six microchiroptera species. Comp. Biochem. Physiol. 147B, 492-497.
Munshi-South, J. and Wilkinson, G. S. (2010). Bats and birds: exceptional longevity
despite high metabolic rates. Ageing Res. Rev. 9, 12-19.
Nussey, D. H., Pemberton, J. M., Pilkington, J. G. and Blount, J. D. (2009). Life
history correlates of oxidative damage in a free-living mammal population. Funct.
Ecol. 23, 809-817.
Pearl, R. (1928). The Rate of Living. London: University of London Press.
Percival, S. S. and Sims, C. A. (2000). Wine modifies the effects of alcohol on
immune cells of mice. J. Nutr. 130, 1091-1094.
Perez-Rodriguez, L., Mougeot, F., Alonso-Alvarez, C., Blas, J., Viñuela, J. and
Bortolotti,
G. R. (2008). Cell-mediated immune activation rapidly decreases plasma
THE JOURNAL OF EXPERIMENTAL BIOLOGY
4519Oxidative stress and immune response in bats
carotenoids but does not affect oxidative stress in red-legged partridges (Alectoris
rufa). J. Exp. Biol. 211, 2155-2161.
R Development Core Team (2010). R: A Language and Environment for statIstical
Computing. Vienna, Austria: R Foundation for Statistical Computing.
http://www.r-project.org
Reth, M. (2002). Hydrogen peroxide as second messenger in lymphocyte activation.
Nat. Immunol. 3, 1129-1134.
Rose, G., Passarino, G., Franceschi, C. and De Benedictis, G. (2002). The
variability of the mitochondrial genome in human aging: a key for life and death? Int.
J. Biochem. Cell Biol. 34, 1449-1460.
Sacher, George A. (1959). Relation of lifespan to brain weight and body weight in
mammals. CIBA Foundation Colloquia on Aging 5, 115-141.
Schneeberger, K., Czirják, G. Á. and Voigt, C. C. (2013). Measures of the
constitutive immune system are linked to diet and roosting habits of neotropical bats.
PLoS ONE 8, e54023.
Sghaier, H., Ghedira, K., Benkahla, A. and Barkallah, I. (2008). Basal DNA repair
machinery is subject to positive selection in ionizing-radiation-resistant bacteria. BMC
Genomics 9, 297.
Sheldon, B. C. and Verhulst, S. (1996). Ecological immunology: costly parasite
defences and trade-offs in evolutionary ecology. Trends Ecol. Evol. 11, 317-321.
Skibska, B., Józefowicz-Okonkwo, G. and Goraca, A. (2006). Protective effects of
early administration of alpha-lipoic acid against lipopolysaccharide-induced plasma
lipid peroxidation. Pharmacol. Rep. 58, 399-404.
Sohal, R. S. and Weindruch, R. (1996). Oxidative stress, caloric restriction, and
aging. Science 273, 59-63.
Sohal, R. S., Svensson, I. and Brunk, U. T. (1990). Hydrogen peroxide production by
liver mitochondria in different species. Mech. Ageing Dev. 53, 209-215.
Speakman, J. R. and Selman, C. (2011). The free-radical damage theory:
accumulating evidence against a simple link of oxidative stress to ageing and
lifespan. Bioessays 33, 255-259.
Tolmasoff, J. M., Ono, T. and Cutler, R. G. (1980). Superoxide dismutase: correlation
with life-span and specific metabolic rate in primate species. Proc. Natl. Acad. Sci.
USA 77, 2777-2781.
Victor, V. M., Rocha, M. and De la Fuente, M. (2004). Immune cells: free radicals
and antioxidants in sepsis. Int. Immunopharmacol. 4, 327-347.
Wibbelt, G., Moore, M. S., Schountz, T. and Voigt, C. C. (2010). Emerging diseases
in Chiroptera: why bats? Biol. Lett. 6, 438-440.
Wilhelm Filho, D., Althoff, S. L., Dafré, A. L. and Boveris, A. (2007). Antioxidant
defenses, longevity and ecophysiology of South American bats. Comp. Biochem.
Physiol. 146C, 214-220.
Wilkinson, G. S. and South, J. M. (2002). Life history, ecology and longevity in bats.
Aging Cell 1, 124-131.
Wood, J. L., Leach, M., Waldman, L., Macgregor, H., Fooks, A. R., Jones, K. E.,
Restif, O., Dechmann, D., Hayman, D. T., Baker, K. S. et al. (2012). A framework
for the study of zoonotic disease emergence and its drivers: spillover of bat
pathogens as a case study. Philos. Trans. R. Soc. B 367, 2881-2892.
THE JOURNAL OF EXPERIMENTAL BIOLOGY
