General and Comparative Endocrinology 250 (2017) 46–53

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General and Comparative Endocrinology

journal homepage: www.elsevier.com/locate/ygcen

Research paper

The nexus of hair corticosterone level, immunocompetence, metabolic

rates and overwinter survival in the root vole, Microtus oeconomus
_ a,⇑, Karol Zub b, Paulina A. Szafrańska b, Monika Wieczorek b, Marek Konarzewski a
Aneta Ksia˛zek
a
Institute of Biology, University of Białystok, Ciołkowskiego 1J Street, 15-245 Białystok, Poland
b
Mammal Research Institute, Polish Academy of Sciences, Waszkiewicza 1, 17-230 Białowiez_ a, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Although corticosterone (CORT) regulates many physiological mechanisms, the associations between
Received 21 July 2016 CORT levels, immunocompetence, energy expenditures and overwinter survival have not been examined.
Revised 11 April 2017 Here, we studied individual variation in CORT level extracted from hair, immunocompetence quantified
Accepted 30 May 2017
as the neutrophil-to-lymphocyte (N/L) ratio, total white blood cells (WBC) and natural antibody levels
Available online 31 May 2017
(NAbs), along with the resting (RMR) and peak metabolic rates (PMR) and mortality during three consec-
utive winter seasons in a natural population of the root vole, Microtus oeconomus. In early winter, hair
Keywords:
CORT level was strongly positively associated with body mass and inversely related to voles’ ability to
Hair corticosterone
N/L ratio
survive. We suggest that the observed association between hair CORT level and body mass may be the
Infestation with Babesia spp. key component of the physiological nexus driving the survivorship of individual rodents. Additionally,
Body mass hair CORT was a significant predictor of variation of the whole body RMR, which in turn enhanced over-
RMR winter survival in the studied population. On the other hand, hair CORT was not significantly associated
Overwinter survival with changes in the blood indices. Interestingly, the analysis carried out only during the first year of study
(2008), which was characterized by a high population density and prevalence of infestation with a blood
protozoan, Babesia spp., showed that the intensity of the infestation was negatively correlated with both
the hair CORT level and the N/L ratio. Because CORT is often considered immunosuppressive, we expected
a positive association between its level and the N/L ratio. However, hair CORT did not significantly cor-
relate with the N/L ratio. We suggest that the lack of an association between hair CORT and the N/L ratio
resulted from a small inter-individual variation in the N/L ratio in 2008, which was much higher and less
variable than in the other years of our study.
Ó 2017 Elsevier Inc. All rights reserved.

1. Introduction documented that CORT also regulates other physiological mecha-

Apart from cortisol, corticosterone (CORT) is a key hormonal
component of the hypothalamo-pituitary-adrenal (HPA) axis gov-
erning physiological and psychological responses to stress in many
vertebrates (Russell et al., 2012; Scorrano et al., 2015). The effect of
CORT on immune responses has been particularly extensively
explored, and numerous studies have documented that long-term
(chronic) stress and/or experimentally elevated CORT level tend
to be immunosuppressive (review in Demas et al., 2010, see also
Bourgeon et al., 2010; McEwen et al., 1997; Martin et al., 2005;
Nelson et al., 2002; Yadav and Haldar, 2014). Apart from the widely
studied relationships between CORT and immunity, it has been
⇑ Corresponding author.
_
E-mail addresses: anetak@uwb.edu.pl (A. Ksia˛zek), karolzub@ibs.bialowieza.pl
(K. Zub), pszafran@ibs.bialowieza.pl (P.A. Szafrańska), monikawieczoreq@wp.pl
(M. Wieczorek), marekk@uwb.edu.pl (M. Konarzewski).
nisms, e.g., the body mass-energy expenditures axis (Buehler
et al., 2012; Dlugosz et al., 2012; Downs et al., 2013; Schmidt
et al., 2012; Versteegh et al., 2012; Wang and Wang, 2000), and
ultimately affects survival rates (Breuner et al., 2008; Cote et al.,
_
2006; Ksia˛zek et al., 2014; MacDougall-Shackleton et al., 2009;
Marasco et al., 2015; Sinclair and Lochmiller, 2000). To our knowl-
edge, however, the nexus between CORT, immunocompetence, the
body mass-energy expenditures axis and survival has not been
examined, even though each of its separate components has
already attracted considerable attention. For example, several
studies have documented that baseline or induced CORT levels
negatively correlated with survival rates, but no direct references
were made to immunocompetence and/or energy expenditures
(Goutte et al., 2010; MacDougall-Shackleton et al., 2009; Ouyang
et al., 2013). On the other hand, Versteegh et al. (2012) found cor-
relations between metabolic rate, various indices of immunity and

http://dx.doi.org/10.1016/j.ygcen.2017.05.021
0016-6480/Ó 2017 Elsevier Inc. All rights reserved.
 A. Ksia˛z_ ek et al. / General and Comparative Endocrinology 250 (2017) 46–53
CORT level at the individual or subspecies level but did not report
simultaneous evaluation of their associations with survival rates.
_
In our previous study (Ksia˛zek et al., 2014), we reported signif-
icant correlations between individual variation in metabolically
costly components of immunocompetence quantified as the
neutrophil-to-lymphocyte (N/L) ratio and total white blood cells
(WBC; see Lee and Klasing, 2004 and Lee, 2006), along with the
resting (RMR) and peak (PMR) metabolic rates and mortality dur-
ing three consecutive winter seasons in a natural population of a
small herbivorous rodent – the root vole, Microtus oeconomus.
More specifically, during the first year of study, which was charac-
terized by a high prevalence of infestation with the blood parasite
Babesia spp., the intensity of infestation was inversely correlated
with the N/L ratio. Moreover, the voles with high N/L ratios were
not only less infested but also more able to survive the second part
of winter (between January and March; Ksia˛zek _ et al., 2014). Thus,
although those analyses were based on a single blood sample taken
in the autumn, they nevertheless provided information on physio-
logical state of individual voles later in the winter season. In the
present study, we link the above findings with individual variation
in CORT level extracted from hair harvested at the beginning of
each of those three consecutive winters. We paid special attention
to possible associations between CORT, infestation with Babesia
spp. and blood indices representing the constitutive arm of innate
(N/L, WBC) and adaptive immunity (natural antibodies; NAbs). We
also analysed those associations taking into account postulated
links between CORT, body mass and metabolic rates. The latter
two have been already shown to affect voles’ winter survival
_
(Ksia˛zek et al., 2014; Zub et al., 2014).
To our knowledge, the present study is among the first to report
CORT variation in hair harvested from a natural population of
rodents. Such an assay offers a number of advantages over alterna-
tive sources of CORT like blood, saliva, urine or faeces (e.g., Accorsi
et al., 2008; Bennett and Hayssen, 2010; Galuppi et al., 2013; Koren
et al., 2002). Hair is safe, readily available, and easy to store and
transport. Most importantly, hair CORT level is insensitive to the
effect of stress episodes of capture and handling and as such is par-
ticularly fitting for studies on wild animals (Koren et al., 2002;
Yang et al., 1998). Like other glucocorticoid hormones, CORT is
deposited in hair by the internal blood vessel that nourishes it
and is subsequently embedded in the hair. Hair samples thus offer
an integrated record of CORT concentration deposited over an
extended time frame (weeks to months), which is distinctly suit-
able for evaluating long-term stress in wildlife populations
(Ashley et al., 2011; Koren et al., 2002).
2. Materials and methods
2.1. Study site and handling of animals
The study was conducted in Biebrza National Park (ca. 53°N,
23°E) in northeastern Poland. The studied population inhabited a
1 ha plot situated in a mire habitat with 85% coverage with fibrous
tussock-sedge, Carex appropinquata, forming a hummock-hollow
structure. Our study site was enclosed with a fence 70 cm high,
covered with plastic and dug down to a depth of 30 cm to prevent
voles from migrating and to protect them from small predators
such as weasels and stoats.
We conducted our study during three consecutive winters
(2008/2009, 2009/2010, and 2010/2011). In November of each
winter, wooden live box traps baited with grain and pieces of car-
rot were used to capture voles on the study plot. Captured animals
were transported to a nearby field station and placed individually
in standard laboratory cages under semi-natural conditions (at an
47
outdoor ambient temperature and photoperiod). Voles resumed
feeding and maintained body mass, so they were not stressed by
captivity. During the next three days, they were subjected to the
resting (RMR) and peak (PMR) metabolic rate measurements, fol-
lowed by blood sampling (200–300 ll) from the retro-orbital sinus
with heparinized capillaries, and then hair sampling for CORT level
determination. Voles were then marked with subcutaneously
implanted microchips (Microchip DS.CO. S. C., Poland), released
near their site of capture, and recaptured later (in January) for sur-
vival rate monitoring. The trapping was continued until there were
no more captures in the enclosure over period of three consecutive
days. If an individual was not present during this recapture session
that we assumed it died because migration from our fenced study
site was not possible. Overall, in November 2008, 2009 and 2010
we captured 202, 117 and 59 root voles, respectively. In January
of the subsequent years, we recaptured 83, 31 and 12 individuals,
respectively.
2.2. Metabolic rate measurements
Resting metabolic rate (RMR) measurements were carried out
during the day, over the 2–4 h trial period at 30 °C, a temperature
within the thermoneutral zone (Wang and Wang, 2000). We used a
computer-controlled positive-pressure, open-circuit respirometry
system (Sable Systems TR-1 setup, Las Vegas, NV, USA) to collect
the measurements. We defined RMR as the 4 min with the lowest
metabolic rate, during which oxygen concentration yielded the
highest value of repeatability and did not change more than
0.01%. Oxygen consumption rate was calculated by using equation
five in Hill (1972), assuming respiratory quotient = 0.8 (Koteja,
1996).
Peak metabolic rate (PMR) was induced by forcing voles to
swim for 10 min. We used the same metabolic set-up as described
above, but fitted with a metabolic chamber that was 75% filled
with a 25 °C water. We defined PMR as the highest oxygen con-
sumption in a two-minute running average during a swim trial
for a given individual. Details of the metabolic rate measurements
are provided in our previous work (Szafrańska et al., 2007; Zub
et al., 2012, 2014).
2.3. Blood parameters and parasites
All blood samples were collected between 18.00 and 20.00 next
day following completion of measurements of metabolic rates. We
did not repeatedly sampled same individuals, because the incurred
stress could adversely affect their condition and therefore their
future survival. However, all blood smears and indices were
screened and subsequently calculated by the same observer (A.
_
Ksia˛zek), so we can safely exclude any observer-related bias and/
or inconsistencies.
For counting leukocytes, a drop of blood was smeared on indi-
vidually marked microscope slides, air-dried, fixed in absolute
methanol, and stained by the May-Grunwald-Giemsa method.
The slides were then scanned under 1000 magnification, and
the absolute WBC count was estimated as the number of leuco-
cytes per 10,000 erythrocytes. To examine the neutrophil/lympho-
cyte (N/L) ratio, we counted 100 leucocytes per sample and
classified them as neutrophils or lymphocytes (Ksia˛zek _ et al.,
2014; Masello et al., 2009; Ots et al., 1998; Parejo and Silva,
2009; Saks et al., 2003).
We measured natural antibody levels (NAbs) following Matson
et al. (2005). To isolate serum, we centrifuged blood at 4 °C and
2500 rpm for 15 min. To develop the agglutination reaction, we
used a suspension of whole rabbit blood in Alsever’s solution
48 A. Ksia˛z_ ek et al. / General and Comparative Endocrinology 250 (2017) 46–53
(Pro Animali, Wrocław, Poland; supplied as 50% whole blood, 50%
Alsever’s) and then prepared the 1% red cells suspension. All serum
samples were run in duplicate.
We analysed infection with the tick-borne protozoan Babesia
spp. in a subset of 39 individual voles randomly selected from
among the animals studied in the autumn 2008 – a year of high
prevalence of Babesia spp. in the studied population (Kloch
et al., 2012). Eight out of those 39 voles have also been included
in our earlier analysis of the association between infestation with
Babesia spp. and immune indices (Ksia˛zek _ et al., 2014). The
remaining 31 individuals were screened for parasites for the first
time. The parasites were identified in the same smears as used for
estimating the N/L ratio and WBC counts under 1000 magnifica-
tion, based on their colouration, size and shape. The intensity of
infestation was quantified as the number of erythrocytes infested
with Babesia spp. counted in 100 fields of view (approx. 100,000
red blood cells) as described in (Kloch et al., 2012; Ksia˛zek _
et al., 2014).
2.4. Hair collection and CORT level determination
Hair CORT level was determined in 209 individuals (52, 112 and
45 in November 2008, 2009 and 2010, respectively). Hair samples
(40 mg) of the same length were manually cut with animal scis-
sors, and were consistently sampled from the upper part of the
right hind leg (opposite to the right groin). All samples were col-
lected at the same time point (between 09.00 and 10.00) next
day following blood sampling, then placed in dry vials, and stored
at 80 °C until further analysis. The protocol for obtaining hair
CORT was slightly modified from Koren et al. (2002), in which
methanol-based CORT extraction was performed using pulveriza-
tion in a ball mill (QIAGEN, Venlo, the Netherlands) instead of son-
ication. Briefly, each sample, consisting of 20–22 mg of hair, was
minced into 3–4 mm pieces with fine scissors and then placed into
a clean tube. Methanol was added, and each sample was pulverized
for 25 min using a single 5 mm stainless steel grinding ball and
then incubated overnight at 50 °C with gentle shaking. The metha-
nol was then pipetted off into glass tubes and evaporated to dry-
ness under a stream of nitrogen. Samples were then
reconstituted with phosphate buffered saline (PBS; pH = 7.0).
The hair CORT concentrations were assayed using the specific
Corticosterone EIA Kit (enzyme-linked immunosorbent assay) in
accordance with the manufacturer’s recommendations (Cayman
Chemical Company, Ann Arbor, MI, USA). The optical density
(OD) of each sample was determined using an automated micro-
plate reader (Bio-Tek Instruments, Inc., Vermont, USA) equipped
with a 405-nm wavelength filter. According to the manufacturer,
cross-reactivity of the antibody used for this kit was as follows:
corticosterone (100%), 11-dehydrocorticosterone (11%), 11-
deoxycorticosterone (7%), progesterone (0.31%) and cortisol
(0.17%). All other hormones and intermediates tested by the
manufacturer exhibited cross reactivity of 0.06%. To validate
EIA corticosterone measurements in hair, parallelism, linearity
and intra- and inter-assay tests were performed. The tests of
parallelism were carried out by comparison of 12 serially diluted
hair extracts to the standard curve generated with kit calibrators
run in the same assay, and exemplified by a representative serial
dilution in Fig. 1S (see Supplementary materials). Visual inspection
shows a close fit between the curves produced by the serially
diluted hair sample and the corticosterone standards, and this
fit was confirmed by regression analysis (r2 = 0.992, P < 0.01). Lin-
earity of dilution was evaluated by assaying 3 different dilutions of
2 independent hair extracts run in duplicate, and then calculating
the rate of recovery by comparison between the observed and the
expected values (e.g. Moya et al., 2013). This procedure assesses
predictability of natural sample recovery for known dilution of
standard range. A good predictability between the observed and
the expected values was found (see Fig. S2 in Supplementary mate-
rials). Rates of recovery from the comparisons between serial
diluted hair samples and standard curve values are summarized
in Table S1 (Supplementary materials). These results provide
strong support for the specificity of our procedure of determination
of hair corticosterone concentrations. The average coefficient of
variation for 2 replicates performed in the same assay (intra-
assay CV) was 6.8%.
For statistical analyses, the hair CORT level was expressed in pg/
mg of hair. The approximate limit of detection of our procedure
was 0.03 pg/mg, based on the lowest standard provided in the
assay kit.
2.5. Statistical methods
Data on blood indices, hair CORT level, metabolic rates, body
mass and Babesia spp. infestation intensity were log-transformed
prior to analyses.
For statistical analyses of CORT effects we used general linear
mixed models (procedure MIXED in SAS software). We first
checked for the intra-assay repeatability of CORT measurements
and possible systematic differences between used assay plates by
means of a model with assay plate as a fixed factor and identity
of voles and year of study as random factors. This corroborated a
highly significant repeatability of CORT estimates within individual
(z = 9.42, P < 0.001) and the lack of significant differences between
assay plates (F6,155 = 0.23, P > 0.9). Consequently we dropped the
effect of assay plates from further analyses.
To estimate the effects of hair CORT on metabolic rates and
blood indices we used models including sex (fixed factor) and year
of study (as a random factor, controlling for, among others, a pos-
sible effect of the between-year variation in population density)
along with hair CORT as a covariate and the respective interactions.
In another set of similarly structured models, we also incorporated
body mass as a covariate in order to analyse body-mass-corrected
effects.
We used a separate set of the models described above to inves-
tigate the associations between hair CORT, blood indices and the
intensity of infestation with Babesia spp. in 2008. Additionally,
2008 was the only study year when, thanks to sufficiently high
summer population densities, we were able to collect data on
pre-winter changes in body mass and age (for further details on
study area and handling of animals see Zub et al., 2014). Here,
we used repeated measurements of body mass obtained for each
individual during a minimum of two subsequent trappings carried
out between March and November and associated them with the
hair CORT level before the winter of 2008. In this mixed-model
analysis, individual body mass change was a response variable,
sex was a fixed factor, and individual ID number was a random fac-
tor, along with hair CORT level and age as covariates.
We applied logistic regression to determine the effect of varia-
tion in hair CORT on winter survival of voles. Initially, we aimed to
analyse this effect on the survival in early (November–January) and
late (January–March) winter. However, the small sample size of
voles re-captured in March forced us to restrict our analysis to only
the first part of the winter season. We constructed a set of candi-
date models including the effects of hair CORT level, body mass,
RMR, PMR and sex on survival probability between November
and January, and we used a model-selection procedure (Burnham
and Anderson, 2002) with models ranked by AICc (corrected
Akaike’s Information Criterion). To compare models, we used delta
AICc, a measure of each model relative to the best model, and as a
rule of thumb, all models for which Di < 2 were regarded as equally
 A. Ksia˛z_ ek et al. / General and Comparative Endocrinology 250 (2017) 46–53 49

supported (Burnham and Anderson, 2002). When more than one

model was supported, we used the confidence set of candidate
models, which included models with Akaike weights that were
within 10% of the value of the highest ranked model. Finally, we
estimated average values of parameters along with confidence
intervals, calculated using unconditional standard errors
(Burnham and Anderson, 2002). The significance of the random
effect (year) was determined using likelihood ratio tests ( 2 times
the difference in log likelihoods between model with and without
random term, estimated using maximum likelihood, tested against
a v2 distribution with one degree of freedom). All above analyses
were carried out using package lme4 of R (Bates et al., 2015).

3. Results

3.1. Between-year variation in hair CORT level

Hair CORT levels did not differ between years of study (z = 0.83,
P = 0.2) but were higher in males (F1,202 = 1.2, P = 0.01) and posi-
tively associated with body mass (F1,202 = 22.5, P < 0.001; Fig. 1).
Hair CORT was a significant predictor of variation of the whole-
body RMR (F1,181 = 16.7, P < 0.001) but not the whole-body PMR
(F1,196 = 1.5, P = 0.2). However, hair CORT did not affect either body
Fig. 2. Correlation between log-transformed hair corticosterone level (corrected for
the effects of sex) and log-transformed neutrophil/lymphocyte ratio during early
winter from three consecutive years of study. Open circles, individuals captured in
2008; filled circles, voles captured in years 2009–2010. Please note that in 2008, the
N/L ratio was much higher than in other years of study (ANCOVA, effect of year:
F1,198 = 126.0, P < 0.001).

mass-corrected RMR (F1,180 = 0.6, P = 0.4) or body mass-corrected

PMR (F1,196 = 0.9, P = 0.3).
When uncorrected for body mass, hair CORT was also insignifi-
cant as a covariate in any of the analyses of the between-year vari- 1.0 0
ation of blood parameters (N/L ratio, WBC and NAbs; in all cases, 30
P > 0.1). However, body-mass-corrected hair CORT was inversely
0.8
probability of survival

correlated with N/L ratio (P = 0.02; Fig. 2). 60

The hair CORT level was inversely related to survival in early
winter (November–January) in models correcting for the effect of

frequency
body mass, as well as in models without body mass (Fig. 3; Table 1). 0.6
In none of the models sex was significant predictor of survival
probability.
0.4
3.2. Hair CORT level variation in 2008
60
0.2
We observed a significant inverse association between the
changes in individual body mass preceding the winter season 30
and hair CORT (F1,32 = 5.8, P = 0.02; Fig. 4), whereas sex was not sig-
0.0 0
nificant as a fixed factor (F1,32 = 1.3, P = 0.3), and age was not signif-
icant as a covariate (P > 0.2). Additionally, hair CORT level was 2.2 2.4 2.6 2.8 3.0 3.2 3.4
hair corticosterone level (pg/mg)
Fig. 3. Probability of vole survival (red line, left axis) and frequency distribution of
corticosterone level (adjusted for the effects of sex and body mass, right axis)
during early winter (November–January). Bars indicate distribution of corticos-
terone for animals that survived (upper part of the figure) and that did not survive
(lower part of the figure). Please note that highest values of corticosterone levels are
associated with decreasing probability of survival. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version
of this article.)

significantly negatively associated with the intensity of infestation

Fig. 1. Correlation between hair corticosterone level (log-transformed, corrected
for the effects of year and sex) and body mass during early winter from three
consecutive years of study. Open circles, females; filled circles, males.
with Babesia spp. (ANCOVA; F1,36 = 6.7, P < 0.01), and body mass
and the body mass  hair CORT interaction were significant as
covariates (F1,36 = 5.5, P = 0.02 and F1,36 = 6.5, P = 0.01, respec-
tively), while sex was not significant as a fixed factor (F1,36 = 1.0,
P = 0.3). On the other hand, hair CORT was not significant as a
covariate in any of the analyses of the variation of blood indices
(P > 0.1 in all cases). In particular, hair CORT was not a significant
predictor of the N/L ratio (F1,36 = 0.82, P = 0.4) even though the
N/L ratio was significantly inversely correlated with the intensity
of infestation (r = 0.39, P = 0.01, n = 37).
50 A. Ksia˛z_ ek et al. / General and Comparative Endocrinology 250 (2017) 46–53
Table 1
Estimates of averaged model parameters with standard error (SE) and 95% confidence
intervals for the effect of hair corticosterone level, body mass, resting (RMR) and peak
(PMR) metabolic rates and sex on the survival probability of voles between November
and January. Coefficients were averaged over 10 equally supported candidate models
according to AICc criterion (with body mass, Table S2 in Supplementary materials)
and over 4 candidate models (without body mass, Table S3 in Supplementary
materials). Metabolic rates were not significant in any set of models and therefore
were omitted in the final calculations.
Predictor Coefficient SE 95% confidence intervals
With body mass
Hair CORT level 1.485 0.788 3.058, 0.005
Sex 0.672 0.425 1.512, 0.175
Body mass 0.349 0.281 0.757, 0.143
Without body mass
Hair CORT level 1.626 0.738 3.100, 0.220
Sex 0.703 0.419 1.541, 0.117
Fig. 4. Correlation between pre-winter body mass changes and log-transformed
hair corticosterone levels in voles captured in November 2008.
4. Discussion
The relevance of the CORT level assayed from hair hinges on the
assumption of its close association with key biological functions
and processes related to energy management, immune response
and behaviour. CORT is most likely deposited in hair over a period
of weeks if not months (e.g., Scorrano et al., 2015), which makes
the hair assay particularly useful as a measure of hormonal activity
averaged over long periods of time. In principle, this measure is
likely to be insensitive to the effect of stress episodes caused by
handling during sampling procedures (Accorsi et al., 2008;
Ashley et al., 2011; Koren et al., 2002) and as such should be par-
ticularly appropriate for studies on wild animals. Due to voles’
short lifespan, the individuals captured in November were no older
than 3–6 months; thus, the CORT extracted from hair represented
the level accumulated during a significant part of their life, cover-
ing periods when voles experienced fluctuations of CORT secretion
resulting from short- and long-term interactions with environmen-
tal stressors. Individuals differ in their perception of environmental
conditions, and the strength of these responses depends on activa-
tion of the HPA axis, which in turn, through activation of the ante-
rior pituitary gland, stimulates the release of glucocorticoids
(Demas et al., 2010). Therefore, in our work CORT assayed from
hair should correspond to an average, rather than the baseline level
of this hormone, thus reflecting the individual susceptibility of the
studied voles to stress.
To our knowledge, there have been no reports validating the
measurement of CORT in hair of wild rodents. Nevertheless, several
studies on captive or domesticated animals demonstrated a posi-
tive correlation between individual hair and faecal, salivary, urine
or blood concentrations of glucocorticoid hormones (including
CORT) in cats and dogs (Accorsi et al., 2008; Bennett and
Hayssen, 2010), rhesus macaques (Davenport et al., 2006), rabbits
(Trocino et al., 2014) and rats and mice (Yu et al., 2015). These
studies lend support to the basic conjecture of the present study
that CORT assayed from hair is a useful marker of the overall indi-
vidual sensitivity of the hypothalamo-pituitary-adrenal (HPA) axis
to environmental stress.
In the case of wild animals, high population densities are con-
sidered to be a strong environmental stressor likely to drive popu-
lation cycles (Dettmer et al., 2014; Novikov and Moshkin, 1998;
Rogovin et al., 2008), mostly through direct competition for food
and high incidence of antagonistic encounters (Judge and De
Waal, 1997; Moshkin et al., 2003; Yamanashi et al., 2013). The
studied population of root voles undergoes cyclic changes in den-
sity, ranging from ca 60 to 300 individuals per hectare (Zub
et al., 2014). Additionally, the years of our study were character-
ized by a wide range of variation in population density, from 202
to 59 individuals/ha in November 2008 and 2010, respectively
_
(Ksia˛zek et al., 2014; present study). However, we did not observe
a significant between-year variation in the hair CORT level, which
suggests the lack of its direct correlation with population density.
Yet, we found a significant inverse association between the hair
CORT level and the probability of individual survival of the first
part of winter, from November to January (Fig. 3 and Table 1).
The hair CORT level was also strongly positively associated with
body mass (Fig. 1), the main predictor of winter survival in the
studied population (Zub et al., 2014).
A direction of a strong positive association between hair CORT
and body mass is consistent with their inverse effects on survival
through the first part of winter (Fig. 3, see also Zub et al., 2014).
Both theoretical Ergon et al. (2004) and field studies (Aars and
Ims, 2002; Gliwicz, 1996; Hansson, 1990) indicate that pre-
winter body mass and size of voles is fine-tuned to the anticipated
overwinter conditions, with body mass being lower during more
demanding conditions (i.e., low food availability or inclement
weather). Interestingly, in a studied population, voles born early
in the summer are significantly heavier/larger than those born
later (Zub et al., 2014). Before winter, however, their body masses
converge, as early- and late-born individuals tend to reduce and
gain mass, respectively (Zub et al., 2014). Our results indicate that
this process is at least partly related to hair CORT, as we observed
an inverse correlation between the magnitude of the body mass
change before winter and the hair CORT level (Fig. 4). This relation-
ship may also partly explain the higher hair CORT levels in larger
individuals. It is also important to note that this relation was unaf-
fected by age, which suggests that the hair CORT level was not
associated with the possible accumulation of the effects of stressful
incidents encountered by older individuals. Our results, therefore,
suggest that the observed association between hair CORT level
and body mass may be the key component of the physiological
nexus driving survivorship of individual rodents.
Our results indicate that low hair CORT enhances survival
(Fig. 3), even when the effect of hair CORT is corrected for body
mass (Table 1). The putative mechanisms, however, remain uncer-
tain. The only significant association found in the analysis of the
between-year hair CORT level variation was a positive relationship
between the hair CORT level and the whole-body RMR. This asso-
ciation can be considered as mostly driven by strong positive links
between body mass and both hair CORT and RMR (Zub et al., 2014;
present study), as it became insignificant when corrected for body
 A. Ksia˛z_ ek et al. / General and Comparative Endocrinology 250 (2017) 46–53
mass. We also did not observe significant associations between
hair CORT and the swim-induced PMR (the latter being a predictor
of probability of survival of root voles living in periodically flooded
sedge meadows; see Zub et al., 2014). However, it is important to
note that in our previous work, which was conducted on a larger
sample of voles from the same population (Ksia˛zek _ et al., 2014),
we showed that individuals with higher body mass-corrected
PMR were more likely to survive early winter (between November
and January). In both laboratory and wild rodents, swimming elic-
its emotional stress, which impairs aerobic performance (Marek
and Szacki, 1988) and elevates serum corticosteroid levels (Beiko
et al., 2004; Rogóz_ et al., 2009, 2012). Thus, although it was not
detected in our study, the well-documented inverse association
between emotional stress and swim-induced PMR, along with a
positive association between stress and CORT, may account for
the observed associations between probability of survival and hair
CORT reported here for early winter, when voles were forced to
swim in their partly flooded habitat (Zub et al., 2012).
At first, the above mechanism is difficult to reconcile with the
negative correlation between body mass-corrected hair CORT
level and the intensity of infestation with Babesia spp. (see
Results), the latter being a mortality factor in the studied popula-
tion (Kloch et al., 2012). Babesia spp. reduced the survival of its
carriers during the second part of winter (between January and
March; Kloch et al., 2012). Early part of this winter (between
November and January) was warm, which caused flooding of vole
habitat and forced them to swim (Zub et al., 2012). This suggests
that in 2008 a positive association between early-winter survival
and high PMR (Ksia˛zek _ et al., 2014; Zub et al., 2014) selected
individual voles characterized by above-average tolerance to
swim-induced stress but also low hair CORT (Fig. 3). Later in the
season, however, individuals with low hair CORT levels were
likely to fall victim to their high levels of infestation with Babesia
spp. (Kloch et al., 2012).
The observed inverse link between hair CORT and infestation
with Babesia spp. clearly requires further discussion and clarifica-
tion because CORT is often considered immunosuppressive (Davis
et al., 2008; Demas et al., 2010), and therefore, a positive, not neg-
ative, correlation between hair CORT and Babesia spp. infestation
could be expected. Furthermore, a positive association between
hair CORT and N/L ratio would be expected because high CORT
levels trigger re-distribution of neutrophils (elevation of counts)
and lymphocytes (reduction of counts, for review, see Davis
et al., 2008). However, this has not been supported by our analysis,
as we found a negative, not positive, association between the hair
CORT level and the N/L ratio across all years of study (Fig. 2). In
particular, we did not find a significant link between the hair CORT
level and the N/L ratio in 2008, which was the year of a high preva-
lence of Babesia spp. in our voles (Kloch et al., 2012). The lack of the
correlation between the hair CORT level and the N/L is unlikely to
stem from limited sample size and/or the low power of the applied
statistical tests, as both the association between body mass-
corrected hair CORT level and infestation and particularly that
between the N/L ratio and infestation (see Results) were highly sta-
tistically significant. It is more likely that the lack of the expected
positive link between these parameters resulted from a small
inter-individual variation in the N/L ratio in 2008, considering that
both voles with a low hair CORT level and those with a high hair
CORT level were characterized by similarly high N/L ratios, much
higher than in the other years of our study (Fig. 2).
Our results add to a growing number of studies chiefly carried
out on birds and showing the inconsistent nature of the links
between corticosterone, parasite infection and related blood
indices (e.g., Kitaysky et al., 2001; Quillfeldt et al., 2004; Raouf
et al., 2006 vs. Bize et al., 2010; Eggert et al., 2010; Lobato et al.,
2008). Those inconsistencies may, however, reflect differences in
51
the conditions of studies rather than genuine differences in basic
physiological mechanisms. For example, apart from an exception-
ally high N/L ratio reported therein for 2008, the lack of a positive
association between hair CORT and N/L ratio may stem from a
stark difference between the monthly time scale represented by
the CORT level in voles’ hair and the short-term fluctuations in
the N/L ratio, which may change in a matter of hours (review in
Davis et al., 2008). However, the deferred association of N/L ratio
with voles’ overwinter survival (Ksia˛zek_ et al., 2014), as well as
results from other studies (e.g., Gilot-Fromont et al., 2012;
Lobato et al., 2005; Xuereb et al., 2012), attest to a significant pre-
dictive power for this blood index. On the other hand, the other
studied blood indices, WBC and NAbs, were not associated with
Babesia spp. infestation intensity, hair CORT level, or voles’ survival
_ et al., 2014; present study). This suggests that Babesia spp.
(Ksia˛zek
was latent at the onset of winter, when those parameters were
evaluated, and was not sensed as a stressor that would increase
the hair CORT level. Therefore, apart from the differences in
time-scales presented above, this putative mechanism may explain
the lack of an expected association between hair CORT and the N/L
ratio.
In conclusion, to our knowledge, we are the first to report a
strong positive correlation between body mass and CORT assayed
in rodents’ hair in a natural population, which points to an involve-
ment of the hair CORT level in pre-winter body mass regulation. As
expected, this body mass-hair CORT relationship significantly
affected winter survival of the root voles, with small individuals
with lower hair CORT levels faring better chances of surviving at
least the first part of the winter season. The above adds to the dis-
cussion on a negative relationship between CORT levels and fitness
(e.g., Bonier et al., 2009). However, we were unable to demonstrate
an unequivocal relationship between the hair CORT level and the
peak or resting metabolic rates or the blood indices examined,
even though all of those factors were shown to significantly affect
overwinter survival in the studied population. Our results suggest
that the associations between the N/L ratio, infestation with Babe-
sia spp. and CORT content in hair are complex and indirect. Never-
theless, our work strongly suggests that studies on CORT deposited
in hair collected under natural conditions may shed light on the
links between blood indices related to parasite infection and life
history traits of wild rodents.
Acknowledgments
This work was supported by a Ministry of Science and Higher
Education (MNiSW) grant [no. N N304 362038] to A.K. This
research has been approved by the Local Ethical Committee on Ani-
mal Testing at the Medical University of Białystok (permits no.
2008/26 and 1/2009).
We thank Staszek Płonowski, Paweł Brze˛k, and Zbigniew Bor-
owski as well as many students and volunteers for their help with
fieldwork and technical assistance.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ygcen.2017.05.
021.
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