Magnetoreception in the wood mouse (Apodemus sylvaticus): Influence of weak frequency-modulated radio frequency fields

Abstract

The mammalian magnetic sense is predominantly studied in species with reduced vision such as mole-rats and bats. Far less is known about surface-dwelling (epigeic) rodents with well-developed eyes. Here, we tested the wood mouse Apodemus sylvaticus for magnetoreception using a simple behavioural assay in which mice are allowed to build nests overnight in a visually symmetrical, circular arena. The tests were performed in the ambient magnetic field or in a field rotated by 906. When plotted with respect to magnetic north, the nests were bimodally clustered in the northern and southern sectors, clearly indicating that the animals used magnetic cues. Additionally, mice were tested in the ambient magnetic field with a superimposed radio frequency magnetic field of the order of 100 nT. Wood mice exposed to a 0.9 to 5 MHz frequency sweep changed their preference from north-south to east-west. In contrast to birds, however, a constant frequency field tuned to the Larmor frequency (1.33 MHz) had no effect on mouse orientation. In sum, we demonstrated magnetoreception in wood mice and provide first evidence for a radical-pair mechanism in a mammal. T he ability to sense the geomagnetic field for use as a global reference during spatial orientation has been demonstrated in more than 30 species of vertebrates 1. The majority of studies have been performed on birds and have yielded valuable insights into the underlying magnetoreception mechanism 2. Extensive research has also been carried out on the detection and use of magnetic cues in amphibians and turtles e.g. 3,4,5

Full text

Magnetoreception in the wood mouse
(Apodemus sylvaticus): influence of
weak frequency-modulated radio
frequency fields
E. Pascal Malkemper
1
, Stephan H. K. Eder
2
, Sabine Begall
1
, John B. Phillips
3
, Michael Winklhofer
4,2
,
Vlastimil Hart
5
& Hynek Burda
1,5,6
1
Department of General Zoology, Faculty of Biology, University of Duisburg-Essen, 45117 Essen, Germany,
2
Department of Earth
and Environmental Sciences, Geophysics, Munich University, 80333 Munich, Germany,
3
Department of Biological Sciences,
Virginia Tech, Blacksburg, Virginia, United States of America,
4
Faculty of Physics, University of Duisburg-Essen, 47057 Duisburg,
Germany,
5
Department of Game Management and Wildlife Biology, Faculty of Forestry and Wood Sciences, Czech University of
Life Sciences, 16521 Praha 6, Czech Republic,
6
Faculty of Science, University of South Bohemia, Branisovska 31, 370 05 Ceske
Budejovice, Czech Republic.
The mammalian magnetic sense is predominantly studied in species with reduced vision such as mole-rats and
bats. Far less is known about surface-dwelling (epigeic) rodents with well-developed eyes. Here, we tested the
wood mouse
Apodemus sylvaticus
for magnetoreception using a simple behavioural assay in which mice are
allowed to build nests overnight in a visually symmetrical, circular arena. The tests were performed in the
ambient magnetic field or in a field rotated by 906. When plotted with respect to magnetic north, the nests were
bimodally clustered in the northern and southern sectors, clearly indicating that the animals used magnetic cues.
Additionally, mice were tested in the ambient magnetic field with a superimposed radio frequency magnetic field
of the order of 100 nT. Wood mice exposed to a 0.9 to 5 MHz frequency sweep changed their preference from
north-southtoeast-west.Incontrasttobirds,however,aconstant frequency field tuned to the Larmor frequency
(1.33 MHz) had no effect on mouse orientation. In sum, we demonstrated magnetoreception in wood mice and
provide first evidence for a radical-pair mechanism in a mammal.
The ability to sense the geomagnetic field for use as a global reference during spatial orientation has been
demonstrated in more than 30 species of vertebrates
1
. The majority of studies have been performed on birds
and have yielded valuable insights into the underlying magnetoreception mechanism
2
. Extensive research
has also been carried out on the detection and use of magnetic cues in amphibians and turtles e.g.
3,4,5
. In mammals,
research on magnetoreception is far less advanced, but the existing data support the idea of a phylogenetically
ancient sense
6,7
, with magnetosensitivity being reported in an increasing number of species including represen-
tatives of at least five different orders (reviewed in
8
).
Early homing and orientation experiments with epigeic rodents suggested that they might possess a magnetic
compass used for navigation
9,10
. Later studies, however, challenged these findings with negative results and failed
experimental replications
11,12
. It was not until the early 1990s that a robust behavioural assay was developed that
provided solid and replicable evidence for magnetoreception in mammals: the nest building assay
13
. This para-
digm, in which the directions (positions) of nests built along the wall of a circular (radially symmetrical) arena
relative to the magnetic field are analysed, has been used ever since with a variety of species, and has yielded the
first insights into the mechanisms of magnetoreception in mammals
14–22
.
The available evidence indicates that there are two biophysically distinct magnetoreception mechanisms in
terrestrial vertebrates: magnetic particles and light-dependent biochemical reactions that involve radical pair
intermediates. The magnetic particle mechanism assumes that the torque or force exerted by the Earth’s magnetic
field on particles of magnetite (or its oxidized form: maghemite) is transduced through direct effects on
membrane conductivity
23
or through the opening of membrane channels mediated by filamentous connections
or via membrane deformation
24–26
. The radical pair mechanism of magnetoreception is based on an effect of an
earth-strength magnetic field on the singlet-triplet interconversion rate of a spin-correlated radical pair formed
OPEN
SUBJECT AREAS:
ANIMAL BEHAVIOUR
NEUROPHYSIOLOGY
ANIMAL MIGRATION
BEHAVIOURAL ECOLOGY
Received
24 November 2014
Accepted
23 March 2015
Published
Correspondence and
requests for materials
should be addressed to
E.P.M. (pascal.
malkemper@uni-due.de)
SCIENTIFIC REPORTS | 5: 9917 | DOI: 10.1038/srep09917 1
29 April 2015

after photo-excitation (reviewed in
27,28
). A specialized class of retinal
photopigments, cryptochromes, the only animal photopigments
known to form radical pair intermediates, are thought to be involved
in the radical pair mechanism, leading to the suggestion that the
magnetic field may be perceived as a visual pattern superimposed
on the animal’s surroundings
29
. As this pattern would be axially
symmetric, it is consistent with the inclination compass found in birds
and amphibians that uses the slope direction of the field lines to distin-
guish polewards and equatorwards, rather than northwards and south-
wardsasinatruepolaritycompass
4,30
. Studies indicate, however, that the
two mechanisms, magnetic particle mechanism and radical pair mech-
anism, are not mutually exclusive, but at least birds and amphibians
might have both mechanisms that they use in different behavioural
contexts and/or to provide spatial and directional information
4,31
.
For rodents (and mammals in general) the majority of behavioural
and histological findings published so far have only provided
evidence for the involvement of a magnetic particle mechanism.
The compass of Ansell’s mole-rats is polarity sensitive (i.e., able to
distinguish magnetic north from south)
21
. Also, the magnetic com-
pass of mole-rats is affected by brief magnetic pulses with an intensity
high enough to re-magnetize magnetite particles
32
. The same prop-
erties were found for the magnetoreceptors of microphthalmic bats
33
.
Importantly, the magnetic compass of Ansell’s mole-rats was un-
affected by a treatment specifically designed to perturb the radical
pair mechanism using a RF field in the low MHz range (broadband as
well as Larmor frequency, i.e., 1.315 MHz)
16
over 300 times stronger
than that shown to cause disorientation in birds
34–39
.
More recent studies, however, raised the question of whether
epigeic rodents (i.e., species that unlike mole-rats are mostly active
above ground) could have a radical pair mechanism-based magnetic
compass. A nest building study of C57BL/6 mice has provided evid-
ence that these mice rely on a magnetic compass that exhibits an
axially symmetrical pattern of response, consistent with a radical pair
mechanism of magnetoreception
18
. In a more recent study, C57BL/6
laboratory mice were trained to remember the magnetic compass
direction of a submerged platform in a variation of the classic
Morris water maze assay, but the training was only successful in
the water maze study after the test room had been electromagnet-
ically shielded against ambient (i.e., man-made) radio frequency
(RF) noise in the frequency range between 0.1 and 100 MHz with
residual values lower than 0.1 nT
40
. Electromagnetic shielding was
also reported to be necessary for the stable compass responses in
the earlier nest building studies of C57BL/6J mice
18
and Siberian
hamsters
22
. Therefore, the current state of knowledge does not allow
firm conclusions to be drawn about the involvement of a radical pair
mechanism in magnetoreception in epigeic rodents, or in other
mammalian orders apart from mole-rats.
In this study we addressed two questions. First, do wood mice,
Apodemus sylvaticus, have a magnetic sense, as suggested by early
orientation experiments
10
? Second, if they have a magnetic sense, is it
sensitive to weak RF magnetic fields, which would point to the
involvement of a radical pair mechanism? To answer these questions,
we tested freshly captured animals with the classic nest building
assay and exposed them to different alignments of an earth-strength
magnetic field and to different low intensity RF-fields.
Results
Nest building preference in Apodemus sylvaticus.The vast majority
of tested mice built a single and clearly distinguishable nest at the wall of
the arena overnight (Figure 1). Plotting the nest directions of nests built
under control conditions (no RF) in the ambient (intensity: 49.03 mT)
and the 90urotated magnetic field (intensity: 49.20 mT) with respect to
magnetic north revealed highly significant bimodal orientation along
the north-northeast and south-southwest magnetic axis (Figure 2a). In
contrast, plotting the nests with respect to topographic north (i.e. the
absolute directions of the nests ignoring the alignment of the magnetic
field) failed to reveal a sig nificant clustering of bearings (Figure 2b). The
distributions of nests built in the two magnetic conditions were
significantly different (Table 1; Watson U
2
:p,0.01, U
2
50.293).
Influence of radio frequency magnetic fields.In both RF conditions
the oscillating fields were aligned vertically at an angle of 25uto the
geomagnetic field lines. Nests built in the ambient magnetic field
(intensity: 49.05 mT) under the influence of a Larmor frequency
oscillating magnetic field (1.33 MHz, 785 – 1,260 nT) showed a
significant bimodal distribution of bearings clustered along the
north-south axis (Figure 3a) that was indistinguishable from
controls (Watson U
2
:p.0.5, U
2
50.05).
In contrast, animals tested in a wideband-FM field (frequency
sweep from 0.9–5 MHz, 25–100 nT, at one msec intervals) displayed
a dramatic change in the distribution of nests relative to the geomag-
netic field, with the axis of orientation rotated by approximately 90u.
The mice now built their nests in the northwest and southeast sectors
with a clear preference for the latter (Figure 3b). Importantly,
the nests built under the two RF conditions showed significantly
different distributions (Watson U
2
:p,0.01, U
2
50.26).
Discussion
The results provide clear evidence for a magnetic sense in wood mice.
When building a nest in an otherwise featureless environment, the
Figure 1
|
The nest building assay. a. The arena (50 cm diameter) was
prepared with hay and saw dust as nest building material and an apple slice
and grain as food. A wood mouse was placed in the arena and the setup was
covered with a frosted white PVC-sheet overnight. b. On the next day the
direction of the nest was measured as from the centre of the arena (arrow).
Figure 2
|
Orientation of wood mice nests built in a visually symmetrical
circular arena. Each data point in the circular diagrams (upper panel)
represents the position of a nest built by an individual mouse. The figure
shows the pooled nest positions of mice tested in the ambient field (filled
circles) and with magnetic north shifted 90ucounterclockwise (open
circles). a. Bearings relative to magnetic north (mN) in the arena.
b. Absolute bearings in the arena. Arrows give the mean vector for the
distribution of the nests, the dotted lines are the 95% confidence intervals
for the mean bearing (m) of non-random distributions (p-value of the
Rayleigh test is given for each distribution). The double-headed arrows
indicate bimodal distributions; the lengths of the arrows represent the
mean vector length r (scaled so the radius of the circles corresponds to
r51), which provides a measure of the degree of clustering in the
distribution of the bearings. n.s. 5not significant
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SCIENTIFIC REPORTS | 5: 9917 | DOI: 10.1038/srep09917 2

animals exhibited a spontaneous preference for the magnetic north-
northeast and south-southwest axis. These findings are consistent
with the earlier report of a magnetic compass sense in this species by
Mather and Baker
10
. A magnetic compass would be highly beneficial
for nocturnal wood mice that occupy comparatively large home
ranges of 1–2 ha
41
, perform regular foraging bouts over distances
of more than 200 m, and show remarkable homing ability from
unfamiliar locations after displacements of up to 350 m
42,43
.
It is unclear whether the observed preference is part of a homing
response or a spontaneous directional preference. However, even
though many animals used in this study were caught north of the
testing site, none were caught south of it. This renders it unlikely that
the observed axial preference is a homing response. However, the
observed directional preference is consistent with the spontaneous
bimodal magnetic alignment observed in other vertebrates (reviewed
in
44
). Concordantly, laboratory mice, in addition to showing learned
compass orientation relative to the magnetic field, also exhibit a
weak, presumably innate (i.e., independent of any learned direction)
axial preference along the magnetic north-south axis
18
. Recently,
such a preference was also revealed in the semi-fossorial bank vole
(Myodes glareolus), which the authors suggested is likely to be
innate
15
. Consequently, although there is insufficient information
to determine if the observed preference in wood mice is innate or
learned, it appears likely that at least some component of the res-
ponse is innate. Although the adaptive significance of spontaneous
magnetic alignment remains an open question
8,44–46
, the widespread
occurrence in epigeic mammals makes this response ideal for initial
studies of the mechanism(s) of magnetoreception.
The available evidence indicates that subterranean, microph-
thalmic mole-rats rely on a light-independent and RF-insensitive
magnetic particle based mechanism of magnetoreception
16
. On the
other hand, the properties of learned magnetic compass orientation
by epigeic rodents are consistent with the involvement of a radical
pair mechanism
40
. This suggests that the visual ecology/physiology
(adaptation to light levels available to diurnal and nocturnal animals
active above ground, rather than to the absence of light in the
subterranean ecotope), rather than phylogenetic relatedness
(membership in the class Mammalia), may be the principle set of
factors influencing the type of magnetoreception mechanism used to
obtain directional (i.e., compass) information. To determine if
macrophthalmic, epigeic wood mice indeed have a radical pair
mechanism, we tested the sensitivity of wood mice to low level radio
frequency fields, using the types of stimuli used in earlier studies
of birds and mole-rats
16,35,47
, i.e., both the Larmor frequency and
wideband-FM (comparable to broadband-RF used in other studies)
oscillating magnetic fields. Contrary to earlier studies of migratory
birds
35,47
, wood mice exhibited non-random directional preferences
in both conditions: The distribution of bearings obtained from mice
tested under the Larmor frequency condition was indistinguishable
from controls (i.e., nests were bimodally distributed approximately
along the north-south magnetic axis; Figure 3a), while that of mice
exposed to the wideband frequency sweep was rotated by roughly 90u
(Figure 3b). As the angle between the static field and the RF fields was
the same in both RF conditions (see Figure 4c), the only differences
were the overall intensity, the temporal pattern, and the frequency
spectrum. The intensities in the Larmor frequency condition had
minimum values of 785 nT in the centre of the arena and therefore
greatly exceeded those shown to affect the inclination compass of
birds
39,47
. In the wideband-FM condition the RF intensities were
lower, due to the frequency response characteristics of the coil.
They varied across the frequency range between 25–50 nT in the
centre of the coil to twice these values at the periphery of the arena.
These field strengths are comparable to the ones used in Engels
et al.
39
, who were able to disrupt the magnetic compass orientation of
European robins by broadband electromagnetic noise with a spectral
intensity of 0.1–0.2 nT per 10 kHz in the range of 600 kHz–3 MHz,
which upon integration over the frequency domain translates into a RF
magnetic field amplitude of 30–35 nT in the time domain.
Table 1
|
Directions of wood mice nests built in different magnetic conditions. Statistically significant differences between the distributions
are indicated in the last column. FM 5frequency-modulated, n 5number of nests, CCW 5counterclockwise
Figure 3
|
Orientation of wood mice nests built in a visually symmetrical
circular arena in the ambient geomagnetic field with superimposed RF
magnetic fields. The oscillating fields were aligned vertically at an angle of
25uto the geomagnetic field lines. Each data point represents the position
of a nest built by an individual mouse. a. Bearings relative to geomagnetic
north (geomN) in the arena in the ambient magnetic field with a
superimposed Larmor frequency oscillating field (785 – 1,260 nT).
b. Bearings relative to geomagnetic north (geomN) in the arena in the
ambient magnetic field with a superimposed wideband (0.9–5 MHz)
oscillating field (25–100 nT). Arrows give the mean vector for the
distribution of the nests, the dotted lines are the 95% confidence intervals
for the mean bearing (m) of non-random distributions (p-value of the
Rayleigh test is given for each distribution). The double-headed arrows
indicate bimodal distributions; the lengths of the arrows represent the
mean vector length r (scaled so the radius of the circles corresponds to
r51), which provides a measure of the degree of clustering in the
distribution of the bearings. The apparent unimodal preference for the
southeast in the wideband-FM condition (b) was not significant (angular
analysis: m5130u678u,r50.398; Rayleigh test: p 50.066, Z 52.696).
Significant differences between the distributions are indicated by the
p- and U
2
-values of the Watson U
2
test. n.s. 5not significant
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SCIENTIFIC REPORTS | 5: 9917 | DOI: 10.1038/srep09917 3

The results are in accordance with a radical pair mechanism of
magnetoreception, providing for the first time positive evidence for
such a mechanism in a mammal. Even though we cannot rule out
completely that the mice were affected by both RF treatments and
oriented topographically in the Larmor frequency field, the similarity
between nest distribution in the latter and the orientation in the
unchanged geomagnetic field suggests that the wood mice magnetic
receptors were unaffected. Future experiments will hopefully verify
this. For now, the fact that we observed an effect on nest positioning
under a low intensity wideband-FM field, but no (behavioural) effect
under the Larmor frequency field at effectively a 15–30 times higher
intensity leads us to speculate about the underlying mechanism. The
response is consistent with a radical pair mechanism in which both
electron spins have an anisotropic coupling to their respective host
molecules, due either to nuclear hyperfine interactions
29,48,49
or, less
likely, to spin-orbit coupling
50
. If we make the assumption that
cryptochrome 1
51,52
is the molecule responsible for the radical pair
mechanism also in mammals, the radical partner of the flavin
adenine dinucleotide (FAD) cofactor thus could not be a reactive
oxygen species (superoxide), as has been suggested for birds
28,53
,
because superoxide is free of hyperfine interactions. The findings
are rather compatible with the radical partner being tryptophan as
in the original radical pair mechanism model
29
or ascorbyl as recently
proposed by Lee et al.
54
. Of course, the results do not exclude that the
host molecule in mammals and perhaps other taxa might differ from
that in birds. In either case, a large number of resonance frequencies,
both below and above the Larmor frequency, are only possible in a
radical pair mechanism in which both members of the radical pair
have hyperfine interactions, which would then have been excited by
the frequencies in the wideband-FM condition.
Interestingly, while RF magnetic fields so far have been found to
cause disorientation in birds, in the present experiments the wide-
band-FM field caused re-orientation in mice, with nest-building
positions shifted by approximately 90urelative to the axis of orienta-
tion observed in the ambient magnetic field. There are two possible
explanations for this re-orientation. First, the wideband-RF might
have disrupted input from the radical pair mechanism, causing
the mice to rely on an alternative source of directional information
(e.g., non-magnetic, or magnetic particle mechanism-based compar-
able to the fixed direction response of birds tested in unnatural light
conditions
37,55
). Alternatively, the wideband-RF may have altered,
rather than eliminated, the pattern of radical pair mechanism res-
ponse, as it has recently been proposed as an explanation for an effect
of a Larmor frequency magnetic field on spontaneous magnetic
alignment in turtles
56
. To our knowledge, the possibility of this type
of RF effect has not yet been addressed by the available models of the
radical pair mechanism. For simple reference-probe radical pair
mechanism models it is the lifetime of the spin-correlated radical
pair (‘‘spin correlation time’’) that determines the magnitude of the
effect of a weak RF magnetic field on the radical pair dynamics: long
correlation times, in the order of 100 microseconds allow weak RF
magnetic fields to fully perturb the singlet-triplet interconversion,
leading to a flattened angular response (suppression of the dir-
ectional dependence required for a compass). Shorter correlation
times alter the absolute values of the yield without flattening the
angular response
50,57
. Theoretically, it is possible that the radical pair
mechanism in rodents is based on a radical pair with shorter spin
coherence time than the ones in migratory birds, so that the effect of a
RF magnetic field could be different in the two taxa. The altered
response in the short-lived radical pair would be equivalent to the
response produced by an intensity shift in the static magnetic field
57
,
which could produce a change in the magnetic visual pattern.
It has been proposed that vertebrates might exploit the visual
pattern of response produced by the radical pair mechanism as a
global reference that could function as a simple spherical grid or
coordinate system fixed in alignment relative to the magnetic field
that appears as a visual pattern superimposed on the animal’s sur-
roundings
58
. Such a reference system would be useful in a variety
of daily challenges from integrating spatial information from
multiple sensory modalities, in novel surroundings, to improving
3-dimensional path stabilization
59
and course control
60
, to placing
multiple locales into register to form a global map of familiar
space
46,58
. If the magnetic field is perceived in this way in epigeic
rodents, mice might position themselves and/or their nests in a spe-
cific alignment with respect to the pattern generated by the radical
pair mechanism. Consequently, a change in the ‘visual’ pattern
caused by RF treatment could result in a corresponding change in
the distribution of nest positions.
It is widely believed that RF magnetic fields influence exclusively a
radical pair mechanism, not a magnetic particle mechanism. This is
certainly true for single-domain magnetite, where the inertia of the
particles surrounded by the viscous cytoplasm is generally believed
to hinder motion and thus transduction of oscillating fields in the
radio frequency range
36,61
. However, according to Shcherbakov &
Winklhofer
25
, a magnetic particle mechanism based on magnetic sus-
ceptibility, such as the maghemite-superparamagnetic magnetite hybrid
magnetoreceptor proposed by Fleissner et al.
62
would convert the
radiation into thermal agitation. As with the putative effect on a radical
pair mechanism, it is not clear why such a heating effect would cause
re-orientation, rather than disorientation. Importantly, however, due to
the higher intensity of the Larmor frequency stimulus compared to the
wideband stimulus, any heating effect would have been more pro-
nounced for the Larmor frequency condition. Consequently, the finding
of an effect of the wideband RF stimulus, but not of the higher intensity
Larmor frequency stimulus, argues against a nonspecific (i.e., thermal)
effect on a mechanism or process other than the radical pair mechanism.
Figure 4
|
Overview of the testing site, the coil-setup and the nest-
building arena. a. Top view of the empty horse stable that consisted of two
compartments. One compartment contained the wooden enclosure, the
other one the testing setup and coil systems. b. Production of the artificial
static magnetic field (top view). In the control condition (mN
ambient
) the
horizontal of the ambient magnetic field (H
ambient
) was left unchanged.
To shift magnetic north by 90ucounterclockwise (mN
west
), an artificial
magnetic field was added with a 135uclockwise aligned Helmholtz-coil
pair (H
artificial
) to produce a 90ushifted resultant field of the same
inclination and total intensity as the ambient magnetic field. c. Profile of
the test arena and the loop coil used to produce the oscillating magnetic
fields in the RF range. The oscillating fields were aligned vertically at an
angle of 25uto the static field lines. The scale only applies to part a. of the
figure.
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SCIENTIFIC REPORTS | 5: 9917 | DOI: 10.1038/srep09917 4

In sum, we show that wood mice possess a magnetic sense that
they use to position their nests along the NNE-SSW axis relative to
the magnetic field. The NNE-SSW preference was not altered by RF
fields delivered at the Larmor frequency, but was shifted by approxi-
mately 90uby a RF frequency sweep (0.9–5 MHz repeated at 1 kHz)
at an intensity of only ,5% that of the Larmor frequency stimulus.
The results point to the involvement of a radical pair mechanism, the
first such evidence for a mammal, although further research is
needed to provide a more thorough characterization of the
underlying mechanism. Finally and importantly, it should be noted,
that the RF magnetic fields applied here have peak intensities
below the ICNIRP guidelines for general public exposure (
63
, i.e.,
B
rms
50.92 mT/f [MHz], or B
peak
51.30 mT/f [MHz]) considered as
harmless for human health. Yet, we show that they are sufficient to
affect behaviour in a mammal.
Methods
Static magnetic fields.We used a pair of double-wrapped Helmholtz-coils (2.15 m
diameter, 10110 turns of 14 gauge copper wire wrapped on a wooden frame, current
1.76 A) powered by a current-regulated power supply (Manson DPD-3030) to alter
the direction of the geomagnetic field. Parallel current flow through both wires on the
coils created a magnetic field, while with the current flowing antiparallel no magnetic
field was created but possible side effects (heat, vibrations, electric fields) where the
same in both conditions
64
. The coils were arranged in such a way that magnetic north
could be shifted by 90ucounterclockwise without changing the intensity or
inclination of the geomagnetic field (Figure 4a,b;
3
). The intensity of the static field and
of extremely low-frequency (mainly 50 Hz) oscillating fields was measured with
a 3-axial magnetometer (Gigahertz NFA 1000) equipped with an additional
magnetostatic probe (Gigahertz MS-NFA). The intensity of the local magnetic field
within the arena was 49.05 mT. With antiparallel current flow the overall intensity
was 49.03 mT and it was 49.20 mT when north was shifted westwards. The output of
the power supply was run through an EMI filter (Schurter 5500.2058) before entering
the coils to reduce low RF fields. Residual oscillating magnetic fields were detectable in
both static field conditions and mainly in the 150–180 Hz range, with intensities
around 60 nT (parallel current flow) and 6 nT (antiparallel current flow).
Radio frequency magnetic fields.Split-shield magnetic-field loops for RF
experiments were constructed using coaxial cable (Aircell 7). One loop was designed
with a sharp resonance at about 1.3 MHz for application of a magnetic field
oscillating at the local Larmor frequency (1.33 MHz) with a 47 Ohm resistance at the
feed for impedance matching, the other cable had a broad resonance between
0.9–5 MHz, with maximum at 4 MHz, where the magnetic field was two times
stronger than at 0.9 MHz. The coils (60 cm diameter) were powered using a Wavetek
144 function generator for a 1.33 MHz continuous sine wave and a Wavetek 193
sweep generator used for generating a wideband-frequency modulated (FM) field,
where the frequency sweep (0.9 MHz to 5 MHz) was repeated at intervals of 1 msec.
The oscillating fields were aligned vertically at an angle of 25uto the static field lines
(Figure 4c). The RF field produced by the coils was measured with an ETS-Lindgren
split-shield magnetic-field probe (7405 E&H 6 cm diameter near field loop probe)
connected through a coax cable to an oscilloscope (Picoscope 4224). The intensities in
the Larmor frequency condition had maximum values of 1.26 mT in the periphery of
the arena and minimum values of 785 nT in the centre, while in the wideband-FM
condition, due to the frequency response characteristics of the coil, the intensities
varied between 25 nT-50 nT in the centre of the coil to twice these values at the
periphery of the arena. An induction coil connected to a HAMEG (HMO 3524)
oscilloscope was used (in FFT mode) to monitor the low-frequency magnetic noise.
Neither single-frequency nor the sweeping conditions were found to cause an
enhanced noise level in the low-frequency range.
Experimental procedure.The experiments were performed in an empty horse stable
in a rural area of the Bohemian Forest, Czech Republic (49u9910.28"N, 13u20956.45"E)
in summer and autumn of 2013. Recently, Engels et al.
39
showed that anthropogenic
electromagnetic noise of spectrum level intensities a low as 1 nT disturbed the
magnetic compass of European robins when being tested in unshielded wooden huts
at the campus of the University of Oldenburg. Although we tested the wood mice in
an unshielded environment, the testing site was remote and comparable to the rural
locality used by Engels and colleagues where the European robins were well oriented.
Wood mice (Apodemus sylvaticus) were live-trapped on private property within
the vicinity of the stable by means of see-saw traps. The trapping and all experiments
were approved by the Institutional Animal Care and Use Committee of the University
of South Bohemia, and the Ministry of Education, Youth and Sports of the Czech
Republic (no. 7946/2010-30) and all experiments were performed in accordance with
their guidelines and regulations. Until testing, the wood mice were kept in a rect-
angular wooden enclosure (approx. 2 m 31m31 m) based in an adjacent part of
the stable (Figure 4a) and fed apples and grain and given access to water ad libitum.
For habituation and stress reduction, each mouse was kept for at least one night but
no longer than three nights in the enclosure before being tested.
The animals were tested in a circular arena (diameter 50 cm) made of black PVC.
The floor of the arena was evenly covered with sawdust and hay which served as nest
building material. An apple slice (1–2 cm thick) and some grain placed in the centre
of the arena served as food and water supply (Figure 1a). All experiments started in
the evening and were conducted overnight. The magnetic conditions (control, 90u
shift, RF) had been set before the mouse was introduced into the arena. The condition
for each day was randomly chosen. The mice were released into the middle of the
arena and the arena was quickly covered with a translucent frosted white PVC-sheet
(Figure 1b). The next morning, the direction of the nest was measured with a hand-
held compass (Figure 1c) and the mouse was released. Only nests built against the wall
of the arena (max. distance 10 cm) were counted. Nights with thunderstorms were
excluded from the analysis (confer
40
). After each test, the arena was emptied and
thoroughly cleaned with 70% ethanol.
To control for a possible observer bias, a subset of the nests (n 524) was analysed
in a double-blind fashion: pictures of the nests were taken with a digital camera from
above and later analysed by a second person unaware of magnetic north and the
experimental conditions. The mean difference between the nest directions obtained
from direct compass measurements and those taken from the pictures was
3.7u(circular SD 57.4u).
Statistical analysis.We used standard circular statistics to analyse the distributions of
the nest positions
65
. All calculations were carried out with Oriana 4.02 (Kovach
Computing). Each nest direction was treated as an independent data point. The
likelihood of retesting a mouse was low because wood mice avoid traps for some time
after they have been captured (unpublished observations). Furthermore, any mouse
that was recaptured had only a 25% chance of being tested in the same experimental
condition. Mean vectors were calculated by vector addition. The method of doubling
the angles was used to convert angular data in axial data prior to statistical analysis.
The Rayleigh test was employed to test the data for significant deviation from random
distribution with p,0.05 as the threshold of statistical significance. The distributions
of the nests in different conditions (group comparisons) were compared with the
Watson U
2
test employed on doubled angles.
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Acknowledgments
We thank Frantisek Sedla
´c
ˇek for assistance in the field, and Lukas Landler and Michael
Painter for careful proof readingof the manuscript. This work was supported by the German
National Academic Foundation (Studienstiftung des deutschen Volkes, PhD fellowship to
EPM), the German Research Foundation (Deutsche Forschungsgemeinschaft), www.dfg.de,
grants Ed258/1-1 (to SHKE) and Wi1828/4-2 (to MW), the Human Frontier Science
Program, www.hfsp.org, grant RGP 13/2013 (to MW), and the Grant Agency of the Czech
Republic,www.gacr.cz, projectno. 15-21840S (to HB, VH, EPM).During analysis and writing
of this paper JBP was supported by theNational Science Foundation, www.nsf.gov, NSFIOS
1349515. The funders had no role in study design, data collection and analysis, decision to
publish, or preparation of the manuscript.
Author contributions
Design of the study: EPM, SHKE, MW. Data collection: EPM, VH. Data analysis: EPM, JBP.
Writing the paper: EPM, SHKE, SB, MW, JBP, HB. All authors read and approved the final
version of the manuscript. We thank three reviewers for comments on an earlier version of
this manuscript.
Additional information
Supplementary information accompanies this paper at http://www.nature.com/
scientificreports
Competing financial interests: The authors declare no competing financial interests.
How t
5
o cite this article: Malkemper, E.P. et al. Magnetoreception in the wood mouse
(Apodemus sylvaticus): influence of weak frequency-modulated radio frequency fields. Sci.
Rep. , 9917; DOI:10.1038/srep09917 (2015).
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