Cone opsins and response of female chamois (Rupicapra rupicapra) to differently coloured raincoats

Abstract

Alpine species are often exposed to intense levels of human recreational activities. Exactly how human disturbances influence the behaviour of these species is still open to much debate. For example, little is known regarding how the colourful clothing often worn by tourists influences the behaviour of animals. Tourists wearing colourful clothing may be more conspicuous to local wildlife and thus cause more disturbances. We therefore investigated this question in female chamois (Rupicapra rupicapra) in the Swiss Alps. We firstly investigated, via a morphological and an immunohistochemical approach, whether chamois are likely to have colour vision and would therefore be more likely to respond to different coloured clothing. We detected evidence of two cone types—short-wavelength-sensitive cones (S-cones, JH 455) and middle-wavelength-sensitive cones (M-cones, JH492) in the chamois retina—suggesting that chamois have dichromatic vision, similar to other ungulates. Secondly, via behavioural assays where a person wearing one of three coloured coats commonly worn by tourists (red, yellow and blue) approached a female chamois, we show that neither the alert and flight initiation distance nor the site of refuge were influenced by the raincoat colour. In addition, behavioural responses of the chamois were neither influenced by animal group size nor the presence of kids nor the time of the experiment. The results suggest that, although chamois possess colour vision, they do not react more strongly towards conspicuous colours worn by hikers. We discuss our results in light of what is already known about chamois biology and suggest implications for future studies.

Full text

ORIGINAL PAPER
Cone opsins and response of female chamois (Rupicapra
rupicapra) to differently coloured raincoats
S. Raveh &W. F. D. van Dongen &C. Grimm &P. Ingold
Received: 10 June 2011 / Revised: 5 March 2012 /Accepted: 12 March 2012
#Springer-Verlag 2012
Abstract Alpine species are often exposed to intense levels
of human recreational activities. Exactly how human dis-
turbances influence the behaviour of these species is still
open to much debate. For example, little is known regarding
how the colourful clothing often worn by tourists influences
the behaviour of animals. Tourists wearing colourful cloth-
ing may be more conspicuous to local wildlife and thus
cause more disturbances. We therefore investigated this
question in female chamois (Rupicapra rupicapra) in the
Swiss Alps. We firstly investigated, via a morphological and
an immunohistochemical approach, whether chamois are
likely to have colour vision and would therefore be more
likely to respond to different coloured clothing. We detected
evidence of two cone types—short-wavelength-sensitive
cones (S-cones, JH 455) and middle-wavelength-sensitive
cones (M-cones, JH492) in the chamois retina—suggesting
that chamois have dichromatic vision, similar to other ungu-
lates. Secondly, via behavioural assays where a person
wearing one of three coloured coats commonly worn by
tourists (red, yellow and blue) approached a female chamois,
we show that neither the alert and flight initiation distance nor
the site of refuge were influenced by the raincoat colour. In
addition, behavioural responses of the chamois were neither
influenced by animal group size nor the presence of kids nor
the time of the experiment. The results suggest that, although
chamois possess colour vision, they do not react more strongly
towards conspicuous colours worn by hikers. We discuss our
results in light of what is already known about chamois
biology and suggest implications for future studies.
Keywords Tourism .Alert distance .Flight initiation
distance .Clothing .Colour vision .Chamois .Rupicapra
rupicapra
Introduction
In the past decades, tourism has increased in the Alpine
Arch of Europe. This rising popularity of recreation (tour-
ism and outdoor leisure sports) in mountain regions often
results in a conflict between human activities and local
wildlife (Arlettaz et al. 2007; Ingold 2005; Stankowich
2008; Taylor and Knight 2003). Many studies have shown,
for example, that alpine animals such as marmots (Marmota
marmota), ibex (Capra ibex) and chamois (Rupicapra rupi-
capra) react strongly to tourist activities, resulting in short
and long-term consequences. These detrimental effects in-
clude loss of grazing time, abandonment of high quality
pastures and increased energy consumption, which leads to
Communicated by C. Gortázar
S. Raveh :P. Ingold
Ethology and Nature Conservation, Department of Zoology,
University of Bern,
Länggassstrasse 27,
3012 Berne, Switzerland
S. Raveh (*):W. F. D. van Dongen
Konrad Lorenz Institute of Ethology, Department of Integrative
Biology and Evolution, University of Veterinary Medicine,
Vienna, Austria
e-mail: shirleyraveh@hotmail.com
C. Grimm
Lab for Retinal Cell Biology, Department of Ophthalmology,
University of Zurich,
Wagistrasse 14,
8952 Schlieren, Switzerland
Present Address:
P. Ingold
Färichweg 1,
3038 Kirchlindach, Switzerland
Eur J Wildl Res
DOI 10.1007/s10344-012-0629-z

a reduction in body condition and reproductive success
(Enggist-Düblin and Ingold 2003; Gander and Ingold
1997; Hamr 1988; Hüppop 1995; MacArthur et al. 1982;
Schaal and Boillet 1992; Schnidrig-Petrig 1994; Zeller
1991; Zimmerli 1993). These negative effects could be
avoided by reducing the encounter rate between human
and wildlife or adapting the tourists' behaviour.
There are several strategies to minimise the impact of
human presence on local wildlife (Keller, 2001). However, it
is firstly essential to identify the factors associated with leisure
activities that elicit responses in wild animals. For example,
several studies have shown that animals react more strongly
when approached off trail and from above than from below
(bighorn sheep: Hicks and Elder 1979; marmots: Mainini et al.
1993; chamois: Zeller 1991). Furthermore, animals such as
wild sheep (Ovis canadensis canadensis)(MacArthuretal.
1982; Martinetto and Cugnasse 2001) and marmots (Mainini
et al. 1993) have been shown to exhibit an increased reaction
when a person was accompanied by a dog. Similarly, the flight
responses of female chamois were longer and more intense
when exposed to loud-speaking, as opposed to silent, hikers
(Kobelt 2004).
In recent times, sportswear has tended to become more
colourful, with brighter designs (such as yellows and reds).
Bright colouration is known to increase the conspicuousness
of objects in relation to drab backgrounds, which are typical
of natural environments (e.g. Endler 1993; Doucet et al.
2007). This may result in an augmented conspicuousness
of alpine tourists, which in turn may have a negative impact
on local wildlife by reducing the chances that humans can
pass the animals without causing a disturbance and by
increasing the time that tourists are visible during the ani-
mal's flight response. In contrast to tourists, clothing worn
by hunters is typically drabber and designed to decrease
conspicuousness at least during the still hunt. However, it
is often unknown whether the game animals can indeed
perceive colour differences. Despite these predictions and
the possible negative consequences of increased conspicu-
ousness of tourists on wildlife, studies investigating the
influence of brightly coloured clothing worn by tourists on
wildlife behaviour are lacking.
An important first step in exploring the effects of colour-
ful clothing on wildlife is to confirm that the species
indeed has the capacity to perceive colour differences.
Immunohistochemistry allows a straightforward approach
to test whether eyes at least have the photoreceptor cells
necessary to detect colour (e.g. Hemmi and Grünert 1999;
Dkhissi-Benyahya et al. 2001; Arrese et al. 2003a). Whereas
rods are responsible for perceiving differences in brightness
and for monochromatic vision in low light levels (Kaskan et
al. 2005), two or more cones are necessary to enable colour
vision. Most mammals possess two cone types, one that is
sensitive to short-wavelengths (blue) and one that is sensitive
to mid-wavelengths (green) and therefore have dichromatic
colour vision (Jacobs 1981;Jacobs2009). In contrast, trichro-
matic colour vision, which requires three cone-types, is re-
stricted to hominoids, Old World monkeys and some New
World monkeys (Jacobs 1993).
Chamois are a common ungulate species inhabiting rocky
and steep mountain areas up to 3,000 m above sea level. In
some areas in the central Alps, chamois are exposed to intense
recreational tourism activities, such as hiking, biking, riding/
trekking and cross-country skiing. Past studies have shown
that increased human activities in chamois habitat can nega-
tively impact on chamois behaviour (Enggist-Düblin and
Ingold 2003; Gander and Ingold 1997;Hamr1988; Schaal
and Boillet 1992). In contrast, it remains unknown whether
the colourful clothing often worn by hikers makes them more
conspicuous to chamois. To test this idea, it is necessary to
show (1) that chamois have colour vision and (2) that they
respond differently to hikers wearing differently coloured
clothing. We therefore firstly used an immunohistochemical
approach, by staining the retina with two cone-specific anti-
bodies, to ascertain whether chamois have at least a dichro-
matic vision system. Although ungulates typically possess
dichromatic vision (e.g. domestic goat, Capra aegagrus
hircus: Siemers et al. 1999;sheep,Ovis aries:Munkenbeck
1982;domesticpig,Sus domesticus: Koba and Tanida 1999;
fallow deer, Dama dama and white-tailed deer, Odocoileus
virginianus:Jacobsetal.1994, Birgersson et al. 2001;horse,
Equus ferus caballus: Carroll et al. 2001), no data currently
exist for colour vision in chamois. Secondly, by using a field
experiment, we investigated whether an approaching person
wearing differently coloured clothing had an influence on the
alert and flight responses and the site of refuge of female
chamois. We predict that conspicuous coloured clothing
would result in (1) an earlier alert response, (2) a longer flight
initiation distance and (3) a longer flight to the site of refuge
than drabber coloured clothing.
Material and methods
Morphological and immunohistochemical analysis
Four enucleated eyes from a dead male and female chamois
were examined in December 2002/January 2003. Both ani-
mals died via authorised culling by a gamekeeper. For light
microscopy examination, two enucleated eyes (retrieved
on-site immediately after the death of the animal) from
different animals were immersed in 2.5 % glutaraldehyde
in 0.1 M cacodylate buffer at pH 7.3 for the transport to the
lab. The cornea and lens were removed, and the eyecup was
further incubated in 2.5 % glutaraldehyde in 0.1 M cacody-
late buffer with pH 7.3 at 4 °C for postfixation (overnight).
The tissue was cut to prepare regions of interest, washed in
Eur J Wildl Res

cacodylate buffer and incubated in osmium tetroxide (1 %)
for 1 h. The tissue was embedded in Epon 812. Sections (0.5
μm) were prepared and counterstained with methylene blue.
For immunohistochemical analysis, the two other eyes
were enucleated, the lenses were removed and the remaining
vitreous bodies, together with the retinas, were preserved in a
4 % paraformaldehyde solution (pH 7.4) for 4 h at 4 °C.
Eyecups were cut in coronal cross sections, washed and kept
overnight at 4 °C in PBS. The retinas were then isolated and
prepared as whole mounts for immunohistochemical analysis.
A standard method was chosen, implementing specific
antibodies that recognise short- and medium-wavelength
opsins to determine the presence of different cone types in
the eye of the chamois. This technique has previously been
used in many similar studies (Wikler et al. 1996; Hemmi and
Grünert 1999; Dkhissi-Benyahya et al. 2001; Arrese et al.
2003a).
Nonspecific binding sites were blocked by incubation in
PBS containing 0.1 % Triton X100 and 10 % normal goat
serum (NGS). Primary antibodies (JH492, 1:4000; JH455,
1:4000) were then applied for 3 h at room temperature in
PBS containing 0.1 % Triton X100 and 3 % NGS. JH492
antibody recognizes middle-wavelength-sensitive cone
opsin (M-cones), whereas JH455 is specific for short-
wavelength sensitive cone opsin. Both antibodies were
raised against the respective human proteins (Chiu and
Nathans 1994) and recognized MWL and S-cone opsins in
a variety of species including the mouse (Mus musculus:
Chiu and Nathans 1994), Syrian hamster (Mesocricetus
auratus: Glosmann and Ahnelt 2002), rabbit (Oryctolagus
cuniculus: Hack and Peichl 1999), marsupials (Arrese et al.
2003b), horses (Sandmann et al. 1996) and primates (Martin
and Grünert 1999). Peanut agglutinin (PNA, 1:250; Sigma)
was used as a general cone marker (Unoki et al. 1988). After
staining, the retinas were washed three times in PBS con-
taining 0.1 % Triton X100. Secondary rabbit antibodies
(1:250, Cy3 coupled; Jackson ImmunoResearch) or peanut
agglutinin were applied for 2 h at room temperature in PBS
containing 0.1 % Triton X100 and 3 % NGS. Retinas were
washed three times in PBS containing 0.1 % Triton X100
before they were transferred to slides and examined by
fluorescent light microscopy (Axioplan, Carl Zeiss AG,
Feldbach, Switzerland). Images were taken using a digital
camera (AxioCam, Carl Zeiss AG, Feldbach, Switzerland).
Behavioural assays
Study area and animals The experiment was carried out on
the northwest flank of the Augstmatthorn game reserve at an
altitude of 1,700 m above sea level (Bernese Oberland,
Switzerland: 46°45′N, 7°55′E). Behavioural observations
were conducted from June to August 2003. The area is
characterised by steep slopes of limestone rocks and screes,
intersected by rocky gullies running down the slopes. A
large number of female chamois with kids can be found at
the northwest side of the Augstmatthorn on sunny days
(Ruckstuhl and Ingold 1998). Natural licks exist in the
gullies, which are regularly visited by chamois to ingest
the mineral-rich water coming out of the rock or marl. The
licks possess a high level of sodium (Marbacher 1989),
which is important for bone structure, growth and digestion
(Robbins 1993). Access to these licks is therefore essential
for suckling females during lactation. One such lick was
chosen as the study site (46°45′46.3″N, 7°55′14.1″E), due
to the ease of observation (no dense vegetation or obstruc-
tions to block the view of the lick) and the reliable avail-
ability of water (Marbacher 1989). The lick is situated
below a rarely used hiking trail that winds along the ridge
of the slope. Only three people were observed using this trail
during the experimental time.
Data collection Experiments were conducted on clear, sun-
ny days between 0600 hours to 1600 hours. To minimise the
effects of group dynamics influencing vigilance behaviour
of the focal individual, experiments were only conducted
when a maximum of three other adult animals were present
in the focal gully. We tested the responses of chamois to
raincoats that were either bright yellow, bright red or azure
blue (red hue: 1°, saturation: 83 %, brightness: 95 %; yellow
hue: 57°, saturation: 72 %, brightness: 97 %; blue hue: 195°,
saturation: 78 % and brightness: 91 %). The colours of the
raincoats were chosen because they were commonly worn
by hikers in the region (S. Raveh: personal observation). An
experiment consisted of a person, wearing one of the three
raincoats approaching a chamois at the lick from a defined
starting point (216 m from the lick). The trail curved around
the lick (distance between path and lick, 45–216 m). The
approach by the investigator to the focal animals was there-
fore indirect (Fig. 1). Any chamois at the lick would per-
ceive the approaching person against a light blue sky
background. The person approached at normal walking
pace, and the same person conducted all the trials alone.
The colour of the raincoat worn in each experiment was
chosen randomly and combined with navy blue trousers
(blue coloured trousers were observed to be often worn by
hikers in the region). When the focal animal first raised its
head and looked towards the investigator, the distance sep-
arating the two was measured in metres (alert distance
(AD)). If a nonfocal female showed an alert or escape
behaviour, before the focal female, then the experiment
was terminated. Experiments were also terminated in cases
where the focal animal took flight before the trial com-
menced. The person then resumed walking on the trail until
the animal took flight. At this time, flight initiation distance
(FID) was measured, defined as the distance between the
focal animal and the person at the moment the animal took
Eur J Wildl Res

flight (Cederna and Lovari 1985). Both AD and FID were
measuredwithalaserrangefinder(Leica7x42BDA,
GEOVID). After the animal took flight, the investigator
visually tracked the animal and then, after a 10-min period,
marked its site of refuge on a 1:25000 map of the study site.
The refuge of the animals was difficult to locate because of
the hilly terrain. In five cases, the observer lost track of the
animals during the flight. The positions of the remaining 25
individuals were recorded on the map.
For each trial, the investigator recorded the time of the day,
the number of animals at the surrounding licks and whether
the focal females were accompanied by kids (all females had
given birth at this point of time). We waited a minimum of
2 h between trials (Marbacher 1989). Trials were conducted
only on sunny days in order to maintain a constant reflection
of the coloured raincoats and when the wind was blowing in
the direction of the investigator to prevent odour cues. We
excluded chamois younger than 2 years from our analyses
because the flight distance in older animals is higher than
that in subadult chamois (Patterson 1988). Age was deter-
mined by the growth rings of the horns (Schröder and
Elsner-Schack 1985). Ten trials per coat colour were
conducted.
Statistical analysis The data were analysed with SPSS 17
statistical software. Kolmogorov–Smirnov test were used to
test for the normality of the data. Levene's test was used to
test for deviations from homogeneity of variances.
Nonparametric tests were used for analysing the AD data,
since these data were not normally distributed. In contrast,
the FID data were normally distributed. The differently
coloured raincoats, experimental time (morning or after-
noon), group size and the presence of kids were treated as
independent variables; whereas the alert distance and the
flight initiation distance were the two dependent variables.
To quantify habituation effects, we tested whether the num-
ber of trials already conducted at the lick was associated
with the AD and FID of the focal individuals by using
Spearman's rho and Pearson correlations, respectively. The
likelihood of testing an individual twice was small due to
the large number of females present in the region (in 2003:
on average078, nmax087) (Willisch et al. unpublished
data). In addition, data collection only occurred on sunny
days, which were often separated by days of high cloud
cover or fog. Sampling, therefore, was scattered over the
3-month period, rendering it unlikely that we constantly
resampled a small number of chamois. Trials were also
conducted at least 2 h apart from each other, and this
decreased the likelihood of measuring a female twice since
females generally did not stay at a lick for such long periods
(10 min–1 h; Raveh, personal observation). Lastly, we
found no effects of habituation (see below), suggesting that
we did not repeatedly test the same subset of animals.
Results
Morphological and immunohistochemical analysis
Morphological sections of the central temporal retina
revealed the presence of two photoreceptor cell types in
the outer retina (Fig. 2a): cells with a long and thin outer
segment and cells with a thicker, cone-like structure (stars in
Fig. 2a, b) in the layer of the photoreceptor inner segments
(PIS) and a shorter outer segment. These cells were most
likely rods and cones, respectively. Using a higher magnifi-
cation, we could clearly distinguish the two photoreceptor
types and also detected the discs (arrow for rod discs,
Fig. 1 Morphology of the chamois retina. aSemithin section of the
outer retina. Asterisk photoreceptor inner segments resembling cones.
ONL, outer nuclear layer; PIS, photoreceptor inner segments; POS,
photoreceptor outer segments; RPE, retinal pigment epithelium. b
Magnification of an area shown in a.Arrow, rod outer segment with
discs containing rhodopsin; arrowhead, cone outer segment with discs
containing cone opsin. Abbreviations and symbols as in a.cSemithin
section through all layers of the retina of chamois. TAP, tapetum; INL,
inner nuclear layer; GCL, ganglion cell layer; NFL, nerve fibre layer.
Other abbreviations as in a.Scale bars 50 (a), 25 (b) and 100 μm(c)
Eur J Wildl Res

arrowhead for cone discs, Fig. 2b) in the outer segments
containing the visual pigment. Spacing of cone cells was
relatively regular with one cone among roughly ten photo-
receptor cells. This distribution was similar in all regions
analysed. Apart from photoreceptors, the chamois retina
showed normal layering of retinal cells with a nerve fibre
layer (NFL), ganglion cell layer (GCL), inner nuclear layer
(INL), outer nuclear layer (ONL) containing five to six rows
of photoreceptor nuclei, the retinal pigment epithelium
(RPE) and the tapetum (TAP) behind the RPE (Fig. 2c).
The spectral reflection of the tapetum during the preparation
of the eyes ranged from greenish-blue to yellow (personal
observation) as described in sheep (Bellairs et al. 1975). Cell
density in GCL and INL was very low (Fig 2), suggesting a
strong convergence of information from photoreceptors to
ganglion cells, which may point to a rather low visual
acuity.
To test the presence of cone photoreceptor cells directly,
we used PNA to stain retinal flat mounts. PNA is reported to
be a specific marker for cones as it interacts with galactose-
galactosamine disaccharides (Blanks and Johnson 1984) and
PNA specifically labelled cone, but not rod cells, in a variety
of species including fish, rabbit, monkey, human, chicken,
guinea pig, mouse, rat and others (Blanks and Johnson
1984; Hageman and Johnson 1986; Krishnamoorthy et al.
2008; Unoki et al. 1988). Staining of chamois retinal flat
mounts revealed a high density of PNA-positive cells (white
arrows in Fig. 3). Due to the specificity of the PNA marker,
these cells are assumed to be cones. Double staining with
PNA and antibodies specific for short-wavelength S (blue)-
cones (staining with JH 455; Fig. 3a) or middle-wavelength
M (green)-cones (staining with JH492; Fig. 3b) confirmed
the presence of at least two cone types. Antibody signals
strictly colocalized with PNA-positive cells (red arrows in
Fig. 3a, b) demonstrate that PNA labels cones also in
chamois. However, not all cells positive for PNA were
positive for one of the two cone-specific antibodies (see
“Discussion”).
Behavioural assay
Alert distance We quantified 30 AD of focal females during
the field season. A median of three females (interquartile range
(IQR), 2–4; n030) was observed in the focal gulley. Offspring
was absent in 16 cases; whereas in the remaining 14 cases, at
least one kid was present. The median AD for blue raincoats
was 214 m (IQR 213.5–215.2 m; n010), for yellow 213.5 m
(IQR 208.8–216 m; n010) and for red 216 (IQR 211.5–216 m;
Fig. 2 Selective staining of cone photoreceptors in the chamois retina.
Immunofluorescence of retinal flatmounts with peanut agglutinin
(green) and with antibodies raised against short-wavelength cone opsin
(JH 455, red)(a) or middle-wavelength cone opsin (JH 492, red)(b),
respectively. The orange colour indicates co-localization. White
arrows correspond to cone outer segments that were positive for peanut
agglutinin only. Red arrows correspond to cone outer segments that
were positive for both peanut agglutinin and short- (a) or middle-
wavelength (b) cone opsins, respectively
Fig. 3 Distribution of the chamois after the animals took flight from
the salt lick (indicated by the black circle) after being exposed to a
hiker wearing coloured coats on the hiking trail (dotted line). The
various symbols represent that position of refuge of chamois exposed
to red raincoats (white triangle), yellow raincoats (black square) and
blue raincoats (grey triangle)
Eur J Wildl Res

n010). The AD was not affected by raincoat colour (Kruskal–
Wallis test χ
2
01.23, df02, p00.54); by the group size at the
lick (Kruskal–Wallis test χ
2
05.17, df04, p00.27) or by the
presence of an offspring at the focal lick (Kruskal–Wallis test
χ
2
01.47, df01, p00.22).
Flight initiation distance During the field season 30 FIDs
were quantified. The average FID in response to a hiker who
was wearing a yellow raincoat was 125.3±12.9 m (mean ±
SE) (median0135, IQR080.2–163.5 m) (n010), a blue
raincoat 111.4± 13.5 m (mean ± SE) (median095.5, IQR0
78.5–142.2 m) (n010) and a red raincoat 118.1 ± 11.6 m
(mean±SE) (median0123.5, IQR 081.5–145.5 m) (n010).
The FID was not significantly influenced by the colour of
the raincoats, time of data collection, the group size at the
lick or the presence of kids (colour: F
2, 21
00.089, p00.91;
presence of kids: F
1, 21
00.023, p00.88; group size at the
lick: F
4, 21
00.931, p00.46; and experimental time: F
1, 21
0
0.276, p00.61). Further, we did not detect an effect of
colour on FID when merging data for the two putatively
conspicuous colours (red and yellow) and comparing
responses to these coats together relative to the presumably
less conspicuous blue jacket (colour: F
1, 22
00.138, p00.71;
presence of kids: F
1, 22
00.064, p00.80; group size at the
lick: F
4, 22
00.936, p00.45; and experimental time: F
1, 22
0
0.315, p00.58).
Position of refuge The site of refuge of the chamois
appeared to vary randomly with the coat colour worn by
the hiker (red n07, blue n09 and yellow n09; Fig. 1). Five
females took refuge in neighbouring gullies, whereas most
of the animals were found in a coppice (n015), another four
focal animals were seen in the bottom of the valley and only
one female escaped into the adjacent slope (Fig. 1). There
was no association between AD and FID and the number of
experiments conducted during the 3 months of the data
collection (AD: Spearman's rho0−0.09, p>0.05, n030;
FID: Pearson correlation0−0.18, p>0.05, n030).
Discussion
We have shown here that the chamois eye possesses both
short-wavelength-sensitive and middle-wavelength-sensitive
cones, suggesting dichromatic colour vision. This indicates
that they are likely to be able to distinguish between dull blue
and yellow colours and are probably able to detect hue differ-
ences in at least the yellow and blue coats worn during the
behavioural experiments (Carroll et al. 2001). Despite this, we
found no effects of the different colours worn by the hiker on
the alert or flight initiation distance of the chamois. In addi-
tion, group size, the presence of kids or the time of day did not
influence the chamois escape behaviour. Based on our data,
therefore, it appears that the specific colour of clothing worn
by tourists is not directly implicated in the flight response of
chamois.
Evidence for colour perception in chamois
Several lines of evidence suggest that, despite not respond-
ing differently to the different coloured coats, the chamois
are able to detect the differences in at least the yellow and
blue coats presented during the behavioural experiments
(red tends to appear as yellow for dichromatic species;
Carroll et al. 2001). Firstly, the histochemical results pre-
sented here, coupled with the general pattern of dichromatic
vision amongst ungulates (e.g. domestic goat: Siemers et al.
1999; sheep: Munkenbeck 1982; domestic pig: Koba and
Tanida 1999; fallow and white-tailed deer: Jacobs et al.
1994, Birgersson et al. 2001; horse: Carroll et al. 2001),
strongly suggests that the chamois also possess dichromatic
vision. The PNA staining suggested a high density of cones
in the retina in the chamois. This was supported by
morphological data showing a regular distribution of cells
with inner and outer segments characteristic for cones.
Antibodies specific for short-wavelength and middle-
wavelength cone opsin, respectively, confirmed the exis-
tence of at least two classes of cone types. However, not
all PNA-positive cells were positive for one of the two
antibodies. Assuming that PNA exclusively stains cones in
the chamois retina as it does in other species including
human, monkey, fish, rabbit, chicken, guinea pig, mouse
rat and others (Blanks and Johnson 1984; Krishnamoorthy
et al. 2008; Hageman and Johnson 1986; Unoki et al. 1988),
this result implies that many cones were not recognized by
the antibodies used here. The reason for this remains unclear
but may include restricted accessibility of the antibodies to
the opsin proteins in some of the antibodies to the cones in
the flat mounted retinas or some unknown protein modifi-
cations preventing the interaction with the antibodies.
Alternatively, chamois retinas may contain cone types that
express a visual pigment not detected by the antibodies
or PNA may stain additional noncone cell classes in the
chamois retina.
The presence of colour vision in chamois can only be
definitively resolved with further physiological experiments
such as the flicker photometric electroretinogram method,
which has been conducted on numerous species to conclu-
sively prove dichromatic vision (goat, sheep and cow, Bos
primigenius:Jacobsetal.1998; fallow and white-tailed
deer: Jacobs et al. 1994; fox, Vulpes vulpes and dog, Canis
lupus familiaris: Jacobs et al. 1993; horse: Carroll et al.
2001). Secondly, independent of their ability to detect col-
ours, it is already known that chamois are able to at least
perceive differences in the brightness of colours (Albrecht
Eur J Wildl Res

1988). Therefore, they should still have been able to detect
the difference in brightness of the different coloured coats.
Brightness contrast between an object and its background is
known to influence its conspicuousness (Endler 1993). For
example, the dark tone of the red coat against a light sky
may appear more achromatically conspicuous than the light
coloured tone of the blue coat.
In addition to detecting two cone types in the chamois
eye, we also found that the eye contains a prominent tape-
tum lucidum in the back of the eyecup. This reflective
structure acts like a mirror, reflecting light back through
the retina. In this way, the retina is provided with two
opportunities to catch and absorb the light entering the
eye. Nocturnal animals or species inhabiting dark environ-
ments typically possess this structure, which is thought to
improve vision in low light conditions (Ollivier et al. 2004).
The detection of a tapetum lucidum in the chamois eye
supports behavioural data that chamois, although largely
diurnal, are also night-active (Ingold et al. 1998).
Responses of chamois to hikers
The two response variables measured during the behaviou-
ral experiments (alert distance and flight initiation distance)
are likely to reflect different stages of response to potential
predators (Schnidrig-Petrig and Ingold 2001). Firstly, alert
distance reflects the moment of detection of the potential
threat. This variable should be correlated with how conspic-
uous the approaching threat is. Therefore, in the context of
our experiments, it would be expected that differences in
alert distances in response to the conspicuousness of differ-
ent jackets would be detected. Similarly, flight distances
may be longer for more conspicuous colours as hikers are
presumed to be visible at greater distances, and hence, for
longer periods of time during the flight response of the
chamois. Despite these predictions, we detected no differ-
ence in the chamois alert and flight initiation distance to the
various jacket colours. Although it was not possible in this
study to quantify the conspicuousness of the three coloured
coats relative to chamois vision, it is at least likely that the
azure blue coat is less conspicuous than the yellow coat,
given that hikers approached the chamois with a sky blue
background. The red coat may also have been inconspicu-
ous, given the lack of long-wavelength-sensitive cones in
the chamois eye. The lack of difference in alert distance
suggests that it may be movement, instead of conspicuous-
ness per se, that alerts the animals to potential threats.
Therefore, if chamois detect the person on the basis of
movement rather than colour, then any effect of raincoat
colour or brightness would be masked by movement of the
hiker.
Our results support a study by Schnidrig-Petrig and
Ingold (2001), which demonstrated that neither the colour
nor brightness of paragliders had an effect on the alert
reactions of chamois. It therefore appears that our findings
are a general trend, in which colours in leisure sport clothes
do not affect the chamois. This is in contrast to other
disturbances which are known to affect chamois, such as
loud voices (Kobelt, 2004) or large group sizes, mountain
biking, cross-country hiking, snowmobiles, cross-country
skiing, aircrafts and approaches from unexpected locations
(Frid 2003;Nayloretal.2009; Neumann et al. 2010;
Reimers et al. 2003; Stankowich 2008). However, our data
do indicate that disturbances by humans are costly to the
chamois. For example, the chamois still covered relative
long and hilly distances to their refuge site after the ap-
proach by the hiker. Along with the increased energetic
costs and stress associated with this flight response, leaving
the salt lick may also be costly in itself (see below). The
flight response may also affect the chamois' feeding activity
(Cederna and Lovari 1985; Hamr 1988).
In addition to the lack of difference in the response to
different coloured rain coats, we also found no affect on
the group size or presence of kids. The former result is
consistent with the findings by Bäbler (2001), but con-
trasts with those by Cederna and Lovari (1985)who
showed that chamois have a more reactive flight re-
sponse when in smaller group sizes. This lack of con-
sensus between studies indicates that additional factors
may influence how vigilant animals are in different
group sizes (e.g. presence of obstructions: Harkin et al.
2000; reproductive state; Klose et al. 2009). For exam-
ple, in contrast to our results, Patterson (1988) showed
that grazing females with kids initiated a flight response
at a greater distance from the threat compared to females
without kids.
Despite following a standard protocol to measure flight
response, our data need to be interpreted with some caution,
as several factors may have masked the effects of raincoat
colouration on the animals' response. For example, the study
was conducted during a summer period which was unchar-
acteristically hot. The experimental lick was one of the very
few in the area that had not completely desiccated.
Therefore, it was an extremely valuable resource for the
chamois, especially for females with kids. Due to the poten-
tially high costs of leaving the salt lick, animals may have
stayed at the lick for as long as possible. Our observation
that several chamois did not take flight from the approach-
ing person towards the end of the season supports this
hypothesis (S. Raveh, personal observation). Several studies
in other taxa have found that the increased costs of leaving a
feeding site influences flight behaviour (Bellman and
Krasne 1983;Cooper2000;Cooperetal.2003;Krause
and Godin 1996; Scrimgeour and Culp 1994; Stankowich
and Blumstein 2005; Tyler 1991). Thus, a female's decision-
makingmayvarywithseasonduetodifferent
Eur J Wildl Res

environmental and individual conditions and thereby influ-
ence risk sensitivities and decision rules (Stankowich 2008).
However, this could not explain why we found no differ-
ences in the alert distances between colours. The lick was
located in an area where few tourist activities occur and
where hunting is not permitted. This may influence the
behaviour of the chamois in this area as hunted populations
show significantly greater flight responses than nonhunted
populations (Stankowich 2008). Although we measured
flight responses of females in an area containing licks,
repeating the study in other contexts (e.g. in pastures where
animals are grazing or flight responses of males) may well
prove to be illuminating. For example, Dalmau et al. (2010)
showed that males are more vigilant than females in the
closely related Pyrenean chamois (Rupicapra pyrenaica
pyrenaica). In addition, repeating the study using natural
greens and browns normally worn by hunters could provide
a more complete picture of how clothing affects the
behaviour of wildlife. Combining our findings with such
additional experiments would be integral to state conclu-
sively that colour plays no role in the fear response of
chamois and to allow appropriate formulation of tourism
management strategies.
Acknowledgments We thank the CantonBern and the Jagdinspektorat
for generously providing the hunting lodge and the Jagdinspektor P. Juesy
and the gamekeepers R. Fuchs and P. Schwendimann for providing the
eyes of the chamois. Furthermore, we thank the Bern University Hospital
Department of Veterinary Medicine, M. Stoffel and his team for providing
chemical additives, as well as K. Bennmann for the demonstration of eye
enucleation. Thanks to Prof. Charlotte Remé (Dr. Med) and Andreas
Wenzel (Dr. sc. nat.) of the Department of Ophthalmology, University
of Zurich for the examination of the chamois retinas and their suggestions
for this paper and also to Jeremy Nathans (Johns Hopkins, Baltimore,
USA) for generously supplying the primary antibodies. We are grateful to
J.-P. Airoldi's statistical help, as well as to C. Raaflaub, A. Imhasly, P.
Enggist, T. Karels, R. Bergmüller, S. Kenyon, A. Nesterova and the AEN
team's comments on the manuscript.
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