Chemical kin label in seabirds: electronic supplementary material
Supplementary methods and results
MATERIALS AND METHODS
Apparatus
Odour tests were performed in a 4 hole-board apparatus (45.0 cm X 45.0 cm X 30.0 cm high)
enclosed by grey Plexiglas. We used 0, 1 or 2 holes among the 4, according to the phase of
the odour test. The hole-board apparatus was placed on the floor of the room (3.0 m X 3.0 m
X 2.4 m high). Holes (3.0 cm diameter X 2.5 cm in depth) were located in each corner of the
board and were 6.0 cm away from the sidewalls, each hole delimiting an open space in which
the odour sample was inserted (See fig 1 for apparatus configuration). When not used, the
holes were sealed. The start box consisted of a grey PVC tube (Ø 20.0 cm X 20.0 cm high)
placed in the centre of the board where the mice were enclosed for 15 seconds at the
beginning of each phase of the test. The apparatus was placed in a room exposed to two lux
lights. Before each mouse was tested, and between each phase of a test, the apparatus was
cleaned with 50% ethanol, then with water. Photocells placed in each hole were used to
automatically evaluate the number of sniff bouts (head-dips) in each hole (Celerier et al.,
2004). When only one hole was active (i.e. contained one odour sample), the parameter used
to evaluate odour investigation was the number of sniff bouts. When two holes were active,
the parameter used to evaluate the investigation of one odour relative to the other was the
percentage of sniff bouts, considering that the percentage expected by chance is 50%. This
parameter allowed us to correct for differences between individual mice in their general
exploratory behaviour, which is probably more dependent on variations of locomotor activity
and personality [1]and stress [2] , across individuals than on the sensorial perception of odour
differences.
Preparation of odour samples
After been collected in the field on cotton swab, the samples were sealed in a glass jar, and
kept at 5-7° C for 3-4 weeks in the field hut and then frozen at -20°C until odour samples
were used. The loss of similarity in samples as a time function for birds’ volatiles on the
Hungarian swabs we used is not known. In [3] authors tested similar swabs with human
odours, and the percentage loss of similarity at room temperature in the dark was about 20%
over 3-4 weeks. Given that our samples were kept in the dark at lower temperature (lower
than 7° C) we can expect a lower percentage loss.
Tests were performed on the odours collected from a subsample of 54 blue petrels (out of the
106 odour samples available) and distributed as follows:
- Experiment 1: odours from 13 individual adult petrel females were used.
- Experiment 2N: odours from four female nestling petrels were used as referent odour
in habituation phase; eight adult females (four mothers and four unrelated females), and six
adult males (four fathers and two unrelated males) were used for discrimination phase.
- Experiment 2F: odours from three female nestling petrels were used as referent
odour in habituation phase; 10 adult females (three mothers and seven unrelated females), and
10 adult males (three fathers and seven unrelated males) were used for discrimination phase.
In Experiments 2a and 2b the number of animals is justified as follow. The year of nestlings
sampling we sampled 11 chicks. The sampled chicks resulted to be four females and seven
males. The year of fledglings sampling, we were able to sample only three chicks out of 11
nests selected in this field campaign. This was due probably to a severe food shortage that led
to desertion (2 nests) or chick death before fledging (1 nest). Moreover four chicks were
predated by skuas (Catharacta skua lönnbergi), a petrel predator, and one chick fledged before
our arrival in the colony. The three chicks sampled resulted to be all female. Having only
fledgling females (n=3) we preferred to use only nestling females (n=4) to avoid a potential
sex effect and have comparable experiments.
An odour sample consisted of a rectangular piece (2 cm ± 0.1 X 1.8 cm ± 0.1) from the cotton
swab impregnated with petrel odours. Each odour sample was placed in a clean phial, inserted
between two squares of gauze (3x3 cm) and held in position by a perforated cap (Ø 2 cm) so
that mice could come into direct contact and perceive both the volatile and non-volatile
components of the odour [4]. The phial was inserted at the bottom of the active hole in the
apparatus and fixed so that it could not be moved by the animals. The odour samples were
stored at 4°C in hermetically sealed bottles and placed at room temperature 10 minutes before
testing. Each odour sample was prepared and manipulated using new nitrile gloves.
Mice and housing
The subjects used as “noses” in our “biological olfactometer” were 131 naïve adult male
Swiss mice (2 months old; 28-32 g) obtained from DEPRE (France). Animals were housed in
groups of 10 in cages (40 cm long X 25 cm large X 15 cm high; ), on a litter made up of
crushed and dried Epicea wood with access to food and water ad libitum. They were kept in a
temperature (21°C) and humidity (50%) controlled facility on a 12 h/12 h light-dark cycle. All
test procedures were conducted during the light phase of the cycle, between 9:00 and 16:00 in
a sound-attenuated and air-regulated experimental room, to which the mice were habituated at
least 12 h before behavioural testing.
Habituation – discrimination tests (Table 1)
Habituation – discrimination is a procedure used to assess the perception of
differences in odour signals [5]. Before each experiment, mice were allowed to freely explore
the apparatus in the absence of odour stimuli for two minutes, in order to get used to the
apparatus. Then the animals were submitted to a habituation phase, followed by a
discrimination phase after a two minutes interval.
Habituation phase: This phase consisted of the display of one referent petrel odour (R) to a
mouse for two trials of 10 minutes each separated by a two-minute interval (the referent odour
was replaced by a fresh odour, i.e. a new piece of swab from the same individual, between
trials 1 and 2 to ensure that habituation was not due to odour degradation). In Exp. 1 the
referent odour belongs to adult female. In Exp.2 the referent odour belongs to chick female
with down in Exp.2N (Nestlings chicks) and with feathers in Exp.2F (Fledglings Chicks). A
mouse was considered habituated when it displayed a reduced interest in the odour between
trials 1 and 2 of habituation phase. Obviously, only habituated mice were tested in the second
discrimination phase.
Discrimination phase (test): In Exp. 1, the mouse was simultaneously presented with two
odours for five minutes: the test odour (the odour of a petrel unrelated to the referent petrel, to
which the mouse was not familiar: odour A) and the referent odour (a fresh sample: odour B).
In Exp. 2, the mouse was simultaneously presented with two odours for five minutes: odour A
belongs to a petrel (adult male or female) unrelated to the referent chick whereas odour B
belongs to a petrel closely related to the referent chick (i.e. its father or mother). For both
Exp.1 and Exp. 2, if the subject perceived odour A as more different in quality from the
referent odour than odour B, it should investigate odour A more often than expected by
chance (>50%). Each odour triad formed by R, A, and B odours was tested by three mice, and
each mouse was used only once. We used the mean of the mice used with the same triads of
odours for the statistical analysis. The habituation and discrimination odours were always
placed in different holes, their locations were counterbalanced across tests, and the
experimenter was blind to the identity of odours.
Statistical Analysis
Data were analysed using either an exact permutation test for paired samples when
N<30 or a student t test for paired samples when N≥30.
For habituation data analysis, the number of sniff bouts in trials 1 and 2 were
compared: the null hypothesis H0 is that the number of sniff bouts is identical in trial 1 and
trial 2; the alternative hypothesis H1 is that the number of sniff bouts in trial 2 is lower than
the number in trial 1.
According to standard habituation-discrimination protocols, in the discrimination
phase, if the subject perceives a greater difference between odour A (unrelated) and the
referent odour than between odour B (related) and the referent odour, it is expected to
investigate odour A more often than expected by chance (50%). Thus, for the discrimination
data analysis, the percentage of sniff bouts of the unrelated odour (odour A) was compared to
the percentage expected by chance (50%): the null hypothesis H0 is that the percentage of
sniff bouts of the unrelated odour is at chance level (50%); the alternative hypothesis H1 is
that the percentage of sniff bouts of the unrelated odour is above chance level.
RESULTS
Habituation phase (see figure 1)
The mice actively investigated all the odours presented and showed no sign of
avoidance to any stimuli in all experiments, during the habituation phase. The mice exhibited
a significant decline of interest for the referent odour between trial 1 and trial 2, showing that
subjects progressively became familiar to petrel odours. More precisely, in Exp.1, mice (N=
46 from 16 odour triads) exhibited 22.3  2.7sniff bouts to the referent petrel odour (R) during
trial 1 and 18.1  2.7 sniff bouts during trial 2 (p = 0.04; Fig. 1a). In Exp.2N, mice (N= 49
from 17 odour triads) exhibited 23.9  2.8 sniff bouts to R during trial 1 and 17.6  2.4 sniff
bouts during trial 2 (p = 0.01; Fig. 2b). Finally, in Exp. 2F, mice (N= 36 from 12 odour triads)
exhibited 29.8  1.5 sniff bouts to R during trial 1 and 22.0  3.4 sniff bouts during trial 2 (p
= 0.04; Fig. 2c). As previously mentioned, when mice did not fulfil habituation criterion (i.e.
a decline in exploration of R between trial 1 and trial 2), they were not tested in the
discrimination phase.
Discrimination phase (see figure 2)
During the discrimination phase, 14 out of 131 mice were excluded from the data
analysis because they did not investigate the 2 holes at least once during the test phase.
Furthermore, 39 mice did not fulfil our habituation criterion in the first phase so they were not
tested in the second discrimination phase. Finally, discrimination phase analysis were
performed on data from 25 mice for Exp.1, 29 mice for Exp.2N and 24 mice for Exp.2F.
These data emanated from :
-
17 pairs “non related female/mother of the referent nestling chick” and 12
pairs “non related male/father of the referent nestling chick” in Exp.2N
-
12 pairs “non related female/mother of the referent fledging chick” and 12
pairs “non related male/father of the referent fledging chick” in Exp.2F
It may be noticed that the discrimination rate of nose mice (55.7-55.9%) is lower than
that in our previous study with baboon odours (60.2-64.4%) [6].The main difference of the
present study is that mice had to discriminate between non-mammals odours. The
phylogenetic distance between mice and birds may explain the lower sensitivity of the mice
discrimination. Indeed, chemicals compounds emitted by birds could be different from those
emitted by mammals and then could be perceived by mice with less acuity, resulting in a
lower resolution power. In agreement with this hypothesis, at least according to human nose,
the musky scent of petrels smells far away from mammals’ odours. An alternative hypothesis
is that a slight degradation of the samples might have smoothed differences/similarities
among samples.
1 Montiglio, P. O., Garant, D., Thomas, D., Reale, D. Individual variation in temporal activity patterns
in open-field tests. Animal Behaviour. 80, 905-912.
2 Hurst, J. L., West, R. S. Taming anxiety in laboratory mice. Nat. Methods. 7, 825-U1516.
3 Hudson, D. T., Curran, A. M., Furton, K. G. 2009 The Stability of Collected Human Scent Under
Various Environmental Conditions. J. Forensic Sci. 54, 1270-1277.
4 Cheetham, S. A., Thom, M. D., Jury, F., Ollier, W. E. R., Beynon, R. J., Hurst, J. L. 2007 The genetic
basis of individual-recognition signals in the mouse. Current Biology. 17, 1771-1777.
5 Halpin, Z. T. 1986 Individual odors among mammals: origins and functions. Adv. Study Behav. 16,
39-70.
6 Celerier, A., Huchard, E., Alvergne, A., Fejan, D., Plard, F., Cowlishaw, G., Raymond, M., Knapp, L. A.,
Bonadonna, F. 2010 Detective mice assess relatedness in baboons using olfactory cues. J. Exp. Biol.
213, 1399-1405.