Familiarity Perception Call Elicited under Restricted
Sensory Cues in Peer-Social Interactions of the Domestic
Mamiko Koshiba1,2*, Yuka Shirakawa1, Koki Mimura1, Aya Senoo1, Genta Karino1, Shun Nakamura1,2
1Tokyo University of Agriculture and Technology, Tokyo, Japan, 2National Center of Neurology and Psychiatry, NCNP, Tokyo, Japan
Social cognitive mechanisms are central to understanding developmental abnormalities, such as autistic spectrum disorder.
Peer relations besides parent-infant or pair-bonding interactions are pivotal social relationships that are especially well
developed in humans. Cognition of familiarity forms the basis of peer socialization. Domestic chick (Gallus gallus) studies
have contributed to our understanding of the developmental process in sensory-motor cognition but many processes
remain unknown. In this report, we used chicks, as they are precocial birds, and we could therefore focus on peer interaction
without having to consider parenting. The subject chick behavior towards familiar and unfamiliar reference peers was video-
recorded, where the subject and the reference were separated by either an opaque or transparent wall. Spectrogram and
behavior correlation analyses based on principal component analysis, revealed that chicks elicited an intermediate contact
call and a morphologically different distress call, more frequently towards familiar versus unfamiliar chicks in acoustic only
conditions. When both visual and acoustic cues were present, subject chicks exhibited approaching and floor pecking
behavior, while eliciting joyful (pleasant) calls, irrespective of whether reference peers were familiar or unfamiliar. Our result
showed that chicks recognized familiarity using acoustic cues and expressed cognition through modified distress calls.
These finding suggests that peer affiliation may be established by acoustic recognition, independent of visual face
recognition, and that eventually, both forms of recognition are integrated, with modulation of acoustic recognition.
Citation: Koshiba M, Shirakawa Y, Mimura K, Senoo A, Karino G, et al. (2013) Familiarity Perception Call Elicited under Restricted Sensory Cues in Peer-Social
Interactions of the Domestic Chick. PLoS ONE 8(3): e58847. doi:10.1371/journal.pone.0058847
Editor: Johan J. Bolhuis, Utrecht University, The Netherlands
Received November 30, 2012; Accepted February 7, 2013; Published March 8, 2013
Copyright: ? 2013 Koshiba et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by MEXT, Grant-in-Aid for Scientific Research on Innovative Areas (21200017), Comprehensive study on making practical
guideline for developmental disorders based on the neuroscientific findings, and by the Intramural Research Grant (22–6) for Neurological and Psychiatric
Disorders of NCNP. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Neurobiological understanding of socio-emotional cognition is
crucial to diagnosis and therapeutic intervention in social-domain
specific developmental disorders, such as autistic spectrum
disorders and other psychiatric illnesses. Recent advances in
behavioral and neurobiological studies on neonatal development
of sensory-motor cognition in animates and specification of social
individuals  have revealed domain-relevant biases toward faces
, and this type of attention bias is not restricted to face
recognition, but also towards self-propelled causal agency  and
biological motion recognition [4–7]. Similar arguments can be put
forward in relation to auditory ,  and olfactory recognition
Domestic chick (Gallus gallus) studies have contributed to the
formulation of a two process theory, namely CONSPEC and
CONLERN. CONSPEC proposes that widely divergent verte-
brates possess similar domain-relevant biases toward faces. A
chick’s natural predisposition mechanisms adhere to CONSPEC.
Additionally, attention biases lead to neuronal based development
of species specific individual cognition or CONLERN. It is
thought that chick, imprinting mechanisms correlate to CON-
LERN, and are processed through the intermediate medial
mesopallium (IMM) area . The neuronal substrates relevant
to CONSPEC and CONLERN comprise the subcortical face-
recognition route, which provides a developmental foundation for
what later becomes the adult cortical ‘social brain’ network .
However, it is uncertain how early neuronal substrates for social
affiliation are integrated as part of the social brain network, a
process that is crucial to understanding socio-emotional develop-
ment and its disorders. Here, we developed a peer social affiliation
chick model, covering a series of developmental stages, and
focused on familiarity cognition using acoustic cues.
In animal communication behavior, the ‘‘call’’ occupies a
unique position, since it is a direct sound transmission of the
sender’s emotional state . The receiver can then decode the
sound and make a response in the form of an action or another
call. This mutual interaction makes a communication loop,
allowing both the sender and receiver to understand the meaning
of calls . Thus, it is crucial to understand and investigate call
behavior in the context of socio-ecological interaction .
Contact calls have been studied extensively and the individuality
of mate and kin relationships in mammals and birds has been
recognized . To date, there are few reports describing
familiarity recognition within con-specific or hetero-specific
groups, beyond mate and kin relationships ,. These
observations suggest that some types of contact calls are learned
and can dynamically change structure during social interactions
PLOS ONE | www.plosone.org1March 2013 | Volume 8 | Issue 3 | e58847
,. However, it is uncertain how the sensory cues of the
social communicator are related to the changes in vocal structure.
Since the pioneering study of chick calls by Collias & Joos ,
spectrograph analysis has revealed great detail in call types, along
with the behavioral and functional relevance of each call.
Domestic fowl chicks emit four different types of calls, a distress
call (cheeps, peeps), pleasure notes (twitterings), an intermediate
call (short peep), and a fear trill .
In this study, two groups of chicks were reared separately and
call behavior was examined in interaction tests observing reactions
between familiar and unfamiliar chicks. All animals were provided
with acoustic cues but at times were deprived of visual cues. To
assess call function, we performed multivariate analysis based on
principal component analysis and visualized the correlation
structure of call types and other behavior parameters , such
as floor pecking. When restricted to acoustic cues, subject chicks
emitted intermediate calls more often to familiar chicks, relative to
unfamiliar chicks, as well as a complex distress call. We found no
significant differences in subject chick call behavior when both
visual and acoustic cues were present in the meetings with familiar
and unfamiliar reference chicks.
1. Call modulation mediated by repeated meetings with
familiar reference chicks
First, we examined call development in subject chicks, over
postnatal days 3 to 16 (P3 to P16) (Figure 1a). The spectrogram
showed a decline in the frequency difference in kHz (f22f1) and
morphological variation (MV) of the first call-component. This
experiment design, shown in Figure 1a, consisted of two contexts.
In Phase I, the subject chick was placed in isolation and in Phase
II, the subject chick was exposed visually and acoustically to the
same reference chicks over time. Our results suggested that call
modulation in subject chicks may be a natural result of
development, as well as an adaptation to repeated exposure to
the same reference chicks in the meeting test. Next, we examined
this assumption by comparing the call frequency difference (f22f1)
between a subject chick exposed repeatedly to the same reference
chicks (repeatedly tested, R) and a subject chick exposed once to
unfamiliar chicks (once-tested, O) (Figure 1b). The regression line
fitting of the O-group chick was slightly negative over time,
although the Pearson’s correlation coefficient was low (R2=0.13).
On the other hand, the regression line (black) slope of the R-group
chick increased (R2=0.50) over time, suggesting an effect for
repetitive exposure to familiar chicks. Next, we compared MV
values between the two test groups (Figure 1c). The linear fit line
slope of the O-group remained almost constant over time
(R2=0.013), while that of the R-group decreased significantly
(R2=0.67). Taken together, these results implied that repeated
meetings with familiar chicks modulated call behavior in subject
chicks, hinting at the existence of social memory. The next step
was to investigate the possible relationship between call morphol-
ogy and social experience, by examining first f22f1 and then MV
2. Social context dependent shifts in call types, towards
familiar and unfamiliar reference chicks
A subject chick met both familiar (Figure 2b, Fam) and
unfamiliar (Figure 2b, Unfam) chicks in a series of tests (Figure 2b).
The series consisted of Familiar and Unfamiliar meetings within
three contexts (isolation, acoustic only cues (v2a+), and visual and
acoustic cues (v+a+), (see more details in Materials and Methods).
To avoid a possible bias in the meeting order, we randomized the
order in which subject chicks met Fam and Unfam reference
chicks. First, we classified three call types according to the
frequency difference (f22f1) within the first component of the call
sonogram (Figure 2c). From 15 birds, we plotted 30-second
periods of data in a call-number histogram, with f22f1 values
from 26 to 2.5[kHz], and with 0.5 kHz bin steps in each context
(Figure 2a). In the isolation context (Figures 2a–1 and –4), a single
peak appeared at around 23[kHz], irrespective of whether the call
was to Fam or Unfam chicks. In the (v2a+) context, twin peaks
appeared in calls to Fam chicks, but a single peak appeared in calls
to Unfam chicks (Figures 2a–2 and –5). On the other hand, in the
(v+a+) context, the difference between calls to Fam and Unfam
chicks was more subtle (Figures 2a–3 and –6). These results
suggested that the dj-call may convey information of familiarity in
the v2a+ context. More detailed analysis of call frequency in each
context is described below.
3. Modulation of call morphological variation depending
on the recognition of familiarity
We introduced call morphological variation (MV) as a
parameter which may correlate with the cognition of familiarity
in Figure 1. We further assessed this point by comparing MV
scores of subject chicks calls to Fam and to Unfam chicks in the
v2a+ context. Since MV scores appeared higher in D- rather than
dj- or J-calls, we compared MV scores for D-calls elicited by
reference chicks. Each subject chick randomly met both Fam and
Unfam peers in separate tests. We then classified the subject call
type elicited for 30 seconds, in response to a call from the
reference chick. From the 15 subject chicks, we noted four
combinations of call types as summarized in Figure 3a. A plot of
MV values from 10 subject D-calls emitted in response to Unfam
(U) and Fam (F) chick calls is shown in Figure 3b. A liner fit line
showed MV values toward familiar peers was significantly higher
than toward unfamiliar peers. To examine this result further, we
compared morphological similarity, using sound analysis software
distributed by Dr. O. Tchernichovski . The similarity scores of
U–F were significantly lower than those for U–U and F–F
(Figure 3c), confirming a morphological difference between D-calls
to Fam and Unfam peers.
4. Transient dj-calls as a parameter of familiarity
Subject chick behavior changed depending on the social
context. Typically, chicks froze or roved, emitting a D-call when
in isolation, roved and pecked the surrounding walls emitting a D-
or dj-call in an acoustic only context, or approached peers, pecking
the floor and emitting a J-call in the visual and acoustic cue
context (see more details in Materials and Methods). In this
analysis, we aimed to identify behavioral parameters that differ
quantitatively in each context by defining the complex correlation
of the three call types and the behavioral parameters, using
principal component analysis (PCA). The social contexts examined
were U v2a+ , U v+a+, F v2a+ and F v+a+, where the capital
letter denotes a meeting with either Unfamiliar (U) or Familiar (F)
reference peers and the lower case letters denote the context of
either acoustic stimulus only (v2a+) or acoustic and visual stimuli
(v+a+). The behavioral parameters were extracted from video
data, as follows; floor-peck, wall-peck, face to peers, and head-
movement (see more details in Materials and Methods). The
correlation between behavioral parameters and D-, dj- and J-call
frequency was then investigated by PCA with correlation matrices,
and visualized as factor loading vectors (Figure 4a). The most
significant difference in behavioral features between Unfam and
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org2March 2013 | Volume 8 | Issue 3 | e58847
Fam peer meetings appeared in the v2a+ context (F=3.53,
p=0.037, Wilks’ lambda), in contrast to the v+a+ context
(Figure 4b, upper panel). The parameters contributing to
differences can be explained by the factor loading vectors. The
distribution of the behavioral vector in the Fam context after PCA
(Figure 4b), expanded into the 4th quadrant and correlated
positively with the dj-call factor loading vector and negatively with
the D-call vector in the v2a+ context. Two sampled t-tests
between Unfam and Fam call behavior showed a significant
difference in dj-call frequency (Figure 4c, djcall). On the other
hand, there was no significant difference between meetings with
Unfam and Fam peers in the v+a+ context (Wilks’ lambda;
F=0.37, p=0.69). Next, we compared context dependent
changes of behavior in Unfam and Fam meetings (Figure 4b
bottom panel). Only in the Fam series, was the context-dependent
behavior shift (from v2a+ to v+a+) statistically significant (Wilks’
lambda; F=3.84, P=0.029), primarily due to the shift in J-call
and floor-pecking parameters in their positive directions, however
J-call was significantly different between v2a+ and v+a+ in Unfam
(Figure 4c). In summary, we found that dj-call frequency
correlated with familiarity cognition in the v2a+ context and
the high frequency of J calls as well as floor pecking in the context
shift from v2a+ to the v+a+ irrespective of whether reference
peers were familiar or unfamiliar.
Matrix-based PCA suggested similar modulations from the
v2a+ to v+a+ contexts, with increasing J-calls and floor pecking
behavior, as positive factors, and with no behaviors related to D-
or dj-calls (Figure 4). To represent the structure of the most
correlated multiple parameters within either the v2a+ or v+a+
contexts, each context was examined by PCA and then the factor
Figure 1. Age- or repetition-dependent call modulation. a. Subject chick behavior was video recorded in the serial social context depicted in
the left scheme (isolation and v2a+ contact). Spectrograms on the right showed typical calls produced by one subject chick on postnatal days 3, 8
and 16 (P3, 8, 16). The subject was exposed to the same reference chicks at P3, P8, and P16. Typical morphological values are indicated by serial
numbers (1–5 at P3, 1–5 at P8 and 1–3 at P16). Calls emitted at P3 and P8 were defined as a ‘‘D-call’’, and calls at P16 as a ‘‘dj-call’’. The call type and
morphology are described in Materials and Methods. b. Modulation of call frequency differences (f22f1 value) over developmental time. The average
of five minimum values of f22f1 (negative values, since frequency f1.f2) during behavioral tests was plotted against the test day (postnatal day).
Black dots represent daily values of subject chicks meeting the same reference chicks during a test, against post natal day of development (the
repeatedly-tested chicks), and red-crosses signify values of subject chicks meeting unfamiliar chicks during a test against post natal day of
development (once tested chicks). The colored lines represent the respective linear regression fitted line. c. Modulation of call morphology over
developmental time. The black dots represent values from repeatedly-tested chicks (see above) and red-crosses, values from once tested chicks (see
above). The colored lines represent the respective linear regression fitted line.
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org3 March 2013 | Volume 8 | Issue 3 | e58847
loadings were represented as positively directed vectors from the
averaged center. Furthermore, to visualize the specific areas
representing subject chicks toward familiar or unfamiliar peers, the
ellipse variance approximation was superimposed. This resulted in
a greater difference between the F and U meetings in the v2a+
context. This difference appeared not in the 1st, but the 4th
component (unpaired two sample T-test for unequal sample sizes
and unequal variance in the 4th component: P=0.0095). In
contrast, the subjects showed very similar behavior towards
familiar and unfamiliar chicks in the v+a+ context. These
combined results suggested that dj-call was a unique behavior
elicited from subject chicks toward familiar peers in the v2a+
context. It may be related to Morton’s motivation-structure rules
 that call type shift from D-call to dj-call during isolation to
v2a+ and dj-call to j-call during v2a+ to v+a+.
In this study, we found that the recognition of familiarity was
expressed in three ways by 15 day old subject chicks. Firstly, the
Figure 2. Changes of call type defined by f22 2f1 frequency in different social contexts. a. Shifting distribution of f22f1frequency in
different social contexts. The series a21 to a26 correspond to the social contexts described in Figure 2b. The peak call number at particular f22f1
values (kHz) is marked by arrows in each social context. Details are described in the text. b. The serial context is as follows: first, isolation; second,
acoustic only exposure to unfamiliar reference chicks (v2a+); third, visual and acoustic exposure (v+a+) to unfamiliar reference chicks; fourth, a
second period of isolation; fifth, v2a+ exposure to familiar chicks and sixth, v+a+ exposure to familiar reference chicks. c. Typical spectrogram of
subject chick calls. D, dj and J, denote the D-call, dj-call and J-call respectively. The D-call is a ‘‘negative’’ expression signifying ‘‘dissatisfaction’’ or
‘‘distress’’, the J-call is a ‘‘positive expression’’ signifying something ‘‘pleasant’’ or ‘‘joyful’’, while the dj call is an intermediate call between the D and J
calls. The black bar denotes 0.2 seconds.
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org4 March 2013 | Volume 8 | Issue 3 | e58847
rate of dj-calls in the v2a+ context increased when emitted to
familiar peers relative to unfamiliar peers. Secondly, D-call
morphology showed greater complexity in calls emitted to familiar
peers in the v2a+ context. Lastly, the rate of J-calls increased
when emitted to familiar peers, with concomitant approach and
floor pecking behavior in the v+a+ context from the v2a+ context.
This result suggested that acoustic cues of familiar peers elicited dj-
calls as well as D-call complexity and that visual cues elicited J-calls
irrespective of familiar or unfamiliar perception. Chicks were
deprived of the somatosensory cues resulting from pecking and
floor scratching, by placing the boxes containing a subject and
reference peers on separate platforms. Similarly the sensory cue of
olfaction was also removed or at least diminished by covering the
test box with a transparent plastic sheet, to prevent air flow
between the cages. These combined results suggested that dj-call
was a unique behavior elicited from subject chicks toward familiar
peers in the v2a+ context. It may be related to Morton’s
motivation-structure rules  that call type shift from D-call to
dj-call during isolation to v2a+ and dj-call to j-call during v2a+ to
v+a+. Our finding suggests that peer affiliation can be established
by acoustic recognition, independent of visual face recognition [1–
2] and when integrated, acoustic recognition is modulated. Widely
divergent vertebrates possess similar domain-relevant biases
toward visual facial cues and similar arguments have been made
for auditory cues . However, the precise mechanisms for
visual recognition dominance are undetermined, The mechanisms
relating to how cross modal sensory integration develops at
behavioral and neurobiological levels, is a subject for further
Subject chicks met reference chicks either through being reared
in the same cage from hatching (familiar reference) or reared in a
different group (unfamiliar reference). In the familiar reference
tests, we observed a decreased call rate and intensity (f22f1 value),
even though the subject and reference chicks had shared the same
cage until the day of testing. The reason for this call modulation in
the repeated familiar reference test may be related to the fact that
in unfamiliar cages, subject chicks suffered stress, as indicated by
roving behavior and an accompanying D-call. In this situation,
meeting familiar peers may reduce this stress, while unfamiliar
peers may impose additional stress ,. As repeated
testing affected call behavior, we used a single test to evaluate chick
behavior towards familiar and unfamiliar references. As men-
tioned earlier, the highly dynamic nature of the contact call has
already been recognized and the development of individual chick
recognition proceeds in parallel with the segregation of inter group
call perception [25–27]. Chicks recognize a number of maternal
calls, including the food call, follow me call, roosting call, predator
call, and fear call ,[28–30]. It is unknown if chicks are capable
of recognizing familiar peer calls based on individual chick
recognition. In this study, we did not address the issue of whether
subject chicks recognized the calls of individual reference chicks in
familiarity cognition, nor did we examine the type of call that
induced calls expressing familiarity. Since our test situation
presented reference chicks in groups of two to four chicks, a
different type of study at a future date is needed examine these
The dj-call was the major call type elicited to familiar
reference chicks in the v2a+ context. The intermediate call has
not been as well characterized in literature as the J-call (pleasure
note, twittering call) or D-call (distress call, peeps). Marx et al.,
 showed that intermediate calls (short peeps) were elicited
during a step-wise isolation paradigm, in a particular starting
group size. Call types in the last step, where subject chicks were
left alone, differed significantly depending on the initial peer
Figure 3. Difference in the morphological variation of D calls
with familiarity. a. Call type combinations from subject chicks to
unfamiliar (U) and familiar (F) reference chicks. Each subject chick met
an unfamiliar or familiar reference chick during this test. We counted
calls from subject chicks for 30 seconds after an initial call from
reference chicks. Only D- and dj-calls were observed in the v2a+
context. U(D)–F(dj) denotes meetings where the test chick emitted D-
call including dj-call to unfamiliar chicks and the same chick emitted dj-
calls without D-calls to familiar chicks b. The correlation plot of the call
number against familiar and unfamiliar chicks in the acoustic only
context. The morphological variation of U(D) calls was plotted against
F(D) calls in the V2A+ context (n=10). Here, the call frequency was
higher towards familiar reference chicks. c. Comparison of D calls
emitted to familiar and unfamiliar reference chicks. Each call was
analyzed by Sound Analysis pro using three pairs; U(D)–U(D), U(D)–F(D)
and F(D)–F(D). The error bar denotes the standard error of mean. The
similarity score for U(D)–F(D) calls was significantly lower than for the
other pairings by One-Way ANOVA, and Two sample independent t-
test. In this analysis, we compared calls from pairs of U–U (10 pairs per
session), U–F (25 pairs per session), and F–F (10 pairs per session)
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org5March 2013 | Volume 8 | Issue 3 | e58847
group size. If the group size was greater than four, the major call
type emitted was a D-call, with no intermediate calls. Where
subject chicks started the step-wise isolation test in groups of two
or three, intermediate and D-calls were emitted. The interpre-
tation of this behavior is currently uncertain. Considering that dj-
calls are emitted from chicks in step-wise isolation and
independent of group size, this call may be related to search
attention  and alert conditions. Electrical stimulation of the
intercollicular nucleus (ICo) in the mesencephalon, induced
distress calls in control chicks, and crowing in testosterone-
treated chicks . However, it is unclear whether D- and dj-
calls are variants, derived from the same call output center, or
whether there is a specified motor control center for each call,
which is regulated differentially by emotional and cognitive
neural networks . Studies examining immediate early gene
expression, and multi-point in vivo recording and micro-dialysis
using awake animals, should shed light on these important
In this study, integrative analysis of multi-behavioral param-
eters was effective in identifying the characteristic structure of
complex expressions, such as social behavior, even though the
analysis is based solely on the extraction
parameters from video recordings, without any assumption of
inter-parameter correlation. It is feasible that PCA could be
appliedtothe objective translation
communication and/or non-social interaction with the environ-
ment by animals, including humans. This method may well
provide support to intra and inter-species communication studies,
as an information processing interface.
Materials and Methods
This experimental protocol was approved by the Ethics review
Committee for Animal.
Experiments of the National Institute of Neuroscience, NCNP
(18–40) and Tokyo University of Agriculture and Technology,
TUAT (19–19). These committees follow the animal care and
experimental guidelines of Japan Neuroscience Society and NIH,
Fertilized eggs from domestic chicks (Gallus gallus domestics),
White Leghorn, Maria strain, were purchased from a local
breeder, Miyake Fukajo (Chiba, Japan). They were kept in a dark
incubator (Showa Furanki) at 37.7 degrees centigrade with
approximately 50% humidity and automatic rolling every hour.
On embryonic day 21 (E21, the start of incubation was defined as
E 1), which usually coincided with one day before hatching (the
day of hatching was defined as post natal day 1, P1), three or four
eggs were moved to incubator-boxes in different rooms, separated
by thick concrete walls and ceilings, thus minimizing communi-
cation between the two sets of birds.
Each incubator box was kept at around 28 degrees centigrade,
with a light bulb (10 watt). Constant lighting was maintained from
E21 until P3, after which a 12 h dark-light cycle was set. We used
three different sized incubator boxes ((width) 6(depth) 6(height)
in cm: 20620620, 35632633 and 52640653) to evaluate the
effects of box size on the development of affiliation behavior. The
peers reared in the same incubator were denoted as familiar peers
(Fam or F), while those from different incubators were denoted as
unfamiliar peers (Unfam or U).
Figure 4. Integrated analysis of social behavior towards familiar and unfamiliar reference peers by PCA. a. Subject behavior was video
recorded in the v2a+ and v+a+ contexts. Seven factors were selected to differentiate responses in behavior towards familiar and unfamiliar peers
after the Two sample independent t-test, and multivariate analysis by correlation matrix-based PCA (see detailed description of behavior parameters
used for PCA in the text). b. The scores from the 1st and 4th component by independent PCA for v2a+ (left-upper) and v+a+ (right-upper) were
approximated, with each variance ellipse by variance, co-variance-based PCA for social behavior towards familiar (Fam: black line or dotted) and
unfamiliar (Unfam: grey out line or dotted) peers. The factor loading positive vectors drawn from the averaged center were adjusted three fold. Clear
differences between Fam and Unfam could be seen, as Fam values distributed lower on the 4th component in the v2a+ but not v+a+ context. The
direction of Fam specific distribution in the v2a+ context could be explained by the following factor loading vectors (see Figure 4a and text). The
statistical significance between Fam and Unfam behavior was evaluated by Wilks’ lambda (see Materials and Methods). c. Call frequency of test chick
towards familiar or unfamiliar reference chicks in the v2a+ and v+a+ contexts. P-values were calculated by two-sample T-test and the asterisks show
significant values. Call frequency of dj calls was significantly higher towards familiar reference chicks only in the v2a+ context. However, J call
frequency in the v2a+ context significantly increased relative to calls in the v+a+ context but only towards unfamiliar reference chicks.
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org6March 2013 | Volume 8 | Issue 3 | e58847
During rearing, we avoided social interaction with chicks, since
handling can induce stress or affiliation effects on chicks , .
Chicks were transferred using a small opaque container during
daily incubator cleaning.
2. Behavioral Tests and the definition of typical behavior
Behavioral tests were conducted in the same room where the
subject birds (7 males and 6 females, age: average-standard
deviation. 12.7–3.1 postnatal day) were reared. The subject chick
was put into one of the two transparent metal net boxes covered
with plastic sheets (29 (w) 629 (d) 629 (h) cm) and the reference
chicks into another. The two boxes were placed adjacent to each
other, separated only by a masking board that was either removed
manually or electrochromically controlled. The box containing the
subject chick was covered with a transparent acrylic board, in
order to reduce olfactory cues from the reference bird and
researchers, and also to increase the specificity of call recordings
from subject birds through a microphone in the test box.
As seen in Figures 2a–1, subject chicks underwent the following
six serial peer-social contexts: context 1; initial isolation period
with no reference chicks, and a masking board in place, context 2;
Unfamiliar or Familiar chicks (U/F) were presented with acoustic
only cues, ensured by a separation board (v2a+) , context 3; U/F
chicks were presented with both visual and acoustic cues, after
removing the separation board (v+a+), context 4; second period of
isolation, context 5; similar to context 2, but chicks met alternative
peers, that is F or U chicks, and context 6; similar to context 3, but
the order of chick presentation was reversed (that is, F or U). Each
context lasted for 1–2 minutes. All behavior was recorded using a
top video camera (SONY handycam) with an external microphone
in the test box. The recorded WMV files were transferred into
WAVE and JPEG files using TMPGEnc-2.5 software (Pegasys
Inc., Tokyo). The subject’s typical behavior in each context was
defined according to the following eight kinds of typical behavior:-
1; Freeze with no call, 2; Freeze with D-call, 3; Freeze with dj-call,
4; Roving with D-call, 5; Roving with dj-call, 6; Roving with J-call,
7; Approach, and 8; G-move. The g-move was defined in this
study as approaching behavior, with the chick moving back and
forth along the separation board in the v+a+ context, thought to
represent grouping behavior. In the parameters of Figure 4a, ‘‘face
to peers’’ defined as beak angle to adjacent cage within 45 degrees
(beak-to-separating wall) refers to subject chick’s preferred head
position towards the reference chicks. We further defined pecking
behavior, pecking floor (floor peck) and pecking cage wall (wall
peck) expressed as frequency of pecking behavior per specified
3. Call analysis
We used free software (Syrinx, version 2.4i) distributed by Dr.
John Burt (University of Washington) to analyze chick calls.
Spectrograms were used to identify call types by analyzing
morphology of the first component (Figure 1 to Figure 3). This
analysis sometimes allowed us to detect unique morphological
features from all call components. We defined three call types
based on the frequency change of the first component over time, as
follows: First, we read the first time point (Figure 1a, t1) where the
frequency was maximum/minimum (f1), and the second time
point (t2) where the frequency minimum/maximum (f2). Next, we
calculated D(delta)f = (f22f1). Finally, if D(delta)f ,22, we
designated this call as a D-call. If D(delta)f .0, as a J-call, and if
22, D(delta)f ,0, as a dj-call. A JPEG file was made of all call
spectrograms and the frequency difference D(delta)f (f22f1) was
measured using Image J software (NIH, USA). Morphological
complexity of the 1st component was measured by counting the
number of negative curvatures moving anti-clockwise along the
edge of the morphological Figure, keeping the Figure on the left
side. The call data from each context covered the duration times
as shown below:
Context 1 and 4, first or second isolation period, respectively;
Context 2, U or F under V2A+ condition; for 30 sec after the
1st U/F J- or dj-call.
Context 3, U or F under V+A+ condition; for 30 sec after the
1st U/F J- or dj-call.
We also confirmed call similarity by Sound Analysis Pro
Statistical analysis was performed using Excel (Microsoft) and
OriginPro ver 7.5 (OriginLab). Linear regression was calculated
using Pearson correlation coefficient.
5. Multi-behavioral parameter assays using principal
In order to integrate multi-behavioral parameters, we used
principal component analysis (PCA) using the free software,
MLVAR95 based on a correlation matrix (http://home.a02.
itscom.net/coffee/takoindex.html). In some cases, results were
confirmed by manual calculations using Excel (Microsoft, Office
2003). The calculated scores were plotted in a two dimensional
(2D) plane defined by PCA components. To compare behavioral
patterns among the different groups, we applied the second PCA
to each group of data and fitted the covariance as an ellipse, where
the long axis was derived from the 1st component and the short
axis from the 2nd component of sub-PCA, using the variance-
covariance matrix. The following 18 parameters were averaged
over the test duration: 1; velocity of head-center movement (head
movement), 2; direction of head movement, 3–6; local preference
(time ratio) at four partial areas, 7; face to peers, 8–10; frequency
of D-, dj- and J-calls [time/sec], 11–13; sonogram detected
numbers of D-, dj- and J-calls, 14; duration ratio of Freeze with D-
call, 15; duration ratio of Freeze with no call; 16–18; duration of
pecking at wall (wall peck), floor (floor peck) or self. The effective
parametric combination for PCA was screened to visualize
variance ellipses with the greatest differences relating to response
behavior between F and U chicks, by identifying unique areas. In
the end, the seven parameters, 1, 7, 11–13, 16 and 18 (see above)
were used in the final analysis and PCA plane was screened to
focus on difference between Fam and Unfam. The statistical
analysis after PCA was performed by Wilks’ lambda .
Authors thank Dr. Masakazu Konishi for his encouragement and valuable
advice. We thank all of our laboratory members at Tokyo University of
Agriculture and Technology and Prof. Toyotoshi Ueda and his students at
NCNP, for their support with behavioral experiments in the initial stage of
Conceived and designed the experiments: MK SN. Performed the
experiments: MK SK. Analyzed the data: MK YS KM AS GK SN.
Contributed reagents/materials/analysis tools: MK YS KM AS GK SN.
Wrote the paper: MK SK.
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org7March 2013 | Volume 8 | Issue 3 | e58847
References Download full-text
1. Morton J, Johnson MH (1991) CONSPEC and CONLERN: A Two-Process
Theory of Infant Face Recognition. Psychological Review 98: 164–181.
2. Salva OR, Farroni T, Regolin L, Vallortigara G (2011) The Evolution of Social
Orienting: Evidence from Chicks (Gallus gallus) and Human Newborns. PLoS
ONE 6: e18802.
3. Mascalzonia E, Regolina L, Vallortigara G (2010) Innate sensitivity for self-
propelled causal agency in newly hatched chicks. PNAS 107: 4483–4485.
4. Vallortigara G, Regolin L, Marconato F (2005) Visually Inexperienced Chicks
Exhibit Spontaneous Preference for Biological Motion Patterns. PLoS Biology 3:
5. Simion F, Regolin L, Bulf H (2008) A predisposition for biological motion in the
newborn baby. PNAS 105: 809–813.
6. Klin A, Lin DJ, Gorrindo P, Ramsay G, Jones W (2009) Two-year-olds with
autism orient to nonsocial contingencies rather than biological motion. Nature
7. Brown J, Kaplan G, Rogers LJ, Vallortigara G (2010) Perception of biological
motion in common marmosets (Callithrix jacchus): by females only. Anim Cogn
8. Vallortigara G (1988) Chicks in a Novel Environment: Effects of Conspecific
Calls. Ethology 78: 241–345.
9. Vallortigara G, Andrew RJ (1994) Olfactory lateralization in the chick.
Neuropsychologia 32: 417–423.
10. Johnson MH (2005) Subcortical face processing. Nat Rev Neurosci 6: 766–774.
11. Andrew RJ (1969) Intracranial self-stimulation in the chick and the causation of
emotional behavior. Annals New York Academy of Sciences 159: 625–639.
12. MacKay DM (1972) Formal analysis of communicative processes. In: Hinde RA
eds. Non-verbal communication. Cambridge: Cambridge University Press. pp.
13. Kondo N, Watanabe S (2009) Contact calls: Information and social function.
Japanese Psychological Research 51: 197–208.
14. Braune P, Schmidt S, Zimmermann E (2008) Acoustic divergence in the
communication of cryptic species of nocturnal primates (Microcebus ssp.). BMC
Biol 6: 19.
15. Boughman JW (1998) Vocal learning by greater spear-nosed bats. Proc Biol Sci
16. Lemasson A, Ouattara K, Petit EJ, Zuberbuhler K (2011) Social learning of
vocal structure in a nonhuman primate? BMC Evol Biol 11: 362.
17. Kondo N, Izawa E, Watanabe S (2012) Crows cross-modally recognize group
members but not non-group members. Proc Biol Sci 279: 1937–1942.
18. Collias N, Joos M (1953) The Spectrographic analysis of sound signals of the
domestic fowl. Behavior 5: 175–188.
19. Koshiba M, Mimura K, Sugiura Y, Okuya T, Senoo A, et al. (2011) Reading
marmoset behavior ‘semantics’ under particular social context by multi-
parameters correlation analysis. Prog Neuropsychopharmacol Biol Psychiatry
20. Tchernichovski O, Lints TJ, Deregnaucourt S, Cimenser A, Mitra PP (2004)
Studying the song development process: rationale and methods. Ann N Y Acad
Sci 1016: 348–363.
21. Morton ES (1977) On the occurrence and significance of motivation-structural
rules in some bird and mammal sounds. The American Naturalist 111: 855–869.
22. Muir DW, Abraham W, Forbes B, Harris LS (1979) The ontogenesis of an
auditory localization response from birth to four months of age. Canadian
Journal of Psychology, 33: 320–333.
23. Feltenstein MW, Sufka KJ (2005) Screening antidepressants in the chick
separation-stress paradigm. Psychopharmacology (Berl) 181: 153–159.
24. Kikusui T, Winslow JT, Mori Y (2006) Social buffering: relief from stress and
anxiety. Philos Trans R Soc Lond B Biol Sci 361: 2215–2228.
25. Bouchet H, Blois-Heulin C, Pellier AS, Zuberbu ¨hler K, Lemasson A (2012)
Acoustic variability and individual distinctiveness in the vocal repertoire of red-
capped mangabeys (Cercocebus torquatus). J Comp Psychol 126: 45–56.
26. Konishi M (1963) The role of auditory feedback in the vocal behavior of the
domestic fowl. Zeitschrift fur Tierpsychologie 20: 349–367.
27. Field SE, Rickard NS, Toukhsati SR, Gibbs ME (2007) Maternal hen calls
modulate memory formation in the day-old chick: the role of noradrenaline.
Neurobiol Learn Mem 88: 321–330.
28. Bolhuis JJ (1999) Early learning and the development of filial preferences in the
chick. Behav Brain Res 98: 245–252.
29. Ellis JM, Riters LV (2012) Vocal parameters that indicate threat level correlate
with FOS immunolabeling in social and vocal control brain regions. Brain
Behav Evol 79: 128–140.
30. Kent JP (1989) On the Acoustic Basis of Recognition of the Mother Hen by the
Chick in the Domestic-Fowl (Gallus-Gallus). Behaviour 108: 1–9.
31. Marx G, Leppelt J, Ellendorff F (2001) Vocalisation in chicks (Gallus gallus
dom.) during stepwise social isolation. Applied Animal Behaviour Science 75:
32. Yazaki Y, Matsushima T, Aoki K (1999) Testosterone modulates stimulation-
induced calling behavior in Japanese quails. J Comp Physiol A 184: 13–19.
33. Panksepp J (2011) Cross-species affective neuroscience decoding of the primal
affective experiences of humans and related animals. PlosOne 6: e21236.
34. Kosfeld M, Heinrichs M, Zak PJ, Fischbacher U, Fehr E (2005) Oxytocin
increases trust in humans. Nature 435: 673–676.
35. Ellis JM, Riters LV (2012) Vocal parameters that indicate threat level correlate
with FOS immunolabeling in social and vocal control brain regions. Brain
Behav Evol 79: 128–140.
36. Bolhuis JJ (1999) Development of perceptual mechanisms in birds: predisposi-
tions and imprinting. In: Bolhuis JJ, Hogan JA eds. The development of animal
behavior, A reader. Oxford: Blackwell Publishers Ltd. pp. 176–191.
Familiarity Perception in Peer Sociality
PLOS ONE | www.plosone.org8 March 2013 | Volume 8 | Issue 3 | e58847