Current Zoology
61 (4): 749–757, 2015
On the deterring effect of a butterfly’s eyespot in juvenile
and sub-adult chickens
Martin OLOFSSON1,2*, Christer WIKLUND1, Anna FAVATI1
1
2
Department of Zoology, Stockholm University, SE-106 91 Stockholm, Sweden
School of Biology, Indian Institute of Science Education and Research Thiruvananthapuram, Thiruvananthapuram 695 016,
India
Abstract Circular patterns, or eyespots, are common anti-predator features in a variety of animals. Two defensive functions
have been documented: large eyespots may intimidate predators, whereas smaller marginal eyespots may divert attacks. However,
a given eyespot potentially serves both functions, possibly depending on the predator's size and/or experience. Naïve predators
are potentially more likely to misdirect their attacks towards eyespots; alternatively, their typically smaller size would make them
more intimidated by the same eyespots. Here we test how juvenile and sub-adult naïve chickens respond to a single eyespot on a
butterfly’s wing. We presented the birds with dead wall brown butterflies, Lasiommata megera, that had their apical eyespot visible or painted over. We assessed the birds’ responses’ by (i) scoring their intimidation reaction, (ii) whether they uttered alarm
calls and, (iii) if they attacked the butterfly and where they targeted their attacks. Results show that both age categories received
higher intimidation scores when offered a butterfly with a visible eyespot. Juveniles were more intimidated by the butterfly than
the sub-adults: they received higher intimidation scores and were more prone to utter alarm calls. Moreover, only sub-adults attacked and did so by preferentially attacking the butterfly’s anterior. We demonstrate an intimidating effect of the type of eyespot
that has previously been shown only to divert attacks. We suggest that one and the same eyespot may serve two functions relative
to different predators; however, further experiments are needed to disentangle the role of predator identity and its link to size, ontogeny and experience [Current Zoology 61 (4): 749–757, 2015].
Keywords
Predator-prey interactions, Prey-attack behaviour, Butterfly, Bird, Eyespot, Ontogeny
Eyespots, defined as concentric circular markings,
are common in a wide range of animal groups such as
fishes, amphibians and insects, and have long been
thought to play a role in anti-predator defense (Blest,
1957; Ruxton et al., 2004; Stevens, 2005; Kodandaramaiah, 2011). In butterflies, eyespots appear to be particularly common, constituting one of the basal wing
pattern elements in the largest butterfly family, the
Nymphalidae (Nijhout, 1991). Moreover, the features of
eyespots vary significantly among butterfly species with
respect to their numbers, size, appearance and placement and are often phenotypically plastic (Monteiro,
2008; Beldalde and Brakefield, 2002; Brakefield et al.,
1996).
Two major hypotheses have been advanced to explain the function of eyespots: large eyespots are typically thought to prevent predators from attacking due to
intimidation (‘Intimidation hypothesis’), whereas small
eyespots on the margins of the prey are thought to divert
their attacks (‘Deflection hypothesis’) (Stevens, 2005;
Kodandaramaiah, 2011). It has been repeatedly demonstrated that birds are deterred by large eyespots. This
has been shown using: (i) live butterfly prey (Blest,
1957; Vallin et al., 2005, 2006, 2007; Olofsson et al.,
2013a), (ii) static and mounted butterfly wings (Kodandaramaiah et al., 2009; Merilaita et al., 2011), (iii)
printed artificial prey (Stevens et al., 2008a, b; Brilot et
al., 2009; Blut et al., 2012) and, (iv) clay caterpillar
dummies (Skelhorn et al., 2014). However, the reason
why large eyespots intimidate birds is debated (Stevens
and Ruxton 2014). One explanation is that eyespots
elicit a fear response because they resemble the eyes of
a potential predator, i.e. the ‘Eye mimicry hypothesis’
(Blest, 1957). Another non-mutually exclusive explanation is that the circular appearance and conspicuousness
of eyespots per se strongly stimulates the visual system
and so causes a neophobic reaction in birds, i.e. the
‘Conspicuous signal hypothesis’ (Stevens, 2005; Stevens et al., 2008a, b; Stevens and Ruxton, 2014).
Evidence for a deflective function of smaller eye-
Received Mar. 5, 2015; accepted July 10, 2015.
 Corresponding author. E-mail: [email protected] or [email protected]
© 2015 Current Zoology
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Current Zoology
spots has been weak (Blest, 1957; Wourms and Wasserman, 1985, Lyytinen et al., 2003, 2004) but the idea
has recently gained momentum in different study systems including dead butterflies/birds (Olofsson et al.,
2010; 2013b), artificial prey/birds (Vallin et al., 2011),
artificial prey/fish (Kjernsmo et al., 2013), live butterflies/invertebrates (Prudic et al., 2015) and via census of
wing damage inflicted by birds on butterflies in the wild
(Pinheiro et al., 2014). Birds such as blue tits Cyanistes
caeruleus show a strong bias in attacking the head and
vulnerable anterior region of butterflies (Olofsson et al.,
2010, 2013b), but the presence of a marginal eyespot
can significantly increase the number of diverted, and
therefore probably failed, attacks (Olofsson et al.,
2013b). Recently, Prudic and colleagues (2015) convincingly demonstrated that praying mantises customarily
misdirect their attacks towards the marginal eyespots on
the wet season form of the squinting bush brown butterfly Bicyclus anynana, whereas butterflies of the dry
season form, which lacks eyespots, received most attacks towards the head, body and thorax.
Studies investigating the protective function of
eyespots have typically tested the deflection hypothesis
or the intimidation hypothesis separately (but see Vallin
et al., 2011). This is probably because the two hypotheses seeking to explain the adaptive function of eyespots suggest a dichotomy in expected size and placement of the patterns on the animal body; deflective
eyespots are assumed to be smaller and more effective if
located as far away from the vulnerable body parts as
possible, whereas intimidating eyespots are expected to
be under positive selection for size and relaxed selection
with regard to their placement (Stevens, 2005; Kodandaramaiah, 2011; Kodandaramaiah et al., 2013). Indeed,
by using a butterfly phylogeny, Kodandaramaiah et al.
(2013) found evidence that small and putatively deflective eyespots in Junonia (Nymphalidae) butterflies were
more likely to be placed closer to the wing margins
compared to large and putatively intimidating eyespots
that had a more central placement on the wing.
Nevertheless, a given eyespot may potentially divert
attacks from one predator species, but be intimidating to
another (Stevens, 2005; Kodandaramaiah, 2011). Moreover, a response to certain characteristics of a prey may
shift with experience (e.g. Lyytinen et al., 2003, 2004)
or merely because of ontogenetic development in the
predator (Smith, 1973). For example, Lyytinen et al.
(2003, 2004) found that naïve juvenile flycatchers Ficedula hypoleuca were less successful than wild-caught
adults in capturing live B. anynana bearing eyespots,
Vol. 61 No. 4
whereas juveniles and adults were equally successful at
capturing butterflies of the dry season form which lacks
eyespots. This difference between juvenile and adult
birds may reflect a learnt component of how prey bearing eyespots should be attacked and handled, in other
words that juvenile birds are more likely to be deceived
by eyespots due to limited experience with prey bearing
such features (Lyytinen et al., 2004). It could also be
due to an ontogenetic shift in response to eyespots, or
both. The intimidating quality of a given eyespot is further expected to vary with regard to the size of the
prospective predator (Vallin et al., 2007) which infers
that juvenile birds should be more likely to be intimidated by a given eyespot stimulus compared to adult
birds because juveniles and adults often differ in size, at
least in precocial species that forage independently from
hatching. Thus, the function (deflection vs. intimidation)
of an eyespot may potentially relate to the size of the
prospective predator (cf. Stevens, 2005; Kodandaramaiah, 2011). Under this framework, we test how naïve
chickens, Gallus gallus domesticus, react to the apical
eyespot of the wall brown butterfly, Lasiommata megera, by testing the birds at the age of 4 and 17 weeks,
respectively. We do this by presenting the birds with
dead specimens of L. megera that either have had their
apical eyespot painted over or have been sham-painted
in a different part of the wing maintaining the presence
of the apical eyespot visible. We investigate the role of
the apical eyespot; in particular, we address (i) the birds’
intimidation reaction to the butterflies, (ii) whether the
birds attack the butterfly or not, and (iii) where the birds
target their attacks.
1
Materials and Methods
1.1 Study animals
Predator
The predators used in the study were 26 female and
26 male domestic fowl from an old Swedish game breed
of chicken, “Gammalsvensk dvärghöna” that were kept
at Tovetorp Zoological Research Station, Stockholm
University. The birds of this breed are similar in behaviour and morphology to their wild ancestor, the red
junglefowl Gallus gallus ssp. (Collias et al., 1966; Collias and Collias, 1996; Pizzari and Birkhead, 2001;
Schütz and Jensen, 2001). Chickens are omnivores, and
include among other things invertebrates such as lepidopterans in their diet (Klasing, 2005).
During the first 4 weeks upon hatching, the birds
were housed indoors in a room measuring 2.5 × 4 × 2.5
m. The room was furnished with a heat source, resting
OLOFSSON M et al.: Deterring effect of a butterfly’s eyespot in chickens
perches and wood shavings on the floor. Commercial
poultry feed and water were supplied ad libitum. After 4
weeks, 28 of the birds were transferred to eight smaller
compartments (2.3 × 1.8 × 1.2 m, 4 compartments × 4
birds, and 2.3 × 1.2 × 1.2 m, 4 compartments × 3 birds);
this was a preparation for another experiment that was
conducted after the current experiment. The smaller
compartments were furnished in the same way as the
large room. During the whole experimental period the
birds were kept naïve with respect to insect prey. Four
birds died between the two experimental occasions, thus
reducing the sample size of 17 weeks old birds to 23
females and 25 males. The light regime where the birds
were housed was 10:14 (Light:Dark) and windows allowed natural daylight to illuminate the rooms.
Prey
The butterflies used in the experiments were from a
laboratory population of the wall brown butterfly L.
megera. Larvae were reared communally on the grasses
Festuca ovina and Dactylis glomerata. After emergence,
adults were euthanized by freezing (-20°C) and were
thereafter allowed to dry in room temperature until they
were used in the experiment. The wall brown butterfly
has a series of small eyespots on the ventral hind wing
surface and a larger eyespot on the apical part of ventral
forewing surface. To test the function of the apical
eyespot, we painted over this eyespot on about half of
the butterflies (‘eyespot painted over’) and sham-painted
an equally large area just beside the eyespot on the rest
of the butterflies (‘eyespot visible’). Both groups had
their smaller hind wing eyespots painted over (Raw
Sienna, Marie’s Acrylic Colour, Shanghai, China). Butterflies were re-used if they were not attacked or damaged during the experiments and we used 30–40 individual butterflies on each of the two experimental occasions. Half of the 4 weeks old birds (n = 26) were offered a butterfly with its eyespot painted over, and the
other half (n = 26) were offered a butterfly which had a
visible eyespot. For 17 weeks old birds, the treatment
distribution was 23 for ‘eyespot painted over’ and 25 for
‘eyespot visible’, randomly assigned with respect to the
juvenile treatment. The sub-adults were justly balanced
with regard to their previous treatment as juveniles: 11
birds: eyespot visible (juvenile treatment) – eyespot
visible (sub-adult treatment); 13 birds: eyespot visible –
eyespot painted over; 14 birds: eyespot painted over –
eyespot visible; 10 birds: eyespot painted over – eyespot
painted over. Moreover, the treatment distribution for
the 17-weeks birds with regards to the two housing regimes was as following: room: n = 12, n = 9 (eyespot
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visible, eyespot painted over); smaller compartments: n
= 13, n = 14 (eyespot visible - eyespot painted over).
1.2 Experimental procedures
The experiments were conducted on two different
occasions (in June and August 2014, respectively); first
when the chickens were 4 weeks old (before 28 of them
were transferred to smaller compartments), and later
when they were 17 weeks old, from here on referred to
as juveniles and sub-adults, respectively. All birds went
through a training session one day before the experiments were conducted; this was done both when the
birds were juveniles and when they were sub-adults. A
training session was prepared by placing a small piece
of egg-white on a piece of cardboard (12 × 7 cm) in one
of the corners of a small room (2.4 × 2.3 × 1.9 m); this
was done to acclimatize the birds to the room and to
make them associate the cardboard with food. Each bird
was released with its head directed towards the piece of
cardboard at a distance of 45 or 90 cm (for juveniles
and sub-adults, respectively). The difference in distance
for juveniles and sub-adults was to account for their size
difference. A few birds froze initially and were reluctant
to explore the room and were guided towards the cardboard by an experimenter, either by pecking with a finger close to the piece of egg-white to attract their attention (for juveniles) or by dropping a piece of egg-white
in front of the bird to make them start walking (for
sub-adults). The training was judged successful once the
birds had consumed the egg-white piece at the cardboard. The room was lit by eight fluorescent tubes (Philips TL-D 90 Graphica Pro 36W/950).
An experiment was prepared by placing a butterfly
specimen on the cardboard. The butterfly (‘eyespot visible’ or ‘eyespot painted over’) was presented flat, with
wings folded, on the cardboard so as to show either the
right or left ventral wing surface. Two small pieces of eggwhite was offered to the birds; one piece was placed 10
or 45 cm in front of the cardboard (for juveniles and
sub-adults, respectively) and one piece was placed on
the cardboard 2 cm above the butterfly specimen. The
piece of egg-white on the cardboard was placed with an
equal distance to the butterfly’s head and the apical part
of the fore wing (Fig. 1).
A trial was started by placing a bird with its head directed towards the butterfly at a distance of 45 or 90 cm
(for juveniles and sub-adults, respectively). The birds
invariably ate the closest piece of egg-white first, and
would then typically walk directly towards the butterfly
and the adjacent piece of egg-white. Behavioural recording started as soon as a bird had eaten the closest
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piece of egg-white and was ended either when the bird
attacked the butterfly or when 60 seconds had elapsed.
The birds’ reaction when in close proximity to the butterfly was graded in four categories: no reaction = 0,
flinching = 1, flinching plus moving away = 2 or flight
including wing flaps and/or jumping = 3. We noted the
strongest reaction displayed by each bird as a measure
of intimidation. All trials were recorded with three video cameras. A Sony camera (DCR-VX100E) was placed
on a tripod and zoomed to capture where attacking birds
targeted the butterfly. Two GoPro Hero 3 cameras were
used to capture the birds’ movement in the room and
reaction when they encountered the butterfly – one was
placed in the ceiling 1.7 m above the butterfly and one
was placed 20 cm above the floor in front of the butterfly at a distance of 40 cm. The trials were also observed by three experimenters of which two were sitting
inside the experimental room in the opposite corners to
where the butterfly was presented, and one sitting outside watching through the door opening. The birds’
reaction was scored by two experimenters by watching
the videos, which at the time were blind with respect to
butterfly treatment (‘eyespot visible’ or ‘eyespot painted
over’). As a second measure of how intimidating the
birds perceived the butterfly we noted whether they
uttered alarm calls which were categorized at three levels: for juveniles, ‘no alarm’ was scored as 0, ‘weak
alarms’ (frrr) as 1 and ‘intense alarms’ (frr-frr-frr or
frrrrrrrr) as 2, and for adults, only weak alarm calls (cut/
cut-cut) were observed and were scored as 1. Adult chickens are known to also utter more intense alarm calls
upon perceiving ground-based threats “cut-cut-cut …
Fig. 1 A schematic illustration of the presentation of the
butterfly
The butterfly was offered to the bird with its head directed to the left
or right, with a small piece of egg-white placed 2 cm above. The piece
of egg-white was placed so as to be equidistant from the butterfly’s
head and its apical forewing where the eyespot is positioned. The
arrow denotes from which direction the bird was released.
Vol. 61 No. 4
cut KAAAH!” (sensu Collias and Joos, 1953) but these
alarm calls were never observed in our experiments. As
a third measure of how intimidating the birds perceived
the butterfly we noted whether a bird attacked the butterfly or not. Moreover, to investigate whether the
eyespot diverted the birds’ attacks we also noted where
on the butterfly’s body the birds targeted their attacks.
The routines for housing of birds and the experimental
procedures described herein have been reviewed and
approved by the regional ethical committee, Linköping
djurförsöksetiska nämnd (Dnr: 114-12).
1.3 Statistics
We performed Ordered Logistic Regressions (OLR)
to analyze the birds’ reaction to the butterfly. For juveniles, we used reaction (four levels: 0–3) as response
variable and treatment (two levels: eyespot visible or
eyespot painted over) and bird sex (two levels: male or
female) as explanatory factors. For sub-adults, we used
reaction (three levels: 0–2) as response variable and
treatment, treatment when they were juveniles and bird
sex as explanatory factors. For sub-adults, we also included whether the birds had been kept in a large group
(24 individuals/room) or in a small group (3–4 individuals/room) between the two experimental occasions
(group; two levels: small or large). In the models for juveniles, we included an interaction term between treatment and sex. For sub-adults, we included following
interaction terms: sex × treatment, sex × juvenile treatment, sex × group, treatment × group and juvenile treatment × group. We also performed an OLR-model on
the juveniles’ propensity to utter alarm calls. We used
alarm call (three levels: 0 = no alarm, 1 = weak alarm
and 2 = intense alarm) as response variable and treatment (two levels: eyespot visible or eyespot painted
over) and bird sex (two levels: male or female) as explanatory factors. We also included an interaction term
between treatment and bird sex. Interaction terms and
factors were stepwise removed from the OLR-models if
P > 0.1 until only significant factors remained; however,
treatment and sex were not removed even if P > 0.1
because these factors were a priori considered important and part of the experimental design. Because only 4
sub-adults uttered alarm calls, we performed a Fisher’s
exact test to test if treatment influenced their propensity
to utter alarm calls.
Whether juveniles and sub-adults differed in their
propensity to attack the butterfly was tested using Mc
Nemar’s chi-squared test. This test takes into account
that the birds were used twice. Mc Nemar’s chi-squared
test was also used to test whether juveniles and sub-
OLOFSSON M et al.: Deterring effect of a butterfly’s eyespot in chickens
adults differed in their propensity to utter alarm calls. To
be able to compare propensity in alarm calling, we
made the alarm calling variable in juveniles binary, i.e.
alarm score 1 and 2 were pooled into one group.
We used a generalized linear model (GLM: binomial
family with logit as link-function) to test whether the
propensity to attack was due to treatment (two levels:
eyespot visible or eyespot painted over), sex (two levels:
male or female), group (two levels: small or large) and
juvenile treatment (two levels: eyespot visible or eyespot painted over). Because no juveniles attacked the
butterfly, this analysis was only carried out on the subadults. We included the following interaction terms in
the model: treatment × group and juvenile treatment ×
group. Bird sex was not included in any interaction
terms because out of the 14 attacking sub-adult birds
only three were females, which means that interpretations of possible interactions would be largely overrated.
Interaction terms and factors were stepwise removed
from the GLM-model if P > 0.1; however, treatment
and sex were not removed even if P > 0.1 because these
factors were a priori considered important and part of
the experimental design.
All analyses were performed in the statistical software R (version 3.1.2, R Core Team 2014) using the
packages MASS, Hmisc and car.
2
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2.2 Sub-adult birds
None of 48 sub-adult birds reacted strongly (i.e.
reaction score = 3) to the butterfly. This means that the
strongest reaction they displayed was an initial flinch,
and after that walking away from the butterfly (i.e. reaction score = 2). However, the birds’ reaction was
stronger if the butterfly had a visible eyespot, compared
to if the eyespot was painted over (Table 3; Figure 3).
There was also a non-significant trend of an interaction
between group size and bird sex (Table 3) suggesting a
weak tendency for female chickens, that had been
housed in smaller groups, to display a somewhat
stronger reaction. All other interactions and factors were
not significant (OLR: all P-values > 0.1).
Results
2.1 Juvenile birds
In general, juvenile birds were intimidated (i.e.
showed a stronger reaction score than 0) by the butterfly
and only 3 of 52 birds showed no reaction at all. However, the birds’ response was stronger if the butterfly
had a visible eyespot, compared to if the eyespot was
painted over (Table 1, Fig. 2). The reaction score did not
differ between males and females (Table 1) and there
was no significant interaction between bird sex and
treatment (OLR: Sex × Treatment: P > 0.1).
Thirty-two of 52 juveniles uttered alarm calls when
perceiving the butterfly (Nweak alarm call (1) = 11, Nintense alarm
call(2) = 21); however, juveniles that confronted a butterfly with a visible eyespot were not more prone to
utter alarm calls (17 of 26), than juveniles that confronted a butterfly which had its eyespot painted over
(15 of 26) (Table 2). The propensity to utter alarm calls
did not differ between males and females (Table 2) and
there was no significant interaction between bird sex
and butterfly treatment with regard to the birds’ propensity to utter alarm calls (OLR: Sex × Treatment: P > 0.l).
None of the 52 juveniles attacked the butterfly.
Fig. 2 The strongest reaction displayed by the juvenile
birds when they were in close proximity to the butterfly
Bars denote the number of birds confronted with a butterfly with
visible eyespots (black bars) or a butterfly that had its eyespot painted
over (grey bars). The reactions were scored as follows: no reaction = 0,
flinching = 1, flinching plus moving away = 2 or flight including wing
flaps and/or jumping = 3.
Table 1 Output from the Ordered Logistic Regression on
the juveniles’ response
Factor
Odds ratio
95% CI-range
t
P
Juvenile
treatment
0.333
0.113‒0.938
-2.046
0.0407
Sex
2.337
0.827‒6.854
1.583
0.113
Reported values are from the final, reduced model. n = 52.
Table 2 Output from the Ordered Logistic Regression on
the juveniles’ alarm calling
Factor
Odds ratio
95% CI-range
t
P
Juvenile treatment
0.66
0.235‒1.825
-0.798
0.425
Sex
1.526
0.551‒4.304
0.81
0.418
Reported values are from the final, reduced model. n = 52.
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Table 3 Output from the Ordered Logistic Regression on
the sub-adults’ response
Factor
Odds ratio
95% CI-range
t
P
Adult treatment
0.251
0.071‒0.813
-2.237
0.0253
Sex
1.118
0.154‒8.664
0.111
0.912
Group size
16.82
2.255‒162.0
2.628
0.00858
Sex × Group size
0.099
0.006‒1.318
-1.704
0.0883
Reported values are from the final, reduced model. n = 48.
Fig. 3 The strongest reaction displayed by the sub-adult
birds when they were in close proximity to the butterfly
Bars denote the number of birds confronted with a butterfly with
visible eyespots (black bars) or a butterfly that had its eyespot painted
over (grey bars). The reactions were scored as follows: no reaction = 0,
flinching = 1, flinching plus moving away = 2 or flight including wing
flaps and/or jumping = 3.
Fourteen of the 48 sub-adult birds attacked the butterfly. Their propensity to attack was significantly affected by sex, since males (11 of 25) were more prone
than females (3 of 23) to attack (GLM: Sex: χ2 = 5.502,
df = 1, P = 0.019). However, we did not detect a significant effect of butterfly treatment on the propensity to
attack; 5 of 25 birds that confronted a butterfly that had
a visible eyespot attacked and 9 of 23 did so when they
confronted a butterfly that had its eyespot painted over
(GLM: Treatment: χ2 = 1.802, df = 1, P = 0.18). All
other interactions and factors were non significant
(GLM: all P-values > 0.1). Ten of 14 birds that attacked
the butterfly attacked the anterior part of the butterfly,
whereas 3 birds attacked the hind-wing and 1 bird attacked the forewing (Fig. 4). The anterior part of the
body constitutes 15 % of the butterfly’s total area (i.e. a
butterfly seen from the side with juxtaposed wings). A
null expectation would be that this area should receive
15 % of the attacks if the birds have no attack prefe-
Vol. 61 No. 4
rence. The observation that 10 of 14 birds attacked this
area by chance is thus unlikely (Exact binomial test: n =
14, hypothesized probability = 0.15, P < 0.0001; Fig. 4),
suggesting that the birds actively chose to target their
attack towards the butterfly’s most vulnerable body
parts. However, the attack distribution was not affected
by butterfly treatment: 2 of 5 birds misdirected their
attacks towards the wings when attacking a butterfly
with a visible eyespot and 2 of 9 birds misdirected their
attacks when attacking a butterfly which had its eyespot
painted over (Fisher’s exact test: P = 0.58). None of the
5 birds that attacked the butterfly which had a visible
eyespot misdirected its attack towards the apical eyespot.
Fig. 4 The black/yellow markings show the distribution of
where on the butterfly’s body the sub-adults directed their
attacks (n = 14)
Ten of 14 sub-adult birds attacked the anterior region which comprises
15% of the butterfly’s body. Note that the figure shows an
un-manipulated wall brown butterfly, and not any of the two manipulated butterflies (‘eyespot visible’ or ‘eyespot painted over’) that were
used in the experiment.
Only 4 sub-adult birds uttered a weak alarm call (cut/
cut-cut) when perceiving the butterfly and there was no
effect of treatment; two of 25 did so when confronting a
butterfly with a visible eyespot, and 2 of 23 did so when
confronting a butterfly which had its eyespot painted
over (Fisher’s exact test: P = 1).
2.3 Differences in reaction and behaviour between
juvenile and sub-adult birds
The reaction of the birds toward the butterfly prey
was stronger when they were juveniles, compared to
when they were sub-adults (mean score juveniles (n = 52) =
1.885 ± 0.273 (95% CI); mean score sub-adults (n = 48) =
0.792 ± 0.193; Paired t-test: n = 48, t = -6.004, df = 47,
P < 0.0001). Note that the mean scores are calculated
including all individuals, but the analysis only includes
the 48 birds that participated both as juveniles and as
sub-adults.
The birds were more prone to utter alarm calls when
OLOFSSON M et al.: Deterring effect of a butterfly’s eyespot in chickens
they were juveniles compared to when they were subadults when perceiving the butterfly. Four birds uttering
alarm calls did so both as a juvenile and as a sub-adult,
26 birds uttered alarm calls only as juveniles, no bird
uttered alarm only as a sub-adult and 18 birds never
uttered alarm calls at all, neither as a juvenile nor as a
sub-adult (McNemar’s Chi-squared test: n = 48, McNemar’s χ2 = 24.039, df = 1, P < 0.0001). The birds
were more prone to attack the butterfly when they were
sub-adults (14 of 48) compared to when they were juveniles (0 of 48) (McNemar’s Chi-squared test: n = 48,
McNemar’s χ2 = 12.071, df = 1, P = 0.000512).
3
Discussion
We have here demonstrated that even a rather small
eyespot on a butterfly wing can have an intimidating
function on both juvenile and sub-adult naïve chickens.
It was further apparent that the juveniles were more
intimidated by the butterfly compared to the sub-adults,
indicating a possible ontogenetic shift in behaviour
when chickens perceive a butterfly. Our experimental
design did not keep the sub-adults perfectly naïve to the
butterfly, which means that previous experience (i.e.
when they performed the experiment as juveniles) may
potentially explain the reduction in response. However,
we want to emphasize that their experience with the
butterfly was limited to a maximum of 60 seconds and
also that 13 weeks separated the two experimental occasions which alone is unlikely to have caused such a
marked change in behavior.
Both juveniles and sub-adults reacted with more intimidation to butterflies with intact eyespots, compared
to butterflies that had their eyespots painted over. However, the presence or absence of an eyespot did not
significantly influence the birds’ propensity to attack the
butterfly, or their propensity to utter alarm calls. In a
recent study, Olofsson et al. (2013a) similarly showed
that adult chickens (age 8–9 months) were more intimidated when confronted with live peacock butterflies
Aglais io, which had visible eyespots, compared to if
the eyespots were painted over. In contrast to the current
study, Olofsson et al (2013a) found an effect on the
probability to utter alarm calls. However, there are two
major differences between these studies that could explain this discrepancy. First, the eyespots of the peacock
are hidden at rest but exposed suddenly upon disturbance by a predator (Blest, 1957; Vallin et al., 2005),
which means that the display includes a surprise/startle
effect which was not the case in the current experiment
where the butterfly was dead and the single eyespot was
755
constantly visible to the birds. Second, peacock butterflies have 4 eyespots on the dorsal wing surface that are
considerably larger than the apical eyespot of the wall
brown butterfly which could elicit a stronger fear response.
It stands to reason that the size of an eyespot is important for its function as a visual signal; in the context
of deterrence or intimidation, evidence suggests that
larger eyespots provide better protection than smaller
ones (Stevens et al., 2008a). However, it is less clear at
what size a given eyespot ceases to intimidate a predator and instead starts to function as a deflection mark.
The apical eyespot of the wall brown butterfly was recently shown to divert attacks from wild caught adult
blue tits when glued onto the hind wing margin on dead
specimens of the closely related speckled wood butterfly Pararge aegeria (Olofsson et al., 2013b). Indeed, the
size and placement of eyespots have often been used to
hypothesize their function in an anti-predator context
(Blest, 1957; Stevens, 2005; Kodandaramaiah, 2011):
large and centrally placed eyespots have been shown to
cause intimidation in birds (e.g. Blest, 1957, Vallin et al.,
2005, Kodandaramaiah et al., 2009; Olofsson et al.,
2013a), whereas smaller eyespots on the margin of prey
to divert predators’ attacks (e.g. Kjernsmo and Merilaita,
2013; Olofsson et al., 2010, 2013b; Prudic et al., 2015).
The observation that the apical eyespot of the wall
brown can have a significant – albeit weak – intimidating effect on naïve chickens is interesting from the
perspective that eyespots on the margin on satyrine butterfly wings are traditionally thought to attract attention
and thus diverting predators’ attacks (i.e. the deflection
hypothesis; Stevens, 2005; Kodandaramaiah, 2011) rather than dissuading predators. Thus, the current study
supports the idea that some eyespots may have a dual
function (cf. Stevens, 2005) and that naïve predators can
be intimidated by the same eyespot that sometimes attracts attention and draws attacks from an experienced
bird predator (Olofsson et al., 2013b).
Overall, the birds were more intimidated by the butterfly as juveniles. The tendency to utter alarm calls
decreased drastically with increasing age, and only
sub-adults attacked the butterfly. It is noteworthy that a
non-negligible number of juvenile birds uttered alarm
calls and reacted with flight behaviour also when perceiving the butterfly that had its eyespot painted over,
suggesting that they are quite deterred when perceiving
a butterfly prey for the first time even if it does not
possesses eyespots. Eyespots are often phenotypically
plastic, as amply shown in the squinting bush brown
756
Current Zoology
butterfly Bicyclus anynana, which comes in two forms:
a wet season form with salient eyespots on the ventral
wing surface and a dry season form with no or only
vestiges of eyespots (Brakefield and Larsen, 1984;
Brakefield et al., 1996). Seasonal differences with respect to surrounding vegetation (i.e background), predator composition and butterfly activity have been hypothesized to account for this plasticity (Brakefield and
Larsen, 1984). Our finding that juvenile predators are
intimidated also by small eyespots opens the possibility
that eyespot polyphenism in tropical satyrines may at
least partly be driven by inexperienced juvenile birds in
the wet season. This has been suggested before, but the
proposed mechanism underpinning the selection pressure has been that the prominent eyespots in the wet
season form are more likely to divert attacks from inexperienced birds (Lyytinen et al., 2003, 2004). A recent
study gives strong support that praying mantises, which
are abundant in the tropics during the wet season, are
unequivocally deceived by marginal eyespots in B. anynana, and misdirect their attacks towards them (Prudic
et al., 2015); thus, their eyespots might simultaneously
serve a deflecting and an intimidating function. However, the role of bird vs. invertebrate predation, the relative strength of eyespots’ intimidating and deflecting
qualities, how it may vary seasonally as well as how it
relates to the size of the prospective predators is largely
unknown and warrants further investigation.
Our study did not find support for the idea that juvenile chickens would be more susceptible to misdirect
their attacks towards the eyespot than are sub-adult
chickens. Indeed, none of the juveniles attacked the
butterfly at all. Moreover, 10 of 14 sub-adults attacked
the 15% most anterior region of the butterfly and none
directed their attack towards the eyespot. The finding
that sub-adults preferentially attacked the anterior is
interesting because it shows that chickens possess an
innate tendency to target their attacks towards the most
vulnerable body parts of a butterfly. Because no chicken
attacked the butterfly at an age of 4 weeks, we do not
know whether the behaviour of attacking the anterior of
a prey is manifested only later in the ontogeny or
whether it is present also during early development.
However, evidence suggests that prey-attack behaviours
in birds may be hard-wired traits and shift quite dramatically during ontogeny; for example, Smith (1973)
demonstrated that loggerhead shrikes Lanius ludovicianus often attacked the tail of mice at a young age but
shifted – without prior learning – to lethal attacks towards the neck when they grew older. In the current
Vol. 61 No. 4
study, the same individual birds were used twice and
sub-adults were thus not perfectly naïve with respect to
the butterfly prey. Nevertheless, the strong propensity in
sub-adults to attack the anterior of the butterfly cannot
be attributed to previous experience because none of
them attacked the butterfly when they were tested as
juveniles. Moreover, for any meaningful learning to
occur in this respect, the birds would need to experience
that attacks directed at the wing margin of a live butterfly would often result in a loss of the prey.
In conclusion, we have demonstrated that a rather
small eyespot on the margin of a butterfly wing can
intimidate naïve chickens. Eyespots of the same appearance and in the same size range have previously
been shown to divert predators’ attacks (Olofsson et al.,
2010, 2013b; Prudic et al., 2015) thus suggesting a dual
function of these eyespots when different predators are
considered. Nevertheless, the current study does not
give support for a dual function with one and the same
predator species. Further experiments designed to fully
disentangle ontogenetic and/or size effects from experience should help us shed more light on the mechanisms shaping eyespot function and evolution in butterflies and other prey.
Acknowledgements This work was supported by grants
from Alice och Lars Siléns fund to M.O. and A.F. and from
the Swedish Research Council to C.W (grant number 6212010-5579). We thank Sara Forslind and Charlotte Rosher for
help with behavioural observations. We also want to thank two
anonymous reviewers for their insightful suggestions and
comments.
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