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Research
Cite this article: Rojas B, Burdfield-Steel E,
Pakkanen H, Suisto K, Maczka M, Schulz S,
Mappes J. 2017 How to fight multiple
enemies: target-specific chemical defences in
an aposematic moth. Proc. R. Soc. B 284:
20171424.
http://dx.doi.org/10.1098/rspb.2017.1424
Received: 26 June 2017
Accepted: 25 August 2017
Subject Category:
Ecology
Subject Areas:
evolution, behaviour, ecology
Keywords:
predator– prey interactions, chemical defences,
aposematism, pyrazines
Author for correspondence:
Bibiana Rojas
e-mail: bibiana.rojas@jyu.fi
†
Denotes equal contribution.
Electronic supplementary material is available
online at https://dx.doi.org/10.6084/m9.
figshare.c.3876109.
How to fight multiple enemies:
target-specific chemical defences in
an aposematic moth
Bibiana Rojas1,†, Emily Burdfield-Steel1,†, Hannu Pakkanen2, Kaisa Suisto1,
Michael Maczka3, Stefan Schulz3and Johanna Mappes1
1
Centre of Excellence in Biological Interactions, Department of Biology and Environmental Sciences, University of
Jyva
¨skyla
¨, PO Box 35, Jyva
¨skyla
¨40001, Finland
2
Department of Chemistry, University of Jyva
¨skyla
¨, Survontie 9, Jyva
¨skyla
¨40500, Finland
3
Technische Universita
¨t Braunschweig, Institute of Organic Chemistry, Hagenring 30, 38106 Braunschweig,
Germany
BR, 0000-0002-6715-7294; EB-S, 0000-0002-8428-5431; JM, 0000-0002-1117-5629
Animals have evolved different defensive strategies to survive predation,
among which chemical defences are particularly widespread and diverse.
Here we investigate the function of chemical defence diversity, hypothesiz-
ing that such diversity has evolved as a response to multiple enemies. The
aposematic wood tiger moth (Arctia plantaginis) displays conspicuous
hindwing coloration and secretes distinct defensive fluids from its thoracic
glands and abdomen. We presented the two defensive fluids from laboratory-
reared moths to two biologically relevant predators, birds and ants, and
measured their reaction in controlled bioassays (no information on colour
was provided). We found that defensive fluids are target-specific: thoracic
fluids, and particularly 2-sec-butyl-3-methoxypyrazine, which they contain,
deterred birds, but caused no aversive response in ants. By contrast, abdomi-
nal fluids were particularly deterrent to ants, while birds did not find them
repellent. Our study, to our knowledge, is the first to show evidence of a
single species producing separate chemical defences targeted to different
predator types, highlighting the importance of taking into account complex
predator communities in studies on the evolution of prey defence diversity.
1. Introduction
Predation is a key agent of natural selection in prey species [1]. In order to sur-
vive in a multi-predator world, animals have evolved different defensive
strategies that vary in their nature and efficacy in relation to predator sensory
abilities and attack tactics [2– 4]. Which strategy, or set of strategies, is used
as a defence depends on the benefits granted and the costs incurred. However,
the strategy employed must ultimately aim to prevent the completion of a pre-
dation event as early as possible in the predation sequence (i.e. detection,
identification, approach, subjugation and consumption sensu; Endler [2]).
Aposematic organisms gain protection from predators by displaying colour-
ful warning signals, which are coupled with some form of unprofitability [5].
This unprofitability is frequently related to the possession of chemical defences
that can be unpalatable or even toxic [1,5 –7]. Predators learn to associate the
warning signal with a bad experience when tasting the prey, and remember
it in subsequent encounters (e.g. [7– 11]), leading to an aversive behaviour
towards that particular prey.
Chemical defences in aposematic species can also vary in composition,
quantity, and quality and, although this variation is relatively common
[12–20], it has been addressed much less frequently than variation in warning
signals [21]. Because these defences are usually effective during the subjugation
and/or consumption stages of the predation sequence [2], chemical defences
are often referred to as secondary defences. They can deter predators in a
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variety of ways, including volatile irritation, distastefulness
or even toxicity [12]. Chemical defences can be costly
[22–24], as they involve processes ranging from the seques-
tration of active compounds, either with or without
subsequent modifications, through to their synthesis
de novo [12,24]. Therefore, these defences are expected to
evolve only if needed, and to be effective against a wide
array of predators [14].
The wood tiger moth (Arctia (formerly Parasemia)plantagi-
nis [25]) is an aposematic arctiid species distributed across the
Holarctic region [26]. Males display either white or yellow
hind wings (except for the Caucasus, where males are
mostly red), whereas females present a hindwing coloration
that varies continuously from yellow through to red. This
warning coloration is coupled with the possession of two
types of seemingly distasteful chemical secretions [27,28].
One type (hereafter ‘neck fluids’) is secreted from the
prothoracic (cervical) glands, and the other one (hereafter
‘abdominal fluids’) is released from the abdominal tract.
These fluids are released under different circumstances
(i.e. seldom simultaneously). While abdominal fluids can be
released in response to subtle disturbances, and mostly
(if not only) during the early stages of adult life, neck fluids
are most frequently secreted in response to the active ‘squeez-
ing’ of the prothoracic glands (i.e. a bird attack; see the
electronic supplementary material, video ESM1). The exact
compounds in the defensive fluids of wood tiger moths
have not yet been fully identified, but many other arctiids
are well known for their chemical defences, which include
pyrrolizidine alkaloids, methoxypyrazines and iridoid glyco-
sides, among others [17–20]. Given the possible costs
associated with insect chemical defences [12,24], it is intri-
guing that wood tiger moths are able to afford two
different types of fluids.
Here, we test the hypothesis that these moths have two
different types of chemical defences because they are targeted
towards different predator types. We collected defensive
fluids from laboratory-reared males, analysed their chemical
composition and examined the reaction of two biologically
relevant predators, birds and ants. We first show that the
two defensive fluids are chemically distinct, and demonstrate
that birds and invertebrate predators react to them differently.
Following the results of these assays we identified a
compound, 2-sec-butyl-3-methoxypyrazine (SBMP), which
explains the target-specific nature of the thoracic defence fluid.
2. Material and methods
(a) Study species and collection of defensive fluids
The wood tiger moth, Arctia plantaginis, is an arctiid species dis-
tributed across the Holarctic region [26]. They are polyphagous
and capital breeders [29], feeding only while larvae. Adults
have a short lifespan (two to three weeks for males, less than
one week for females) and produce only one generation per
year in the wild. Under laboratory conditions, wood tiger
moths can be relatively easily bred and kept on a diet consisting
mostly of dandelion (Taraxacum sp.) leaves, and can produce
three generations per year. The individuals used in the present
experiments were obtained from two laboratory stocks, estab-
lished in 2010 and 2011, from wild moths collected from
central and southern Finland, and reared at the University of
Jyva
¨skyla
¨(Finland) under natural light conditions and a
temperature ca 238C.
Fluids for the bird experiments were collected in 2012 from
approximately 120 males, 60 white and 60 yellow, taken from
the laboratory stock founded in 2011. Fluids for the ant exper-
iments were collected in 2014 from 45 males from the same
stock (see details about collection of defensive fluids in the elec-
tronic supplementary material, S2). There are no differences
between wild and laboratory-reared moths in the volume of
their defensive fluids, which appear to be produced de novo [30].
(b) Chemical analyses
For the preliminary chemical analysis, neck and abdominal fluids
from five individuals were pooled. Five hundred microlitres of
dichloromethane (DCM) was added to thoracic fluids and vor-
texed, and 20 ml of the abdominal fluid was pipetted into 500 ml
DCM. The DCM was then evaporated under constant nitrogen
flow and the dried samples re-dissolved with 250 ml pyridine
and 250 ml silylation reagent (BSTFA þ1% TMCS, Regisil).
Extracted fluid samples were analysed with an Agilent 6890 gas
chromatograph– 5973 mass spectrometer (GC/MS) system. A
sample volume of 1 ml from both thoracic and abdominal fluid
samples was injected into the injector using a pulsed, splitless
mode and the temperature was set to 2908C. Compounds were
separated with a HP-5 ms column (30 m 0.25 mm internal diam-
eter with a film thickness of 0.25 mm; J&W Scientific Inc.). Helium
was used as a carrier gas at a constant flow (1 ml min
21
). The oven
temperature was programmed as follows: 2 min at 808C, then
ramped to 1808C at the rate of 88Cmin
21
and from 1808Cto
2908C at the rate of 78C min
21
, and kept at that temperature for
an additional 10 min. Electron ionization (70 eV) mass spectra
were used for identification. Chromatograms and mass spectra
were evaluated using Agilent Chemstation (version G1701CA)
software, and the Wiley 7th edition mass spectral database.
A further chemical analysis was performed at TU Bransch-
weig. The samples were collected using Supelco Red (100 mm
Polydimethylsiloxane, PDMS) and Black (75 mm CarboxenTM/
PDMS) solid phase micro extraction (SPME) fibres with neck
fluids (1 –10 ml) of freshly eclosed moths. Fibres were placed
into the neck fluid and immediately transferred to the injection
port of the GC/MS system. GC/MS analyses were carried out
on an Agilent GC 7890B system connected to a 5977A mass-selec-
tive detector (Agilent) fitted with a HP-5 MS fused-silica
capillary column (30 m 0.25 mm i.d., 0.22 mm film; Hewlett –
Packard). Conditions were as follows: carrier gas (He):
1.2 ml min
21
; injector: 2508C; transfer line from injector to
column: 3008C. The gas chromatograph was programmed as fol-
lows: 508C (5 min isothermal ), increased at 58C min
21
to 3208C,
and operated in splitless mode. The identification of compounds
was performed by comparison of mass spectra and retention
times with those of reference compounds (see the electronic
supplementary material, S3).
(c) Bird response to moths’ chemical defences
Blue tits (Cyanistes caeruleus) were observed through a mesh-
covered window in one of the experimental cage’s sides, and
video-recorded with a digital camera (Sony DSC-HX1). The
experimental cages were placed in a dark room, such that the
observer was not noticeable to the birds (see details on bird hous-
ing and training in the electronic supplementary material, S2).
Each bird was randomly assigned to one of five different
groups, each with 13 birds. Groups were tested with either
abdominal (A) fluids from yellow (Y) or white (W) moths; and
neck (N) fluids from yellow or white moths. The fifth and final
group was a control (C), tested with water only.
Each assay consisted of five trials, the first and last of which
were done with water-soaked oats to ensure that the birds were
feeding at the beginning of the experiment, and were not satiated
at the end; in trials 2, 3 and 4 the birds were offered the treatment
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oats, which contained one type of the defence fluids. Therefore,
only trials 2, 3 and 4 were included in the analysis. Each of
these three trials was carried out with 25 ml of a specific blend
of the fluids of three males of the same colour (see the electronic
supplementary material, S2 for details on fluid collection) mixed
with distilled water. Each blend was used twice (i.e. for two
different birds). The 25 ml of fluids (or water, in case of the con-
trol group (C)), were distributed among three oat flakes, which
were presented simultaneously to the birds, each of which had
been food-deprived for a period no longer than two hours in
order to ensure motivation to feed. During the experiment we
recorded the ‘latency to approach’, defined as the time taken
by the bird to approach and peck/eat the oats after seeing
them, and recorded the number of oats eaten by the bird in a
maximum trial duration of 5 min. The duration of the trial,
taken as the time taken by the bird to finish the oats, was
recorded in those cases where the birds ate all the oat flakes
before the 5 min limit.
We ran two separate statistical analyses, one to test for differ-
ences in bird reaction towards the abdominal (A) or neck (N)
fluids in comparison to the controls (C), and a second one to
compare bird reactions to the defence fluids of white (W) and
yellow (Y) morphs. For the first analysis the differences in bird
latency to approach the oats among treatments were analysed
using a mixed-effects Cox model. The time before the bird started
to eat the oats (i.e. time to event) was used as the response vari-
able, and type of fluid (C, N or A), trial number and the
interaction between the two were taken as explanatory variables,
with bird identity (ID) as a random factor. Then, we ran a gener-
alized linear mixed model (GLMM) with a Poisson distribution
including the total number of oats eaten as response variable,
using the same predictor variables as mentioned above. Trial
duration was included as a covariate to account for the time it
took for the birds to consume the oats, and bird ID was entered
again as a random factor. Once we confirmed that bird reaction
to the moths’ chemical defences was different from that of con-
trols, we ran the second analysis excluding the individuals
from the control (C) group, using the same models described
above, but with moth colour rather than fluid type as an expla-
natory variable. In order to see whether bird reaction changed
over the course of the experiment, we compared trials 3 and 4
to trial 2, as birds were exposed for the first time to the moths’
defences during trial 2. Model simplification (see the electronic
supplementary material, S2) was done on the basis of differences
in Akaike information criterion (AIC).
(d) Ant response to moths’ chemical defences
The assays with ants were done in September 2014 in a forest
patch in the vicinity of Jyva
¨skyla
¨(62.193 N, 25.699 E), Finland.
We identified 15 ant nests (Formica sp.) and their associated
trails; two different trails per nest were chosen on the basis of
their traffic (number of ants following the trail) in order to
test ant response to the two different chemical defences of
A. plantaginis following a protocol modified from previous
studies [31,32]. Once a trail was chosen, an acetate disc of
approximately 9 cm diameter was placed on the ground,
making sure that the ants would walk over it. Three drops of
10 ml each were added to the disc at similar distances from each
other, two containing a blend of chemical fluids coming from
three different males of the same colour, mixed with a 20%
sugar solution (sucrose), and one with only the sugar solution,
acting as a control. Using a sugar solution combined with a
blend (in a 10% concentration) of the chemical defences ensured
that the ants would have the motivation to drink despite the
bad taste. We drew marks on the acetate disc with three different
randomly assigned colours to identify the fluid type in each dro-
plet. Two discs were used for each nest, one for each type of
chemical defence. Both discs had fluids from both colour
morphs plus a control droplet (i.e. NY, NW and C were presented
simultaneously in one disc, and AY, AW and C were presented in
the other one).
Ants were allowed to come to the disc and drink from the
droplets for 5 min after which the disc was removed. Each
assay was filmed with a digital camera (SONY DSC-HX1), and
the videos were analysed in detail after the final experiment.
For each disc we counted the number of drinking events (an
ant approaches the droplet and drinks from it) and rejections
(an ant approaches the droplet, tastes it and leaves immediately)
in each droplet. With this we calculated an ‘acceptance score’ as
the number of drinking events divided by the sum of drinking
events and rejections, where values closer to 0.5 mean the ants
have no preference or repulsion, values closer to 1 mean the
ants drank the fluid more than they rejected it, and values
close to 0 indicate that ants reject the fluid more than they
drink it. Additionally, we did scans every 30 s to count the
number of ants drinking from each droplet, and on the disc,
and took the maximum number of ants over the 5 min period
as a proxy for ant traffic.
We ran a GLMM with binomial distribution where the accep-
tance score was the response variable, and the interaction between
morph and type of fluid was included as the explanatory variable.
We also included ant traffic as acovariate, and nest ID as a random
factor. Main effects were not included, as neck and abdominal fluid
were not presented to the ants simultaneously and, therefore, are
not directly comparable. For this and all other analyses we took a
full-model approach. The variance explained by random effects
was calculated following [33]. This and all statistical analyses
were carried out with the software R STUDIO [34], using the
packages coxme [35] and lmer4 [36].
(e) Bird and ant response to pure pyrazine
Following the results of the second chemical analysis (see below)
we performed a second assay with ants (June 2016) and birds
(November 2016) to determine whether the pyrazine detected
in the neck fluids was capable of eliciting aversive reactions on
its own, and in the concentrations found. The procedures fol-
lowed the protocols described above for each predator type.
For details on the methods of these assays see the electronic
supplementary material, S2.
3. Results
(a) Preliminary chemical analysis
We found that the two types of defensive fluids differ in their
composition (electronic supplementary material, S4). In
addition to containing a greater number of peaks, the peak
areas obtained from the neck fluids were essentially larger
(electronic supplementary material, S4a) compared to
abdominal fluids (electronic supplementary material, S4b).
The main compound groups in neck defensive fluids were
amino and carboxylic acids (see table S1 in the electronic sup-
plementary material, S2). The methods used in this first
analysis did not allow for the identification of highly volatile
compounds because it aimed to identify as many compounds
as possible using a silylation derivatising step, in which the
very volatile compounds are lost.
(b) Bird response to moths’ chemical defences
Birds were overall significantly more deterred by the neck
fluids than by the abdominal ones. This was reflected in a
higher latency to approach oats soaked with neck fluids
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compared to control oats across trials (table 1; figure 1a),
whereas no differences were found between the latency to
approach oats soaked with abdominal fluids and controls
(table 1).
Likewise, birds ate oats soaked with neck fluids at a sig-
nificantly lower rate than controls (i.e. either took longer to
finish the three oats presented, or ate less of them within
the maximum length (5 min) of each trial; estimate +
s.e. ¼20.409 +0.152, z¼22.689, p¼0.007; figure 2b), and
then oats soaked with abdominal fluids (estimate +
s.e. ¼20.317 +0.131, z¼22.408, p¼0.016; figure 2b);
however, there was no difference between the number of
oats eaten when soaked with abdominal fluids and water
(estimate +s.e. ¼20.092 +0.124, z¼20.740, p.0.05;
figure 2b). Oat eating rate did not differ either between trial 3
(estimate +s.e. ¼20.058 +0.124, z¼20.473, p.0.05) or
trial 4 (estimate +s.e. ¼20.031 +0.125, z¼20.247, p.0.05)
and trial 2.
Having found that neck fluids repel birds whereas abdomi-
nal fluids do not, we checked with a second analysis whether
there were differences between the colour morphs in the effi-
ciency of their neck defensive fluids. This analysis revealed a
significant interaction between moth colour and trial, so that
the latency to approach in the fourth trial was significantly
higher in response to the neck fluids of yellow males than to
those of white males (morph (Y) trial (4): estimate+
s.e. ¼22.057 +0.128, z¼23.16, p¼0.002; figure 2a;table
S2 in the electronic supplementary material, S2), indicating
that latency increases with time in response to fluids of
yellow males (figure 2a), but not in response to white males’
fluids. The rate at which birds presented with neck fluids ate
oats was not affected by moth colour (estimate +s.e. ¼
0.057 +0.265, z¼20.215, p.0.05; figure 2b).
(c) Ant response to moths’ chemical defences
Ants reacted in a different way to the two types of moth
fluids. Compared to the controls, neck fluids had a higher
acceptance score, whereas abdominal fluids had a lower
one (figure 3). As expected, there was no significant differ-
ence between the acceptance score of the controls in the
discs containing abdominal fluids and those of discs contain-
ing neck fluids (fluid (A) morph (C): estimate +s.e. ¼
0.07 +0.24, z¼0.30, p¼0.77; figure 3). Nest ID accounted
only for 5.3% of the variance in acceptance score. There was
a significant interaction between the type of fluid and
colour morph indicating that, compared to controls, abdomi-
nal fluids of both colour morphs are rejected more often than
neck fluids (fluid (A) morph (W): estimate +
s.e. ¼21.09 +0.16, z¼26.77, p,0.001; fluid (A) morph
(Y): estimate +s.e. ¼21.40 +0.17, z¼28.31, p,0.001;
figure 3). Taking a closer look at the disks of each fluid
type, we found that the abdominal fluids of yellow males
are rejected more often than those of white males (estimate +
s.e. ¼20.459 +0.14, z¼23.26, p¼0.001; figure 3), whereas
no significant differences in acceptance score were found
between the neck fluids of white males and those of yellow
males (estimate +s.e. ¼20.459 +0.14, z¼23.26, p¼
0.001; figure 3). Neck fluids of white males, however, were
accepted significantly more than the pure sugar solution con-
tained in controls (estimate +s.e. ¼0.505 +0.22, z¼2.27,
p¼0.023; figure 3).
(d) Further chemical analysis
Further chemical analysis of the neck fluids by SPME without
derivatisation proved the presence of the volatile SBMP
(figure 4), which was not detected in abdominal fluids. The
SBMP concentration in individual samples of neck fluids
ranged from 0.1 to 1 ng ml
21
. As methoxypyrazines are
known to be deterrent for birds [37], and they are commonly
found in the defensive fluids of lepidopterans [38], we further
tested bird reaction to oats coated with SBMP.
(e) Bird and ant response to pure pyrazine
Birds (n¼10) showed a strong aversion to pure SBMP even at
the lowest concentration (0.1 ng ml
21
), reflected in the signifi-
cantly lower amount of oats eaten when soaked with the
pyrazine than with water (estimate +s.e.: 20.560 +0.177,
t¼23.163, p¼0.005; electronic supplementary material,
S5a). Birds exposed to pyrazine-soaked oats also showed a
tendency to hesitate for a longer time before approaching
than did birds exposed to controls (estimate +s.e. ¼21.143,
0.604, z¼21.89, p¼0.059; electronic supplementary
material, S5b). By contrast, we did not find pure SBMP to
have a deterrent effect on ants. There were no differences in
acceptance score between a sugar solution containing
1ngml
21
SBMP and the control solution (estimate+s.e. ¼
0.139 +0.235; z¼0.589; p.0.056; electronic supplementary
material, S5c).
4. Discussion
Many animals are prey to multiple species, spread across
numerous taxa. This predator diversity poses a significant
problem for the effectiveness of anti-predator defences, as
different taxa have different sensory capabilities, tolerances,
and hunting strategies. Thus, different predator types may
produce differential selection pressures on the same prey
[7,39], which may explain why defence chemicals vary so
greatly between and within species [21]. This variation in
selection pressures could even result in prey evolving
defences targeted at particular predators. Our experiments
reveal a case of animal target-specific chemical defences.
Wood tiger moths produce two types of defensive fluids,
which differ in function and composition. While neck fluids
Table 1. GLMM showing the effect of fluid type on bird latency to approach
during the three trials with defensive fluids (fluid C and trial 2 are included
in the intercept). (A ¼abdominal, N ¼neck, C ¼control (only water).
Numbers in bold denote significant parameters at the p,0.05 level.)
variable estimate +++++ s.e. zp
fluid (A) 20.577 +0.53 21.08 0.280
fluid (N) 20.511 +0.52 20.98 0.330
trial 3 20.328 +042 20.77 0.440
trial 4 20.524 +0.42 21.25 0.210
fluid (A): trial 3 0.867 +0.52 1.66 0.098
fluid (N): trial 3 21.182 +0.54 22.20 0.028
fluid (A): trial 4 0.200 +0.52 0.38 0.700
fluid (N): trial 4 21.051 +0.35 21.97 0.049
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successfully deter birds, abdominal fluids repel ants. In both
cases, the chemical defences of yellow individuals elicited a
stronger aversion than those of white males.
Previous studies on the chemical defences of several lepi-
dopteran species have revealed that their active compounds,
mostly pyrrolizidine alkaloids, cardenolides and cardiac gly-
cosides [17,18,40 – 46], are unpalatable to a wide array of
predators, including ants [31,47], spiders [48], bats [49], and
birds [50–52]. Our findings suggest, however, that having
only one type of chemical defence would not be enough to
deter all the different predator types that wood tiger moths
could encounter.
The two defence types found in A. plantaginis seem well
suited for the different contexts in which these moths may
encounter avian and invertebrate predators. Because neck
fluids are secreted when the prothoracic glands are com-
pressed, birds could be exposed to these chemicals when
attacking the moth, regardless of whether the moth is
control
234
trial
00
0.10
0.20
0.30
0.40 *
*
*
100
200
300
latency to approach (s)
oats eaten per second
abdominal neck
control
abdominal
neck
(b)(a)
Figure 1. (a) Latency to approach (time taken for blue tits to start eating the oat flakes) is higher in response to neck fluids; and (b) birds eat oats soaked in neck
fluids at a significantly lower rate. Asterisks indicate significant differences. Boxes show the median and the 25th and 75th percentiles of data distribution. Vertical
lines indicate data range. Diamonds and circles denote extremes and outliers in data distribution, respectively.
234
trial
234
trial
0
100
200
300
latency to approach (s)
(a)
0
0.05
0.10
0.20
0.15
0.25
yellow
white
oats eaten per second
(b)
Figure 2. (a) Latency to approach oats soaked in neck fluids (time taken for blue tits to start eating the fluid-soaked oat flakes) increases with time for neck fluids
coming from yellow males; and (b) oat flakes are eaten at similar rates when soaked with neck fluids of yellow or white males. Asterisk indicates significant
differences. Boxes show the median and the 25th and 75th percentiles of data distribution. Vertical lines indicate data range. Diamonds and circles denote extremes
and outliers in data distribution, respectively.
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flying or resting on the vegetation. Additionally, previous
observations have revealed that birds tend to attack the
moths by their heads, which means an almost immediate
exposure to the neck fluids (see the electronic supplementary
material, S1). Abdominal fluids, on the other hand, may be
particularly useful for protection from terrestrial predators
(i.e. ants) at moments when the moths are resting on the veg-
etation (especially females; J. Mappes 2013, personal
observation), or when fleeing is difficult, for example when
the moth is coming out of the pupa and its wings are not
yet fully extended, or when the temperature is too low to
initiate flight. Indeed, the abdominal fluids may not be pro-
duced solely for adult defence against predators, but might
rather be the remains of the pupae liquid (i.e. meconium),
and hence available primarily at the very early stages of
adult life, when individuals are most vulnerable. Laboratory
observations support this idea, as abdominal fluid is typically
(but not always) produced during the first few days of adult-
hood, and individuals frequently release it if disturbed
(E. Burdfield-Steel 2015, personal observation).
Ants were, as expected, motivated to drink from the three
droplet types, presumably because of their content of sucrose,
which they prefer over other sugar kinds [53]. However, the
clear differences in acceptance scores show that not only are
abdominal fluids distasteful, but also that neck fluids tend
to be more accepted than the control solution. It is possible
that neck fluids have valuable nutrients for the ants in
addition to sugar. For instance, some ant species find a
mixed solution of sugar and a blend of amino acids more
appealing than a pure sugar solution [53]. Indeed, our pre-
liminary chemical analysis showed high levels of amino
acids, particularly in the neck fluids (table S2 in the electronic
supplementary material, S2; electronic supplementary
material, S4a), as is the case for some zygaenid moths [15].
Future research into the wood tiger moth defences could
therefore focus on understanding why they invest in such
costly products not related to the defence, or whether those
are instead just by-products of the haemolymph.
While the initial chemical analysis shows that the abdomi-
nal fluids contain fewer compounds and are generally more
dilute, it also shows that many of the major components of
the two fluids are the same. These included many acids, such
as citric acid. However, the pH of the fluids is close to neutral
(E. Burdfield-Steel 2015, personal observation), suggesting that
acidity is unlikely to be contributing to the predator response.
Although there do appear to be some compounds present in
the abdominal fluids that are missing from the neck fluids,
mostly notably glutamic acid, it is still not clear what
compound is responsible for the deterrent effect against ants.
Birds were significantly more deterred by neck fluids than
by abdominal fluids. Furthermore, their latency towards neck
fluids from yellow individuals was the highest by the end of
the three trials (figure 2a). Because in our experiment bird
predators did not have information on prey coloration, their
response was based purely on the odour and taste of the
chemicals they were exposed to. This might indicate that
the odour of neck fluids from yellow males is more of a deter-
rent than that of white males. While warning colours are
always ‘on’, taste and smell are hidden to predators until
they come closer to the prey and/or attack them, in a similar
fashion to ultrasonic clicks emitted by tiger moths in response
to echolocating bats [54].
As our initial chemical analysis did not detect any clear
source of the strong odour and taste associated with the
neck fluids, we performed a second analysis to identify vola-
tile candidate compounds that may be contributing to the
predator aversive response. This resulted in the discovery of
SBMP. Pyrazines, most specifically methoxypyrazines, have
been previously found in the chemical defences of some arc-
tiids [38,55], and we believe SBMP is one of the major
components explaining the anti-predator effect of the neck
fluids. It has been suggested that the odour of methoxypyra-
zines, which are responsible for some of the strongest and
most haunting odours known [56], could serve a warning
function towards predators which use smell to locate prey,
in the same way that certain colours or colour patterns
would work as warning signals for visual predators [38]. Pre-
vious studies have indeed convincingly shown that odours
from methoxypyrazines can reinforce aversive responses of
predators to certain colours [37,57], or elicit taste-avoidance
learning on their own [58]. Domestic chicks have even been
shown to be able to detect the methoxypyrazine odour
from a distance and to associate such smell with a bitter
taste provoking an aversive reaction [56]. However, there is
little prior evidence that methoxypyrazines are in themselves
strongly aversive to birds. Here we demonstrate that birds
exposed to pure SBMP indeed find it very repellent, even at
the lower end of the concentration range detected from the
moths defences.
By contrast, much less is known about the role of pyra-
zines in invertebrate signalling (but see [59] for an
illustration of the deterrent effect of SBMP against tropical
invertebrate predators). We therefore also tested the effect
of pure SBMP on ants and found, in keeping with the results
0
0.25
0.50
0.75
1.00
Y
C
W
acceptance score
abdominal neck
*
*
*
Figure 3. Acceptance score of ants (see methods section for details on cal-
culation) is lower for abdominal fluids, especially from yellow males, which
tend to be more rejected than accepted. The variation in the acceptance score
of abdominal fluids from yellow males, however, is the greatest. Boxes show
the median and the 25th and 75th percentiles of data distribution. Vertical
lines indicate data range, circles denote outliers and asterisks highlight
statistically significant differences.
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from the neck fluid trials, that it did not deter them. Thus,
SBMP seems the key behind the target-specific nature of the
neck fluids, effective against bird predators, but not against
insect predators such as ants.
Neck fluids of yellow males appear to be more effective
than those of white males. Stronger defences in white males
would have indicated a trade-off between warning signal effi-
cacy and the strength of chemical defences that would help
explain why, against theoretical expectations, white and
yellow males can coexist in the same populations. With a
more efficient warning signal [28] and somewhat better
chemical defences (i.e. neck fluids that elicit bird increasing
latency to approach with time (figure 2a), and abdominal
fluids that are more often rejected than accepted by invert-
ebrate predators (figure 3)), the reason(s) why the yellow
morph has not reached fixation remains puzzling. These
between-morphs differences in chemical defence quality are
unlikely to be because of differences in larval diet between
the two morphs, as larvae present no detectable differences
in food choice (K. Suisto 2014, personal observation).
Recent studies suggest that variation in the composition in
predator communities [60], combined with differential
mating success [61] and sufficient gene flow [61,62], could
contribute to the maintenance of this colour polymorphism.
Further research should thus assess the relative importance
of warning signals versus chemical defences in wood tiger
moths, and evaluate whether either defence overrides the
other, or whether they have a synergistic effect and form a
redundant multimodal display (sensu Partan & Marler [63]).
Chemical defences can vary in several ways, yet this has
not been studied as thoroughly as variation in coloration
[21]. Here we demonstrate that the existence of two different,
seemingly costly ([28]; K. Suisto et al. 2011, unpublished;
E. Burdfield-Steel et al. 2015, unpublished), defensive fluids
is justified by their predator specificity. Although the mech-
anisms by which these chemicals are produced are not yet
known, our findings will hopefully stimulate research on
the possible life-history trade-offs and fitness-related conse-
quences faced by species with one type of chemical
defences versus those faced by species with two (or more).
Comparative phylogenetic analyses could be a useful and
interesting approach to track the origin and evolution of gen-
eral versus specific chemical defences. We also show that
there are differences between yellow and white males in
chemical defence quality. This aspect of variation in chemical
defences is not trivial for aposematic species [64]. Exper-
iments are needed where the probability of survival of
individuals with different levels of chemical defence is
recorded, in order to gain a better understanding of the
mechanisms underlying intraspecific variation in chemical
defences.
Our study not only highlights the largely overlooked
importance of invertebrate predators as selective agents on
prey defences [65], despite their abundance in nature, but
also stresses the need to choose relevant predator species
when studying the efficacy of chemical defences, and draw-
ing conclusions about the selective agent shaping prey
defences. The presence of enemy-specific chemical defences
in a same prey animal hints at the importance of predator
community in shaping prey evolution, and suggests that
selection on chemical defence may be far more complex
than we have previously assumed.
Ethics. Wild birds were used with permission from the Central Finland
Centre for Economic Development, Transport and Environment and
licence from the National Animal Experiment Board (ESAVI/9114/
04.10.07/2014) and the Central Finland Regional Environment
Centre (VARELY/294/2015), and used according to the ASAB guide-
lines for the treatment of animals in behavioural research and teaching.
Data accessibility. The datasets have been uploaded as part of the
electronic supplementary material.
Authors’ contributions. Study design: B.R., E.B.-S., K.S., J.M.; implemen-
tation of bioassays: B.R., E.B.-S., K.S. Chemical analyses: H.P., E.B.-
S., S.S., M.M., K.S.; video analyses: B.R.; statistical analyses and
first draft of the paper: B.R., E.B.-S.; all co-authors contributed to
final editing, and approved the submitted version of the manuscript.
Competing interests. We have no competing interests to declare.
Funding. Centre of Excellence in Biological Interactions (Academy of
Finland, project no. 284666 to J.M.).
Acknowledgements. We are indebted to Helina
¨Nisu for help with birds,
to the greenhouse workers at the University of Jyva
¨skyla
¨for moth
rearing; to Janne Valkonen and Sebastiano De Bona for statistical
34 000
26 000
22 000
18 000
14 000
10 000
6000
2000
5.00 7.00 9.00 11.00 13.00
retention time (min)
SBMP
15.00 17.00 19.00
30 000
abundance
(b)(a)
Figure 4. (a) Results of GC-MS analysis monitoring ions m/z124, 138 and 151 (electronic supplementary material, figure S3); and (b) structure of 2-sec-butyl-3-
methoxypyrazine (SBMP), the compound responsible for bird deterrence towards wood tiger moths’ neck fluids.
rspb.royalsocietypublishing.org Proc. R. Soc. B 284: 20171424
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advice; and to Catherine Soler and Morgan Brain for help with
assays. J.V. and S.D.B. filmed the bird attack. J.V., Rose Thorogood,
Candy Rowe and three anonymous referees provided thoughtful
comments that greatly improved the manuscript.
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