ArticlePDF Available

Abstract and Figures

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, hypothesizing 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 laboratoryreared 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, abdominal 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.
Content may be subject to copyright.
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:
Received: 26 June 2017
Accepted: 25 August 2017
Subject Category:
Subject Areas:
evolution, behaviour, ecology
predator– prey interactions, chemical defences,
aposematism, pyrazines
Author for correspondence:
Bibiana Rojas
Denotes equal contribution.
Electronic supplementary material is available
online at
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
Centre of Excellence in Biological Interactions, Department of Biology and Environmental Sciences, University of
¨, PO Box 35, Jyva
¨40001, Finland
Department of Chemistry, University of Jyva
¨, Survontie 9, Jyva
¨40500, Finland
Technische Universita
¨t Braunschweig, Institute of Organic Chemistry, Hagenring 30, 38106 Braunschweig,
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
[1220], 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
&2017 The Author(s) Published by the Royal Society. All rights reserved.
on September 28, 2017 from
variety of ways, including volatile irritation, distastefulness
or even toxicity [12]. Chemical defences can be costly
[2224], 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 [1720]. 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
¨(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
). The oven
temperature was programmed as follows: 2 min at 808C, then
ramped to 1808C at the rate of 88Cmin
and from 1808Cto
2908C at the rate of 78C min
, 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
; 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
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 Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
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
¨(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 Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
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
. 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
), 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
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 Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
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 [5052]. 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
0.40 *
latency to approach (s)
oats eaten per second
abdominal neck
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.
latency to approach (s)
oats eaten per second
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. Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
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
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. Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
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
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
¨for moth
rearing; to Janne Valkonen and Sebastiano De Bona for statistical
34 000
26 000
22 000
18 000
14 000
10 000
5.00 7.00 9.00 11.00 13.00
retention time (min)
15.00 17.00 19.00
30 000
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. Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
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.
1. Edmunds M. 1974 Defence in animals: a survey of
antipredator defences. New York, NY: Longman.
2. Endler JA. 1986 Defense against predators. In
Predator prey relationships. Perspectives and
approaches from the study of lower vertebrates
(ed. MELGV Feder), pp. 109134. Chicago, IL:
University of Chicago Press.
3. Hoverman JT, Relyea RA. 2007 The rules of
engagement: how to defend against combinations
of predators. Oecologia 154, 551– 560. (doi:10.
4. Sih A, Englund G, Wooster D. 1998 Emergent
impacts of multiple predators on prey. Trends Ecol.
Evol. 13, 350– 355. (doi:10.1016/S0169-
5. Poulton EB. 1890 The colours of animals: their
meaning and use, pp. 558 612. London, UK: Kegan
Paul, Trench, Trubner.
6. Cott HB. 1940 Adaptive coloration in animals.
London, UK: Methuen.
7. Ruxton GD, Sherratt TN, Speed MP. 2004 Avoiding
attack: the evolutionary ecology of crypsis, warning
signals and mimicry, i p. Oxford, UK: Oxford
University Press.
8. Alatalo RV, Mappes J. 1996 Tracking the evolution
of warning signals. Nature 382, 708– 710. (doi:10.
9. Guilford T. 1990 The secrets of aposematism:
unlearned responses to specific colors and patterns.
Trends Ecol. Evol. 5, 323. (doi:10.1016/0169-
10. Mappes J, Marples N, Endler JA. 2005 The complex
business of survival by aposematism. Trends
Ecol. Evol. 20, 598603. (doi:10.1016/j.tree.2005.
11. Skelhorn J, Halpin CG, Rowe C. 2016 Learning about
aposematic prey. Behav. Ecol. 27, 955– 964. (doi:10.
12. Bowers MD. 1992 The evolution of unpalatability
and the cost of chemical defense in insects. In
Insect chemical ecology. An evolutionary approach
(eds BD Roitberg, MB Isman), pp. 216244.
London, UK: Chapman & Hall.
13. Maan ME, Cummings ME. 2012 Poison frog colors
are honest signals of toxicity, particularly for bird
predators. Am. Nat. 179, E1– E14. (doi:10.1086/
14. Pasteels JM, Gregoire JC, Rowellrahier M. 1983 The
chemical ecology of defense in arthropods. Annu.
Rev. Entomol. 28, 263289. (doi:10.1146/annurev.
15. Pentzold S, Zagrobelny M, Khakimov B, Engelsen
SB, Clausen H, Petersen BL, Borch J, Møller BL, Bak
S. 2016 Lepidopteran defence droplets: a composite
physical and chemical weapon against potential
predators. Sci. Rep. 6, 22407. (doi:10.1038/
16. Ritland DB. 1994 Variation in palatability of queen
butterflies (Danaus gilippus) and implications
regarding mimicry. Ecology 75, 732– 746. (doi:10.
17. Rothschild M, Aplin RT, Cockrum PA, Edgar JA,
Fairweather P, Lees R. 1979 Pyrrolizidine alkaloids
in arctiid moths (Lep.) with a discussion on host
plant relationships and the role of these secondary
plant substances in the Arctiidae. Biol. J. Linn. Soc.
12,305 326. (doi:10.1111/j.1095-8312.1979.
18. Trigo JR. 2000 The chemistry of antipredator
defense by secondary compounds in neotropical
Lepidoptera: facts, perspectives and caveats.
J. Brazil. Chem. Soc. 11, 551561. (doi:10.1590/
19. Triponez Y, Naisbit RE, Jean-Denis JB, Rahier M,
Alvarez N. 2007 Genetic and environmental sources
of variation in the autogenous chemical defense of
a leaf beetle. J. Chem. Ecol. 33, 20112024.
20. Weller SJ, Jacobson NL, Conner WE. 1999 The
evolution of chemical defences and mating systems
in tiger moths (Lepidoptera: Arctiidae). Biol. J. Linn.
Soc. 68, 557 578. (doi:10.1111/j.1095-8312.1999.
21. Speed MP, Ruxton GD, Mappes J, Sherratt TN. 2012
Why are defensive toxins so variable? An
evolutionary perspective. Biol. Rev. 87, 874– 884.
22. Reudler JH, Lindstedt C, Pakkanen H, Lehtinen I,
Mappes J. 2015 Costs and benefits of plant
allelochemicals in herbivore diet in a multi enemy
world. Oecologia 179, 11471158. (doi:10.1007/
23. Skelhorn J, Ruxton GD. 2008 Ecological factors
influencing the evolution of insects’ chemical
defenses. Behav. Ecol. 19, 146153. (doi:10.1093/
24. Zvereva EL, Kozlov MV. 2016 The costs and
effectiveness of chemical defenses in herbivorous
insects: a meta-analysis. Ecol. Monogr. 86, 107
124. (doi:10.1890/15-0911.1)
25. Ro
¨, K., Mappes J, Kaila L, Wahlberg N. 2016
Putting Parasemia in its phylogenetic place: a
molecular analysis of the subtribe Arctiina
(Lepidoptera). Syst. Entomol. 41, 844– 853. (doi:10.
26. Hegna RH, Galarza JA, Mappes J. 2015 Global
phylogeography and geographical variation in
warning coloration of the wood tiger moth
(Parasemia plantaginis). J. Biogeogr. 42, 1469–
1481. (doi:10.1111/jbi.12513)
27. Lindstedt C, Eager H, Ihalainen E, Kahilainen A,
Stevens M, Mappes J. 2011 Direction and strength
of selection by predators for the color of the
aposematic wood tiger moth. Behav. Ecol. 22,
580587. (doi:10.1093/beheco/arr017)
28. Nokelainen O, Hegna RH, Reudler JH, Lindstedt C,
Mappes J. 2012 Trade-off between warning signal
efficacy and mating success in the wood tiger moth.
Proc. R. Soc. B 279, 257265. (doi:10.1098/rspb.
29. Tammaru T, Haukioja E. 1996 Capital breeders and
income breeders among Lepidoptera: consequences
to population dynamics. Oikos 77, 561– 564.
30. Burdfield-Steel E, Pakkanen H, Rojas B, Galarza JA,
Mappes J. Submitted. De novo synthesis of chemical
defences in an aposematic moth.
31. Molleman F, Whitaker MR, Carey JR. 2010 Rating
palatability of butterflies by measuring ant feeding
behaviour. Entomol. Bericht. 70, 52– 62.
32. Mu¨ller C, Boeve
´, J.-L, Brakefield PM. 2002 Host
plant derived feeding deterrence towards ants in
the turnip sawfly Athalia rosae.Entomol. Exp. Appl.
104, 153157. (doi:10.1046/j.1570-7458.2002.
33. Nakagawa S, Schielzeth H. 2010 Repeatability for
Gaussian and non-Gaussian data: a practical guide
for biologists. Biol. Rev. 85, 935– 956. (doi:10.1111/
34. RStudio. 2015 RStudio: Integrated development
environment for R (version 0.99.441) [Computer
software], Boston, MA. See
35. Therneau TM. 2015 coxme: mixed effects Cox
models. (2.2-5 edn). See
36. Bates D, Maechler M, Bolker B, Walker S. 2015
Fitting linear mixed-effects models using lme4.
J. Stat. Softw. 67, 1– 48. (doi:10.18637/jss.v067.i01)
37. Rowe C, Guilford T. 1996 Hidden colour aversions in
domestic clicks triggered by pyrazine odours of
insect warning displays. Nature 383, 520522.
38. Rothschild M, Moore BP, Brown WV. 1984 Pyrazines
as warning odour components in the monarch
butterfly, Danaus plexippus, and in moths of the
genera Zygaena and Amata (Lepidoptera).
Biol. J. Linn. Soc. 23, 375 380. (doi:10.1111/j.
39. Vencl FV, Srygley RB. 2013 Enemy targeting, trade-
offs, and the evolutionary assembly of a tortoise
beetle defense arsenal. Evol. Ecol. 27, 237252.
40. Cogni R, Trigo JR, Futuyma DJ. 2012 A free lunch?
No cost for acquiring defensive plant pyrrolizidine
alkaloids in a specialist arctiid moth (Utetheisa Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
ornatrix). Mol. Ecol. 21, 6152– 6162. (doi:10.1111/
41. Moranz R, Brower LP. 1998 Geographic and
temporal variation of cardenolide-based chemical
defenses of queen butterfly (Danaus gilippus)in
Northern Florida. J. Chem. Ecol. 24, 905 932.
42. Rothschild M, Euw JV, Reichstein T. 1973 Cardiac
glycosides (heart poisons) in the polka-dot moth
Syntomeida Epilais Walk. (Ctenuchidae: Lep.) with
some observations on the toxic qualities of Amata
(¼Syntomis)phegea (L.). Proc. R. Soc. Lond. B 183,
227247. (doi:10.1098/rspb.1973.0015)
43. Hartmann T, Theuring C, Beuerle T, Bernays EA,
Singer MS. 2005 Acquisition, transformation and
maintenance of plant pyrrolizidine alkaloids by the
polyphagous arctiid Grammia geneura.Insect
Biochem. Mol. Biol. 35, 10831099. (doi:10.1016/j.
44. Hartmann T, Theuring C, Beuerle T, Ernst L, Singer MS,
Bernays EA. 2004 Acquired and partially de novo
synthesized pyrrolizidine alkaloids in two
polyphagous arctiids and the alkaloid profiles of their
larval food-plants. J. Chem. Ecol. 30, 229254.
45. von Nickisch-Rosenegk E, Wink M. 1993
Sequestration of pyrrolizidine alkaloids in several
arctiid moths (Lepidoptera, Arctiidae). J. Chem.
Ecol. 19, 18891903. (doi:10.1007/BF00983794)
46. Roque-Albelo L, Schroeder FC, Conner WE,
Bezzerides A, Hoebeke ER, Meinwald J, Eisner T.
2002 Chemical defense and aposematism: the case
of Utetheisa galapagensis.Chemoecology 12, 153
157. (doi:10.1007/s00012-002-8341-6)
47. Molleman F, Kaasik A, Whitaker MR, Carey JR. 2012
Partitioning variation in duration of ant feeding
bouts can offer insights into the palatability of
insects: experiments on African fruit-feeding
butterflies. J. Res. Lepidopt. 45, 6575.
48. Carrell JE. 2001 Response of predaceous arthropods
to chemically defended larvae of the pyralid moth
Uresiphita reversalis (Guene
´e) (Lepidoptera:
Pyralidae). J. Kansas Entomol. Soc. 74, 128135.
49. Hristov N, Conner WE. 2005 Effectiveness of tiger
moth (Lepidoptera, Arctiidae) chemical defenses
against an insectivorous bat (Eptesicus fuscus).
Chemoecology 15, 105 113. (doi:10.1007/s00049-
50. Brower LP, Ryerson WN, Coppinger LL, Glazier SC.
1968 Ecological chemistry and the palatability
spectrum. Science 161, 13491350. (doi:10.1126/
51. Cardoso MZ. 1997 Testing chemical defence based
on pyrrolizidine alkaloids. Anim. Behav. 54,985–
991. (doi:10.1006/anbe.1997.0505)
52. Massuda K, Trigo J. 2009 Chemical defence of the
warningly coloured caterpillars of Methona themisto
(Lepidoptera: Nymphalidae: Ithomiinae).
Eur. J. Entomol. 106, 253 259. (doi:10.14411/eje.
53. Blu¨thgen N, Fiedler K. 2004 Preferences for sugars
and amino acids and their conditionality in a
diverse nectar-feeding ant community. J. Anim.
Ecol. 73, 155166. (doi:10.1111/j.1365-2656.2004.
54. Ratcliffe JM, Nydam ML. 2008 Multimodal warning
signals for a multiple predator world. Nature 455,
96. (doi:10.1038/nature07087)
55. Moore BP, Brown WV, Rothschild M. 1990
Methylalkylpyrazines in aposematic insects, their
hostplants and mimics. Chemoecology 1, 4351.
56. Guilford T, Nicol C, Rothschild M, Moore BP. 1987
The biological roles of pyrazines: evidence for a
warning odour function. Biol. J. Linn. Soc. 31, 113
128. (doi:10.1111/j.1095-8312.1987.tb01984.x)
57. Lindstro
¨m L, Rowe C, Guilford T. 2001 Pyrazine
odour makes visually conspicuous prey aversive.
Proc. R. Soc. Lond. B 268, 159162. (doi:10.1098/
58. Roper TJ, Marples NM. 1997 Odour and colour as
cues for taste-avoidance learning in domestic chicks.
Anim. Behav. 53, 1241– 1250. (doi:10.1006/anbe.
59. Vencl FV, Ottens K, Dixon MM, Candler S, Bernal XE,
Estrada C, Page RA. 2016 Pyrazine emission by a
tropical firefly: an example of chemical
aposematism? Biotropica 48, 645– 655. (doi:10.
60. Nokelainen O, Valkonen J, Lindstedt C, Mappes J.
2014 Changes in predator community structure
shifts the efficacy of two warning signals in Arctiid
moths. J. Anim. Ecol. 83, 598 605. (doi:10.1111/
61. Gordon SP, Kokko H, Rojas B, Nokelainen O, Mappes
J. 2015 Colour polymorphism torn apart by
opposing positive frequency-dependent selection,
yet maintained in space. J. Anim. Ecol. 84, 1555–
1564. (doi:10.1111/1365-2656.12416)
62. Galarza JA, Nokelainen O, Ashrafi R, Hegna RH,
Mappes J. 2014 Temporal relationship between
genetic and warning signal variation in the
aposematic wood tiger moth (Parasemia
plantaginis). Mol. Ecol. 23, 4939 4957. (doi:10.
63. Partan S, Marler P. 1999 Communication goes
multimodal. Science 283, 12721273. (doi:10.
64. Rowland HM, Ihalainen E, Lindstro
¨m L, Mappes J,
Speed MP. 2007 Co-mimics have a mutualistic
relationship despite unequal defences. Nature 448,
6467. (doi:10.1038/nature05899)
65. Peka
´rS, Petra
´L, Bulbert MW, Whiting MJ,
Herberstein ME. 2017 The golden mimicry complex
uses a wide spectrum of defence to deter a
community of predators. Elife 6, e22089. (doi:10.
7554/eLife.22089) Proc. R. Soc. B 284: 20171424
on September 28, 2017 from
... These chemical defenses are typically considered secondary defenses, which act to prevent consumption after subjugation has occurred or to dissuade predators from attacking such prey in the future . However, chemical defenses may also be detected before subjugation and influence the predator's likelihood or latency to approach or attack the prey (Guilford et al., 1987;Rowe and Halpin, 2013;Rojas et al., 2017Rojas et al., , 2019. Therefore, the dichotomy between primary and secondary defenses is not perfect, and it is possible for a single defense mechanism to protect prey across multiple stages of a predator's attack. ...
... Chemical diversity may help prey defend themselves against multiple enemies, whereby different compound types are used to target different predators. For example, in A. plantaginis neck fluids defend against bird predators (but not invertebrates) and abdominal fluids defend against invertebrates (but not birds) (Rojas et al., 2017). It may also be more difficult for predators to evolve immunity to a suite of toxins compared to just one (Zhao et al., 2003). ...
... Smell and taste may also act at different stages of attack. Smell can be used to detect volatile odorants from a distance, potentially allowing predators to perceive chemical defenses before prey capture Rojas et al., 2017Rojas et al., , 2019. Whereas non-volatile compounds require predators to first capture prey before the chemical defense can be perceived via taste receptors. ...
Full-text available
Aposematic organisms warn predators of their unprofitability using a combination of defenses, including visual warning signals, startling sounds, noxious odors, or aversive tastes. Using multiple lines of defense can help prey avoid predators by stimulating multiple senses and/or by acting at different stages of predation. We tested the efficacy of three lines of defense (color, smell, taste) during the predation sequence of aposematic wood tiger moths ( Arctia plantaginis ) using blue tit ( Cyanistes caeruleus ) predators. Moths with two hindwing phenotypes (genotypes: WW/Wy = white, yy = yellow) were manipulated to have defense fluid with aversive smell (methoxypyrazines), body tissues with aversive taste (pyrrolizidine alkaloids) or both. In early predation stages, moth color and smell had additive effects on bird approach latency and dropping the prey, with the strongest effect for moths of the white morph with defense fluids. Pyrrolizidine alkaloid sequestration was detrimental in early attack stages, suggesting a trade-off between pyrrolizidine alkaloid sequestration and investment in other defenses. In addition, pyrrolizidine alkaloid taste alone did not deter bird predators. Birds could only effectively discriminate toxic moths from non-toxic moths when neck fluids containing methoxypyrazines were present, at which point they abandoned attack at the consumption stage. As a result, moths of the white morph with an aversive methoxypyrazine smell and moths in the treatment with both chemical defenses had the greatest chance of survival. We suggest that methoxypyrazines act as context setting signals for warning colors and as attention alerting or “go-slow” signals for distasteful toxins, thereby mediating the relationship between warning signal and toxicity. Furthermore, we found that moths that were heterozygous for hindwing coloration had more effective defense fluids compared to other genotypes in terms of delaying approach and reducing the latency to drop the moth, suggesting a genetic link between coloration and defense that could help to explain the color polymorphism. Conclusively, these results indicate that color, smell, and taste constitute a multimodal warning signal that impedes predator attack and improves prey survival. This work highlights the importance of understanding the separate roles of color, smell and taste through the predation sequence and also within-species variation in chemical defenses.
... Yellow males are subject to lower levels of predation in the wild (Nokelainen 69 et al., 2012(Nokelainen 69 et al., , 2014, while white males have a positive frequency-dependent mating advantage 70 . Yellow morphs have stronger chemical defences (Rojas et al., 2017), 71 but show reduced flight activity compared to white males, although yellows may fly at more 72 selective times, i.e. at peak female calling periods (Rojas, Gordon and Mappes, 2015). In 73 summary, there is a trade-off between natural selection through predation and reproductive 74 success, which contributes to the maintenance of this polymorphism (Rönkä et al., 2020). ...
Full-text available
Colour is often used as an aposematic warning signal, with predator learning expected to lead to a single colour pattern within a population. However, there are many puzzling cases where aposematic signals are also polymorphic. The wood tiger moth, Arctia plantaginis , uses bright hindwing colours as a signal of unpalatability, and males have discrete colour morphs which vary in frequency geographically. In Finland, both white and yellow morphs can be found, and these colour morphs also differ in behavioural and life-history traits. Complex polymorphisms such as these are often explained by supergenes. Here, we show that male colour is linked to an extra copy of a yellow family gene that is only present in the white morphs. This white-specific duplication, which we name valkea , is highly upregulated during wing development, and could act to reduce recombination, thus potentially representing a supergene. We also characterise the pigments responsible for yellow, white and black colouration, showing that yellow is partly produced by pheomelanins, while black is dopamine-derived eumelanin. The yellow family genes have been linked to melanin synthesis and behavioural traits in other insect species. Our results add to only a few examples of seemingly paradoxical and complex polymorphisms which are associated with single genes.
... Predation is a key agent of natural selection in prey species (Edmunds, 1974;Ruxton et al., 2018). To survive in the real world with multiple predators, animals evolve different defense capabilities that vary in their nature and efficacy in relation to predator sensory abilities and attack tactics (Hoverman & Relyea, 2007;Rojas et al., 2021). ...
Full-text available
Prey evolve antipredator strategies against multiple enemies in nature. We examined how a prey species adopts different predation avoidance tactics against pursuit or sit-and-wait predators. As prey, we used three strains of Tribolium beetles artificially selected for short (short strain) or long (long strain) duration of death feigning, and a stock culture (base population). Death feigning is known to be effective for evading a jumping spider in the case of the long strains, while the present study showed that the long-strain beetles used freezing against a sit-and-wait type predator, Amphibolus venator, in this study. The short- strain beetles were more easily oriented toward predators. The time to predation was also shorter in the short strains compared to the long strains. The results showed that, as prey, the short strains displayed the same behavior, escaping, against both types of predators. Traditionally, death feigning has been thought to be the last resort in a series of antipredator avoidance behaviors. However, our results showed that freezing and death feigning were not parts of a series of behaviors, but independent strategies against different predators, at least for long-strain beetles. We also examined the relationship between a predator's starvation level and its predatory behavior. In addition, the orientation behavior toward and predation rate on the prey were observed to determine how often the predatory insect attacked the beetles.
... Odoriferous and distasteful pyrazines are widespread in nature (97) and are better known as predator deterrents in aposematic insects (60,117). However, they do not always confer protection, especially against invertebrate predators (28,115), and may have other functions. For example, (100), which is rich in amino acids that likely react enzymatically to form pyrazines (94). ...
Ants have outstanding capacity to mediate inter- and intraspecific interactions by producing structurally diverse metabolites from numerous secretory glands. Since Murray Blum's pioneering studies dating from the 1950s, there has been a growing interest in arthropod toxins as natural products. Over a dozen different alkaloid classes have been reported from approximately 40 ant genera in five subfamilies, with peak diversity within the Myrmicinae tribe Solenopsidini. Most ant alkaloids function as venom, but some derive from other glands with alternative functions. They are used in defense (e.g., alarm, repellants) or offense (e.g., toxins) but also serve as antimicrobials and pheromones. We provide an overview of ant alkaloid diversity and function with an evolutionary perspective. We conclude that more directed integrative research is needed. We suggest that comparative phylogenetics will illuminate compound diversification, while molecular approaches will elucidate genetic origins. Biological context, informed by natural history, remains critical not only for research about focal species, but also to guide applied research. Expected final online publication date for the Annual Review of Entomology, Volume 67 is January 2022. Please see for revised estimates.
... Adults were chosen because they don't feed and thus taking up bacteria from the environment is unlikely at this life stage. Moreover, the secretion is an important survival trait for the species [32,33], and thus it is expected to be highly conserved, including its associated microbiota. The defensive secretion was collected under a laminar flow to minimize contamination using a sterile capillary and placed in a 1.5 ml Eppendorf tube containing 30 ul of autoclaved ddH20. ...
Full-text available
Antibiotics have long been used in the raising of animals for agricultural, industrial or laboratory use. The use of subtherapeutic doses in diets of terrestrial and aquatic animals to promote growth is common and highly debated. Despite their vast application in animal husbandry, knowledge about the mechanisms behind growth promotion is minimal, particularly at the molecular level. Evidence from evolutionary research shows that immunocompetence is resource-limited, and hence expected to trade off with other resource-demanding processes, such as growth. Here, we ask if accelerated growth caused by antibiotics can be explained by genome-wide trade-offs between growth and costly immunocompetence. We explored this idea by injecting broad-spectrum antibiotics into wood tiger moth ( Arctia plantaginis ) larvae during development. We follow several life-history traits and analyse gene expression (RNA-seq) and bacterial (r16S) profiles. Moths treated with antibiotics show a substantial depletion of bacterial taxa, faster growth rate, a significant downregulation of genes involved in immunity and significant upregulation of growth-related genes. These results suggest that the presence of antibiotics may aid in up-keeping the immune system. Hence, by reducing the resource load of this costly process, bodily resources may be reallocated to other key processes such as growth.
... Defensive compounds are generally not target specific within a studied predator-prey system since they often act against ants as well as birds, and they can also possess antimicrobial and antifungal activities 47,48 . But some insect species contain chemicals that are target specific towards guilds of natural enemies, acting for instance on predators but not parasitoids 49 , or even towards predator types by acting on ants but not birds, or vice-versa 50 . Thus, this study, which pits a prey group against one predator type, constitutes a simplified situation compared with prey species facing multiple and various antagonists in natural conditions. ...
Full-text available
The sawfly larvae of most Argidae and Pergidae (Hymenoptera: Symphyta) species contain toxic peptides, and these along with other traits contribute to their defense. However, the effectiveness of their defense strategy, especially against ants, remains poorly quantified. Here, five Arge species, A. berberidis, A. nigripes, A. ochropus, A. pagana, A. pullata, plus three Pergidae species, Lophyrotoma analis, Lophyrotoma zonalis, Philomastix macleaii, were tested in laboratory bioassays on ant workers mainly of Myrmica rubra. The experiments focused on short-term predator–prey interactions, sawfly survival rate after long-term interactions, and feeding deterrence of the sawfly hemolymph. The larvae of Arge species were generally surrounded by few ants, which rarely bit them, whereas larvae of Pergidae, especially P. macleaii, had more ants around with more biting. A detailed behavioral analysis of Arge-ant interactions revealed that larval body size and abdomen raising behavior were two determinants of ant responses. Another determinant may be the emission of a volatile secretion by non-eversible ventro-abdominal glands. The crude hemolymph of all tested species, the five Arge species and L. zonalis, was a strong feeding deterrent and remained active at a ten-fold dilution. Furthermore, the study revealed that the taxon-specific behavior of ants, sting or spray, impacted the survival of A. pagana but not the large body-sized A. pullata. The overall results suggest that the ability of Arge and Pergidae larvae to defend against ants is influenced by the body size and behavior of the larvae, as well as by chemicals.
... Progress in understanding the ecology of chemical defences as they relate to prey sampling and survival has been more gradual (though unabating, e.g. [88][89][90]), and some fundamental questions remain. The extent to which Daphnia spp. ...
Full-text available
The combined use of noxious chemical defences and conspicuous warning colours is a ubiquitous anti-predator strategy. That such signals advertise the presence of defences is inherent to their function, but their predicted potential for quantitative honesty-the positive scaling of signal salience with the strength of protection-is the subject of enduring debate. Here, we systematically synthesized the available evidence to test this prediction using meta-analysis. We found evidence for a positive correlation between warning colour expression and the extent of chemical defences across taxa. Notably, this relationship held at all scales; among individuals, populations and species, though substantial between-study heterogeneity remains unexplained. Consideration of the design of signals revealed that all visual features, from colour to contrast, were equally informative of the extent of prey defence. Our results affirm a central prediction of honesty-based models of signal function and narrow the scope of possible mechanisms shaping the evolution of aposematism. They suggest diverse pathways to the encoding and exchange of information, while highlighting the need for deeper knowledge of the ecology of chemical defences to enrich our understanding of this widespread anti-predator adaptation.
Chemical defences often vary within and between populations both in quantity and quality, which is puzzling if prey survival is dependent on the strength of the defence. We investigated the within- and between-population variability in chemical defence of the wood tiger moth (Arctia plantaginis). The major components of its defences, SBMP (2secbutyl3methoxypyrazine) and IBMP (2isobutyl3methoxypyrazine) are volatiles that deter bird attacks. We expected the variation to reflect populations predation pressures and early-life conditions. To understand the role of the methoxypyrazines, we experimentally manipulated synthetic SBMP and IBMP and tested the birds reactions. We found a considerable variation in methoxypyrazine amounts and composition, both from wild-caught and laboratory-raised male moths. In agreement with the cost of defence hypothesis, the moths raised in the laboratory had a higher amount of pyrazines. We found that SBMP is more effective at higher concentrations and that IBMP is more effective only in combination with SBMP and at lower concentrations. Our results fit findings from the wild: the amount of SBMP was higher in the populations with higher predation pressure. Altogether, this suggests that, regarding pyrazine concentration, more is not always better, and highlights the importance of testing the efficacy of chemical defence and its components with relevant predators, rather than relying only on results from chemical analyses
Defensive chemicals are used by plants and animals to reduce the risk of predation through different mechanisms, including toxins that cause injury and harm (weapons) and unpalatable or odiferous compounds that prevent attacks (deterrents). However, whether effective defences are both toxins and deterrents, or work in just one modality is often unclear. In this study, our primary aim was to determine whether defensive compounds stored by nudibranch molluscs acted as weapons (in terms of being toxic), deterrents (in terms of being distasteful), or both. Our secondary aim was to investigate the response of different taxa to these defensive compounds. To do this, we identified secondary metabolites in 30 species of nudibranch molluscs and investigated their deterrent properties using anti‐feedant assays with three taxa: rock pool shrimp, Palaemon serenus, and two fish species: triggerfish Rhinecanthus aculeatus and toadfish Tetractenos hamiltoni. We compared these results to toxicity assays using brine shrimp Artemia sp. and previously published toxicity data with a damselfish Chromis viridis. Overall, we found no clear relationship between palatability and toxicity, but instead classified defensive compounds into the following categories: Class I & II ‐ highly unpalatable and highly toxic; Class I ‐ weakly unpalatable and highly toxic; Class II ‐ highly unpalatable but weakly toxic; WR (weak response) ‐ weakly unpalatable and weakly toxic. We also found eight extracts from six species that did not display activity in any assays indicating they may have very limited chemical defensive mechanisms (NR, no response). We found that the different classes of secondary metabolites were similarly unpalatable to fish and shrimp, except extracts from Phyllidiidae nudibranchs (isonitriles) that were highly unpalatable to shrimp but weakly unpalatable to fish. Our results pave the way toward better understanding how animal chemical defences work against a variety of predators. We highlight the need to disentangle weapons and deterrents in future work on anti‐predator defences to better understand the foraging decisions faced by predators, the resultant selection pressures imposed on prey, and the evolution of different anti‐predator strategies.
Chemical secretions are an effective means by which insects can deter potential enemies. Several terrestrial insects spray these liquids directionally toward enemies, but little is known about spraying behavior in aquatic and semiaquatic insects. The larvae of Osmylus hyalinatus (Neuroptera: Osmylidae) are semiaquatic, inhabiting the edges of small streams and ponds where they encounter multiple enemies on land and in water. The larvae of this osmylid sprayed a hyaline liquid from the anal opening if disturbed in either air and water, although the spray appeared slightly viscous in water. The liquid was stored in the posterior half of the hindgut and sprayed directionally toward an artificial stimulus. Spraying allowed the larvae to escape biting by ants, and to repel them in 90% of encounters. Spraying caused the regurgitation of 71% and 60% of all larvae swallowed by terrestrial frogs and aquatic newts, respectively. Aquatic fishfly larvae released 30% of captured larvae due to spraying. Most of the larvae that repelled ants or were regurgitated by amphibians survived, but those released by fishfly larvae were killed by heavy biting with the mandibles. This is the first report of effective liquid spraying by insects in water, and also within the order Neuroptera.
Full-text available
Many animals protect themselves from predation with chemicals, both self-made or sequestered from their diet. The potential drivers of the diversity of these chemicals have been long studied, but our knowledge of these chemicals and their acquisition mode is heavily based on specialist herbivores that sequester their defenses. The wood tiger moth (Arctia plantaginis, Linnaeus, 1758) is a well-studied aposematic species, but the nature of its chemical defenses has not been fully described. Here, we report the presence of two methoxypyrazines, 2-sec-butyl-3-methoxypyrazine and 2-isobutyl-3-methoxypyrazine, in the moths' defensive secretions. By raising larvae on an artificial diet, we confirm, for the first time, that their defensive compounds are produced de novo rather than sequestered from their diet. Pyrazines are known for their defensive function in invertebrates due to their distinctive odor, inducing aversion and facilitating predator learning. While their synthesis has been suspected, it has never previously been experimentally confirmed. Our results highlight the importance of considering de novo synthesis, in addition to sequestration, when studying the defensive capabilities of insects and other invertebrates.
Full-text available
Mimicry complexes typically consist of multiple species that deter predators using similar anti-predatory signals. Mimics in these complexes are assumed to vary in their level of defence from highly defended through to moderately defended, or not defended at all. Here, we report a new multi-order mimicry complex that includes at least 140 different putative mimics from four arthropod orders including ants, wasps, bugs, tree hoppers and spiders. All members of this mimicry complex are characterised by a conspicuous golden body and an ant Gestalt, but vary substantially in their defensive traits. However, they were similarly effective at deterring predators - even mildly defended mimics were rarely eaten by a community of invertebrate and vertebrate predators both in the wild and during staged trials. We propose that despite the predominance of less defended mimics the three predatory guilds avoid the mimics because of the additive influence of the various defensive traits.
Full-text available
Full-text available
Despite being popular among amateur and professional lepidopterologists and posing great opportunities for evolutionary research, the phylogenetic relationships of tiger moths (Erebidae: Arctiinae) are not well resolved. Here we provide the first phylogenetic hypothesis for the subtribe Arctiina with the basic aim of clarifying the phylogenetic position of the Wood Tiger Moth Parasemia plantaginis Hübner, a model species in evolutionary ecology. We sampled 89 species in 52 genera within Arctiina s.l., 11 species of Callimorphina and two outgroup species. We sequenced up to seven nuclear genes (CAD, GAPDH, IDH, MDH, Ef1α, RpS5, Wingless) and one mitochondrial gene (COI) including the barcode region (a total of 5915 bp). Both maximum likelihood and Bayesian inference resulted in a well-resolved phylogenetic hypothesis, consisting of four clades within Arctiina s.s. and a clade comprising spilosomine species in addition to Callimorphina and outgroups. Based on our results, we present a new classification, where we consider the Diacrisia clade, Chelis clade, Apantesis clade, Micrarctia Seitz and Arctia clade as valid genera within Arctiina s.s., whereas Rhyparia Hübner syn.n. and Rhyparioides Butler syn.n. are synonymized with Diacrisia Hübner; Neoarctia Neumoegen & Dyar syn.n., Tancrea Püngeler syn.n., Hyperborea Grum-Grshimailo syn.n., Palearctia Ferguson syn.n., Holoarctia Ferguson syn.n., Sibirarctia Dubatolov syn.n. and Centrarctia Dubatolov syn.n. are synonymized with Chelis Rambur; Grammia Rambur syn.n., Orodemnias Wallengren syn.n., Mimarctia Neumoegen & Dyar syn.n., Notarctia Smith syn.n. and Holarctia Smith syn.n. are synonymized with Apantesis Walker; and Epicallia Hübner syn.n., Eucharia Hübner syn.n., Hyphoraia Hübner syn.n., Parasemia Hübner syn.n., Pericallia Hübner syn.n., Nemeophila Stephens syn.n., Ammobiota Wallengren syn.n., Platarctia Packard syn.n., Chionophila Guenée syn.n., Eupsychoma Grote syn.n., Gonerda Moore syn.n., Platyprepia Dyar syn.n., Preparctia Hampson syn.n., Oroncus Seitz syn.n., Acerbia Sotavalta syn.n., Pararctia Sotavalta syn.n., Borearctia Dubatolov syn.n., Sinoarctia Dubatolov syn.n. and Atlantarctia Dubatolov syn.n. are synonymized with Arctia Schrank, leading to 33 new genus-level synonymies. Our focal species Arctia plantaginis comb.n. is placed as sister to Arctia festiva comb.n., another widespread aposematic species showing wing pattern variation. Our molecular hypothesis can be used as a basis when adding more species to the tree and tackling interesting evolutionary questions, such as the evolution of warning signalling and mimicry in tiger moths.
Full-text available
Insects often release noxious substances for their defence. Larvae of Zygaena filipendulae (Lepidoptera) secrete viscous and cyanogenic glucoside-containing droplets, whose effectiveness was associated with their physical and chemical properties. The droplets glued mandibles and legs of potential predators together and immobilised them. Droplets were characterised by a matrix of an aqueous solution of glycine-rich peptides (H-WG11-NH2) with significant amounts of proteins and glucose. Among the proteins, defensive proteins such as protease inhibitors, proteases and oxidases were abundant. The neurotoxin β-cyanoalanine was also found in the droplets. Despite the presence of cyanogenic glucosides, which release toxic hydrogen cyanide after hydrolysis by a specific β-glucosidase, the only β-glucosidase identified in the droplets (ZfBGD1) was inactive against cyanogenic glucosides. Accordingly, droplets did not release hydrogen cyanide, unless they were mixed with specific β-glucosidases present in the Zygaena haemolymph. Droplets secreted onto the cuticle hardened and formed sharp crystalline-like precipitates that may act as mandible abrasives to chewing predators. Hardening followed water evaporation and formation of antiparallel β-sheets of the peptide oligomers. Consequently, after mild irritation, Zygaena larvae deter predators by viscous and hardening droplets that contain defence proteins and β-cyanoalanine. After severe injury, droplets may mix with exuding haemolymph to release hydrogen cyanide.
Although famous for photic courtship displays, fireflies (Coleoptera: Lampyridae) are also notable for emitting strong odors when molested. The identity of volatile emissions and their possible role, along with photic signals, as aposematic warnings of unpalatability have been little explored, especially in tropical species. Pursuant to the observation that the widespread Neotropical fireflies, Photuris trivittata and Bicellonycha amoena, emit pungent odors, glows, and flashes when handled, we investigated their cuticular and headspace chemistry. Gas chromatography–mass spectrometry analyses revealed that both fireflies have species-specific cuticular hydrocarbon profiles. Photuris trivittata headspace was dominated by 2-methoxy-3-(1-methylpropyl) pyrazine (hereafter, pyrazine), on the order of 1.59 ng/individual and a suite of sesquiterpenes, while B. amoena emitted 3-methoxy-2-butenoic acid methyl ester and a few ketones. This is the first report of such compounds in fireflies. We investigated the role of pyrazine in P. trivittata's interactions with potential predators: sympatric ants, toads, and bats. Solvent-washed P. trivittata painted with pyrazine incurred lower ant predation than did their solvent-washed counterparts. Pyrazine significantly repelled ants at baits in concentrations as low as 9.8 9 10 À4 ng/ll. The toad, Rhi-nella marina, readily accepted intact fireflies, pyrazine-coated and uncoated mealworms. Both Myotis nigricans and Molossus molossus bats rejected fireflies, but accepted both pyrazine-coated and uncoated mealworms. While pyrazine repels ants, its role as an aposematic signal warning other potential predators of firefly distastefulness requires further investigation. Our results underscore the idea that multiple enemies exert conflicting selection on firefly defenses. Abstract in Spanish is available with online material.
Extensive observations of aposematic Uresiphita reversalis (Guenee) larvae feeding on sky-blue lupine Lupinus cumulicola Small in February in south Florida revealed a low incidence of predation by natural arthropod enemies. Three species of spiders, the wolf spiders Lycosa ceratiola Gertsch & Wallace and L osceola Gertsch & Wallace and the crab spider Misumenops sp., rejected U. reversalis larvae that were offered to them in laboratory predation tests. However, the green lynx spider Peucetia viridans (Hentz) and the assassin bug Zelus longipes (L.) were found to feed on the caterpillars. Bioassays with the wolf spider L. ceratiola confirmed previous studies showing that the larval integument possess potent antifeedant properties, most likely because it contains quinolizidine alkaloids of dietary origin.
The question, “Why should prey advertise their presence to predators using warning coloration?” has been asked for over 150 years. It is now widely acknowledged that defended prey use conspicuous or distinctive colors to advertise their toxicity to would-be predators: a defensive strategy known as aposematism. One of the main approaches to understanding the ecology and evolution of aposematism and mimicry (where species share the same color pattern) has been to study how naive predators learn to associate prey’s visual signals with the noxious effects of their toxins. However, learning to associate a warning signal with a defense is only one aspect of what predators need to do to enable them to make adaptive foraging decisions when faced with aposematic prey and their mimics. The aim of our review is to promote the view that predators do not simply learn to avoid aposematic prey, but rather make adaptive decisions about both when to gather information about defended prey and when to include them in their diets. In doing so, we reveal what surprisingly little we know about what predators learn about aposematic prey and how they use that information when foraging. We highlight how a better understanding of predator cognition could advance theoretical and empirical work in the field.
The evolution of defensive traits and strategies depends on the intensity of selection imposed by natural enemies and on the fitness costs of defenses against these enemies. We tested several hypotheses about the evolution of chemical defenses in plant-feeding insects using a meta-analysis. We analyzed the effectiveness (in terms of prey survival; 159 publications) and costs (in terms of reduction in performance due to defense production; 33 publications) of chemical defenses in various prey-predator systems (140 herbivore species and 124 enemy species). The chemical defenses of insect herbivores, on average, were effective against generalist predators, were not effective against specialist predators and generalist parasitoids, and increased the risk of parasitism by specialist parasitoids. The defenses were more effective against vertebrate than against invertebrate predators and most effective against birds. Defensive compounds synthesized de novo and derived from the herbivore's food plants did not differ in the magnitude of their effects. Externalization of chemical defenses enhanced their effects on naive vertebrate predators but simultaneously increased the risk of parasitism. The defenses of specialist herbivores were more effective than those of generalists, mostly due to species that sequestered plant allelochemicals for their own defenses. Advertising of chemical defenses by warning display enhanced their effectiveness only against vertebrate predators. Aposematic colors and patterns were more effective warning signals than other types of conspicuous coloration against both experienced and naive vertebrate predators, suggesting that certain colors and/or patterns were more important than conspicuousness for both learning and innate avoidance. The meta-analysis did not reveal physiological costs of the production of chemical defenses across 22 herbivore species, although the results varied strongly with the method used to measure these costs. We conclude that the cost-benefit trade-offs driving the evolution of chemical defenses in herbivorous insects are affected by ecological costs (i.e., increased susceptibility to parasitoids) more than by costs in terms of resources. Still, a favorable cost-benefit ratio, i.e., great effects for a small expenditure, may partly explain the prevalence of chemical anti-predator defenses in insects.