Antipredator strategies of pupae: How to avoid predation in an immobile life stage?

Abstract

Antipredator strategies of the pupal stage in insects have received little attention in comparison to larval or adult stages. This is despite the fact that predation risk can be high during the pupal stage, making it a critical stage for subsequent fitness. The immobile pupae are not, however, defenceless; a wide range of antipredator strategies have evolved against invertebrate and vertebrate predators. The most common strategy seems to be ‘avoiding encounters with predators' by actively hiding in vegetation and soil or via cryptic coloration and masquerade. Pupae have also evolved behavioural and secondary defences such as defensive toxins, physical defences or deimatic movements and sounds. Interestingly, warning coloration used to advertise unprofitability has evolved very rarely, even though the pupal stage often contains defensive toxins in chemically defended species. In some species, pupae gain protection from conspecifics or mimic chemical and auditory signals and thereby manipulate other species to protect them. Our literature survey highlights the importance of studying selection pressures across an individual's life stages to predict how ontogenetic variation in selective environments shapes individual fitness and population dynamics in insects. Finally, we also suggest interesting avenues for future research to pursue.
This article is part of the theme issue ‘The evolution of complete metamorphosis’.

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royalsocietypublishing.org/journal/rstb
Review
Cite this article: Lindstedt C, Murphy L,
Mappes J. 2019 Antipredator strategies of
pupae: how to avoid predation in an immobile
life stage? Phil. Trans. R. Soc. B 374: 20190069.
http://dx.doi.org/10.1098/rstb.2019.0069
Accepted: 21 May 2019
One contribution of 13 to a theme issue ‘The
evolution of complete metamorphosis’.
Subject Areas:
behaviour, ecology, evolution
Keywords:
pupal defence, protective coloration, chemical
defence, physical defence, predator–prey
interactions
Author for correspondence:
Carita Lindstedt
e-mail: carita.a.lindstedt@jyu.fi
Antipredator strategies of pupae: how to
avoid predation in an immobile life stage?
Carita Lindstedt, Liam Murphy and Johanna Mappes
Department of Biological and Environmental Sciences, P.O. Box 35, FI‐40014 University of Jyväskylä, Jyväskylä,
Finland
CL, 0000-0001-8176-3613
Antipredator strategies of the pupal stage in insects have received little
attention in comparison to larval or adult stages. This is despite the fact that
predation risk can be high during the pupal stage, making it a critical stage
for subsequent fitness. The immobile pupae are not, however, defenceless; a
wide range of antipredator strategies have evolved against invertebrate and
vertebrate predators. The most common strategy seems to be ‘avoiding
encounters with predators’by actively hiding in vegetation and soil or via
cryptic coloration and masquerade. Pupae have also evolved behavioural
and secondary defences such as defensive toxins, physical defences or deimatic
movements and sounds. Interestingly, warning coloration used to advertise
unprofitability has evolved very rarely, even though the pupal stage often
contains defensive toxins in chemically defended species. In some species,
pupae gain protection from conspecifics or mimic chemical and auditory
signals and thereby manipulate other species to protect them. Our literature
survey highlights the importance of studying selection pressures across an
individual’s life stages to predict how ontogenetic variation in selective
environments shapes individual fitness and population dynamics in insects.
Finally, we also suggest interesting avenues for future research to pursue.
This article is part of the theme issue ‘The evolution of complete
metamorphosis’.
1. Introduction
‘I spent my time investigating insects. At the beginning, I started with silk worms
in my home town of Frankfurt. I realized that other caterpillars produced
beautiful butterflies or moths, and that silkworms did the same. This led me to
collect all the caterpillars I could find in order to see how they changed’. This
is quoted from the foreword by Maria Sibylla Merian in her ‘Metamorphosis insec-
torum Surinamensium’published in 1705 [1, p. 3]. The idea of the complete
metamorphosis was developed by Aristotle 2000 years earlier [2], but Merian
was the first entomologist who described insect metamorphosis in detail, includ-
ing pupation, which makes her one of the most significant contributors to the
field of entomology. It is intriguing that more than 300 years after Merian’s fun-
damental observations about insect metamorphosis and the discovery of pupae,
this is the life stage, along with the egg stage [3], that we still know almost
nothing about compared to the adult and larval stages in insects.
Merian’s butterflies, along with the other holometabolous insects, have mor-
phologically distinct immature life stages, larva and pupa, which they need to
live through to reach the final reproductive adult stage. Holometabolous insects
are thought to have evolved from hemimetabolous insectsthat have only two life
stages: the nymph and the adult [4]. Ancestral forms of immobile ‘compact’
pupae were most likely mobile nymph-resembling pupae similar to pupa of,
for example, the snakeflies [5]. Vulnerability to natural enemies in each of these
life stages has resulted in the evolution of an extensive diversity of adaptations
protecting individuals against different types of predators and parasitoids [6,7].
These adaptations can vary dramatically in each life stage depending on the indi-
vidual’s lifestyle (e.g. sessile versus mobile life stages), reproductive stage (larval
stage versus adult stage) and ontogenetic shifts in resource use [8,9].
© 2019 The Author(s) Published by the Royal Society. All rights reserved.

Traditionally, the evolution of antipredator defence mech-
anisms is considered on a scale of one life stage focusing on,
for example, larval instars or adult stages [6]. Research has
traditionally focused much less on the antipredatory strategies
of egg or pupal stages. However, even though the selective
environment can change dramatically between each life
stage, an individual’s fitness (e.g. reproductive success and
ability to survive until and beyond a given reproductive
stage) is the sum of conditions experienced during the pre-
vious life stages [10–12]. To predict how different conditions
shape individual fitness or population dynamics of insects,
it is therefore not enough to know the factors that contribute
to the survival in the larval stage or what affects reproductive
success as an adult. We also need to understand what happens
between these two stages: what kind of antipredator defences
individuals have during the pupal stage, how it is affected by
the conditions experienced during earlier life stages or how
behaviour during the larval stage affects pupal predation
risk. This kind of information helps us to understand the
evolution of defensive traits in general, but may also have
applied importance in predicting factors that shape popu-
lation dynamics in potential pest species or in species at risk
of extinction. At present, we have surprisingly little data tack-
ling these issues and behavioural and evolutionary research
on the topic is scarce.
Here, we first review the literature on predation on the
pupal stage to find what attacks insect pupae, how high preda-
tion risk is for them, and what different types of antipredator
defence mechanisms have evolved for insect pupae as a result
of selection by predation. We acknowledge that parasitism is
also an important source of mortality in the pupal stage, and
it is likely that many defensive mechanisms against predators
(e.g. defensive toxins, camouflage, protection gained from
other species) can have dual function against both predators
and parasitoids [7,13,14]. Therefore, we will take into account
parasitism where relevant. However, the main focus of our
review will be on antipredator defence strategies. Finally,
we discuss the gaps in our knowledge and outline some
promising directions for future research to pursue.
2. Predation risk is often high during the pupal
stage
Predation risk during the pupal stage has received the most
attention in species that have some economical value such
as many forest pest insects. Owing to their outbreak
dynamics, pupae of forest pest insects can form an abundant
food source during the high-density population peaks for
small mammals such as voles, mice and shrews, along with
invertebrate predators such as ground beetles, ants and ear-
wigs [15–17]. For example, in gypsy moths (Lymantria
dispar), small mammal predators such as the white-footed
mouse in North America, invertebrate predators such as
ground beetles Carabidae, and ants have been suggested to
be among the most important predators during the pupal
stage [15]. Similarly, in ground-dwelling pupae of winter
moths (Operophtera brumata) and autumnal moths (Epirrita
autumnata), moles, mice, voles and shrews together with
invertebrate predators (larvae of Carabidae, Elateridae and
Saphylinidae beetles that prey upon pupae in the soil) have
been suggested to be important sources of mortality in
Europe [17,18]. Birds have also been reported to feed on
pupae, but depending on the insect species, their importance
varies from moderate [15] to high [16,19].
Generalist predators of insect cocoons can have a major
impact on many insect populations, for example, in stabiliz-
ing population cycles [16,18,20]. Based on the mortality
rates reported in the literature, the magnitude of predation
risk at the pupal stage can be surprisingly high: estimates
from studies with gypsy moths report predation rates as
high as 90–100% [15]. Studies on winter moths and autumnal
moths report pupal predation rates ranging from 20 to 72%
[17,18]. In Neodiprion sertifer pine sawflies, small mammals
caused 70% mortality on the ground and bird predation
caused 70–85% mortality [16]. In another forest pest species,
the pine processionary caterpillars (Thaumetopea pityocampa),
predation by Hoopoes (Upupa epops) can result in up to
68.3–74.1% mortality in the pupal stage [19]. Much less infor-
mation from the pupal predation risk exists for the species
that do not have cyclic population dynamics and economic
value. In two moth species from the family Limacodidae
(slug caterpillars), cocoon predation resulted in intermediate
mortality ranging from 22 to 29% depending on the moth
species [21].
Based on these estimates, predation risk can vary consider-
ably from moderate to very high, making survival through the
pupal stage likely to be a critical step for the majority of
the insect species. From an evolutionary point of view, the
strength of selection on mechanisms enhancing survival at
this stage should therefore be extremely strong. It is, however,
important to note that these mortality risk estimates are
species-specific, perhaps over-estimating the predation risk
to some extent. For example, how ‘naturally’pupae are
located on experimental plots varies among studies and
thus, predation risk may sometimes be over-estimated.
The second common feature arising from the literature is
that predators that prey on insect pupae are very diverse,
including both visual and non-visual predators with, per-
haps, the emphasis on latter [15,17,18]. This is different
from the larval stage where visual predators such as insecti-
vorous birds are often considered to be the most important
predator group [22]. Physical and chemical antipredator strat-
egies play an important role in defence against non-visual
predators and could therefore be assumed to be selected for
in the pupal stage [6].
3. Avoiding detection by visual predators
The most common strategy to avoid attack is via cryptic
coloration that makes prey difficult for a predator to detect
(e.g. camouflage). This is also true for insect pupae [8,23].
Cryptic coloration of the pupa is adaptive as it increases sur-
vival against visually hunting predators when the colour
matches with the visual background [24–26] (figure 1). In
some taxonomic groups, variation in the visual background
has favoured environmentally plastic variation in pupal color-
ation such as colour polyphenism. Polyphenism in cryptic
pupal coloration has evolved independentlyat least in Papilio-
nidae, Pierinae, Satyrinae and Nymphalinae [24,25,27,28]. In
many of these cases, there are two types of pupae where one
form is green–yellow and other brown–black.
Pupal colour variation has been shown to be mainly an
environmentally plastic trait, even though individual sensi-
tivity to environmental cues triggering the change in pupal
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2

colour can also vary genetically [25,28]. For example, tactile
signals such as roughness of the background can impact on
whether the pupae develop green (smooth surfaces) or
brown (rough textures) in Papilio xuthus [29]. In addition to
substrate texture, several environmental conditions have
been shown to affect pupal colour (reviewed in [30]) such
as background colour, relative humidity [30], photoperiod
length, temperature, wavelength of light and diet quality
[26]. Pupal colour polyphenism can also be influenced by
the developmental pathway of the individual. In bivoltine
or multivoltine species whose pupal stage length varies
considerably depending whether the individual overwinters
in a pupal diapause (pupal stage takes several months) or
develops directly (pupal stage is only a week or two long),
the hormonal state of the pupae has a strong effect on its
coloration. For example, directly developing summer pupae
have a higher tendency to become green, whereas the over-
wintering pupae have a higher tendency to become brown
[31]. Often, several of the factors listed above can affect the
pupal coloration simultaneously [32,33].
Interestingly, the adaptive function of cryptic pupal color-
ation can also be linked to life-history traits in adult and
larval stages [28,34]. In the speckled wood butterfly Pararge
aegeria, individuals eclosing from the green coloured pupae
are larger as adults and have a larger thorax mass, which is
relevant to flight behaviour, in comparison to individuals
eclosing from the brown pupae. This suggests that there is
a trade-off between protection against predators during the
pupal stage and adult life stages, maintaining variation
both in pupal and adult traits [28].
Attack risk can also decrease if organisms deceive visual
predators by mimicking something uninteresting for the
potential predator such as sticks or bird droppings [35].
Masquerade is a relatively common defence mechanism in
the larval stage in many insect groups, but has sometimes
evolved in the pupal stage as well [36] (figure 1). For example,
Neochlamisus leaf beetles’larvae build a ‘faecal case’under
which they hide during the larval and pupal stage. In addition
to faeces, they also add trichomes from their host plant on
the surface of the case and closer to pupation they build a tri-
chome-filled chamber under the outer layer of faeces. Survival
against invertebrate predators is significantly improved owing
to these structures. These faecal cases covered with plant-
derived thricomes could help to masquerade the pupae on
the plants. Cases with trichomes can also offer physical protec-
tion against predators as, for example, solder bugs were not
able to penetrate the case with their mouth parts [36]. Faeces
and plant trichomes can also contain deterrent compounds
and therefore have an additional function as a secondary
chemical defence. Bagworm moths (Psychidae) build cases
that protect them during the larval and pupal stages [37].
These cases often contain material from their environment
(sand, twigs, rocks) that potentially makes them uninteresting
objects or cryptic for avian and insect predators.
In addition to morphological and physical traits, insects
have different types of behavioural adaptations that decrease
their detectability to predators and parasites during the pupal
stage. For example, many species burrow into the ground to
pupate, which offers a refuge from the predators preying
above the ground such as birds [17]. Before individuals
enter the pre-pupal stage and start to spin their cocoon,
they often disperse from their larval host plant or food
source [9,24,38,39]. Staying close to the host plant can
increase the risk of being detected by natural enemies that
often use the host plant as a cue to locate their potential
prey [13,40]. These behavioural adaptations before entering
the pupal stage can also vary within species depending on
the life-history strategy of an individual. For example, most
(a)
(c)(d)
(b)
Figure 1. Insects have evolved an extensive diversity of protective coloration strategies in the pupal stage, although their function and mechanisms have largely
remained unstudied. (a) Pupae of the Peacock butterfly (Aglais io) are well camouflaged. (b) Pupae can also masquerade themselves to resemble something unin-
teresting to potential predators such as leaf-mimicking chrysalises of the Common Maplet butterfly (Chersoneria risa). (c) In some species, such as Harlequin ladybird
(Harmonia axyridis), pupae may advertise their defensive toxins with conspicuous warning colours. (d) Pupae can also have shiny coloration such as chrysalides of
the Common Crow butterflies (Euploea core), which could both function as a warning signal or conceal the pupa from the predator’s eye. (Photos: a–dAdobe Stock.)
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3

Lepidopteran larvae have a wandering phase prior to pupa-
tion when larvae move away from their host plant while
seeking a suitable substrate for pupation [38]. In the European
swallowtail butterfly, Papilio machaon, this wandering phase is
longer in the overwintering generation, which has a longer
pupal stage, in comparison to larvae that are under direct
development (non-overwintering larvae), which have a
pupal stage of only one to two weeks. Owing to increased
mobility and distances moved, the wandering phase is risky
for a larva [9,38] and this may suggest that individuals
trade-off host-related predation risk by wandering less when
the pupal stage is shorter [38].
4. Avoiding detection by non-visual predators
Individuals do not always need to mimic visual components
in their environment. Instead, if the most important predators
are non-visual, it can be more effective for a prey to use
chemical ‘camouflage’[41]. Most of the literature on the
use of chemical compounds in confounding the detection of
prey by predators comes from species that live in a close
association with ants [41]. For example, myrmecophilous
Lycaeides argyrognomon butterflies larvae have organs that
they use to produce nectar for attending ants. In return, the
ants protect them against different types of natural enemies.
Individuals lose this organ during the pupal stage, but
instead start to produce cuticular compounds that supress
attacks from the ants and make the pupae less prone to ant
predation [42]. In this way, L. argyrognomon butterflies can
pupate in ant nests where they are protected from natural
enemies. Since non-visual predators are an import predator
group during the pupal stage, these kind of tactics relying
on chemical communication for protection against predators
could be more common than previously thought.
5. High detectability cost as a constraint for
the evolution of warning coloration in the
pupal stage?
The opposite strategy for hiding from the predators’senses is
aposematism. Aposematic individuals can advertise their
unprofitability as prey (e.g. toxicity) to predators with con-
spicuous signals (i.e. aposematism), which can include
colours, sounds or odours. Predators learn to associate the
signal with the unprofitability of the prey and avoid attacking
individuals sharing a similar appearance in future encounters
[6,43]. Intriguingly, the aposematic defence strategy seems to
be rare during the pupal stage [8,23] ( figure 1). For example,
in swallowtail butterflies (family Papilionidae), all species
have a cryptically coloured pupal stage even though their
larval and adult defence strategies vary from cryptic and
masquerade to aposematism [23].
The evolution of aposematic defence strategies may be
constrained in the pupal stage owing to some physiological
or developmental characteristics. However, developmental
constraints at least are unlikely; studies on the pupal color-
ation of Papilio species have shown that brown or green
pupal coloration is based on a blend of brightly coloured pig-
ments (red, yellow, black, blue) [44], which excludes the
possibility that lack of bright pigments would constrain the
development of colourful pupae. However, background
matching in the pupal stage combined with chemical
defences can be favoured under conditions where the detect-
ability risk for conspicuously coloured aposematic prey is
high. In their mathematical model, Endler & Mappes [45]
showed that low conspicuousness can evolve for a chemically
defended prey if there is variation in the cognitive and per-
ceptual capacity among predators or in their sensitivity to
the secondary defences of prey. This increases the detectabil-
ity costs for a prey, favouring lower conspicuousness. This
could also be true for insect pupae that are vulnerable for
long periods of time to a diverse predator community that
contains both visual and non-visual predators. In addition,
a conspicuous appearance can also invite attacks from
specialist parasitoids that have evolved to tolerate, or even
benefit, from the defensive toxins of the pupal stage [40].
Cryptic coloration or masquerade can also guarantee
highest survival during an immobile life stage [46,47]: apose-
matism can be favoured if it facilitates the mobility of the
organisms among different visual environments and back-
grounds [48]. This is because individuals with camouflage
are always dependent on their visual background to conceal
them from the predator’s eyes, but aposematism is expected
to function irrespective of the background. This enables apose-
matic individuals to acquire resources more effectively. For
example, in Acronicta alni, the larval mobility increases towards
the later instars when larvae grow larger and need to move
more to feed [49]. This dual benefit in terms of predator avoid-
ance and resource collection can be one factor that explains
why many Lepidopteran larvae switch strategy from crypsis
or masquerade to aposematism when they grow larger or
why immobile pupae rely on cryptic coloration [8,47,50].
6. Chemical defence
Chemical defences are one of the most widespread defensive
mechanisms against predators [51]. Among insects, chemical
defences can be defensive secretions that are actively released
in the presence of a predator, decreasing the likelihood that
the predator needs to taste (and potentially kill) the prey
before it finds it unpalatable. Chemical defences can also be
stored in body parts such as wings in many toxic butterflies
[6,51]. In both of these cases, the efficacy of chemical defence
is based on shared predator education costs where predators
learn to avoid the chemically defended prey sharing a similar
appearance [52]. If prey species offer conspicuous cues
(aposematism) associated with a chemical defence, this
avoidance learning rate becomes even more effective [52].
At present, the focus of research has been on chemical
defence in larval and adult stages and much less is known
about the role of chemical defences in protection against
predators during the pupal stage. In species whose larvae
and adults contain defensive compounds, pupal stages are
also often chemically defended [53–55]. For example, both
larvae and pupae in Delphastus catalinae ladybirds have
minute secretory hairs that produce defensive secretions
deterrent to their predators [56]. Eclosion fluid in pupae
can also be bitter and play role in chemical defence (J.M.
2017, personal observation) even though its function in
chemical defence has not been tested.
Since the selective environment and lifestyle are likely to
change during the larval, pupal and adult stages, we can
expect qualitative and quantitative changes in defensive
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chemistry during development [57,58]. In leaf beetles (Oreina
gloriosa), individuals contain defensive compounds (car-
denolides) during all the life stages [57]. However, the
composition of the chemical cocktail changes from larval to
pupal and pupal to adult stage. The authors suggest that
these differences can partly be explained by changes in the life-
style and respective predator and parasite community
structures. Studying this hypothesis experimentally would
offer a promising route to test how changes in predator com-
munity structure shape and maintain diversity in chemical
protection [51].
The quality and quantity of defensive compounds in the
pupal stage can also depend on the developmental environ-
ment during the larval stage. For example, Junonia coenia
(Nymphalidae) larvae sequester iridoid glycosides from
their host plant (Plantago lanceolata) [54]. These compounds
have been shown to be deterrent against different types of
invertebrate predators such as ants [59]. Both host plant gen-
otype and the predator type present have been shown to
affect the quality and quantity of defensive chemical content
in the pupal stage [54]. Catalpol content was higher during
the pupal stage in individuals that grew in the presence of
stinkbugs in comparison to individuals that had suffered pre-
dation by wasps. Thus, J. coenia individuals can alter their
chemical protection in the pupal stage to match the predation
risk from the most important predator species (target-specific
defence; see also [60]).
Sometimes possessing defensive compounds in the
pupal stage can increase predation risk from conspecifics.
Pyrrolizidine alkaloids are plant-derived compounds that
are widely used in chemical defence and communication,
especially among Arctiid species [53,61]. Utetheisa ornatrix
larvae derive pyrrolizidine alkaloids (PAs) from their host
plants Crotalaria spp. and retain alkaloids through life
stages and metamorphosis. These compounds also occur
during the pupal stage, offering potential protection against
natural enemies. However, these same compounds can
make the pupal stage vulnerable to cannibalism. Before
pupation, larvae tend to wander further from the host plant
as U. ornatrix larvae cannibalize pupae with high PA content
to acquire PAs for themselves [62]. In addition to protection
against predators such as spiders or birds, PAs play an
important role in sexual selection in U. ornatrix, offering
one explanation for the cannibalistic behaviour in the larval
stage [61,62].
7. Physical and behavioural defences
Pupae can also offer physical defence against potential preda-
tors (figure 2). Hairs and spines have been shown to be
especially effective defence mechanisms against invertebrate
predators [63–65] during the larval stage and may play a
potentially important role in pupal defence. For example,
pine processionary caterpillars are covered by urticating
hairs that can cause strong allergic reactions in humans.
Thus, it is possible that they are unprofitable to other preda-
tors too. In addition, their cocoons contain exuvia of the last
(a)
(c)
(d)
(b)
Figure 2. Pupae can defend against predators with different kinds of morphological and chemical defences or even rely on protection from other individuals. (a)
Aposematic Heliconius melpomene butterflies’pupae are covered with spines. (b) Many moth species produce a silk cocoon or loose silk nets to protect the pupa
(white satin moth, Leucoma salicis). (c) Larval, pupal and adult stage in the common buckeye (J. coenia) contain iridoid glycosides that give them chemical protec-
tion predators. (d)Maculinea rebeli butterfly pupae use vocal communication to recruit ants to guard them. (Photos: a–cAdobe Stock, D. Marco Gherlenda.)
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larval moult, bearing urticating hairs twined into the cocoon
walls [19]. Gonometa-moth species such as African wild silk
moth (Gonometa postica) larvae have urticating hairs that they
incorporate into the pupal cocoon walls [66]. Cocoon walls
can also be enforced with other structures that make them
less likely to break when attacked: Veldtman et al. [66]
suggest that in G. postica, cocoons appear conspicuous against
their background. They also suffer much lower bird predation
risk (2%) in comparison to Gonometa rufobrunnea (50%),
whose cocoons are otherwise similar but appear more cryptic
for the human eye [66]. Rather than aposematic defence,
authors suggest that lower predation risk in G. postica is
likely to be owing to the difference in the structure of the
cocoon. Gonometa postica cocoons are covered by small cal-
cium crystals that make the cocoon more difficult to break,
in addition to differences in cocoon colour. The function
and origin of these different types of physical protective
mechanisms in defence against predators in the pupal stage
have not, however, been tested experimentally. For example,
larvae may need to get rid of the hairs before successful
pupation. Incorporating hairs into the cocoon walls may
therefore be a physiological necessity offering additional
benefits in defence against predators or parasitoids.
Many holometabolous insects spin their cocoon before
metamorphosis. The silk cocoon can protect them against pre-
dators and parasitoids, but also protect against desiccation or
microparasites [67,68]. The silk that is the primary constituent
of Lepidopteran cocoons is also thought to play a defensive
role for pupae. Although the majority of silk is made up of
proteins that combine to maintain the silk’s structural proper-
ties, there are a number of other proteins present in different
species’silk [69]. These alternative roles are thought to include
contributing to unpalatability of the cocoons and defending
their contents against microbes [70,71]. The majority of studies
on these non-structural proteins have focused on the silk of the
silkmoth, Bombyx mori; however, the silk components of moths
from different families have been shown to be very similar to
one another [69]. The silk of the silkmoth cocoons contains
protease inhibitors that could act against a range of bacterial-
and fungal-derived proteases. Yet, Kaur et al. [72] have argued
that many of the purported antibiotic properties of silk were
owing to experiments using contaminated silk. These con-
taminants originated from the processes of breaking apart
components of the silk prior to the experiments they were
used in. So, the extent to which the silk of cocoons defends
the pupae against bacterial or fungal infections or makes
them unpalatable for predators is far from clear cut and
requires further experimental studies.
Pupae can also display defensive movements that can
potentially have a deimatic function against predators [73]
or, they can make attacks by predators and parasitoids phys-
ically difficult [74]. For example, pupae of Tenebrio molitor
and Zophobas atratus rotate their abdominal segments in
response to tactile stimulation and this behaviour has been
shown to decrease the risk of cannibalism by the larvae of
the same species [75]. Pupae of the small tortoiseshell (Aglais
urticae) start to wriggle very intensively when the parasitoid
tries to land on it, often preventing the parasitoid from depos-
iting its egg into the pupa [74]. In the same paper, Cole [74]
reported higher oviposition success of parasitoids with Ledi-
dopteran species P. aegeria and Pieris brassicae, whose pupae
are not able to wriggle as intensively as A. urticae pupae.
8. Intraspecific interactions and survival
throughout the pupal stage
Predation risk is often quoted as a major selective force
favouring sociality [76], including cooperative protection of
offspring during the pupal stage. In many socially behaving
insects, such as eusocial hymenoptera, different types of
cooperative breeding strategies have evolved where adults
take care of the immature stages, including pupae, and
defend them against different types of natural enemies [77].
However, in eusocial species such as ants it is also the case
that pupae are not safe from predators: for example, Asiatic
black bears are more likely to forage on ant nests that have
plentiful pupae [78] (but see [79]).
In some species, individuals can form aggregations in the
pupal stage that decrease mortality risk. These aggregations
can be passive: for example, owing to features in the land-
scape (if suitable habitats for pupation are scattered) that
lead to the clumping of individuals. Alternatively, aggrega-
tions can be active when individuals actively maintain
contact with each other. Aggregating in the pupal stage (or
in any life stage) can function in three ways: first, it minimizes
encounters with random search predators, but may increase
vulnerability to, for example, visual predators that are good
at detecting groups of prey [16,80]. Second, it dilutes the
effect of the predator after it has been encountered (safety
in numbers owing to predator satiation and handling times)
[80–82]. For example, in a stream-dwelling trichopteran,
Rhyacophila vao, pupal aggregations were disadvantageous
in terms of encounter risk with the predators. However,
grouping was beneficial in terms of dilution of predation
risk. When evaluated together (attack-abatement effect
[80]), grouping in the pupal stage offered higher net benefits
against predation risk [83].
The third way in which aggregation can lower predation
risk is by enhancing the avoidance learning of predators if the
prey is unprofitable [81]. Predators have been shown to learn
to avoid chemically defended cryptic (immobile) artificial
and real prey items more effectively when these prey items
are in groups compared to solitary prey [81,84]. This mechan-
ism has not been experimentally tested at the pupal stage, but
it could play an important role in species whose pupae pos-
sess chemical defences and are often aggregated spatially and
temporally. For example, in the gregarious chemically
defended pine sawfly species, pupae are often aggregated
in the vicinity of the host trees where larvae feed gregariously
in actively maintained groups. In Neodiprion pine sawflies
[39], individuals switch from gregarious behaviour to solitary
in non-feeding final instar where larvae disperse to spin their
cocoons on the ground. Yet, cocoons are often clumped in the
space under host trees and can form attractive feeding sites
for mammalian predators such as shrews and voles [20] or
birds [16]. Pine sawfly pupa include defensive chemicals as
the defensive glands of larvae are disposed into the pupa
during metamorphosis [85]. However, during the pre-adult
stage, individuals actively turn inside the cocoon if the
cocoon wall is pierced and move the disposed defensive
gland sacs towards the potential attacker [85]. Whether this
chemical defence mechanism of pupa has any effect on the
predators’behaviour has not been directly tested [16,20]
and its role might be more important in defence against
macroparasites [85].
royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20190069
6

9. Protection from other species and survival
throughout the pupal stage
Individuals can gain protection against predators from other
species during the pupal stage [9,14,86]. These interactions
can vary from mutualistic (both partners benefit from it) to
parasitic (costly for the other partner without any gained
benefits). Sometimes, the relationship can also be commensa-
listic: for example, Platyperpia virginalis (Arctiidae) caterpillars
shift host plant and habitat during pupation. Individuals
prefer to pupate within a spiny plant species, which are in
a different habitat and not the host plant they feed upon
during the larval stage. Furthermore, individual survival
during the pupal stage was higher in these physically
defended plants. This was confirmed experimentally: in
plants where the physical defence structures were removed,
survival of pupae decreased [9]. Similarly, in papilionidae
Battus polydamas archidamas, pupae have a higher probability
of survival when they are on cacti in comparison to shrubs,
rocks or the ground [26].
Some species can even manipulate other species to gain
protection from them [87]. Lycaenidae and Riodinidae butter-
fly families are famous for their relationships with ants, which
vary from mutualistic to parasitic (reviewed in [86,88]). In gen-
eral, larvae of several species of this group get protection from
ants against parasites and predators. Ants in return receive a
nutritious secretion from the larvae. How dependent these
species are on ants varies among species. Some of the species
are obligatory myrmecophiles such as Jalmenus evagoras,which
are practically unable to survive without ants. Some other
species are facultative myrmechophiles.
Interestingly, not only larvae but also pupae of this group
of butterflies have evolved mechanisms to manipulate the
ants’behaviour. All Lycaenid pupae produce sound via
stridulation (sound-producing organs) regardless whether
they are ant-associated or not [86]. These pupal sounds can
be considered to have a deimatic function [73], as these
sounds are produced when pupae are disturbed [89]. How-
ever, in some species, pupal sounds are involved in ant
recruitment. For example, in J. evagoras pupae, sound pro-
duction can attract ants and maintain ant guard. These
examples show that the pupal stage is not ‘a passive and
inactive’life stage but pupae can actively communicate
with their environment. Perhaps the most fascinating
example of this comes from the parasitic Maculinea rebeli
butterflies (Lycaenidae). Both their larvae and pupae produce
sound that accurately mimics the sound of its host, the queen
ants of Myrmica schenki [90]. Sound produced by pupae is
actually more accurate than the sound produced by larvae.
This pupal sound elicits a similar response to sounds made
by queens, giving M. rebeli the ability to manipulate its host
and gain protection from ants.
10. Conclusion and future questions
Our literature survey shows that an extensive diversity of
defensive mechanisms have evolved to increase protection
against predators through the pupal stage in insects. These
strategies include different types of protective coloration
strategies, defensive toxins, protection gained from conspeci-
fics and manipulation of host species’sensitivity to specific
chemical or auditory cues to gain their protection. Thus, the
pupal stage is not an ‘inactive’stage but can protect against
predators with an extensive diversity of morphological,
chemical and behavioural mechanisms. However, consider-
ing the literature available from the larval or adult stage,
understanding of the defensive strategies at the pupal stage
is still in its early stages. Therefore, predation risk, and how
to protect from it in the pupal stage will evidently offer an
interesting and important topic for future research. Determin-
ing these selection pressures across life stages is critical to
forming a realistic view of the evolution of life-history strat-
egies in species with complex lifecycles and to predict factors
that shape their population dynamics. This information can
even provide insights to mechanismsbehind dramatic declines
in insect populations [91,92] and be beneficial for effective con-
servation planning and management. With these aspects in
mind, we have outlined several potentially interesting and
important routes for future research to follow.
(a) How does variation in predator community
composition shape the evolution of prey defences
throughout an individual’s life cycle?
Variation in the predator community structure can be an
important selective agent that shapes the evolution of prey
defences. At present, these effects have been mainly considered
within the individual life stage (e.g. larval or adult) [60,93].
However, based on our literature review, predator community
structure is also likely to change across different life stages
[15,17]. How it shapes the function and diversity of defensive
mechanisms between life stages, including the pupal stage, is
not known [8]. For example, variation in the composition of
predator and parasitoid community structure could explain
why we sometimes observe variation in the quantity and qual-
ity of defensive compounds in larval, pupal and adult stages.
To study those unstudied aspects, we can benefit from
representative model systems for prey species [54,55,61],
where we already have accumulating information on the mul-
tiple factors that shape individual defensive strategies in
different life stages. We also need to focus on relevant predator
species as a recent study suggest that chemical defences can be
target-specific [60], addressing the importance of choosing
the correct focal predator species when the efficacy of
chemical defences is studied. With the accumulating empirical
research information from different species, we can then
perform systematic analyses to evaluate how much defences
across different life stages are linked or whether they evolve
independently.
(b) Function of chemical defences during the pupal
stage: can non-visual predators learn to avoid
cryptically coloured pupae based on chemical,
tactical or auditory signals?
Aposematism has repeatedly evolved in larval and adult
stages, but very rarely in the pupal stage [8,23]. However, a
general assumption is that defensive compounds also occur
in the pupal stage when larval and adult stages are chemi-
cally defended. This is also supported by empirical data in
species where defensive chemical content in the pupal stage
has been analysed [54,61]. As conspicuous signals should
enhance the predator’s avoidance learning efficiency, the
royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20190069
7

occurrence of these defended prey species with low signal
intensity is puzzling [45]; this is especially true if chemical
defences are costly to produce and maintain and the individ-
ual is already well protected by camouflage [94]. If a predator
always needs to bite the pupa to find it unpalatable (i.e. pre-
dators do not learn to avoid chemically defended pupae),
defensive chemicals in the pupal stage do not benefit the indi-
vidual, which will die anyway after the cocoon shell is
pierced. For example, Wiklund & Sillén-Tullberg [23]
suggested that pupae might even be innately more vulner-
able to handling by predators because the hard cuticle of
pupae is more likely to crack broken in comparison with
more flexible larval and adult cuticles.
What cues could predators then use to learn to avoid
chemically defended pupae? First, conspicuousness may
not be as important as long as defended prey are sufficiently
distinctive from the palatable prey [95]. Second, most of the
research on pupal coloration has focused on conspicuousness
to human eyes. However, we know very little about the con-
spicuousness of pupa to predators with different types of
visual systems, such as birds that also perceive UV wave-
lengths [96]. Some species have metallic golden or silver
shiny pupae (figure 1) [97]. This kind of shiny coloration
may function either as a warning signal in a similar way to
iridescent colours, or as a camouflage reflecting the visual
environment [97–100]. Third, our literature review shows
that pupae have evolved many other ways to communicate
such as movements, sounds and chemical cues. These kind
of cues could function as effective warning signals of defen-
sive toxins [101], especially for non-visual predators.
Currently, we lack experimental studies that test if these
non-visual predators are able to learn to avoid chemically
defended pupae based on the cues available. Finally, it is
possible that defensive compounds occur in the pupal stage
simply because they need to be transported from the larval
stage to adult stage without any adaptive function during
the pupal stage. Future research could try further exper-
iments with dummy prey, similar to those that are widely
used to test predation risk during the larval [22] and adult
stages [102]. This would offer a way to test how different phe-
notypic traits such as size, coloration or pupation site are
under selection by predation.
(c) Life-history trade-offs across life stages and how
they link with predation risk during the pupal stage
Another less studied aspect is potential life-history trade-offs
between pupal and adult stages [28,34]. For example, if allo-
cation to effective camouflage pigmentation during the pupal
stage trades-off with the size or flight capacity during the
adult stage, selective predation during the pupal stage will
shape phenotypic variation in adults indirectly. Similarly, if
predation favours either large or small size during the
pupal stage [21], it is also likely to reflect traits such as size
in adults. Defensive traits, such as chemical defence, can
also be costly to produce and maintain during the larval
stage [12,103,104] and constrain performance during the
pupal and adult stages. For example, Lindstedt et al. [103]
found that when the costs of chemical defence were high,
Diprion pini pine sawfly individuals were less likely to
reach the pupal stage and grew more slowly. In P. brassicae
larvae, higher contribution to chemical defence decreased
their likelihood of reaching the pupal stage and they were
smaller in the pupal stage [12]. Finally, allocating resources
to building up the protective cocoon during the pupal stage
can also be costly and constrain resources available during
the adult stage [105].
How pupal traits are associated with adult or larval traits
could be especially interesting questions to test with species
that are polymorphic at some life stage. For example, if
adult and pupal traits are correlated and predation risk
varies accordingly with life‐stage, selection during the
pupal stage could be an important factor explaining variation
in the frequencies of adult morphs. Studies that link phenoty-
pic variation in pupal traits with the variation in larval and
adult traits are, however, very scarce, and would therefore
be important in understanding the evolution of life-history
strategies in insects.
(d) Do defensive traits have multiple functions in
defence against multiple enemies?
Even though our main focus in this literature survey has been
on predation, we want to address the fact that many of the
defensive mechanisms listed above can be equally critical in
protection against parasitoids and pathogens [13,85,106]. Cur-
rently, there is accumulating evidence that studying these two
selection pressures simultaneously in ‘multienemy-frame-
work’can help to understand how defensive traits have
evolved initially [106–109]. For example, some defensive com-
pounds can have a dual function and serve multiple purposes
in protection against predators and parasites [106,107] (but see
[40]). Thus, it is possible that the same defensive compounds
that play an important role in the protection of the larval
stage against predators may play a more important role in pro-
tection against fungi and pathogens during the pupation. If
defensive toxins have evolved primarily against parasitoids
or diseases, it will offer one more evolutionary explanation
for the weak visual signals of chemically defended pupae.
Similarly, defensive movements during the pupal stage or
camouflage can increase survival both against predators and
parasitoids [13]. Again, there is a clear need for experimental
studies where the importance of visual and chemical cues in
protection against parasitoids and predators can be tested.
(e) How often do individuals switch habitats or rely on
protection from other species during the pupal
stage?
Our review shows that individuals can switch habitats [9],
host plants [9,26] or even evolve parasitic or cooperative
interactions with other species [14,68,90] to gain enemy-free
space during the pupal stage. How often pupae actually
rely on protection from other species in their defence [14]
or how dependent insect species are from multiple habitats
during their lifecycle would offer an interesting topic for
future research to consider. This would require natural
history data and behavioural observations combined with
experimental manipulations and phylogenetic studies [110].
This information could also have applied importance in plan-
ning of conservation areas targeted to protect certain species:
often efforts in conservation are allocated on protection of
areas abundant in larval host plants. However, if insect
species are dependent on other species to survive through
the pupal stage [14], or even need to change habitats to
royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20190069
8

pupate successfully, effective conservation needs to take these
requirements into account.
Data accessibility. This article has no additional data.
Authors’contributions. C.L. led the writing of the manuscript. All authors
contributed critically to the drafts and gave final approval for
publication.
Competing interests. We declare no competing interests.
Funding. This study was funded by the Academy of Finland via Centre
of Excellence in Biological Interactions.
Acknowledgements. We thank Paul Johnston, Stuart Reynolds and Jens
Rolff for the invitation to the ‘Evolution of complete metamorpho-
sis’theme issue. We thank an anonymous reviewer, Dirk
Mikolajewski, Jens Rolff, Tapio Mappes and Emily Burdfield-Steel
for commenting the earlier versions of the manuscript. Francesca
Barbero together with Marco Gherlenda kindly provided the
photo of M. rebeli pupae.
References
1. Merian MS. 1705 Metamorphosis insectorum
Surinamensium. Amsterdam, The Netherlands: Lannoo
Publishers and National Library of the Netherlands.
2. Reynolds S. 2019 Cooking up the perfect insect:
Aristotle’s transformational idea about the complete
metamorphosis of insects. Phil. Trans. R. Soc. B 374,
20190074. (doi:10.1098/rstb.2019.0074)
3. Guerra-Grenier E. 2019 Evolutionary ecology of
insect egg coloration: a review. Evol. Ecol. 33,1–19.
(doi:10.1007/s10682-018-09967-8)
4. Belles X. 2019 The innovation of the final moult and
the origin of insect metamorphosis. Phil. Trans. R. Soc.
B374, 20180415. (doi:10.1098/rstb.2018.0415)
5. Suzuki Y, Truman JW, Riddiford LM. 2008 The role
of Broad in the development of Tribolium
castaneum: implications for the evolution of the
holometabolous insect pupa. Development 135,
569–577. (doi:10.1242/dev.015263)
6. Ruxton GD, Sherratt TN, Speed MP. 2004 Avoiding
attack: the evolutionary ecology of crypsis, warning
signals and mimicry. Oxford, UK: Oxford University
Press.
7. Critchlow JT, Norris A, Tate AT. 2019 The legacy of
larval infection on immunological dynamics over
metamorphosis. Phil. Trans. R. Soc. B 374,
20190066. (doi:10.1098/rstb.2019.0066)
8. Gaitonde N, Joshi J, Kunte K. 2018 Evolution of
ontogenic change in color defenses of swallowtail
butterflies. Ecol. Evol. 8, 9751–9763. (doi:10.1002/
ece3.4426)
9. Grof-Tisza P, Holyoak M, Antell E, Karban R. 2015
Predation and associational refuge drive ontogenetic
niche shifts in an arctiid caterpillar. Ecology 96,
80–89. (doi:10.1890/14-1092.1)
10. Lindstedt C, Talsma JHR, Ihalainen E, Lindström L,
Mappes J. 2010 Diet quality affects warning
coloration indirectly: excretion costs in a generalist
herbivore. Evolution 64,68–78. (doi:10.1111/j.
1558-5646.2009.00796.x)
11. Burdfield-Steel E, Brain M, Rojas B, Mappes J. 2019 The
price of safety: food deprivation in early life influences
the efficacy of chemical defence in an aposematic
moth. Oikos 128,245–253. (doi:10.1111/oik.05420)
12. Higginson AD, Delf J, Ruxton GD, Speed MP. 2011
Growth and reproductive costs of larval defence in
the aposematic lepidopteran Pieris brassicae.
J. Anim. Ecol. 80, 384–392. (doi:10.1111/j.1365-
2656.2010.01786.x)
13. Gross P. 1993 Insect behavioral and morphological
defenses against parasitoids. Annu. Rev. Entomol.
38, 251–273. (doi:10.1146/annurev.en.38.010193.
001343)
14. Hammer TJ, Moran NA. 2019 Links between
metamorphosis and symbiosis in holometabolous
insects. Phil. Trans. R. Soc. B 374, 20190068.
(doi:10.1098/rstb.2019.0068)
15. Elkinton JS, Liebhold AM. 1990 Population dynamics
of gypsy moth in North America. Annu. Rev.
Entomol. 35, 571–596. (doi:10.1146/annurev.en.35.
010190.003035)
16. Hanski I, Parviainen P. 1985 Cocoon predation by small
mammals, and pinesawfly populationdynamics. Oikos
45,125–136. (doi:10.2307/3565230)
17. East R. 1974 Predation on the soil-dwelling stages
of the winter moth at Wytham Woods, Berkshire.
J. Anim. Ecol. 43, 611–626. (doi:10.2307/3526)
18. Heisswolf A, Klemola N, Ammunét T, Klemola T.
2009 Responses of generalist invertebrate predators
to pupal densities of autumnal and winter moths
under field conditions. Ecol. Entomol. 34, 709–717.
(doi:10.1111/j.1365-2311.2009.01121.x)
19. Battisti A, Bernardi M, Ghiraldo C. 2000 Predation
by the hoopoe (Upupa epops) on pupae of
Thaumetopoea pityocampa and the likely influence
on other natural enemies. BioControl 45, 311–323.
(doi:10.1023/A:1009992321465)
20. Kollberg I, Bylund H, Huitu O, Björkman C. 2014
Regulation of forest defoliating insects through
small mammal predation: reconsidering the
mechanisms. Oecologia 176, 975–983. (doi:10.
1007/s00442-014-3080-x)
21. Murphy SM, Lill JT. 2010 Winter predation of
diapausing cocoons of slug caterpillars (Lepidoptera:
Limacodidae). Environ. Entomol. 39, 1893–1902.
(doi:10.1603/EN10094)
22. Mappes J, Kokko H, Ojala K, Lindström L. 2014
Seasonal changes in predator community switch the
direction of selection for prey defences. Nat.
Commun. 5, 5016. (doi:10.1038/ncomms6016)
23. Wiklund C, Sillén-Tullberg B. 1985 Why distasteful
butterflies have aposematic larvae and adults, but
cryptic pupae: evidence from predation experiments
on the monarch and the European swallowtail.
Evolution 39, 1155–1158. (doi:10.1111/j.1558-5646.
1985.tb00456.x)
24. Wiklund C. 1975 Pupal colour polymorphism in
Papilio machaon L. and the survival in the field of
cryptic versus non-cryptic pupae. Trans. R. Entomol.
Soc. Lond. 127,73–84. (doi:10.1111/j.1365-2311.
1975.tb00555.x)
25. Hazel W, Brandt R, Grantham T. 1987 Genetic
variability and phenotypic plasticity in pupal colour
and its adaptive significance in the swallowtail
butterfly Papilio polyxenes.Heredity 59, 449–455.
(doi:10.1038/hdy.1987.155)
26. Piñones-Tapia D, Rios RS, Gianoli E. 2017 Pupal
colour dimorphism in a desert swallowtail
(Lepidoptera: Papilionidae) is driven by changes in
food availability, not photoperiod. Ecol. Entomol.
42, 636–644. (doi:10.1111/een.12430)
27. Starnecker G, Hazel W. 1999 Convergent evolution
of neuroendocrine control of phenotypic plasticity in
pupal colour in butterflies. Proc. R. Soc. Lond. B
266, 2409–2412. (doi:10.1098/rspb.1999.0939)
28. Van Dyck H, Matthysen E, Wiklund C. 1998 Phenotypic
variation in adult morphologyand pupal colour within
and among families of the speckled wood butterfly
Pararge aegeria.Ecol. Entomol. 23,465–472. (doi:10.
1046/j.1365-2311.1998.00151.x)
29. Hiraga S. 2006 Interactions of environmental factors
influencing pupal coloration in swallowtail butterfly
Papilio xuthus.J. Insect Physiol. 52, 826–838.
(doi:10.1016/j.jinsphys.2006.05.002)
30. Mayekar HV, Kodandaramaiah U. 2017 Pupal colour
plasticity in a tropical butterfly, Mycalesis mineus
(Nymphalidae: Satyrinae). PLoS ONE 12, e0171482.
(doi:10.1371/journal.pone.0171482)
31. West DA, Snellings WM, Herbek TA. 1972 Pupal
color dimorphism and its environmental control in
Papilio polyxenes asterius Stoll (Lepidoptera:
Papilionidae). J. N Y Entomol. Soc. 80, 205–211.
32. Smith AG. 1978 Environmental factors influencing
pupal colour determination in Lepidoptera
I. Experiments with Papilio polytes,Papilio demoleus
and Papilio polyxenes.Proc. R. Soc. Lond. B 200,
295–329. (doi:10.1098/rspb.1978.0021)
33. Gerard SA, Cyril C. 1980 Environmental factors
influencing pupal colour determination in
Lepidoptera. II. Experiments with Pieris rapae,Pieris
napi and Pieris brassicae.Proc. R. Soc. Lond. B 207,
163–186. (doi:10.1098/rspb.1980.0019)
34. Galarza JA, Dhaygude K, Ghaedi B, Suisto K,
Valkonen J, Mappes J. 2019 Evaluating responses to
temperature during pre-metamorphosis and carry-
over effects at post-metamorphosis in the wood
tiger moth (Arctia plantaginis). Phil. Trans. R. Soc. B
374, 20190295. (doi:10.1098/rstb.2019.0295)
35. Skelhorn J, Rowland HM, Speed MP, Ruxton GD.
2010 Masquerade: camouflage without crypsis.
Science 327, 51. (doi:10.1126/science.1181931)
royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20190069
9

36. Brown CG, Funk DJ. 2010 Antipredatory properties
of an animal architecture: how complex faecal cases
thwart arthropod attack. Anim. Behav. 79,
127–136. (doi:10.1016/j.anbehav.2009.10.010)
37. Rhainds M, Davis DR, Price PW. 2009 Bionomics of
Bagworms (Lepidoptera: Psychidae). Annu. Rev.
Entomol. 54, 209–226. (doi:10.1146/annurev.ento.
54.110807.090448)
38. Wiklund C, Stefanescu C, Friberg M. 2017 Host plant
exodus and larval wandering behaviour in a
butterfly: diapause generation larvae wander for
longer periods than do non-diapause generation
larvae. Ecol. Entomol. 42, 531–534. (doi:10.1111/
een.12409)
39. Terbot JW, Gaynor RL, Linnen CR. 2017
Gregariousness does not vary with geography,
developmental stage, or group relatedness in
feeding redheaded pine sawfly larvae. Ecol. Evol. 7,
3689–3702. (doi:10.1002/ece3.2952)
40. 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)
41. Wang B, Lu M, Cook JM, Yang D-R, Dunn DW, Wang
R-W. 2018 Chemical camouflage: a key process in
shaping an ant–treehopper and fig–fig wasp
mutualistic network. Sci. Rep. 8, 1833. (doi:10.1038/
s41598-018-20310-7)
42. Mizuno T, Hagiwara Y, Akino T. 2018 Chemical tactic
of facultative myrmecophilous lycaenid pupa to
suppress ant aggression. Chemoecology 28,
173–182. (doi:10.1007/s00049-018-0270-8)
43. Mappes J, Marples N, Endler JA. 2005 The
complex business of survival by aposematism.
Trends Ecol. Evol. 20, 598–603. (doi:10.1016/j.tree.
2005.07.011)
44. Futahashi R, Fujiwara H. 2005 Melanin-synthesis
enzymes coregulate stage-specific larval cuticular
markings in the swallowtail butterfly, Papilio
xuthus.Dev. Genes Evol. 215, 519–529. (doi:10.
1007/s00427-005-0014-y)
45. Endler JA, Mappes J. 2004 Predator mixes and the
conspicuousness of aposematic signals. Am. Nat.
163, 532–547. (doi:10.1086/382662)
46. Skelhorn J, Rowland HM, Ruxton GD. 2010 The
evolution and ecology of masquerade: review
article. Biol. J. Linn. Soc. 99,1–8. (doi:10.1111/
j.1095-8312.2009.01347.x)
47. Merilaita S, Tullberg BS. 2005 Constrained
camouflage facilitates the evolution of conspicuous
warning coloration. Evolution 59,38–45. (doi:10.
1111/j.0014-3820.2005.tb00892.x)
48. Speed MP, Brockhurst MA, Ruxton GD. 2010
The dual benefits of aposematism: predator
avoidance and enhanced resource collection.
Evolution 64, 1622–1633. (doi:10.1111/j.1558-5646.
2009.00931.x)
49. Valkonen JK, Nokelainen O, Jokimäki M, Kuusinen E,
Paloranta M, Peura M, Mappes J. 2014 From
deception to frankness: benefits of ontogenetic shift
in the anti-predator strategy of alder moth Acronicta
alni larvae. Curr. Zool. 60, 114–122. (doi:10.1093/
czoolo/60.1.114)
50. Higginson AD, Ruxton GD. 2010 Optimal defensive
coloration strategies during the growth period of
prey. Evolution 64,53–67. (doi:10.1111/j.1558-
5646.2009.00813.x)
51. Speed MP, Ruxton GD, Mappes J, Sherratt TN. 2012
Why are defensive toxins so variable? An
evolutionary perspective. Biol. Rev. 87, 874–884.
(doi:10.1111/j.1469-185X.2012.00228.x)
52. Lindström L, Alatalo RV, Mappes J, Riipi M,
Vertainen L. 1999 Can aposematic signals evolve by
gradual change? Nature 397, 249–251. (doi:10.
1038/16692)
53. Hartmann T, Theuring C, Beuerle T, Bernays EA.
2004 Phenological fate of plant-acquired
pyrrolizidine alkaloids in the polyphagous arctiid
Estigmene acrea.Chemoecology 14, 207–216.
(doi:10.1007/s00049-004-0276-2)
54. Bowers MD, Stamp NE. 1997 Effect of hostplant
genotype and predators on iridoid glycoside content
of pupae of a specialist insect herbivore, Junonia
coenia (Nymphalidae). Biochem. Syst. Ecol. 25,
571–580. (doi:10.1016/S0305-1978(97)00058-6)
55. Bowers MD, Stamp NE. 1997 Fate of host-plant
iridoid glycosides in lepidopteran larvae of
Nymphalidae and Arcthdae. J. Chem. Ecol. 23,
2955–2965. (doi:10.1023/A:1022535631980)
56. Deyrup ST, Eckman LE, Lucadamo EE, McCarthy PH,
Knapp JC, Smedley SR. 2014 Antipredator activity
and endogenous biosynthesis of defensive secretion
in larval and pupal Delphastus catalinae (Horn)
(Coleoptera: Coccinellidae). Chemoecology 24,
145–157. (doi:10.1007/s00049-014-0156-3)
57. Eggenberger F, Rowell-Rahier M. 1993 Production of
cardenolides in different life stages of the
chrysomelid beetle Oreina gloriosa.J. Insect Physiol.
39, 751–759. (doi:10.1016/0022-1910(93)90050-2)
58. Despland E, Simpson SJ. 2005 Food choices of
solitarious and gregarious locusts reflect cryptic and
aposematic antipredator strategies. Anim. Behav.
69, 471–479. (doi:10.1016/j.anbehav.2004.04.018)
59. 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, 1147–1158. (doi:10.1007/
s00442-015-3425-0)
60. 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. (doi:10.1098/rspb.2017.1424)
61. Eisner T, Meinwald J. 1995 The chemistry of sexual
selection. Proc. Natl Acad. Sci. USA 92,50–55.
(doi:10.1073/pnas.92.1.50)
62. Bogner F, Eisner T. 1992 Chemical basis of pupal
cannibalism in a caterpillar (Utetheisa ornatrix).
Experientia 48,97–102. (doi:10.1007/BF01923618)
63. Dyer LA. 1995 Tasty generalists and nasty
specialists? Antipredator mechanisms in tropical
lepidopteran larvae. Ecology 76, 1483–1496.
(doi:10.2307/1938150)
64. Murphy SM, Leahy SM, Williams LS, Lill JT. 2010
Stinging spines protect slug caterpillars
(Limacodidae) from multiple generalist predators.
Behav. Ecol. 21, 153–160. (doi:10.1093/beheco/
arp166)
65. Sugiura S, Yamazaki K. 2014 Caterpillar hair as a
physical barrier against invertebrate predators.
Behav. Ecol. 25, 975–983. (doi:10.1093/beheco/
aru080)
66. Veldtman R, McGeoch MA, Scholtz CH. 2007 Can
life-history and defence traits predict the population
dynamics and natural enemy responses of insect
herbivores? Ecol. Entomol. 32, 662–673. (doi:10.
1111/j.1365-2311.2007.00920.x)
67. Craig CL. 1997 Evolution of arthropod silks. Annu.
Rev. Entomol. 42, 231–267. (doi:10.1146/annurev.
ento.42.1.231)
68. Armitage SAO, Fernández-Marín H, Wcislo WT,
Boomsma JJ. 2012 An evaluation of the possible
adaptive function of fungal brood covering by attine
ants. Evolution 66, 1966–1975. (doi:10.1111/j.1558-
5646.2011.01568.x)
69. Fedic R, Zurovec M, Sehnal F. 2002 The silk of
lepidoptera. J. Insect Biotechnol. Sericology 71,
1–15. (doi:10.11416/jibs2001.71.1)
70. Danks HV. 2004 The roles of insect cocoons in cold
conditions. Eur. J. Entomol. 101, 433–437. (doi:10.
14411/eje.2004.062)
71. Nirmala X, Kodrík D, Žurovec M, Sehnal F. 2001
Insect silk contains both a Kunitz-type and a unique
Kazal-type proteinase inhibitor. Eur. J. Biochem.
268, 2064–2073. (doi:10.1046/j.1432-1327.2001.
02084.x)
72. Kaur J, Rajkhowa R, Afrin T, Tsuzuki T, Wang X.
2014 Facts and myths of antibacterial properties of
silk. Biopolymers 101, 237–245. (doi:10.1002/bip.
22323)
73. Umbers KDL, De Bona S, White TE, Lehtonen J,
Mappes J, Endler JA. 2017 Deimatism: a neglected
component of antipredator defence. Biol. Lett. 13,
20160936. (doi:10.1098/rsbl.2016.0936)
74. Cole LR. 1959 On the defences of Lepidopterous
pupae in relation to the oviposition behaviour of
certain Ichneumonidae. J. Lepid. Soc. 13,1–10.
75. Ichikawa T, Kurauchi T. 2009 Larval cannibalism and
pupal defense against cannibalism in two species of
tenebrionid beetles. Zool. Sci. 26, 525–529. (doi:10.
2108/zsj.26.525)
76. Groenewoud F, Frommen JG, Josi D, Tanaka H,
Jungwirth A, Taborsky M. 2016 Predation risk drives
social complexity in cooperative breeders. Proc. Natl
Acad. Sci. USA 113, 4104–4109. (doi:10.1073/PNAS.
1524178113)
77. Hölldobler B, Wilson EO. 1990 The ants. Cambridge,
MA: Harvard University Press.
78. Fujiwara S, Koike S, Yamazaki K, Kozakai C, Kaji K.
2013 Direct observation of bear myrmecophagy:
relationship between bears’feeding habits and ant
phenology. Mamm. Biol. 78,34–40. (doi:10.1016/J.
MAMBIO.2012.09.002)
79. Swenson JE, Jansson A, Riig R, Sandegren F. 1999
Bears and ants: myrmecophagy by brown bears in
central Scandinavia. Can. J. Zool. 77, 551–561.
(doi:10.1139/z99-004)
80. Turner GF, Pitcher TJ. 1986 Attack abatement: a
model for group protection by combined avoidance
royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20190069
10

and dilution. Am. Nat. 128, 228–240. (doi:10.1086/
284556)
81. Riipi M, Alatalo RV, Lindström L, Mappes J. 2001
Multiple benefits of gregariousness cover
detectability costs in aposematic aggregations.
Nature 413, 512–514. (doi:10.1038/35097061)
82. Hamilton WD. 1971 Geometry for the selfish herd.
J. Theor. Biol. 31, 295–311. (doi:10.1016/0022-
5193(71)90189-5)
83. Wrona FJ. 1991 Group size and predation risk: a
field analysis of encounter and dilution effects. Am.
Nat. 137, 186–201. (doi:10.1086/285153)
84. Jones R, Davis S, Speed MP. 2013 Defence cheats
can degrade protection of chemically defended prey.
Ethology 119,52–57. (doi:10.1111/eth.12036)
85. Eisner T, Johnessee JS, Carrel J, Hendry LB,
Meinwald J. 1974 Defensive use by an insect of a
plant resin. Science 184, 996–999. (doi:10.1126/
science.184.4140.996)
86. Pierce NE, Braby MF, Heath A, Lohman DJ, Mathew
J, Rand DB, Travassos MA. 2002 The ecology and
evolution of ant association in the Lycaenidae
(Lepidoptera). Annu. Rev. Entomol. 47, 733–771.
(doi:10.1146/annurev.ento.47.091201.145257)
87. Mokkonen M, Lindstedt C. 2016 The evolutionary
ecology of deception. Biol. Rev. 91, 1020–1035.
(doi:10.1111/brv.12208)
88. Atsatt PR. 1981 Lycaenid butterflies and ants:
selection for enemy-free space. Am. Nat. 118,
638–654. (doi:10.1086/283859)
89. Travassos MA, Pierce NE. 2000 Acoustics, context
and function of vibrational signalling in a lycaenid
butterfly–ant mutualism. Anim. Behav. 60,13–26.
(doi:10.1006/anbe.1999.1364)
90. Barbero F, Thomas JA, Bonelli S, Balletto E,
Schönrogge K. 2009 Queen ants make distinctive
sounds that are mimicked by a butterfly social
parasite. Science 323, 782–785. (doi:10.1126/
science.1163583)
91. Hallmann CA et al. 2017 More than 75 percent
decline over 27 years in total flying insect biomass
in protected areas. PLoS ONE 12, e0185809. (doi:10.
1371/journal.pone.0185809)
92. Lister BC, Garcia A. 2018 Climate-driven
declines in arthropod abundance restructure
a rainforest food web. Proc. Natl Acad. Sci. USA
115, E10 397–E10 406. (doi:10.1073/pnas.
1722477115)
93. 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/
1365-2656.12169)
94. Leimar O, Enquist M, Sillen-Tullberg B. 1986
Evolutionary stability of aposematic coloration and
prey unprofitability—a theoretical analysis. Am.
Nat. 128, 469–490. (doi:10.1086/284581)
95. Merilaita S, Ruxton GD. 2007 Aposematic signals
and the relationship between conspicuousness and
distinctiveness. J. Theor. Biol. 245, 268–277.
(doi:10.1016/j.jtbi.2006.10.022)
96. Stevens M, Ruxton GD. 2012 Linking the evolution
and form of warning coloration in nature.
Proc. R. Soc. B 279, 417–426. (doi:10.1098/rspb.
2011.1932)
97. Steinbrecht RA, Mohren W, Pulker HK, Schneider D.
1985 Cuticular interference reflectors in the golden
pupae of danaine butterflies. Proc. R. Soc. Lond. B
226, 367–390. (doi:10.1098/rspb.1985.0100)
98. Pegram KV, Rutowski RL. 2016 Effects of
directionality, signal intensity, and short-wavelength
components on iridescent warning signal efficacy.
Behav. Ecol. Sociobiol. 70, 1331–1343. (doi:10.1007/
s00265-016-2141-z)
99. Kjernsmo K, Hall JR, Doyle C, Khuzayim N, Cuthill
IC, Scott-Samuel NE, Whitney HM. 2018 Iridescence
impairs object recognition in bumblebees. Sci. Rep.
8, 8095. (doi:10.1038/s41598-018-26571-6)
100. White TE. 2018 Illuminating the evolution of
iridescence. Trends Ecol. Evol. 33, 374–375. (doi:10.
1016/J.TREE.2018.03.011)
101. Rowe C, Guilford T. 1999 Novelty effects in a
multimodal warning signal. Anim. Behav. 57,
341–346. (doi:10.1006/anbe.1998.0974)
102. Willmott KR, Willmott JCR, Elias M, Jiggins CD. 2017
Maintaining mimicry diversity: optimal warning
colour patterns differ among microhabitats in
Amazonian clearwing butterflies. Proc. R. Soc. B
284, 20170744. (doi:10.1098/rspb.2017.0744)
103. Lindstedt C, Miettinen A, Freitak D, Ketola T,
López-Sepulcre A, Mäntylä E, Pakkanen H. 2018
Ecological conditions alter cooperative behaviour
and its costs in a chemically defended sawfly.
Proc. R. Soc. B 285, 20180466. (doi:10.1098/rspb.
2018.0466)
104. Grill CP, Moore AJ. 1998 Effects of a larval
antipredator response and larval diet on adult
phenotype in an aposematic ladybird beetle.
Oecologia 114, 274–282. (doi:10.1007/
s004420050446)
105. Stevens DJ, Hansell MH, Freel JA, Monaghan P.
1999 Developmental trade-offs in caddis flies:
increased investment in larval defence alters adult
resource allocation. Proc. R. Soc. Lond. B 266,
1049–1054. (doi:10.1098/rspb.1999.0742)
106. Johnson PTJ et al. 2018 Of poisons and parasites—
the defensive role of tetrodotoxin against infections
in newts. J. Anim. Ecol. 87, 1192–1204. (doi:10.
1111/1365-2656.12816)
107. Laurentz M, Reudler JH, Mappes J, Friman V, Ikonen
S, Lindstedt C. 2012 Diet quality can play a critical
role in defense efficacy against parasitoids and
pathogens in the Glanville fritillary (Melitaea cinxia).
J. Chem. Ecol. 38, 116–125. (doi:10.1007/s10886-
012-0066-1)
108. Friman V-P, Lindstedt C, Hiltunen T, Laakso J,
Mappes J. 2009 Predation on multiple trophic levels
shapes the evolution of pathogen virulence. PLoS
ONE 4, e6761. (doi:10.1371/journal.pone.0006761)
109. Nokelainen O, Lindstedt C, Mappes J. 2013
Environment-mediated morph-linked immune and
life-history responses in the aposematic wood tiger
moth. J. Anim. Ecol. 82, 653–662. (doi:10.1111/
1365-2656.12037)
110. Johnston PR, Paris V, Rolff J. 2019 Immune gene
regulation in the gut during metamorphosis in a
holo- versus a hemimetabolous insect. Phil.
Trans. R. Soc. B 374, 20190073. (doi:10.1098/rstb.
2019.0073)
royalsocietypublishing.org/journal/rstb Phil. Trans. R. Soc. B 374: 20190069
11