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Multimodal Aposematic Defenses Through the Predation Sequence

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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.
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ORIGINAL RESEARCH
published: 16 August 2021
doi: 10.3389/fevo.2021.657740
Edited by:
Julien Terraube,
University of the Sunshine Coast,
Australia
Reviewed by:
Alice Exnerova,
Charles University, Czechia
Piotr Jablonski,
Seoul National University,
South Korea
*Correspondence:
Anne E. Winters
a.e.winters@exeter.ac.uk
Specialty section:
This article was submitted to
Behavioral and Evolutionary Ecology,
a section of the journal
Frontiers in Ecology and Evolution
Received: 23 January 2021
Accepted: 14 July 2021
Published: 16 August 2021
Citation:
Winters AE, Lommi J, Kirvesoja J,
Nokelainen O and Mappes J (2021)
Multimodal Aposematic Defenses
Through the Predation Sequence.
Front. Ecol. Evol. 9:657740.
doi: 10.3389/fevo.2021.657740
Multimodal Aposematic Defenses
Through the Predation Sequence
Anne E. Winters1,2*, Jenna Lommi1, Jimi Kirvesoja1, Ossi Nokelainen1and
Johanna Mappes1,3
1Department of Biological and Environmental Science, University of Jyväskylä, Jyväskylä, Finland, 2Centre for Ecology
and Conservation, College of Life and Environmental Sciences, University of Exeter, Penryn, United Kingdom, 3Organismal
and Evolutionary Biology Research Programme, Faculty of Biological and Environmental Sciences, University of Helsinki,
Helsinki, Finland
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.
Keywords: aposematism, Arctia plantaginis,Cyanistes caeruleus, defense mechanisms, multimodal signaling,
predator-prey interactions, chemical defense, warning signals
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Winters et al. Multimodality and the Predation Sequence
INTRODUCTION
Predation is one of the main threats to an organism’s survival.
As a result, there are many different traits that have evolved
to help organisms avoid predation and most organisms use
more than one line of defense. In some cases, these multiple
defenses can act simultaneously (Ruxton et al., 2018). For
example, prey may evolve behaviors such as background choice
(Sargent, 1966;Kang et al., 2012;Kjernsmo and Merilaita, 2012;
Green et al., 2019) or body orientation (Kang et al., 2012;
Rowland et al., 2020) and also to have a color pattern that is
camouflaged against their surroundings, all of which help avoid
detection by predators (Stevens and Ruxton, 2018). However,
many animals have defense mechanisms that act sequentially
by impeding different stages of attack (Endler, 1991;Caro,
2005;Ruxton et al., 2018). Primary defenses act to prevent
physical contact between predator and prey (i.e., at the encounter,
detection, identification and approach stages of an attack),
whereas secondary defenses deter attack after or just before the
predator has made physical contact with the prey (i.e., at the
subjugation and consumption stages of attack) (Ruxton et al.,
2018). Whether selection favors investment in primary and/or
secondary defenses depends on the relative properties of those
defenses such as their energetic cost and efficacy against predators
(Broom et al., 2010).
Aposematism is a defense strategy that relies on
communication to signal unprofitability to predators (Poulton,
1887,Poulton, 1890;Cott, 1940;Stevens, 2013). Aposematic
prey (the signaler) use a warning signal to inform predators
(the receiver) of unpleasant or harmful defenses to reduce the
likelihood or extent of attack by the predator and to promote,
enhance, or maintain learned avoidance of that prey type
in future encounters (Poulton, 1887,Poulton, 1890;Cott,
1940). Such warning signals may act as a primary defense if
the predator has an innate color bias (Smith, 1975;Roper,
1990;Schuler and Roper, 1992;Mastrota and Mench, 1995;
Lindström et al., 1999) or has learned to avoid the warning
signal through prior experience (Gittleman and Harvey,
1980;Roper and Wistow, 1986;Alatalo and Mappes, 1996;
Ham et al., 2006;Green et al., 2018). Conversely, warning
signals may act as a secondary defense if increased predator
wariness improves the chance that prey will escape or reduces
harm to prey after subjugation (Halpin et al., 2008;Ruxton
et al., 2018) or if the warning signal is “switchable” and only
becomes apparent after the predator has engaged with the
prey (Blest, 1964;Sivinski, 1981;Grober, 1988;Broom et al.,
2010;Umbers and Mappes, 2015;Kang et al., 2016;Umbers
et al., 2017;Song and Jablonski, 2020). Often, visual or auditory
warning signals are combined with chemical defenses, which
deter predators through some combination of taste (Marples
et al., 1994;Skelhorn and Rowe, 2006, 2010), smell (Rowe
and Guilford, 1996, 1999;Lindström et al., 2001;Jetz et al.,
2001;Kelly and Marples, 2004;Rojas et al., 2019), or toxicity
(Cortesi and Cheney, 2010;Arenas et al., 2015). 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 (Ruxton et al., 2018). 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.,
2017, 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.
Aposematism is formed by multimodal signaling (Rowe and
Halpin, 2013). That is, it involves the use of signal components
that are received through two or more sensory modalities by
a single receiver (Stevens, 2013). Warning signals are usually
conspicuous visual or auditory signals that are combined
with some form of chemical defense (either sequentially or
simultaneously), which predators perceive through certain
smell or taste receptors. However, smell and taste reception
are thought to have evolved largely to help animals avoid
the inadvertent consumption of harmful, toxic food (Shi
et al., 2003;Fischer et al., 2005;Chandrashekar et al., 2006;
Reed and Knaapila, 2010). In this way, smell and taste can
also be considered signals that warn predators of toxicity
(Eisner and Grant, 1981;Weldon, 2013). However, as with
Batesian mimics, which imitate visual aposematic signals, the
information content of such chemical signals may not always
be truthful, as not all chemicals that are perceived to have
an unpleasant smell or taste are toxic (Ruxton and Kennedy,
2006;Nissim et al., 2017;Winters et al., 2018;Lawrence
et al., 2019). In addition, there is evidence that defensive
smell and bitter taste alone are not necessarily sufficient to
prevent successful attack from predators (Eisner and Grant, 1981;
Guilford et al., 1987;Moore et al., 1990;Rowe and Guilford,
1996;Kelly and Marples, 2004;Siddall and Marples, 2011),
therefore the function of defense chemicals as honest signals
of toxicity within aposematic systems requires further study
(Holen, 2013).
Many chemically defended species use complex chemical
mixtures that contain different types of chemicals, and utilizing
multiple defensive compounds can be an adaptive strategy
in a number of different ways. 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). In addition, multiple defense compounds may
be used as a multimodal signal if a single predator uses
both smell (of volatile compounds) and taste (of non-volatile,
bitter compounds) to assess the toxicity of chemically defended
prey (Marples et al., 1994). 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 (Rowe and
Halpin, 2013;Rojas et al., 2017, 2019). Whereas non-volatile
compounds require predators to first capture prey before the
chemical defense can be perceived via taste receptors. Despite
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this, the effect of smell and taste is rarely differentiated in
studies of multimodal aposematic displays, but see Marples
et al. (1994), and therefore it remains unclear how interactions
between smell and taste fit within the theoretical framework of
multimodal aposematic signals (Rowe and Halpin, 2013). Thus,
measuring the individual and combined effects of smell, taste, and
warning coloration is essential for understanding the evolution
and maintenance of both chemical diversity and multimodal
warning signals.
Here, we investigate a multi-modal aposematic defense
(visual warning signal, smell, taste) in the polymorphic wood
tiger moth Arctia plantaginis to blue tit (Cyanistes caeruleus)
predators. In a recent study, blue tits used the potent smell of
methoxypyrazines as context-setting signals for the aposematic
colors of A. plantaginis (Rojas et al., 2019). Birds only
delayed attack in response to the white model color when the
methoxypyrazine smell was present. This finding differed from
previous studies that found the yellow morph to be better
protected when live moths were offered to blue tits in the
lab (Nokelainen et al., 2012), when dead moths were placed
in the field (Nokelainen et al., 2012) and when moth models
(i.e., dummies) were placed in the field (Nokelainen et al.,
2014). One explanation for the difference between the response
of blue tits to yellow and white morphs under laboratory
conditions could be the use of models (Rojas et al., 2019)
rather than live prey (Nokelainen et al., 2012). To address
this issue, we use live moths in this study. In addition, Rojas
et al. (2019) found that chemically treated models differed to
controls in terms of proportion of moths body eaten and beak
wiping behavior (a common disgust response), suggesting the
presence of both an aversive smell and taste in this species
and highlighting the need to disentangle these two modalities
from the defense fluid (Rojas et al., 2019). A. plantaginis
also have the ability to sequester pyrrolizidine alkaloids, which
they distribute to all tissues including neck fluids (Anne
Winters unpublished data), which might explain the aversive
taste. Pyrrolizidine alkaloids are well documented to defend
against invertebrate predators (Brown, 1984;Dussourd et al.,
1988;Masters, 1990;Eisner and Eisner, 1991;Conner et al.,
2000;Eisner et al., 2000). However, evidence for their defense
against vertebrates is less robust (Ritland, 1991;Rowell-Rahier
et al., 1995;Yosef et al., 1996;Cardoso, 1997). Therefore,
while lepidopterans that sequester pyrrolizidine alkaloids widely
exhibit conspicuous coloration (Nishida, 2002), further evidence
is needed to support the role of pyrrolizidine alkaloids in
aposematic defenses against vertebrates (Nishida, 2002). In
the present study, live moths of each color morph that were
manipulated to have only the smell (methoxypyrazines), only
the bitter taste (pyrrolizine alkaloids) or with both present,
were offered to birds to test whether color, smell and taste
constitute a multimodal warning signal in A. plantaginis. We
test whether combined modalities improve discrimination of
toxic prey by predators and enhance aversion learning. We
also investigate at which stage of attack (approach, attack,
subjugation, consumption) each defense modality is effective
at influencing predator behavior and whether multimodality
improves prey survival.
MATERIALS AND METHODS
Study Species A. plantaginis
The aposematic wood tiger moth (Arctia plantaginis, formerly
Parasemia plantaginis) is a member of the Erebidae family
(Rönkä et al., 2016) and widely distributed across the Northern
hemisphere (Hegna et al., 2015). There is geographical variation
in warning coloration (Hegna et al., 2015). In Europe, male
hindwings are either yellow or white and female hindwings
vary continuously from yellow to red (Lindstedt et al., 2011;
Nokelainen et al., 2012;Hegna et al., 2015). Hindwings are often
exposed at rest in this species, particularly if moths are alerted,
preparing to fly, or if the weather is cool. The discrete variation in
male hindwing coloration follows a one-locus two allele model,
where the yellow allele (y) is recessive to the white (W), resulting
in three genotypes (WW, Wy, yy). Both homozygous white
(WW) and heterozygous (Wy) genotypes have white hindwing
coloration, while the homozygous recessive genotype (yy) has
yellow (Suomalainen, 1938;Nokelainen et al., in prep a). Therea
are also differences between genotypes in the white hue of the
forewings, which is perceptible to birds (Nokelainen et al., in prep
a). The color polymorphism is under selection by bird predators
in the wild (Rönkä et al., 2020). In predation experiments, birds
respond differently toward the hindwing morphs, avoiding either
yellow (Nokelainen et al., 2012, 2014) or white (Rojas et al., 2019),
but see Rönkä et al. (2018).Rojas et al. (2019) speculate that the
variable response by predators could be due to differences in cues
between the moths and their model stimuli, differences in light
environment between experiments (Nokelainen in prep b), or the
presence or absence of methoxypyrazine odor.
Arctia plantaginis is chemically defended, with two uniqe
defense secretions that target different predators (Rojas et al.,
2017). One secretion is released between the head and thorax
when the thorax is grabbed or pinched and deters birds (neck
fluid), and a second secretion is released from the abdomen
when the moth is disturbed and deters ants (abdominal fluid)
(Rojas et al., 2017). Two main methoxypyrazine compounds
are released from the neck fluids: 2-sec-butyl-3-methoxypyrazine
(SBMP) and 2-isobutyl-3-methoxypyrazine (IBMP) (Burdfield-
Steel et al., 2018). These are produced de novo by the moth when
raised on an artificial diet (Burdfield-Steel et al., 2018). These
methoxypyrazines emit a potent odor that is aversive to blue tit
predators, causing delayed attack, increasing disgust behaviors
such as beak wiping, and reducing the amount or likelihood of
consumption (Rojas et al., 2017, 2019). In addition, A. plantaginis
is efficient at sequestering pyrrolizidine alkaloids from their diet
(Table 1). These alkaloids are present in wild-caught moths, and
distributed to all body parts of the moths including both neck and
abdominal defense fluids of moths (Anne Winters, unpublished
data). The efficacy of pyrrolizidine alkaloids sequestered by
A. plantaginis in defense against predation has not yet been tested.
Manipulation of Color, Smell, and Taste
of A. plantaginis
The wood tiger moth (Arctia plantaginis) is well suited to
examine the role of color, smell and taste in the multimodal
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TABLE 1 | Quantification of pyrrolizidine alkaloids (PA) seneciphylline and
senecionine in the larvae, food, and feces of A. plantaginis raised on an artificial
diet with 10% freeze-dried Senecio vulgaris.
PA Sample µg/mg s.e n
Seneciphylline Larvae 2.42 0.48 6
Food 0.24 0.02 2
Feces 0.01 0 2
Senecionine Larvae 0.14 0.03 6
Food 0.05 0.04 2
Feces 0 0 2
This may differ from the final concentration at adulthood.
aposematic display of a live insect because each of these three
components can be independently manipulated (Figure 1).
Importantly, the use of live prey accounts for the actual
nutritional value of prey (Halpin et al., 2014) and natural
delivery mechanism(s) of the chemical defense secretions (Eisner
and Meinwald, 1966), both of which improve the ecological
significance of results compared experiments using models as
stimuli (Rowe and Halpin, 2013).
Color Manipulation
To control the color morph of male A. plantaginis used in
this experiment, moth families were purpose bred from 3rd
generation 2019 lab stock of known (color morph) genotype
at the University of Jyväskylä. Moths were paired to produce
offspring of WW, Wy, and yy genotypes and mate pairings
were staggered so that adults would continuously emerge from
November-March, providing a sufficient time period to conduct
the behavioral experiment.
Taste Manipulation
To control the pyrrolizidine alkaloid “taste” of the moth, each
clutch was then split between two artificial diet treatments: a
control diet with no dietary source of pyrrolizidine alkaloids (4.6
agar, 8.58 g yeast, 32.1 g semolina, 8.3 g wheat germ, 150 ml
boiling water, 1.76 g Vanderzant vitamin mix, 1800 µl nipagen
and 180 µl acetic acid) and an artificial diet with 10% freeze-dried
Senecio vulgaris, as a dietary source of pyrrolizidine alkaloids
(4.6 g agar, 8.15 g yeast, 30.5 g semolina, 7.89 g wheat germ,
150 ml boiling water, 2.5 g freeze-dried Senecio vulgaris, 1.76 g
Vanderzant vitamin mix, 1800 µl nipagen and 180 µl acetic acid).
Larvae of each family/diet treatment were housed together in
plastic containers until pupation and fed daily with fresh food
spooned onto small squares of baking paper. To confirm the
sequestration of pyrrolizidine alkaloids from the second diet, six
larvae were selected and subject to chemical analysis along with
2 samples of their diet treatment, food and feces. Briefly, the
samples were first freeze-dried and then weighed to the nearest
0.1 mg. Samples were then homogenized, extracted and processed
through LC-MS/MS following the protocol outlined in Reinwaldt
et al. (2017). Seneciphylline and Senecionine were identified
as major compounds and quantified using a stock solution of
standards (2 mg each of Monocrotaline, Monocrotaline N-oxide,
Jacobine, Jacobine N-oxide, Intermedine, Intermedine N-oxide,
Retrorsine, Seneciphylline, Seneciphylline N-oxide, Senecionine,
Senecionine N-oxide, and Senkirkine, in 20 ml of 5% methanol
solution). A. plantaginis efficiently sequestered pyrrolizidine
alkaloids. Both major compounds identified were accumulated,
rather than excreted by the larvae, resulting in a greater
concentration of pyrrolizidine alkaloid in the moth compared to
their dietary source. Seneciphylline was 10×as concentrated in
the larvae compared to their food, while only trace amounts were
excreted in the feces. Senecionine was 3×as concentrated in the
larvae compared to their food, while only trace amounts were
excreted in the feces (Table 1).
Smell Manipulation
After pupation, individuals were placed singly in vials with a
sponge cap, which was sprayed daily with water to prevent
desiccation until they eclosed. After the moth eclosed, it was
stored in a refrigerator at 4C until use in the experiment
(12 days ±0.5 SE). To manipulate the methoxypyrazine “smell”
of the moth, neck fluids were removed from a subset of the
emerging adults by squeezing the thorax between the fingers and
collecting the resultant fluid using a microcapillary. Moths were
squeezed the day before they were used in the experiment so
that the majority of the methoxypyrazine smell could be released
and dissipated and then again 15 min before the experiment on
the day of the trial (see below for further details), to remove
any remaining methoxypyrazines. The moth was removed from
the refrigerator 30 min before each sampling and the sponge
cap was sprayed with water to allow the moth to warm up and
hydrate for 30 min. Moths that retained their neck fluids for the
experiment underwent the same protocol except the neck fluids
were not collected. Neck fluids were sampled in a separate room
with closed doors so that the odor was not pervasive in the bird
housing or experimental enclosures.
From these manipulations, 251 adult male moths were spread
between 12 treatment groups with at least n= 9 moths per
treatment (Table 2). Treatments with moths of the yellow morph
have lower sample size for two reasons: (1) Moths of the
yellow morph have poor fecundity compared to other genotypes
(Nokelainen et al., 2012;Gordon et al., 2018); poor mating
success and small clutch sizes resulted in fewer offspring with
yellow hindwings. (2) The experiment was ended prematurely
due to COVID-19, and the recently eclosed and yet to eclose
yellow males that could have increased the sample size had
to be discarded.
C. caeruleus Capture and Husbandry
In total, 84 wild blue tits (C. caeruleus) were trapped from a
feeding station at Konnevesi Research Station in Central Finland
between November 2019 and March 2020. Birds were weighed
on the day of capture and then individually housed in plywood
enclosures (65 cm ×50 cm ×80 cm) on a 11 h : 13 h (light : dark)
cycle for at least one day (8 days ±0.5 SE) before the experiment
started so that they acclimatized to captive conditions. During
this time, birds had ad libitum access to sunflower seeds,
peanuts, a vitamin enriched food supplement and water. After
the experiment, birds were ringed for identification purposes,
aged and sexed according to established methods published in:
Svensson (1992) Identification Guide to European Passerines
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Winters et al. Multimodality and the Predation Sequence
FIGURE 1 | Illustration of the source for each component (color, smell, and taste) in the multimodal warning signal of A. plantaginis and brief description of the
method used to manipulate that component in live moths for this experiment.
TABLE 2 | Number of birds (and moths) in each of treatment group including those of each hindwing phenotype: white and yellow, genotype: WW, Wy, or yy, those with
(+) and without () methoxypyrazine smell (MP), and those with (+) and without () pyrrolizidine alkaloid taste (PA).
Hindwing Genotype Chemical Treatment
Phenotype MP smell + MP smell
PA taste + PA taste PA taste + PA taste
White WW 12 (36) 7 (21) 11 (33) 8 (23)
White Wy 6 (18) 6 (18) 5 (15) 7 (21)
Yellow yy 7 (21) 3 (9) 6 (18) 6 (18)
ISBN: 9789163011184 Publisher: British Trust for Ornithology,
and then released at their site of capture. Birds were captured and
housed with permission of Central Finland Centre for Economic
Development, Transport and Environment (VARELY/294/2015)
and a license from the National Animal Experiment Board
(ESAVI/9114/04.10.07/2014).
Behavioral Experiment
Birds were transported to a separate experimental room and
placed inside masonite enclosures (50 cm ×50 cm ×70 cm),
which were equipped with a perch and water bowl and lit
with an Exo Terra Repti Glo 25 W 5.0 UVB compact light
bulb (see Waldron et al., 2017 Supplementary Material for
irradiance measurements). Spectral reflectance measurements
of the masonite background along with the forewings and
hindwings for each genotype are included in the Supplementary
Material (Supplementary Figure 1). Birds were observed
through a mesh-covered opening at the front of the cage and
by a video camera (Sony DSC-HX1) at the top of the cage
(Figure 2). During an acclimation/training period, birds were
offered two sunflower seeds through a hatch behind a visual
barrier, which was used to accurately measure when the moth
was seen, approached, and attacked. The first sunflower seed was
offered immediately after the bird was placed in the cage. After
1 h, if the bird ate the first sunflower seed, it was then offered
a second sunflower seed and monitored every 15 min until the
second seed was eaten. If after 1 h the bird had not eaten the
first seed, it was monitored every 15 min and offered the second
seed only after the first was eaten. To ensure the birds were
sufficiently hungry, the behavioral experiment was initiated 1 h
after the bird ate the second sunflower seed. To measure predator
avoidance learning across trials, each bird was presented with 3
moths (one moth per day for three consecutive days) from one of
the treatment groups (Table 2).
During the experiment, the observing room was kept dark
and silent to reduce the effect of the researcher on bird behavior.
Moths were held with forceps by the forewing and then placed
into the experimental enclosure using the same hatch used for
the sunflower seeds (Figure 2). We observed moths to hold
their wings slightly open, partially exposing their polymorphic
hindwings, which is a common natural resting position for the
moth. After the bird saw the moth, it had 15 min to attack, if it
did not attack, the experiment ended. After the bird attacked the
moth, the experiment ended when the bird showed no further
interest in any part of the moth for one full minute. During
the assay, birds and moths were observed by two authors (JL
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FIGURE 2 | Experimental setup of the behavioral experiment demonstrating the placement of perch, water, camera, light source, mesh opening for observation, and
hatch for inserting moths into the enclosure.
and AEW) and data was recorded to measure the following
16 variables (Supplementary Table 1): (1) approach probability
measured whether or not the bird approached the moth (yes/no,
for all moths) (2) approach latency was measured as the time
(in seconds) from seeing the moth (tilts head to look down at
it) to approaching it (landing beside the moth, usually on the
movable platform the moth was placed unless the moth moved)
(3) attack probability measured whether or not the bird attacked
the moth (yes/no, for moths that were approached) (4) attack
latency measured the time (in seconds) from approaching the
moth to grabbing it (5) drop probability measured whether or not
the bird dropped the moth at least once before eating it (yes/no,
for moths that were attacked) (6) prey drop latency measured
the time (in seconds) from grabbing the moth to dropping it
for the first time for birds that ate <50% of the moth (7) prey
dropping counted the number of times the bird dropped the
moth before beginning to eat it (8) handing duration was a
sum of the time (in seconds) the bird spent holding the moth
(grabbing the moth until dropping the moth). Includes each
occurrence the moth was held and includes eating duration (9)
eating probability measured whether or not the bird ate at least
part of the moth (yes/no, for moths that were attacked) (10)
eating duration was a sum of the time (in seconds) the bird
spent eating the moth (started eating the moth until stopped
eating the moth). Includes each occurrence the moth was eaten.
(11) proportion eaten was calculated by adding together the
proportion of each of six body parts eaten (antennae, head,
thorax, abdomen, legs, wings), as estimated by eye, and dividing
by six to calculate the total proportion of the moth that was
eaten by the bird (12) kill latency was measured as the time (in
seconds) from seeing the moth to killing it (usually by eating or
removing the head) (13) beak wiping, which is a common disgust
behavior (Evans and Waldbauer, 1982;Skelhorn and Rowe, 2009;
Rowland et al., 2015;Rojas et al., 2017, 2019) was measured as
the number of bouts of beak wiping the bird performed after
grabbing the moth until the end of the trial, (14) water drinking,
which may increase after the bird has consumed something
distasteful (Burdfield-Steel et al., 2019), was measured as the
number of “sips” taken from the water bowl after grabbing the
moth until the end of the trial. In addition, (15) moth activity,
sum of the time (in seconds) the moth spent crawling, flying, or
flexing which includes each occurrence the moth was active, and
(16) moth survival (yes/no, for all moths) were also recorded.
These behaviors were first recorded on datasheets during the
experiment using a stopwatch (to nearest second) and then
confirmed by JL watching the video afterward. If there was a
discrepancy between the video and the original observation in
terms of the timing or counts of a behavior, the video observation
was used because these behaviors could be measured more
accurately using the video. However, kill latency was always
measured using the original observation because it is difficult to
ascertain the time of death from the video. Birds remained under
observation for 30 min following the experiment to monitor for
ill effects from moth consumption, but none were observed. After
the observation period, birds were offered 8 g of meal worms. The
weight of mealworms eaten within 10 min was used as a measure
of the bird’s hunger level (Stevens et al., 2010). If the bird did not
eat the moth or the mealworms (2 individuals), it was excluded
from the experiment.
Statistical Analysis
All analyses were conducted using R version 4.0.3 (R Core
Team, 2011). All models include the fixed effects of moth
morph (white, yellow), methoxypyrazine smell (present, absent),
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pyrrolizidine alkaloid taste (present, absent), and trial number
(1, 2, 3). We then used a forward stepwise selection process
to include interactions and co-variates based on AICc and a
threshold of 12. As there are a large number of interactions
in our multimodality study to consider that may have potential
to be biologically meaningful, we used the dredge function in
the MuMin Package (Barton and Barton, 2015) for this step. In
all cases the simplest model within 12 of the top model was
selected. Then, we compared models using genotype (WW, Wy,
yy) or color morph (w, y). If genotype improved the AICc score of
the model by greater than 12, moth genotype was used instead.
Finally, after selecting interactions and morph or genotype,
additional relevant co-variates were selected to be included in the
model if they improved the AICc score of the model by greater
than 12. These co-variates that have potential to influence the
predation sequence include: moth activity which can influence
the bird’s required effort, hunger level which can influence the
bird’s motivation, bird age which may relate to experience,
bird sex where physiological differences may influence behavior
and motivation, and bird weight which may relate to body
condition and motivation. In all models, except for moth survival
probability, bird ID was included as a random factor to account
for multiple trials per bird. Model assumptions were checked
and distributions were chosen accordingly. Follow-up analyses
were conducted to determine which treatments differed from
the control. Tables detailing model selection (Supplementary
Table 2) and model summaries (Supplementary Table 3) are
provided in the Supplementary Material.
First, we tested whether the probability that blue tits
would progress through the predation sequence (binomial
response variables = approach probability,attack probability, drop
probability,eating probability, or moth survival) differed among
treatments. To do this, we used generalized linear mixed-effects
models (GLMM) with binomial distributions using the package
lme4 (Bates et al., 2015). Bird weight improved the AICc score
for the models of drop probability, eating probability, and moth
survival by more than 12, so it was included as a co-variate in
those models (Supplementary Table 2).
Next, we tested whether timed bird behaviors (approach and
attack latencies, eating and handling durations, drop latency, and
kill latency) differ among treatments to using cox proportional
hazards models (Therneau and Therneau, 2015). Moth genotype
improved the AICc score for the models of approach latency,
attack latency,drop latency, and eating duration by more than 1
2 AICc, so moth genotype was used instead of moth morph for
these models (Supplementary Table 2). For the model of attack
latency, the interaction between moth genotype and pyrrolizidine
alkaloid taste improved the AICc score by more than 12,
and for kill latency the interaction between methoxypyrazine
smell and pyrrolizidine alkaloid taste improved the AICc score
by more than 12, so these interactions were included in the
models (Supplementary Table 2). Based on AICc comparison,
bird age was selected to be included as a co-variate in the
model of attack latency, and bird weight was included in the
models of drop latency, handling duration, and eating duration,
while bird hunger level was included in the model of kill latency
(Supplementary Table 2).
Then, we tested whether counts of bird disgust behaviors after
attacking the moth (prey dropping,beak wiping,water drinking)
differed among our treatments using GLMM with poisson
distributions (except for beak wiping). The sum of squared
Pearson residuals indicated that the model for beak wiping
behavior was overdispersed, so a negative binomial distribution
was used instead. For the model of water drinking, the interaction
between methoxypyrazine smell and trial number improved the
AICc score by more than 12, so it was included in the model
(Supplementary Table 2). Bird weight improved the AICc score
for the model of prey dropping by more than 12, so it was
included as a co-variate in that model (Supplementary Table 2).
Finally, we tested whether the proportion eaten differed among
treatments. Model residuals were normally distributed, therefore
we used a linear mixed effects model with a Gaussian distribution.
Bird weight improved the AICc score by more than 12, so it was
included as a co-variate in the model (Supplementary Table 2).
RESULTS
Approach
Birds approached the moths in each of the 251 trials (except
for one case, trial 2, Wy, white morph, both chemical
defenses). Independent of neck fluids, approach latency
was longer for moths of the WW genotype, but not the
Wy genotype, compared to moths of the yy genotype
(estimate ±SE = 0.6958 ±0.2143, z=3.25, p= 0.001;
Figure 3A and Supplementary Table 3). However, when
neck fluids with methoxypyrazine smell were present, birds
approached both white morph genotypes more slowly compared
to yy moths (WW estimate ±SE = 0.8214 ±0.2998,
z=2.4, p= 0.006; Wy estimate ±SE = 0.6445 ±0.3324,
z=1.94, p= 0.052, Figure 3A and Supplementary Table 3)
suggesting that the methoxypyrazine smell of heterozygote
moths is especially important at this stage of attack. Approach
latency was longer for juvenile birds compared to adults
(estimate ±SE = 0.4321 ±0.2054, z=2.10, p= 0.035;
Supplementary Figure 2A and Supplementary Table 3).
Overall, approach latency decreased as the trials progressed
(estimate ±SE = 0.2899 ±0.0873, z= 3.32, p= 0.001;
Supplementary Figure 3A and Supplementary Table 3).
Approach latency was not affected by the presence of neck fluids
or pyrrolizidine alkaloids alone (Supplementary Table 3).
Attack
Birds attacked the moths in 94% of the trials (236 out of
251), with a trend for attack probability to increase with
trial number (estimate ±SE = 0.8207 ±0.4833, z= 1.698,
p= 0.089, Supplementary Figure 3B and Supplementary
Table 3). There was no effect of moth morph, methoxypyrazine
smell, or pyrrolizidine alkaloid taste on attack probability
(Supplementary Table 3).
The interaction between moth genotype and
pyrrolizidine alkaloid taste influenced bird attack latency
(estimate ±SE = 1.1902 ±0.3563, z=3.34, p= 0.001,
Figure 3B and Supplementary Table 3). The pyrrolizidine
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FIGURE 3 | (A) Approach latency in response to each moth genotype with (blue outline) and without (black outline) methoxypyrazine smell. Latency to approach
moths of the Wy genotype (white hindwings) morph was dependent on the presence of methoxypyrazine smell. Whereas, birds hesitated longer to approach moths
of the WW genotype (white hindwings) compared to moths of the yy genotype (yellow hindwings) irrespective of methoxypyrazine smell. *Indicates significant
differences (. = trend) from yy moths without methoxypyrazine smell. (B) Attack latency in response to each moth genotype with (red outline) and without (black
outline) pyrrolizidine alkaloid taste. Latency to attack moths of the yy genotype (yellow hindwings) was lower for moths on the pyrrolizidine alkaloid diet, while latency
to attack moths of the WW genotype (white hindwings) was higher for moths on the pyrrolizidine alkaloid diet. Pyrrolizidine alkaloid diet did not impact attack latency
for moths of the Wy genotype (white hindwings) *Indicates significant differences from yy moths without pyrrolizidine alkaloid taste.
alkaloid diet increased bird attack latency for moths of the WW
genotype, but decreased bird attack latency for moths of the
yy genotype (Figure 3B), suggesting the diet treatments affect
the primary anti-predator defenses of genotypes in different
ways. Bird attack latency decreased as the trials progressed
(estimate ±SE = 0.3244986 ±0.08131366, z= 3.99, p<0.001,
Supplementary Figure 3C and Supplementary Table 3). Attack
latency was not affected by methoxypyrazine smell.
Subjugation
Following attack, birds dropped the moth at least once in 28%
of the trials (65 out of 236), and independent of moth morph,
bird drop probability was higher for moths that had neck fluids
than those that did not (estimate ±SE = 1.2688 ±0.5532,
z= 2.294, p= 0.0218; Figure 4A and Supplementary Table 3).
However, when investigated separately, it was only white moths
with methoxypyrazine smell that significantly differed from
yellow moths with none (estimate ±SE = 2.4375 ±1.0445,
z= 2.334, p= 0.0196; Figure 4A and Supplementary
Table 3). Drop probability was positively associated with
bird weight, heavier birds were more likely to drop the
moth (estimate ±SE = 1.4527 ±0.4463, z= 3.255,
p= 0.0011, Supplementary Figure 2B and Supplementary
Table 3). Independently, there was no effect of moth morph,
pyrrolizidine alkaloid taste, or trial number on drop probability
(Supplementary Table 3).
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FIGURE 4 | (A) % of moths that were dropped at least once after being grabbed in response to moth morph (yellow or white) and methoxypyrazine smell. Moths
with white hindwings and methoxypyrazine smell were more likely to be dropped and with (blue outline) or without (black outline) methoxypyrazine smell. Panel (B)
were also dropped a greater number of times compared to yellow moths without methoxypyrazine smell. *Indicates significant differences from yellow moths without
methoxypyrazine smell. (C) Drop latency (time between grabbing and dropping the moth for the first time for birds that ate<% 50 of the moth) was quicker for moths
of the Wy genotype with methoxypyrazine smell compared to yellow moths without (*indicates significant differences from moths of the yy genotype without
methoxypyrazine smell), suggesting a more potent defense upon contact and (D) was slower for moths that were raised on a pyrrolizidine alkaloid diet compared to
moths with no chemical defenses, suggesting toxin sequestration is costly to other (primary) defenses (*indicates significant difference from moths without
methoxypyrazine smell or pyrrolizidine alkaloid taste). Bar graphs show mean ±SE [or percent (Dropped = yes)].
In addition, prey dropping behavior (number of times the
bird dropped the moth before eating it) increased if the moth
had methoxypyrazine smell (estimate ±SE = 0.9679, ±0.4286,
z= 2.258, p= 0.0239, Figure 4B and Supplementary Table 3)
and heavier birds exhibited more prey dropping behavior
(estimate ±SE = 0.8179 ±0.3261, z= 2.508, p= 0.0121,
Supplementary Figure 2D). However, again it seems likely this
effect is driven by moths of the white morph, as this was the only
treatment to independently differ from yellow moths without
methoxypyrazine smell (estimate ±SE = 1.9527 ±0.8343,
z= 2.341, p= 0.0193, Figure 4B and Supplementary Table 3)
in a separate analysis. Independently, there was no effect of
moth morph, pyrrolizidine alkaloid taste, or trial number on prey
dropping behavior.
Of the birds that ate less than half of the moth, birds had
a shorter drop latency for moths with methoxypyrazine smell
compared those without (estimate ±SE = 0.6173 ±0.2360,
z= 2.62, p= 0.0089, Figure 4C and Supplementary Table 3),
and moths of the Wy genotype compared to those of the WW
(estimate ±SE = 0.9142 ±0.2886, z= 3.17, p= 0.0015) or
yy (estimate ±SE = 0.7022 ±0.3040, z= 2.31, p= 0.0210,
Supplementary Table 3) genotypes, with a shorter period of
time between grabbing and abandoning Wy moths (Figure 4C).
This effect is likely being driven by the drop latency for
Wy moths with methoxypyrazine smell, which was the only
treatment to significantly differ from yellow moths without
methoxypyrazine smell (estimate ±SE = 1.5433, 0.4460, z= 3.46,
p= 0.0005, Figure 4C and Supplementary Table 3). Surprisingly,
drop latency was quicker for moths raised on the control
diet compared to those raised on a diet with pyrrolizidine
alkaloids (estimate ±SE = 0.5087 ±0.2288, z=2.2,
p= 0.0260, Figure 4D and Supplementary Table 3), which is
likely being driven by drop latency for moths with pyrrolizidine
alkaloids but without methoxypyrazine smell, which was the only
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treatment to significantly differ from moths with no chemical
defenses (estimate ±SE=0.8317 ±0.3934, z=2.11,
p= 0.0350), suggesting a trade-off between pyrrolizidine alkaloid
sequestration and synthesis of methoxypyrazine reserves. Heavier
birds had quicker drop latency (estimate ±SE = 0.6337 ±0.1748,
z= 3.63, p= 0.0003, Supplementary Figure 2C). There was no
effect of trial number on drop latency (Supplementary Table 3).
Handling duration decreased as the trials progressed
(estimate ±SE = 0.6538 ±0.0978, z= 6.68, p<0.001;
Supplementary Figure 3D and Supplementary
Table 3). Heavier birds had a shorter handling duration
(estimate ±SE = 0.62076875 ±0.20082539, z= 3.09, p= 0.002,
Supplementary Figure 2E and Supplementary Table 3). Moth
morph, methoxypyrazine smell, and pyrrolizidine alkaloid taste
did not influence handling duration (Supplementary Table 3).
Consumption
Birds ate at least part of the moth in 81% of the trials (48 out of
251), and were less likely to eat moths that had methoxypyrazine
smell than those that did not (estimate ±SE = 2.2490 ±0.9158,
z=2.456, p= 0.0141; Figure 5A and Supplementary Table 3).
There was no effect of moth morph, pyrrolizidine alkaloid taste,
or trial number on eating probability (Supplementary Table 3).
There was a significant effect of genotype on eating duration,
where birds took longer to eat moths of the WW genotype
compared to the yy genotype (estimate ±SE = 0.6592 ±0.2532,
z=2.60, p= 0.0092, Figure 5B and Supplementary
Table 3). Eating duration decreased with trial number
(estimate ±SE = 0.44751035 ±0.1026699, z= 4.36, p<0.001,
Supplementary Figure 3E), and heaver birds had a shorter
eating duration (estimate ±SE = 0.46967725 ±0.1540265,
z= 3.05, p= 0.0023, Supplementary Figure 2G). Neither
methoxypyrazine smell nor pyrrolizidine alkaloid taste affected
eating duration (Supplementary Table 3).
In addition, the proportion eaten decreased if the moth
had methoxypyrazine smell compared to those without
[t(79) = 2.405621, p= 0.0185; Figure 5C and Supplementary
Table 3]. However, it seems likely this effect is driven by the
treatment where moths that have both methoxypyrazine smell
and pyrrolizidine alkaloid taste in their neck fluids. When
analyzed separately, the proportion eaten was smaller from
moths with both defenses compared to moths with no defenses
[t(78) = 2.2654, p= 0.0263; Figure 5C and Supplementary
Table 3], but the proportion eaten did not differ between
moths with only methoxypyrazine smell and those with no
chemical defenses [t(78) = 1.2821, p= 0.2036; Figure 5C and
Supplementary Table 3]. The proportion eaten increased with
trial number [t(151) = 2.0533, p= 0.0418]; Supplementary
Figure 3F and decreased with bird weight [t(78) = 3.6513,
p= 0.0005; Supplementary Figure 2H and Supplementary
Table 3]. There was no effect of pyrrolizidine alkaloid taste or
moth morph on the proportion eaten (Supplementary Table 3).
Disgust
Beak wiping behavior decreased with trial number
(estimate ±SE = 0.7963 ±0.1089, z=7.311, p<0.001;
Supplementary Figure 3G and Supplementary Table 3).
There was no effect of moth morph, methoxypyrazine
smell, or pyrrolizidine alkaloid taste on beak wiping
(Supplementary Table 3).
The interaction between methoxypyrazine smell and
trial number influenced bird water drinking behavior
(estimate ±SE = 0.6299 ±0.2814, z= 2.238, p= 0.0252,
Figure 5D and Supplementary Table 3). Water drinking
decreased with trial number if the moth did not have neck
fluids (Figure 5D). Neither the moth morph nor pyrrolizidine
alkaloid taste affected water drinking behavior in the birds
(Supplementary Table 3).
Survival
The interaction between methoxypyrazine smell and
pyrrolizidine alkaloid taste influenced bird kill latency
(estimate ±SE=1.1483162 ±0.4830955, z=2.38,
p= 0.017, Figure 6A and Supplementary Table 3). Kill latency
was quicker if the moth did not have a methoxypyrazine smell
and was raised on a diet with pyrrolizidine alkaloids, suggesting a
trade-off between toxin sequestration and methoxypyrazine
synthesis. Kill latency decreased with trial number
(estimate ±SE = 0.6360943 ±0.1096281, z= 5.80, p<0.001;
Supplementary Figure 3H and Supplementary Table 3), and
with hunger level (estimate ±SE = 1.0167007 ±0.2755151,
z= 3.69, p<0.001; Supplementary Figure 2J and
Supplementary Table 3). There was no effect of moth morph on
kill latency (Supplementary Table 3).
Moths survived in only 43 (17%) trials. Moth
survival increased if they had methoxypyrazine smell
(estimate ±SE = 1.25092 ±0.40567, z= 3.084, p= 0.0021;
Figure 6B and Supplementary Table 3), and moth survival
increased with bird weight (estimate ±SE = 1.48241 ±0.33329,
z= 4.448, p<0.001, Supplementary Figure 2I and
Supplementary Table 3). When analyzed separately, white
moths with methoxypyrazine smell were the only moths
with higher survival compared to yellow moths without
(estimate ±SE = 1.6612 ±0.7887, z= 2.106, p= 0.0352,
Figure 6B and Supplementary Table 3), and moths with
both methoxypyrazine smell and pyrrolizidine alkaloid taste
were the only moths with higher survival compared to moths
with no chemical defenses (estimate ±SE = 1.1442 ±0.4999,
z= 2.289, p= 0.0221, Figure 6C and Supplementary Table 3),
however, there was a trend for moths with only methoxypyrazine
smell to also have higher survival compared to moths with no
chemical defenses (estimate ±SE = 0.9899 ±0.5201, z= 1.903,
p= 0.0570). Neither pyrrolizidine alkaloid taste alone nor trial
number affected moth survival (Supplementary Table 3).
DISCUSSION
Summary
This study investigated multimodal anti-predator defenses
through the predation sequence (Figure 7), and how
multimodality impacts predator avoidance learning and
moth survival. Bird approach latency toward moths of the Wy
(white hindwings) morph was dependent on the presence of
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FIGURE 5 | (A) The probability for birds to eat at least part of the moth in response to the presence (blue) or absence (white) of methoxypyrazine smell. Birds were
more likely to eat moths without methoxypyrazine smell. (B) The amount of time birds spent eating moths of each genotype. Birds ate moths of the WW (white
hindwings) genotype more slowly compared to moths of the yy genotype (yellow hindwings). *Indicates significant differences from the yy genotype. (C) The
proportion of the moth’s body eaten in response to methoxypyrazine smell and pyrrolizidine alkaloid taste. The proportion eaten was smaller if the moth had both
types of chemical defense. *Indicates significant difference from moths without methoxypyrazine smell or pyrrolizidine alkaloid taste. (D) The number of times birds
took a drink of water in response to trial number and methoxypyrazine smell. Birds reduced their water drinking as trials progressed to a greater degree for moths
that did not have methoxypyrazine smell compared to those that did, suggesting that moths with methoxypyrazine smell maintain aversion across trials. Bar graphs
show mean ±SE [or percent (Eaten = yes)].
neck fluids with methoxypyrazine smell, but approach latency
was longer for moths of the WW (white hindwings) compared
to moths of the yy genotype (yellow hindwings) irrespective of
methoxypyrazine smell. Color and smell had additive effects on
dropping behavior, where moths of the white morph with neck
fluids were more likely to be dropped and were also dropped a
greater number of times. Drop latency (time between grabbing
and dropping the moth for the first time) was quickest for moths
of the Wy genotype that had neck fluids, suggesting a more
potent defense upon contact for this genotype. Furthermore,
taste alone did not deter bird predators. Birds were less likely
to eat moths with neck fluids compared to those without,
but only responded to the presence of pyrrolizidine alkaloids
(taste and toxicity) after they had started to eat the moth and
when the methoxypyrazine smell was also present, causing
them to eat a smaller proportion of the moth’s body with both
chemical defenses. Surprisingly, the pyrrolizidine alkaloid diet
had a detrimental effect on predator deterrence in the early
stages of attack including attack latency,drop latency, and kill
latency (Figure 7), suggesting a possible trade-off between
secondary (toxin sequestration) and primary (methoxypyrazine
synthesis and/or wing pigmentation) defenses. Overall, moths
of the white morph with methoxypyrazine smell had the
greatest chance of survival. However, even though pyrrolizidine
alkaloids had a negative impact on attack stage progression,
toxin sequestration did not negatively impact survival. Indeed,
moths with both methoxypyrazine smell and pyrrolizidine
alkaloid taste had the highest survival overall. We did not
find support for predator aversion learning, although birds
adjusted their water drinking behavior across trials in response
to methoxypyrazine smell. 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.
The effect of each modality on each stage of attack (Figure 7)
is detailed below.
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FIGURE 6 | (A) Bird latency to kill the moth in response to methoxypyrazine
smell and pyrrolizidine alkaloid taste. Moths raised on a pyrrolizidine alkaloid
diet, but without methoxypyrazine smell were killed more quickly than moths
without either chemical defense. *Indicates significant difference from moths
without either chemical defense. (B) Moth survival in response to moth morph
(yellow or white) and methoxypyrazine smell. Moths of the white morph with =
blue methoxypyrazine smell had higher survival compared to moths of the
yellow morph without = black methoxypyrazine smell. *Indicates significant
difference from yellow moths without methoxypyrazine smell. (C) Moth survival
in response to methoxypyrazine smell and pyrrolizidine alkaloid taste. Moths
with both methoxypyrazine smell and pyrrolizidine alkaloid taste had higher
survival than moths without either chemical defense. *Indicates significant
differences (. = trend) from moths without either chemical defense. Bar graph
shows percent (Survived = yes)±.
Approach
At the approach stage (Figure 7), birds hesitated longer to
approach moths of the WW genotype compared to the yy
genotype but only hesitated to approach moths of the Wy
genotype if they had a methoxypyrazine smell. In addition, adult
birds approached moths more quickly than juveniles, which
suggests juveniles were more cautious with their prey. In a
previous study, Rojas et al. (2019) found that blue tits took longer
to approach models with white wings when neck fluids were
present, regardless of whether they were coated with fluids from
yellow (Y) or white (W) males, suggesting that methoxypyrazine
smell did not differ between the morphs. While, neck fluids
from Wy and WW males were not differentiated in Rojas
et al. (2019), examination of Figure 3 suggests that variation in
approach latency is largest for white morphs, which is consistent
with the idea that fluid properties may differ between WW
and Wy genotypes. Further chemical analysis is necessary to
determine genotype differences in the type or quantity of de novo
synthesized methoxypyrazines.
The difference in approach latency between WW and Wy
moths without neck fluids, both of which have white hindwings,
suggests that there is a perceptible visual difference between these
two genotypes to blue tits. Ultraviolet (UV) components of the
color pattern differ between these two genotypes especially in the
forewings (Nokelainen in prep a), and therefore it is possible that
birds are responding to UV-reflectance by delaying their attack of
prey. Indeed, UV reflective white color is used as a warning signal
in other lepidopteran species (Corral-Lopez et al., 2020), although
in some earlier experiments UV-reflectance was found to invite
rather than deter attacks by birds (Lyytinen et al., 2001, 2004).
Our results support the findings of Rojas et al. (2019) that, in
the presence of neck fluid odor, birds take longer to approach
A. plantaginis with white hindwings compared to those with
yellow hindwings. Rojas et al. (2019) presented moths on a
green background, while we presented moths against brown
masonite (Supplementary Figure 1), which suggests that the
white morph elicits longer approach hesitation even when
presented against different colored backgrounds. These findings
are at odds with previous studies where the yellow morph was
found to be better protected (Nokelainen et al., 2012, 2014),
but confirms that this discrepancy is not simply a difference in
cues between model stimuli and natural prey. As suggested by
Rojas et al. (2019) and experimentally tested by O. Nokelainen
et al. (in prep b) there is an interaction between color pattern
and light environment on predator response to A. plantaginis
hindwing coloration. This could explain differences between
experiments. Furthermore, natural prey that are not alive, such
as some of those used in the Nokelainen et al. (2012) field
experiment, may lack chemical delivery mechanisms to effectively
release methoxypyrazine odors, and these volatile compounds
may not have been present when the moths were presented
to birds. These results highlight the importance of considering
the interplay between multiple modalities, but also variation in
natural environmental conditions, such as light environment,
that can influence predator responses to defended prey.
Attack
Surprisingly, bird attack latency (time from approaching to
attacking the moth) depended on the interaction between moth
genotype and the pyrrolizidine alkaloid diet treatment (Figure 7),
with birds hesitating longest to attack homozygous yellow moths
from the control diet and homozygous white moths if they
were raised on the pyrrolizidine alkaloid diet. Birds could be
more motivated to attack larger prey, however, diet did not
impact pupal weight for the WW and yy genotypes and attack
latency did not differ between diet treatments for heterozygous
moths, which were heavier when raised on the pyrrolizidine
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Winters et al. Multimodality and the Predation Sequence
alkaloid diet (Supplementary Figure 4). It is puzzling that birds
could perceive these non-volatile toxins before they are tasted
and that the presence of toxins could cause birds accelerate
their attack (for homozygous yellow moths). A more likely
explanation is that sequestering toxins from the diet can be
costly, limiting investment in other defense mechanisms such as
methoxypyrazine synthesis or wing pigmentation, and that this
cost differs between the genotypes. For example, it is possible
that small reserves of methoxypyrazines are present even in
moths that had their neck fluids removed, that the potency
of these reserves is impacted by diet, and that the detection
of these reserves requires close range (between approach and
attack). Similarly, Lindstedt et al. (2010) found that a diet
with iridoid glycoside toxins was costly to primary defenses of
female A. plantaginis, resulting in hindwings with a lighter hue.
The cost of pyrrolizidine alkaloid sequestration, and particularly
its impact on other anti-predator defenses in A. plantaginis
warrants further study.
Subjugation
After attack, bird likelihood to drop the moth and the number
of times they dropped the moth differed in response to the
additive effects of methoxypyrazine smell and color morph. Birds
increased dropping behavior when moths had methoxypyrazine
smell, and even more so when the moth had white hindwings.
It is possible that these behaviors vary in response to the
combination of visual and chemical signals. Such a relationship
between methoxypyrazines and warning coloration is common
in the literature, where it is suggested that methoxypyrazines
act as context-setting signals (Marples and Roper, 1996;Rowe
and Guilford, 1996, 1999;Lindström et al., 2001;Jetz et al.,
2001;Kelly and Marples, 2004;Rowe and Halpin, 2013;Vickers
and Taylor, 2018, 2020). However, it is also possible that white
morph moths, and in particular those that are heterozygous for
hindwing coloration, have more potent chemical defense, which
causes differences in predator response between the genotypes.
These effects are not mutually exclusive. Indeed, Rojas et al.
(2019) found that moths of the white morph may have a
more aversive taste.
Bird drop latency (the time between grabbing and dropping
the moth) was quicker for heterozygous moths and moths
with methoxypyrazine smell, but slower for moths from the
pyrrolizidine alkaloid diet, and particularly for moths with
pyrrolizidine alkaloids but no methoxypyrazines (Figure 7).
Again, one explanation for the apparent eagerness for birds
to pursue moths that have pyrrolizidine alkaloids is that toxin
sequestration may be costly and reduce investment in other
defenses, such as methoxypyrazines, which in turn may reduce
defense potency in the earlier stages of attack before the bird has
encountered the taste of pyrrolizidine alkaloids.
Consumption
Birds were less likely to eat moths with a methoxypyrazine smell
than those without. In addition, birds took longer to eat moths
of the WW genotype compared to moths of the yy genotype
(Figure 7). Bird water drinking, which was correlated with eating
duration (Supplementary Figure 5), decreased across trials, but
only if the moths did not have neck fluids. However, despite
being the only component that is intrinsically linked to the
concentration of hepatotoxic pyrrolizidine alkaloids, taste alone
did not deter bird predators. Birds only reduced the proportion
eaten of the moth’s body with pyrrolizidine alkaloids compared to
other treatments when neck fluids containing methoxypyrazines
were also present. One possibility is that methoxypyrazines alert
predators to the presence of bitter toxins. In the attention-altering
hypothesis ‘one signal can increase the degree to which a receiver
focuses attention on another sensory field, and by doing so,
improves discrimination within that field’ (Hebets and Papaj,
2005). For instance, Guilford (1994) first suggested that visual
warning signals might be ‘go-slow’ signals that alert predators to
pay better attention in their assessment of prey palatability. Our
findings suggest that smell may also provide a ‘go-slow’ signal for
taste and toxicity.
Fluid secretion may be an important mechanism for the
delivery of chemical defenses, discharging a distasteful chemical
cocktail into the bird’s mouth before the bird has had a chance to
bite into and taste the more nutritious tissues of the moth (Eisner
and Meinwald, 1966). Indeed, in a study of leaf beetles, birds
were more likely to reject prey that had their defense secretion
intact compared to those that only had pyrrolizidine alkaloids
sequestered into their body tissues (Rowell-Rahier et al., 1995).
Therefore, it is possible that we have underestimated the effect
of pyrrolizidine alkaloid defense, and that pyrrolizidine alkaloids
in the neck fluids (Anne Winters Unpublished data) might
contribute to moth defense at earlier stages of the attack sequence.
The role of pyrrolizidine alkaloids in defense against
invertebrates is well-documented (Brown, 1984;Dussourd et al.,
1988;Masters, 1990;Eisner and Eisner, 1991;Hare and Eisner,
1993;Conner et al., 2000;Eisner et al., 2000). Rojas et al. (2017)
found that A. plantaginis abdominal fluids were deterrent to ants,
but not to birds, but the compounds in the abdominal fluid
were not identified. Pyrrolizidine alkaloids are present in the
abdominal fluids of A. plantaginis (Anne Winters unpublished
data) and it is possible that these contribute toward defense
against invertebrates. Birds did not find the abdominal fluids
(which do not contain methoxypyrazines) aversive, and this is
in line with our finding that birds only reduced consumption of
pyrrolizidine alkaloids when methoxypyrazines are also present.
Invertebrate predators may also respond to visual aposematic
signals. Similar to findings with birds, jumping spiders alter their
response to visual signals in response to odor (Vickers and Taylor,
2018, 2020). Therefore, multimodal displays of color, smell, and
taste are likely under selection from multiple, taxonomically
distinct, predators. Defenses may asymmetrically target these
predators, providing marginal protection for some types of
predators and strong protection against others. Thus, multiple
predators may create different selection pressures that shape the
evolution of multimodal aposematic signals.
Survival
Moths raised on the pyrrolizidine alkaloid diet that had
their neck fluids (methoxypyrazine smell) removed were killed
more quickly than moths with no defenses (Figure 7),
which, as mentioned above, suggests a trade-off between toxin
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Winters et al. Multimodality and the Predation Sequence
FIGURE 7 | Summary of the results indicating the bird behaviors (approach latency, drop probability, drop latency, prey dropping, eating probability, eating duration,
proportion eaten, kill latency, moth survival) that were influenced by each defense modality (color, color + smell, smell, color + taste, smell + taste) and the stage of
the attack sequence that was inhibited. *Indicates that genotype rather than moth color morph was included in the model based on AICc. Text in red indicates
reduced defense efficacy for that defense treatment/bird behavior (cost of pyrrolizidine alkaloid toxin sequestration may trade-off with primary defenses), while black
texts indicates improved efficacy.
sequestration and investment in other anti-predator defenses
such as methoxypyrazine reserves and/or wing pigmentation. In
addition, while moth activity was not selected to be included in
the model based on AICc, moths on the pyrrolizidine alkaloid
diet were less active compared to moths raised on the control
diet (Supplementary Figure 6), which could reduce the amount
of time needed for birds to capture and kill them. Overall,
moth survival was highest for moths of the white morph with
methoxypyrazine smell (Figure 7) and, despite the detrimental
effects of pyrrolizidine alkaloid sequestration in terms of attack
latency, drop latency, and kill latency, pyrrolizidine alkaloids did
not negatively impact moth survival (Figure 7). Indeed, moths
with both methoxypyrazine smell and pyrrolizidine alkaloid taste
had the highest survival.
Predator Learning
We did not find evidence for aversion learning in this study.
Instead, the time birds took to approach, attack, and handle the
moths decreased with trials and birds were more likely to attack
the moths as the trials progressed. Birds were quicker and more
likely to attack moths in all treatments, including the treatment
with no chemical defenses, suggesting a protective benefit of prey
novelty that decreases with predator experience.
Birds that are no longer surprised by chemical defenses might
still be expected to avoid them if those defenses are toxic or
cause harm. However, birds in our experiment did not learn to
avoid moths that contained toxic pyrrolizidine alkaloids. There
are a number of reasons birds might decide to consume toxic
prey, even after they have been warned about it (Barnett et al.,
2007, 2012). For example, Hämäläinen et al. (2020) found that
great tits differ in taste perception, but that their decision to
eat toxic prey depended on the bird’s body condition, and not
taste perception. Similarly, we found that bird body weight (as
a proxy for condition), impacted behaviors across the predation
sequence including dropping, handling, killing and eating the
moth. Likewise, decisions about eating chemically defended prey
may also relate to the presence and nutritional value of alternative
food sources (Brower et al., 1968;Turner and Speed, 1999;
Kokko et al., 2003;Sherratt, 2003). It is possible that birds
would have learned to avoid toxic moths if they were given the
choice of a nutritious and non-toxic alternative. In addition, these
experiments took place in the winter, when food, and especially
live insects, are scarce and the ambient temperature is cooler
compared to summer months, which could influence the choice
to consume toxic prey. Indeed, Chatelain et al. (2013) found
that starlings increased consumption of prey that they knew to
contain toxins when the ambient temperature was cooler. Stevens
et al. (2010) found that birds are more likely to eat unpalatable,
aposematic prey when they are hungry, and similarly, we found
that hungrier birds killed the moths more quickly.
Intra-Specific Differences in Chemical
Defense
As described above, bird approach latency changed based
on the presence of methoxypyrazines, but only for the Wy
genotype. In addition, birds abandoned Wy moths with
methoxypyrazine smell more quickly than other genotypes. And,
contrary to our expectation, moth genotype also interacted
with pyrrolizidine alkaloid taste at the attack latency stage (the
time from approaching to attacking the moth). Together, these
results suggest that the neck fluid defenses of moths that are
heterozygous for hindwing coloration may be particularly potent,
and that the potential cost of toxin sequestration is unequal across
the genotypes. Using life history data of moths obtained from this
experiment, male moths of the Wy genotype were the only moths
that were differentially impacted by the diet manipulation in
terms of pupal weight. The pupae of Wy males were heavier when
raised on the diet containing pyrrolizidine alkaloids compared to
the diet without (Supplementary Figure 4), suggesting the Wy
genotype may perform better on this diet compared to the other
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Winters et al. Multimodality and the Predation Sequence
genotypes. Further research is required to determine whether
the Wy genotype differs in the quantity or ratio of de novo
synthesized methoxypyrazines SBMP and IBMP and whether the
Wy genotype more efficiently sequesters or differentially utilizes
pyrrolizidine alkaloids from their diet. However, our findings
suggest that heterozygotes may have an advantage when it comes
to the dietary sequestration of chemical defenses and in defense
against predation, which could help to explain the persistence of
color polymorphism in this species (see also Gordon et al., 2018).
Conclusion
Altogether these results suggest that color, smell, and taste
function as a multimodal warning signal, and that there may
be trade-offs between defense modalities, which impact different
stages of attack such that primary defenses may dishonestly
signal pyrrolizidine alkaloid content. Color and smell provided
protection from a distance and during the initial encounter,
while during consumption, methoxypyrazine smell may alert
predators to the presence of pyrrolizidine alkaloids, reducing
the proportion eaten in the treatment with both chemical
defenses compared to the control. Overall, moth survival was
highest for moths of the white morph with methoxypyrazine
smell and, despite the detrimental effects of pyrrolizidine
alkaloid sequestration on defense in the early attack stages,
toxin sequestration did not negatively impact moth survival.
Indeed, of the chemical defense treatments, moths with both
methoxypyrazine smell and pyrrolizidine alkaloid taste had the
highest survival. The smell of methoxypyrazines seems to be
an especially important signal, facilitating predator responses to
both color and taste perception.
DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included
in the article/Supplementary Material, further inquiries can be
directed to the corresponding author/s.
ETHICS STATEMENT
The animal study was reviewed and approved by The Central
Finland Centre for Economic Development, Transport and
Environment, a license from the National Animal Experiment
Board (ESAVI/9114/04.10.07/2014) and the Central Finland
Regional Environmental Centre (VARELY/294/2015).
AUTHOR CONTRIBUTIONS
JL participated in the design of the study, lab work, fieldwork,
data analysis, and drafting the manuscript. JK participated
in the lab work, fieldwork, and drafting the manuscript. ON
participated in the design of the study and drafting the
manuscript. JM participated in the conception and design of the
study and drafting the manuscript. AW conceived, coordinated,
and designed the study, participated in lab work, field work, data
analysis, and drafting the manuscript. All authors approved the
submitted version.
FUNDING
This work was supported by the Academy of Finland to
JM (#320438) and the Grant (#21000038821) to ON and by
the European Union’s Horizon 2020 research and innovation
program under the Marie Skłodowska-Curie Grant agreement
(#840944) to AW.
ACKNOWLEDGMENTS
We thank Marjut Mähönen and Teemu Tuomaala for assistance
raising moth lab stock, Kaisa Suisto for assistance and advice
with moth care, Helinä Nisu for help catching and caring for
birds at Konnevesi Research Station, Riccardo Tambornini for
assistance with behavioral experiments, and Hannu Pakkanen for
quantifying pyrrolizidine alkaloids. We also thank two reviewers
for helpful comments on an earlier draft of this manuscript.
SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fevo.2021.
657740/full#supplementary-material
Supplementary Figure 1 | Spectral reflectance (% reflectance from 300 to
700 nm wavelengths) of the masonite enclosure (background) and for the
forewings (FW) and hindwings (HW) and black portion of the wing (BK) of each
genotype: WW, white hindwings; Wy, white hindwings; yy, yellow hindwings.
Supplementary Figure 2 | Effect of co-variates: (A) approach latency in
response to bird age, (B–I), drop probability, drop latency, prey dropping, handling
duration, eating probability, eating duration, proportion eaten, and moth survival in
response to bird weight, (J) kill latency in response to hunger level.
Supplementary Figure 3 | Approach latency (A), attack probability (B), attack
latency (C), handling duration (D), eating duration (E), proportion eaten (F), beak
wiping (G), and kill latency (H) in response to trial number.
Supplementary Figure 4 | Pupal weight in response to diet for male
A. plantaginis raised on a diet with (AG) and without (ART) the addition of
pyrrolizidine alkaloids (10% freeze dried Senecio vulgaris). Moths of the Wy
genotype raised on the pyrrolizidine alkaloid diet were heavier compared to Wy
moths raised on the artificial diet.
Supplementary Figure 5 | Bird water drinking (number of sips) increased in
response to eating duration.
Supplementary Figure 6 | Moth activity in response to pyrrolizidine alkaloid
taste. “no,” moths that were not raised on a pyrrolizidine alkaloid diet; “yes,”
moths that were raised on a pyrrolizidine alkaloid diet. Moths raised on a
pyrrolizidine alkaloid diet were less active.
Supplementary Table 1 | General definition, operative definition and unit of
measure for each type of variable.
Supplementary Table 2 | Model selection using AICc for each response variable.
Interactions and co-variates were selected to be included in the model and
genotype replaced color morph if it improved the AICc score by more than 12.
Supplementary Table 3 | Model summaries and follow up analyses for each
response variable.
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... Previous studies have indeed shown that multiple selective pressures act on the male coloration. The two male morphs are differently protected against predators (Nokelainen et al., 2014;Rojas et al., 2017;Winters et al., 2021), with yellow males generally having higher survival (Nokelainen et al., 2012; Rojas et al., 2017). In addition, male morph mating advantage is dependent on the morph frequency and males that origin from "mixed-morph lines" have higher mating success compared to the moths that originated from more monomorphic lines (Gordon et al., 2018), which suggests that heterozygote advantage may also contribute to the color polymorphism in this species. ...
... The advantage of the dominant (W) allele in our species does not appear to change for fitness-related measures supported by the general advantage of Wy (and WW genotype) and over the general disadvantage of the yy genotype throughout the reproductive output, a pattern somewhat opposite to the wolf of the Yellowstone National Park (Coulson et al., 2011;Hedrick et al., 2014). In contrast, the heterozygosity advantage in the wood tiger moth may be context dependent: in mating probability either due to female choice or intrasexual competition (Gordon et al., 2018), in the reproductive output (this study), or as defense against predators (Winters et al., 2021), which suggests the importance of considering both natural and sexual selective processes. ...
... This suggests that other mechanisms and selective forces are at play. The extensive literature on this study system shows indeed that male morphs experience a multitude of morph-specific selective pressures, from predation (Nokelainen et al., 2012(Nokelainen et al., , 2014Rojas et al., 2017, Winters et al., 2021 linked also to light environment (Nokelainen et al., 2022a), to immune response (Nokelainen et al., 2013), and density-dependent effects . This likely affects the expected ratio of white and yellow morphs in natural populations. ...
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The persistence of intrapopulation phenotypic variation typically requires some form of balancing selection since drift and directional selection eventually erode genetic variation. Heterozygote advantage remains a classic explanation for the maintenance of genetic variation in the face of selection. However, examples of heterozygote advantage, other than those associated with disease resistance are rather uncommon. Across most of its distribution, males of the aposematic moth Arctia plantaginis have two hindwing phenotypes determined by a heritable one locus-two allele polymorphism (genotypes: WW/Wy = white morph, yy = yellow morph). Using genotyped moths we show that the presence of one or two copies of the yellow allele affects several life-history traits. Reproductive output of both males and females, and female mating success are negatively affected by two copies of the yellow allele. Females carrying one yellow allele (i.e. Wy) have higher fertility, hatching success, and offspring survival than either homozygote, thus leading to strong heterozygote advantage. Our results indicate strong female contribution especially at the postcopulatory stage in maintaining the color polymorphism. The interplay between heterozygote advantage, yellow allele pleiotropic effect and morph-specific predation pressure may exert balancing selection on the color locus, suggesting that color polymorphism may be maintained through complex interactions between natural and sexual selection. This article is protected by copyright. All rights reserved.
... For example, resources need to be allocated between defense and reproduction, sequestered plant secondary compounds need to be handled, modified and stored in the body, and certain traits may be advantageous against some predators, but not against others. Hence, for each species the balance between the costs and benefits of expressing defensive traits needs to be met [3]. In addition, potentially suitable secondary plant compounds to be sequestered for defense are not equally available in all host plants. ...
... In contrast, many other Arctiinae species have wing patterns with colorful bright red, orange, yellow or green-blue spots often contrasting with a dark ground color (Figure 1). Their aposematic appearance is usually coupled with chemical defense, either using secondary plant metabolites sequestered from their larval host plants [11], or collected during the adult stage through pharmacophagy (e.g., [12,13]), or by de novo synthesis of toxic compounds by the insects [3]-yet detailed information is unavailable for most tropical species so far. As a result, colorful tiger and lichen moths are often times unpalatable, or even toxic, to their predators, and they signal their unpalatability through their visual appearance [9], in some cases also supplemented by acoustic signals that address bats as nocturnal predators [2,14]. ...
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On tropical mountains, predation pressure decreases with elevation. Accordingly, one expects an elevational decay in the prevalence of costly defensive traits such as aposematic coloration. Using light-trap catches of Arctiinae moths (353 species, 4466 individuals), assembled along a forested gradient in the megadiverse tropical Andes of southern Ecuador, we show that the incidence of aposematic coloration decreases strongly between 1040 and 2670 m asl. While over 60% of Arctiinae moths were warningly colored at lowest sites, this fraction decreased to less than 20% in montane forest, yet increased slightly again at the highest sites in the very open Purdiaea nutans forest. In parallel, the incidence of hymenopteran mimics and of species that mimic chemically defended beetles decreased with elevation. Hymenopteran mimics accounted for less than 5% of Arctiinae moths at sites above 2100 m, and beetle mimics were essentially lacking at high elevations. These patterns coincide with a change in gross taxonomic composition of Arctiinae ensembles and with an increase in average body size towards higher elevations. Representatives of Euchromiina and Ctenuchina became scarce with altitude, whereas the prevalence of Lithosiinae increased. Our findings suggest that the variable selective pressures along the elevational gradient favor warning coloration primarily at lower sites, whereas cryptic appearance of adult moths dominates in the tropical upper montane forest.
... ; https://doi.org/10.1101/2022.05.28.493811 doi: bioRxiv preprint 26 invest more in the deterrent olfactory cue when they are raised with a constant amount of 541 resources (aka in the laboratory) and on a diet from which they cannot sufficiently sequester 542 defensive toxins such as pyrrolizidine alkaloids (PAs). Predators can indeed use more than 543 one cue to assess the toxicity of prey, so multiple defensive compounds can be used as a PAs confers better defences to the moths (Winters et al., 2021). Because the laboratory-raised 548 moths in the current study did not sequester PAs from their diet, it is possible that they 549 invested more in the production of pyrazines. ...
Preprint
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
... 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. ...
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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.
Thesis
Les insectes, comme les autres animaux, sont dotés d’un système gustatif qui leur permet d’analyser les molécules sapides présentes dans leurs aliments, comme des sucres (qui stimulent l’alimentation) ou bien des molécules amères (qui l’inhibent). L’amertume est généralement considérée comme permettant à l’animal d’éviter d’ingérer des substances toxiques. L’amertume d’un composé est testée la plupart du temps en mélangeant un composé potentiellement amer à un aliment et en mesurant la quantité consommée en conditions de choix ou de non-choix. Un tel type de protocole ne permet cependant pas de distinguer entre les effets liés à l’amertume proprement dite de ceux qui sont liés à la toxicité.Dans cette thèse, nous avons essayé d’évaluer si l’amertume était liée à la toxicité, en étudiant les réponses alimentaires et la survie de Drosophila melanogaster vis-à-vis de 7 molécules, choisies pour leur amertume et leur toxicité (quinine, berbérine, benzoate de dénatonium, paraquat, nicotine, escine, caféine). Afin de quantifier le comportement alimentaire, nous avons d’abord établi un protocole de mesure semi-automatique du volume de liquide ingéré par des mouches individuelles. Lorsque ces mouches ont le choix entre un liquide sucré et un liquide contenant une substance amère, nous avons pu observer que les mouches diminuaient leur consommation avec la concentration en molécule amère, et que cette réduction de consommation pouvait également impacter la consommation de solution sucrée. Nous avons également évalué l’impact de ces molécules sur la durée de vie des mouches, lorsqu’elles sont mélangées au milieu alimentaire (liquide ou agar), ou injectée dans l’abdomen de l’animal. Nous avons pu constater que la durée de vie des mouches était fortement corrélée à la quantité de molécules de sucre ingérées plus qu’à la quantité de molécules amères. L’ensemble de ces résultats suggère que l’amertume des molécules inhibe fortement l’alimentation des insectes, plus que la toxicité proprement dite. Par ailleurs, nous nous sommes intéressés à la possibilité de masquage de l’amertume d’une molécule modèle, l’oleuropéine qui est le principal composé de l’amertume de l’huile d’olive, par interaction avec une protéine laitière, la β-lactoglobuline. L’efficacité du masquage de l’amertume a été évaluée par consommation de mélange oleuropéine/β-lactoglobuline par les mouches. L’amertume de l’oleuropéine semble être masquée par la protéine pour des concentrations en oleuropéine inférieures ou égales à 0,1 mM.
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The definition of colour polymorphism is intuitive: genetic variants express discretely coloured phenotypes. This classification is, however, elusive as humans form subjective categories or ignore differences that cannot be seen by human eyes. We demonstrate an example of a 'cryptic morph' in a polymorphic wood tiger moth (Arctia plantaginis), a phenomenon that may be common among well-studied species. We used pedigree data from nearly 20,000 individuals to infer the inheritance of hindwing colouration. The evidence supports a single Mendelian locus with two alleles in males: WW and Wy produce the white and yy the yellow hindwing colour. The inheritance could not be resolved in females as their hindwing colour varies continuously with no clear link with male genotypes. Next, we investigated if the male genotype can be predicted from their phenotype by machine learning algorithms and by human observers. Linear discriminant analysis grouped male genotypes with 97% accuracy, whereas humans could only group the yy genotype. Using vision modelling, we also tested whether the genotypes have differential discriminability to humans, moth conspecifics and their bird predators. The human perception was poor separating the genotypes, but avian and moth vision models with ultraviolet sensitivity could separate white WW and Wy males. We emphasize the importance of objective methodology when studying colour polymorphism. Our findings indicate that by-eye categorization methods may be problematic, because humans fail to see differences that can be visible for relevant receivers. Ultimately, receivers equipped with different perception than ours may impose selection to morphs hidden from human sight.
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