Acoustical mimicry in a predatory social parasite of ants

Abstract

Rapid, effective communication between colony members is a key attribute that enables ants to live in dominant, fiercely protected societies. Their signals, however, may be mimicked by other insects that coexist as commensals with ants or interact with them as mutualists or social parasites. We consider the role of acoustics in ant communication and its exploitation by social parasites. Social parasitism has been studied mainly in the butterfly genus Maculinea, the final instar larvae of which are host-specific parasites of Myrmica ants, preying either on ant grubs (predatory Maculinea) or being fed by trophallaxis (cuckoo Maculinea). We found similar significant differences between the stridulations of model queen and worker ant castes in both Myrmica sabuleti and Myrmica scabrinodis to that previously reported for Myrmica schencki. However, the sounds made by queens of all three Myrmica species were indistinguishable, and among workers, stridulations did not differ significantly in two of three species-pairs tested. Sounds recorded from the predatory caterpillars and pupae of Maculinea arion had similar or closer patterns to the acoustics of their host Myrmica sabuleti than those previously reported for the cuckoo Maculinea rebeli and its host Myrmica schencki, even though Maculinea rebeli caterpillars live more intimately with their host. We conclude that chemical mimicry enables Maculinea larvae to be accepted as colony members by worker ants, but that caterpillars and pupae of both predatory and cuckoo butterflies employ acoustical mimicry of queen ant calls to elevate their status towards the highest attainable position within their host's social hierarchy.

Full text

4084
INTRODUCTION
Effective communication is fundamental to the ability of social
insects to live in complex hierarchical societies, in which different
castes or individuals perform different activities, yet each type of
behaviour is so well integrated that the colony functions as a
‘superorganism’ (Hölldobler and Wilson, 2009). In ants, the primary
method of communication involves chemical and, to a lesser extent,
tactile cues (Hölldobler and Wilson, 1990; Hölldobler and Wilson,
2009). Acoustics also plays a role among the adults of four ant
subfamilies (Ponerinae, Nothomyrmecinae, Pseudomyrmecinae,
Myrmicinae) that can stridulate (Markl, 1973; Taylor, 1978), and
others that drum the substrate, by inducing specific behaviours in
receiving individuals (Markl, 1965; Barbero et al., 2009) or by
amplifying or modulating the effects of pheromones (Markl and
Hölldobler, 1978; Baroni-Urbani et al., 1988; Hölldobler, 1999).
Although acoustics has generally been regarded as ‘weakly
developed’ in ants (Hölldobler and Wilson, 1990; Hölldobler and
Wilson, 2009; Keller and Gordon, 2009), it is also the least studied
of their communication systems (Barbero et al., 2009). Indeed,
knowledge of the structures involved in the production and
reception of signals is incomplete. Most sounds or vibrations are
produced by a minutely ridged stridulating organ (pars stridens)
situated on the mid-dorsal edge of the fourth ‘abdominal’ segment,
and by an embossed spike (plectrum) projecting from the rear
edge of the post-petiole (Giovannotti, 1996; Grandi, 1966; Pavan
et al., 1997; Grasso et al., 1998; Ruitz et al., 2006). When an ant
raises and lowers her gaster, the two structures rub together and
emit a series of ‘chirps’ (Hölldobler and Wilson, 1990; Roces
and Hölldobler, 1996; Ruitz et al., 2006). Stridulations are
defined by the transmitting medium, and may be sounds
transmitted by air or vibrations transmitted by the substrate. Many
myrmecologists maintain that ants cannot hear the aerial
component of stridulations and perceive only substrate-transmitted
vibrations (Fielde and Parker, 1904; Roces and Tautz, 2001;
Hölldobler and Wilson, 2009), an idea supported by the discovery
of a subgenual organ in Camponotus ants (Menzel and Tautz,
1994). However, evidence for the perception of air-transmitted
sounds is accumulating, at least over distances of a few
centimetres (Hickling and Brown, 2000; Hickling and Brown,
2001; Roces and Tautz, 2001). Possible receptors of air-borne
sounds are the Johnston’s organ (Masson and Gabouriaut, 1972)
or the trichoid organs on the tips of the antennae (Dumpert, 1972),
although the latter, in ants, differ in structure from the filiform
hair trichoid sensilla used by other insects and spiders for auditory
reception (Tautz, 1977; Kumagai et al., 1998; Barth, 2000).
However sounds are detected, ant acoustics frequently signal
antagonistic or distress behaviours between workers in a colony,
including alarm (Markl, 1965; Markl, 1985; Roces and Hölldobler,
1995), intimidation (Stuart and Bell, 1980; Ware, 1994), aposematic
‘threatening’ (Santos et al., 2005), or a call for rescue after a cave-
in of the nest (Markl, 1985). In addition, when combined with
pheromones or in isolation, several species stridulate or drum during
foraging to enhance worker recruitment to food sources (Markl and
Hölldobler, 1978; Baroni-Urbani et al., 1988; Roces et al., 1993;
Hölldobler and Roces, 2000): in Atta stridulations are most frequent
when leaves of the highest quality for fungal cultures are found
(Hölldobler and Roces, 2000). Myrmica workers frequently
stridulate during trophallaxis, particularly the receiving worker when
The Journal of Experimental Biology 212, 4084-4090
Published by The Company of Biologists 2009
doi:10.1242/jeb.032912
Acoustical mimicry in a predatory social parasite of ants
F. Barbero1,2, S. Bonelli1, J. A. Thomas3, E. Balletto1and K. Schönrogge2
1Department of Animal and Human Biology, University of Turin, 10123 Turin, Italy, 2Centre for Ecology and Hydrology, Maclean
Building, Wallingford, Oxfordshire, OX10 8BB, UK and 3Department of Zoology, University of Oxford, Tinbergen Building, Oxford,
OX1 3PS, UK
Author for correspondence (simona.bonelli@unito.it)
Accepted 3 June 2009
SUMMARY
Rapid, effective communication between colony members is a key attribute that enables ants to live in dominant, fiercely
protected societies. Their signals, however, may be mimicked by other insects that coexist as commensals with ants or interact
with them as mutualists or social parasites. We consider the role of acoustics in ant communication and its exploitation by social
parasites. Social parasitism has been studied mainly in the butterfly genus
Maculinea
, the final instar larvae of which are host-
specific parasites of
Myrmica
ants, preying either on ant grubs (predatory
Maculinea
) or being fed by trophallaxis (cuckoo
Maculinea
). We found similar significant differences between the stridulations of model queen and worker ant castes in both
Myrmica sabuleti
and
Myrmica scabrinodis
to that previously reported for
Myrmica schencki.
However, the sounds made by
queens of all three
Myrmica
species were indistinguishable, and among workers, stridulations did not differ significantly in two of
three species-pairs tested. Sounds recorded from the predatory caterpillars and pupae of
Maculinea arion
had similar or closer
patterns to the acoustics of their host
Myrmica sabuleti
than those previously reported for the cuckoo
Maculinea rebeli
and its
host
Myrmica schencki
, even though
Maculinea rebeli
caterpillars live more intimately with their host. We conclude that chemical
mimicry enables
Maculinea
larvae to be accepted as colony members by worker ants, but that caterpillars and pupae of both
predatory and cuckoo butterflies employ acoustical mimicry of queen ant calls to elevate their status towards the highest
attainable position within their host’s social hierarchy.
Key words: Lycaenidae butterfly,
Myrmica
ant,
Maculinea
,
Phengaris
, acoustic mimicry, stridulation, cuckoo, predatory parasite.
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4085Social parasite mimics ant acoustics
food decreases (Stuart and Bell, 1980; Zhantiev and Sulkanov,
1977).
Inter-caste acoustical communication has been recorded in only
a few instances. Mating queens of Pogonomyrmex badius stridulate
to signal to males when their spermathecae are full (Markl et al.,
1977), whereas in Atta those workers that cut leaves stridulate when
they are ready to return to the nest. This recruits individuals of the
smallest minim caste to climb onto the leaf fragment from where
they protect their larger sisters from attack by phorid flies during
the journey home (Roces and Hölldobler, 1995). Until recently, there
was no direct evidence that different members of an ant society
produced distinctive caste-specific sounds to induce appropriate
patterns of behaviour in fellow or other castes, although this was
implied when Markl (Markl, 1968) found that the major workers
of Atta cephalotes produce more intense sounds, that carry further,
than their smaller nestmates, and by Grasso and colleagues’ (Grasso
et al., 1998) demonstration that the space between the ridges of the
pars stridens in queens of Messor species was greater than in the
workers. Working with Myrmica schencki, we reported that the
difference in width between the ridges of workers and queens was
larger than in Messor, and that despite a considerable overlap, the
queens made distinctive sounds from the workers (Barbero et al.,
2009). When recordings of unstressed adult M. schencki were played
back to laboratory cultures of workers, the sounds of both castes
induced benign responses including aggregation and antennation at
the speaker. Moreover, when workers were played their queen’s
sounds, they stood ‘on guard’ on the speaker to a much greater extent
than when worker sounds were played, each holding the
characteristic posture adopted by a Myrmica worker when protecting
an object of high value to the colony (Barbero et al., 2009).
The chemical component of ant communication systems is highly
specific, enabling colony members to recognise kin ants as well as
conspecifics. Nevertheless, approximately 10,000 other invertebrate
species live as social parasites within ant colonies, where they exploit
the rich resources concentrated inside nests (Thomas et al., 2005a).
The mechanism whereby they penetrate and often integrate with
their host society has been studied in very few social parasites, but
generally involves corrupting the honest signals of the ants
(Hölldobler and Wilson, 1990; Akino et al., 1999; Lenoir et al.,
2001; Thomas et al., 2005a; Hojo et al., 2009). European species
of Maculinea butterflies are among the better understood examples.
The adult butterfly is free-living and oviposits on a specific food
plant(s), on which the first three larval instars feed. On entering the
fourth and final instar, weighing just 1–2% of its ultimate biomass,
the small larva (hereafter called a caterpillar) falls from its plant
and secretes semio-chemicals that mimic the surface hydrocarbons
of Myrmica ants, causing any forager that encounters it to carry the
caterpillar to the brood chambers of the underground ant nest
(Thomas, 1984; Thomas, 2002; Elmes et al., 1991a). Two strategies
have evolved in Maculinea to exploit Myrmica resources.
Caterpillars of the cuckoo species, Maculinea rebeli and M. alcon,
remain in the brood chambers for 11–23months, where they are
tended and fed directly by the nurse ants on regurgitations and other
food (Elmes et al., 1991a; Elmes et al., 1991b). Caterpillars of M.
arion and M. teleius are predators of ant larvae, and inhabit
peripheral cells from which they periodically foray to binge-feed
in the brood chambers (Thomas and Wardlaw, 1992).
With a few apparent exceptions (Barbero, 2007; Tartally et al.,
2008), all Maculinea species or populations are specific to one
primary host species or local genotype of Myrmica at a regional
scale (Thomas et al., 1989; Thomas et al., 2005a; Thomas et al.,
2005b; Nash et al., 2008). In cuckoo species, host specificity is
explained by chemical mimicry of the host’s communication system.
Pre- and newly adopted caterpillars of cuckoo Maculinea are weak
chemical mimics of their regional host (Akino et al., 1999; Nash et
al., 2008), but within a few days are given preferential care. Thus,
in western Europe where its host is Myrmica schencki, Maculinea
rebeli caterpillars are rescued ahead of the ant brood when a colony
is disturbed, and are fed in preference to M. schencki larvae when
food is scarce (Thomas et al., 1998). This deeper infiltration
coincides with the secretion of additional hydrocarbons that more
closely mimic the distinctive odour of M. schencki, but inevitably
make this genotype of M. rebeli a poor (and seldom tolerated) mimic
of other Myrmica species (Elmes et al., 2004; Schönrogge et al.,
2004).
Neither chemical mimicry nor their begging behaviour explains
why M. rebeli caterpillars are treated in preference to host ant brood.
Instead, we have suggested that acoustical cues are employed
(Barbero et al., 2009). It is well known that certain pupae and
caterpillars of Lycaenidae produce sounds; the former from tooth-
and-comb stridulatory organs between the fifth and sixth segments
(Downey, 1966; Downey and Allyn, 1973; Downey and Allyn,
1978; Pierce et al., 2002), whereas caterpillar sounds probably
emanate from muscular contraction and air compression through
the trachaea (Schurian and Fiedler, 1991). The acoustics of
mutualistic lycaenid species do not obviously mimic ant
stridulations, and an attraction to ants has been demonstrated only
in the pupae of one extreme mutualist (Travassos and Pierce, 2000;
Pierce et al., 2002). The calls of socially parasitic Maculinea
caterpillars, however, more closely resemble Myrmica worker
stridulations, although the putative mimicry appeared to be modelled
on the genus rather than a host species of Myrmica (DeVries et al.,
1993). The same study showed that Myrmica larvae are mute,
suggesting that in this trait Maculinea caterpillars are mimicking
an adult ant cue.
The recordings by DeVries et al. (DeVries et al., 1993) were
restricted to distressed worker ants and caterpillars, and were not
played back to the ants. Using modern equipment, we recently found
that unstressed Maculinea rebeli caterpillars and pupae were close
acoustical mimics of Myrmica schencki, and that the sounds
produced by both butterfly stages were significantly closer to those
of the queen ants than the workers (Barbero et al., 2009). Playing
M. rebeli sounds back to laboratory cultures of M. schencki workers
resulted in similar enhanced ‘on guard’ (and other) benign
behaviours as when the ants were played the stridulations of their
own queens, especially when pupal calls were played. We concluded
that although chemical mimicry is used to gain and maintain
acceptance in a M. schencki society, the social parasite
simultaneously employs acoustical mimicry inside the nest to
advance its status to that of the highest member in its host’s
hierarchy.
DeVries et al. (DeVries et al., 1993) showed that caterpillars of
predatory Maculinea species also produce sounds that appear to
mimic Myrmica (worker) stridulations, although in nature they are
less closely integrated with their host’s society (Thomas et al.,
2005a). We speculated that they might be less perfect acoustical
mimics of their hosts. Here we test this idea by comparing the
acoustics of unstressed Maculinea arion caterpillars and pupae with
those of the queens and workers of its host ant, Myrmica sabuleti,
and with our published data for Maculinea rebeli and Myrmica
schencki. We also compare the worker and queen sounds of M.
sabuleti, and those of another ant Myrmica scabrinodis, to determine
whether the distinctive acoustical communication made by different
castes of M. schencki exists in its congeners.
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MATERIALS AND METHODS
Materials
Myrmica colonies were excavated in the field and set up as laboratory
ant colonies of >100 workers in 28cm⫻15cm⫻10cm Perspex
containers and maintained on a diet of sugar and Drosophila larvae. We
collected Myrmica schencki Emery 1894 colonies (N12) in May 2006
at Colle di Tenda, Piedmont, Italy where this species is host to
Maculinea rebeli (Barbero et al., 2009). Two colonies of M. sabuleti
Meinert 1861 were collected from Italy and four from Somerset, UK
from sites where they are host to Maculinea arion. Eleven colonies of
M. scabrinodis Nylander 1846 were taken from Dorset, UK from sites
with no Maculinea parasites, although this ant is host to Maculinea
alcon and M. teleius elsewhere in Europe (Thomas et al., 2005a). The
post-adoption larvae and pupae of Maculinea arion Linnaeus 1758
were collected in Somerset, UK and kept with M. sabuleti colonies until
they were recorded.
Stridulation organ morphology
M. sabuleti and M. scabrinodis ants (three queens and three workers
of each species) were kept in 70% ethanol and dissected between
the post-petiolum and the abdomen to expose the pars stridens and
the plectrum. The two ant parts were mounted on the same steel
stub, coated with gold, and the distances between adjacent ridges
of the pars stridens were measured automatically (10 measurements
per individual) using a Cambridge Stereoscan S360 scanning
electron microscope (SEM). The SEM operated at 20–25kV.
F. Barbero and others
Sound recording
Recordings were made of individual workers and queens of the three
Myrmica species (Table1) using the procedures described by
Barbero et al. (Barbero et al., 2009). We also recorded four pre-
adoption caterpillars of Maculinea arion, i.e. before they came into
contact with the ants, three post-adoption caterpillars and three
pupae. The sounds for M. rebeli and Myrmica schencki used in the
analyses are the same as those used in Barbero et al. (Barbero et
al., 2009) (Table1).
The recording equipment consisted of a 12.5cm⫻8cm⫻2cm
recording chamber with a moving-coil miniature microphone
attached through the centre. A second microphone of the same type
was used to record ambient noise but in anti-phase. An amplifier
was attached to each microphone and calibrated to maximise the
noise cancellation of ambient noise from the two microphones,
leaving the signal from the recording chamber. The resulting signal
was processed through a two-stage low-noise amplification before
being recorded digitally on a laptop computer using Audacity v.
1.2.4 (http://audacity.sourceforge.net/). To further reduce ambient
noise and interference, the equipment was powered by a 12V gel
cell battery, and the recording chamber and microphones were placed
inside an anechoic chamber. Sounds were recorded for 15min
periods starting 5min after an insect was introduced and had become
calm.
Statistical analysis
The sound parameters used by DeVries et al. (DeVries et al., 1993)
and Barbero et al. (Barbero et al., 2009), dominant frequency (DF;
Hz), pulse repetition frequency (the reciprocal of the duration of
one pulse; PRF; s–1) and pulse length (PL; s), were measured using
Audacity 1.2.4. To test whether sound differed between groups
we calculated the pairwise normalized Euclidean distance over
all three parameters and used a nested (‘Individual’ within
‘Group’) ANalysis Of SIMilarity implemented in Primer v.
6.1.12 (Primer-E Ltd.). Groupings were visualised using non-
parametric multi-dimensional scaling (nMDS) with sound
fragments averaged within individuals. To further test whether
the overall sounds produced by butterfly pupae and larvae were
more similar to queens or workers of the two host ants M. sabuleti
and M. schencki, we estimated mean Euclidean distances between
groups and used Student’s t-test to estimate the significance of
the differences. Student’s t-test was also used to establish
the differences in the morphometric measurements on the
stridulation organs of M. sabuleti and M. scabrinodis queens and
workers.
Table1. Source and number of the sound fragments analysed in
this study
No. of No. of sound
Species Group individuals fragments
Maculinea arion
Pre-adoption larvae 4 98
Post-adoption larvae 3 89
Pupae 3 89
Maculinea rebeli
Post-adoption larvae 10 292
Pupae 4 115
Myrmica sabuleti
Queens 8 226
Workers 8 240
Myrmica schencki
Queens 11 285
Workers 13 345
Myrmica scabrinodis
Queens 6 180
Workers 5 180
Sounds from
Maculinea rebeli
and
Myrmica schencki
are the identical
fragments used in Barbero et al., 2009.
Table2. Average values of the three sound parameters for the casts and stages of the three
Myrmica
species and
Maculinea arion
and
M. rebeli
Group Pulse length (s) Pulse repetition frequency (s–1) Dominant frequency (Hz)
M. arion
pre-adoption larvae 0.029±0.002 10.08±0.71 561.6±8.79
M. arion
post-adoption larvae 0.038±0.005 4.55±0.34 240.6±29.9
M. arion
pupae 0.012±0.003 13.74±0.66 1070.0±47.6
M. rebeli
larvae 0.036±0.005 13.32±1.40 404.3±39.23
M. rebeli
pupae 0.040±0.005 12.84±1.02 530.9±13.51
M. sabuleti
queens 0.019±0.001 36.99±1.96 832.8±33.18
M. sabuleti
workers 0.017±0.001 40.39±1.99 1285.3±80.6
M. scabrinodis
queens 0.023±0.003 31.62±4.04 810.7±31.1
M. scabrinodis
workers 0.021±0.002 29.93±2.61 1480.3±161.4
M. schencki
queens 0.021±0.001 34.50±2.53 812.2±40.1
M. schencki
workers 0.021±0.002 39.03±3.37 1132.3±105.2
Values are means ± s.e.m.
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4087Social parasite mimics ant acoustics
RESULTS
Worker and queen acoustics in three species of
Myrmica
ant
The workers of all Myrmica species recorded to date have been
shown to stridulate. We recorded both workers and queens of
Myrmica sabuleti, M. scabrinodis and M. schencki to compare
species and casts. Average measurements for the three sound
parameters PL, PRF and DF are listed in Table2. Using a
multivariate approach over the three sound parameters, the
normalised Euclidean distance within each group were (mean ±
s.e.m.): M. sabuleti queens: 0.68±0.05, workers: 0.97±0.08; M.
scabrinodis queens: 1.26±0.19, workers: 1.38±0.22; M. schencki:
queens: 0.97±0.06, worker: 1.74±0.05. The sounds of M. schencki
workers are quite diffuse (Fig.1), whereas those of the other
workers are more clustered. However, the sounds of queens of all
three species were very similar and closely overlap, as seen in
Fig.1. This is also reflected in the results of a nested ANOSIM
to test for pairwise differences between groups. Among the ant
groups, M. schencki workers were significantly different only from
their own queens (ANOSIM R0.12, P0.04), and did not separate
from the workers or queens of the other species (R<0.12, P>0.17).
More intriguingly, there was no significant difference in the sounds
made by the queens of the three species tested (M. sabuleti versus
M. scabrinodis R0.05, P0.22; M. sabuleti vs M. schencki
R0.002, P0.43; M. scabrinodis vs M. schencki R0.11, P0.15).
All other pairwise comparisons among groupings show significant
separation (R≥0.49, P≤0.01). In other words, as previously
demonstrated in M. schencki, the queens of both M. sabuleti and
M. scabrinodis made distinctive sounds that were significantly
different from those of their workers. This difference reflects the
distinct stridulatory organ morphology of the two castes (Fig.2).
As shown in M. schencki, the distance between the pars stridens
ridges in M. sabuleti (queens: 1.61±0.18m, workers: 1.35±
0.11m) and M. scabrinodis (queens: 1.86±0.50m, workers:
1.27±0.48m) differs between queen and worker ants (two-
sample t-test tM.sabuleti6.53, d.f.58, P<0.001; tM.scabrinodis4.66,
d.f.58, P<0.001).
Sound similarities of larvae and pupae of the predatory
Maculinea arion
and workers and queens of its host ant
Myrmica sabuleti
Using a nested ANOSIM we found that sounds from M. arion pupae
differ from those of pre-adoption larvae (R1, P0.029), yet they
apparently do not differ from the post-adoption larvae (R1, P0.1;
Fig.3). However, we interpret the results in Fig.3, as an artefact of
small sample size. Since data were available for only three pupae
and three post-adoption larvae, there were only 10 possible
permutations, and in one instance R, after permutation, was greater
than the observed R, giving a level for P of 0.1.
Group
M. sabuleti Q
M. sabuleti W
M. scabrinodis Q
M. scabrinodis W
M. schencki Q
M. schencki W
Distance
1.55
1.8
2D Stress: 0.03
Fig.1. MDS plot of the normalised Euclidean distances of the
queens and workers of the three model ants,
Myrmica
sabuleti
,
M. scabrinodis
and
M. schencki
. Solid symbols
indicate data from queens (Q), open symbols are data from
workers (W). The contours indicate the normalised Euclidean
distance separating the groups.
Fig.2. The
pars stridens
on the stridulatory organs of a
M. sabuleti
queen (A), a worker (B), and of a
M. scabrinodis
queen (C) and worker (D).
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4088
The acoustical signals of all three groups of butterfly differed
significantly from both M. sabuleti queens and workers (Fig.3). t-
Tests comparing the between-group normalised Euclidean distances
of the butterfly to workers and queens showed that the acoustics
made by all three stages of M. arion were significantly closer to
the stridulations of the host queens rather than workers (Pupae:
distancequeens1.98±0.07, distanceworker3.33±0.09, t11.3, d.f.41,
P<0.001; pre-adoption larvae: dqueens2.32±0.08, dworker3.13±0.08,
t7.18, d.f.61, P<0.001; and post-adoption larvae: dqueens
3.33±0.09, dworker4.22±0.11, t6.13, d.f.45, P<0.001).
Comparison of
Maculinea arion
(predator) and
M. rebeli
(cuckoo) as mimics of their host ants
Owing to their cuckoo feeding life style, M. rebeli larvae, although
not the pupae, are in more frequent and closer contact with ant
workers than those of M. arion. We therefore predicted that M. rebeli
might be a closer acoustical mimic of its host M. schencki than M.
arion of M. sabuleti. In the absence of behavioural data, we
compared the normalised Euclidean distances of sounds of the larval
and pupal stages of both Maculinea species to the stridulations of
both Myrmica species’ queens and workers (Table3).
We found no evidence that M. rebeli is a closer mimic of M.
schencki than M. arion is to M. sabuleti. In fact, with one exception
(M. arion pre-adoption larvae and M. rebeli pupae are equidistant
to M. schencki workers), in all comparisons between M. arion pupae
F. Barbero and others
or pre-adoption larvae and M. rebeli larvae or pupae with queens
and workers of either ant species, the acoustics of M. arion were
significantly closer to Myrmica stridulations than were those of M.
rebeli. Only the acoustics of M. arion post-adoption larvae were
found to be more distant than either stage of M. rebeli to both castes
of either ant species (Table3). Overall, the acoustics made by the
immature stages of M. arion were 19.7% closer to Myrmica
stridulations than those from M. rebeli (max39.3% M. arion pupa
and M. rebeli larva vs M. sabuleti workers, min6.5% M. arion pre-
adoption larva and M. rebeli larva vs M. sabuleti workers). This
comfortably encompasses the range of acoustical variation found
between M. rebeli larvae and pupae, where differences in the worker
responses to butterfly sounds have been demonstrated (Barbero et
al., 2009).
DISCUSSION
Our results demonstrate that stridulating queens from two additional
Myrmica species make distinctive sounds to those of their workers
using morphologically distinct organs. Indeed, intra-specific inter-
caste differences were more clear-cut in M. scabrinodis and M.
sabuleti than we previously reported for M. schencki (Barbero et
al., 2009). Less expected was the fact that queen stridulations from
the three Myrmica species were indistinguishable, as were worker
stridulations in two of the three pairs of species tested. This suggests
that acoustics plays little or no part in the cues used by Myrmica
Group
M. arion P
M. arion post-ad L
M. arion pre-ad L
M. sabuleti Q
M. sabuleti W
Distance
1.7
2.1
2D Stress: 0.03
Fig.3. MDS plot of the normalised Euclidean distances of the
developmental stages of the predatory social parasite
Maculinea
arion
and the queens and workers of its host ant
Myrmica
sabuleti
. Open symbols indicate the pupal stage of
M. arion
and
the workers of
M. sabuleti
. The final instar larval stages of
M.
arion
are separated into pre-adoption (pre-ad), before the larvae
had contact with ants, and post-adoption (post-ad) recorded
after they were in contact with ants. The contours indicate the
normalised Euclidean distance separating the groups. ANOSIM
indicates significant separation between ant queens and workers
and the larval and pupal stages of
M. arion
(see text). Pupae
also differed from
M. sabuleti
queens (
R
0.99,
P
0.006) and
workers (
R
0.99,
P
0.006), as did pre-adoption larvae (queens:
R
1,
P
0.002; workers:
R
1,
P
0.002) and post-adoption larvae
(queens:
R
1,
P
0.006; workers:
R
1,
P
0.006). P, pupa; L,
larva; Q, queen; W, worker.
Table3. Pairwise
t
-test of normalized Euclidian distances between the predatory social parasite
Maculinea arion
, the cuckoo feeding
parasite
Maculinea rebeli
and the queens and workers of their respective host ants
Myrmica sabuleti
and
Myrmica schencki
M. arion
pre-adoption
M. arion
post-adoption
M. sabuleti
queens
M. arion
pupa (t; d.f.;P) larva (t; d.f.;P) larva (t; d.f.;P)
M. rebeli
pupae 5.61; 46; <0.001* 3.19; 50; 0.002* 3.36; 53; 0.001†
M. rebeli
larvae 4.85; 101; <0.001* 2.59; 108; 0.011* 3.60; 94; 0.001†
M. sabuleti
workers
M. rebeli
pupae 9.99; 46; <0.001* 2.94; 55; 0.005* 4.09; 53; <0.001†
M. rebeli
larvae 9.96; 101; <0.001* 3.07; 109; 0.003* 3.65; 86; <0.001†
M. schencki
queens
M. rebeli
pupae 4.47; 69; <0.001* 3.15; 79; 0.002* 3.41; 74; 0.001†
M. rebeli
larvae 4.22; 137; <0.001* 2.90; 147; 0.004* 3.69; 115; <0.001†
M.
schencki workers
M. rebeli
pupae 4.17; 74; <0.001* 1.63; 100; n.s. 2.62; 84; 0.011†
M. rebeli larvae 5.76; 160; <0.001* 2.35; 125; 0.02* 2.49; 77; 0.015†
*The mean distance of the
M. arion
pupae or larvae is closer; †the mean distance to
M. rebeli
larvae or pupae is closer; n.s., not significant.
THE JOURNAL OF EXPERIMENTAL BIOLOGY

4089Social parasite mimics ant acoustics
to distinguish between non-kin or other species of ant and members
of their own society, and indeed numerous studies demonstrate the
predominant role of chemical cues and the gestalt odour in colony
recognition or physiological states within an ant society (Hölldobler
and Wilson, 1990). However, our recent results suggest that
acoustical communication, in isolation, is capable of signalling at
least the caste and the status of a colony member, and of inducing
appropriate behaviour towards it by the workers (Barbero et al.,
2009). It is possible that caste differences are yet more distinctive
than the data presented here (Figs1, 2), since to date we have
measured and compared only the three attributes most commonly
studied in ant–butterfly acoustics: dominant frequency, pulse
repetition frequency and pulse length. Furthermore, when analysing
our recordings of Myrmica acoustics, we noted that a wide variety
of sound sequences was made by an individual queen or worker
(see Barbero et al., 2009) (Fig.2). During bioassays, we played the
full repertoire of a test ant to cultures of workers, but it is possible
that different component phrases induce one or other of the three
behaviours observed in these tests (aggregation, antennation or guard
attendance). It is also probable that individual ants can alter the
rhythms, speed and intensity of stridulations to communicate other
information under more natural or different conditions to those
imposed by our experiments. For example, our equipment and
procedures ensured that the ants were unstressed both at the time
of recording and when receiving signals; and we observed only
benign responses, with none of the antagonistic or alarm behaviours
induced in early experiments, when ants were often distressed; and
vice versa (Hölldobler and Wilson, 1990; Barbero et al., 2009).
Yet our experimental procedures were far from natural, and we
suspect that a wider spectrum of information might involve
acoustical communication. To draw a parallel from vertebrates, the
main benefit of acoustical communication is its flexibility, allowing
many short-lived signals to convey different information in a short
time, by changing the pitch, harmonies or volume of the sound
(Krebs and Davies, 1993; Greenfield, 2002). We have not yet studied
whether different castes of Myrmica ant respond differently when
exposed to the same sounds, although this seems probable, because
queen Myrmica schencki respond aggressively when introduced to
Maculinea rebeli pupae (which mimic queen sounds) whereas the
workers tend them gently (Barbero et al., 2009) (Supporting Online
Material: http://www.sciencemag.org/cgi/content/full/323/5915/
782/DC1). And it is of course probable that the distinctive acoustical
signals made by different members of an ant society modulate
chemical and tactile cues in different ways.
Confirmation that the queens in other Myrmica societies make
distinctive sounds from their workers indicates that this model is
available to other mimetic Maculinea species or races that parasitise
other Myrmica species, as we confirm here for Maculinea arion
and Myrmica sabuleti. However, the demonstration that queens
make the same sound in all three host ants studied suggests that
acoustical mimicry functions strictly to raise the hierarchical status
of a social parasite once it has been successfully accepted as a
chemical mimic by a host society. In other words, acoustical mimicry
is genus rather than species specific, as DeVries et al. (DeVries et
al., 1993) concluded, albeit after recording highly stressed ants and
Maculinea caterpillars. Although this presumably conveys a
considerable benefit when resources in a nest are scarce – a frequent
occurrence when Myrmica colonies are parasitised by
supernumerary caterpillars of predatory (Thomas and Wardlaw,
1992) or cuckoo Maculinea caterpillars (Thomas et al., 1993) – it
does not influence host specificity. For example, despite their similar
acoustics, mortality of Maculinea arion caterpillars is more than
five times greater in Myrmica scabrinodis nests than when adopted
by M. sabuleti, whereas mortality of (western) Maculinea rebeli
caterpillars is more than 30 times higher when adopted by M. sabuleti
than by their primary host, M. schencki (Thomas et al., 1989; Thomas
et al., 2005a).
In our earlier study of M. rebeli–M. schencki interactions, we
found that the pupal stage of the social parasite was a closer
acoustical mimic of host queens than the caterpillar. This was
demonstrated by worker ant behaviour, where pupal and queen
sounds elicited characteristic ‘on guard’ behaviour at equal
frequency, as did larval sounds, but to a slightly lesser degree
(Barbero et al., 2009). That and this study also illustrate the
amount of variation in acoustic signals found within and between
the groups. The only guidance currently available of how much
difference in acoustical similarity results in a difference in worker
behavioural response comes from the original study, where, for
instance, there were 27% more differences between the pupal and
the worker calls than between the pupal and the queen calls,
resulting in behaviour frequencies that were the same as towards
queens, but significantly different from workers (Barbero et al.,
2009). In this study, between-group differences in acoustical
similarity ranged from ~6% to ~40%, and those of M. arion pupae
and larvae tended to be closer to the queens than those of M.
rebeli pupae and larvae, suggesting that the predatory species is
at least as good a mimic as the cuckoo-feeding one. Further
behavioural work should focus on the response norm of the
receiving partner, i.e. the variation in the cue that is still accepted
to trigger a behaviour, which in this case would be both workers
and queens. This could be achieved by varying particular aspects
of the sounds used for behavioural assays by computer
manipulation. That M. arion caterpillars are apparently closer
mimics of queen Myrmica would suggest that this interaction
evolved as a basal trait in the Phengaris–Maculinea clade of
Lycaenidae, for molecular studies suggest that the predatory life
style preceded the evolution of the cuckoo forms (Als et al., 2004).
Behavioural assays and further recordings will allow acoustic
signalling to be used to address these evolutionary hypotheses.
As in ant communication, we suspect that the role of acoustics
has been underestimated in the few studies made to date of the
adaptations with which an estimated 10,000 species of invertebrate
social parasite succeed in cheating ant societies. Promising taxa for
future research are Lepidochrysops and 11 other lines of lycaenid
butterfly that have independently evolved social parasitism from
mutualistic, presumably sound-producing, ancestors (Fiedler, 1998):
Myrmecophila species of cricket; the stridulations of staphilinid
beetle social parasites; and the many inquiline ‘queen’ ants that
parasitise other ants, including their close relatives.
We thank Luca Casacci for help during fieldwork and Mark Charles for designing
and building the acoustic equipment. This study was supported by the Italian
Ministry of the Environment and the Biodiversa project CLIMIT (CLimate change
impacts on Insects and their MITigation).
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