Chemical and vibratory signals used in alarm communication in the termite Reticulitermes flavipes (Rhinotermitidae)

Abstract

Termites have evolved diverse defence strategies to protect themselves against predators, including a complex alarm communication system based on vibroacoustic and/or chemical signals. In reaction to alarm signals, workers and other vulnerable castes flee away while soldiers, the specialized colony defenders, actively move toward the alarm source. In this study, we investigated the nature of alarm communication in the pest Reticulitermes flavipes. We found that workers and soldiers of R. flavipes respond to various danger stimuli using both vibroacoustic and chemical alarm signals. Among the danger stimuli, the blow of air triggered the strongest response, followed by crushed soldier head and light flash. The crushed soldier heads, which implied the alarm pheromone release, had the longest-lasting effect on the group behaviour, while the responses to other stimuli decreased quickly. We also found evidence of a positive feedback, as the release of alarm pheromones increased the vibratory communication among workers and soldiers. Our study demonstrates that alarm modalities are differentially expressed between castes, and that the response varies according to the nature of stimuli.

Full text

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Insectes Sociaux
https://doi.org/10.1007/s00040-018-00682-9
RESEARCH ARTICLE
Chemical andvibratory signals used inalarm communication
inthetermite Reticulitermes flavipes (Rhinotermitidae)
O.Delattre1,2· J.Šobotník1· V.Jandák3· J.Synek1· J.Cvačka4· O.Jiříček3· T.Bourguignon1,5· D.Sillam‑Dussès2,6
Received: 22 November 2017 / Revised: 3 December 2018 / Accepted: 4 December 2018
© International Union for the Study of Social Insects (IUSSI) 2018
Abstract
Termites have evolved diverse defence strategies to protect themselves against predators, including a complex alarm com-
munication system based on vibroacoustic and/or chemical signals. In reaction to alarm signals, workers and other vulnerable
castes flee away while soldiers, the specialized colony defenders, actively move toward the alarm source. In this study, we
investigated the nature of alarm communication in the pest Reticulitermes flavipes. We found that workers and soldiers of R.
flavipes respond to various danger stimuli using both vibroacoustic and chemical alarm signals. Among the danger stimuli,
the blow of air triggered the strongest response, followed by crushed soldier head and light flash. The crushed soldier heads,
which implied the alarm pheromone release, had the longest-lasting effect on the group behaviour, while the responses to
other stimuli decreased quickly. We also found evidence of a positive feedback, as the release of alarm pheromones increased
the vibratory communication among workers and soldiers. Our study demonstrates that alarm modalities are differentially
expressed between castes, and that the response varies according to the nature of stimuli.
Keywords Communication· Defence· Pheromones· Positive feedback· Vibratory behaviour
Introduction
Alarm communication is common in social animals, and it
increases rates of survival (Wyatt 2003; Hunt and Richard
2013). Almost all social insects use alarm communication
to coordinate the defensive activities of the entire colony
(Leonhardt etal. 2016). These alarm signals are shared
through different communication channels, with vibratory
and pheromonal communication being the most common
(Greenfield 2002; Cocroft and Rodríguez 2005). In ter-
mites, as well as their sister group, the subsocial roaches
Cryptocercus (Seelinger and Seelinger 1983; Connétable
etal. 1999; Röhrig etal. 1999; Reinhard and Clément 2002;
Hager and Kirchner 2013; Delattre etal. 2015), vibratory
and/or chemical signals are produced by disturbed colony
members and induce the retreat of workers and other vulner-
able castes while attracting soldiers (Reinhard and Clément
2002; Šobotník etal. 2008a, 2010). These mechanisms are
essential for termite colony defence, and greatly contribute
to their ecological success. For example, tamandua anteaters
are specialized predators of Nasutitermes, which they find
in wood pieces, but they are unable to feed on this termite
directly in the nest where the soldier proportion is higher
(Lubin and Montgomery 1981).
Insectes Sociaux
Electronic supplementary material The online version of this
article (https ://doi.org/10.1007/s0004 0-018-00682 -9) contains
supplementary material, which is available to authorized users.
* J. Šobotník
sobotnik@fld.czu.cz
1 Faculty ofForestry andWood Sciences, Czech University
ofLife Sciences, Kamýcká 129, 16521Prague6Suchdol,
CzechRepublic
2 Institute ofResearch forDevelopment, Sorbonne Universités,
Institute ofEcology andEnvironmental Sciences ofParis, U
242, 32 Avenue Henri Varagnat, 93140Bondy, France
3 Faculty ofElectrical Engineering, Czech Technical
University inPrague, Technická 2, 16627Prague6,
CzechRepublic
4 Institute ofOrganic Chemistry andBiochemistry, Academy
ofSciences oftheCzech Republic, Flemingovo nám.,
16610Prague, CzechRepublic
5 Okinawa Institute ofScience andTechnology Graduate
University, 1919-1 Tancha, Onna-son, Kunigami-gun,
Okinawa904-0495, Japan
6 Laboratory ofExperimental andComparative Ethology,
EA4443, University Paris 13, Sorbonne Paris Cité, 99
Avenue Jean-Baptiste Clément, 93430Villetaneuse, France

O.Delattre et al.
1 3
Many termite species produce substrate-borne vibra-
tions by hitting their heads and/or abdomens on the sub-
strate (Connétable etal. 1999; Röhrig etal. 1999; Hager
and Kirchner 2013). All colony members are able to detect
vibrations with their subgenual organs, specialized chordo-
tonal vibroreceptors, located on the leg tibiae (Howse 1962,
1965a; Chapman 1998). Such unique alarm communica-
tion mechanism may have evolved only once in the com-
mon ancestor of termites and Cryptocercus (Seelinger and
Seelinger 1983), probably after its diet switched from loose
substrates to wood, a material through which vibratory sig-
nals easily spread. The vibratory alarm signals are generated
using several types of oscillatory movements in termites.
In this paper, we will follow the terminology introduced by
Hill (2014), who recognized two categories of vibrational
mechanisms: (1) drumming, commonly used in termites to
produce substrate-borne vibrations; (2) tremulation, a body
movement performed without any hit on the substrate, used
to propagate the alarm selectively to calm nestmates (Kettler
and Leuthold 1995; Šobotník etal. 2008b).
Another mean of alarm signalling is the chemical chan-
nel. Social insects evolved a rich set of exocrine glands (Bil-
len and Šobotník 2015) producing a wide range of infoche-
micals. In termites, alarm pheromones are produced either
by the frontal or labial glands, which are the defensive
glands of soldiers (Šobotník etal. 2008a, 2010; Delattre
etal. 2015). Alarm compounds are mostly mono- or sesqui-
terpenes (see Šobotník etal. 2010 for a review), except in
Mastotermes darwiniensis, that uses benzoquinone (Delattre
etal. 2015).
In certain termite species, the efficiency of alarm signal-
ling is enhanced by positive feedback, a mechanism imply-
ing amplification of the signal by newly alerted specimens
(Vrkoč etal. 1978; Roisin etal. 1990; Röhrig etal. 1999;
Hager and Kirchner 2013; Cristaldo etal. 2015; Delattre
etal. 2015). Vibratory positive feedback has been demon-
strated in M. darwiniensis (Delattre etal. 2015), in several
Macrotermitinae (Termitidae; Connétable etal. 1999; Hager
and Kirchner 2013; Röhrig etal. 1999), and in Constric-
totermes cyphergaster (Termitidae: Nasutitermitinae; Cris-
taldo etal. 2015). Chemical positive feedback relies upon
the alarm pheromone present in soldier defensive secretion
and is known in M. darwiniensis (Delattre etal. 2015) and in
several Nasutitermes species (Termitidae: Nasutitermitinae)
(Roisin etal. 1990; Vrkoč etal. 1978).
While the nature of vibratory or chemical alarm signals
is well-known and has been the focus of many studies, the
alarm transmission mechanisms have seldom been investi-
gated. Cristaldo etal. (2015) demonstrated that individuals
react to alarm pheromone stimulation by vibratory alarm sig-
nalling in C. cyphergaster, and Delattre etal. (2015) showed
that artificial vibratory alarm signals trigger the release of
the alarm pheromones in M. darwiniensis. In this study, we
carried out a careful examination of alarm communication
mechanisms in Reticulitermes flavipes (Kollar, 1837) (Rhi-
notermitidae), including a vibrational and chemical analy-
sis of this behaviour. Since only the basal termites and the
most derived termites have been studied, this study aims to
improve our understanding of the evolution of alarm com-
munication in termites. Reticulitermes is a temperate genus
that includes many pest species responsible for billions of
dollars of damage annually worldwide (Su and Scheffrahn
2000). We characterized the vibratory and chemical com-
munication in workers and soldiers, and determined their
responses to particular danger stimuli, including the alarm
pheromones of their colony mates.
Materials andmethods
Insect material
All the experiments were carried out on one colony of Retic-
ulitermes flavipes collected in Île d’Oléron (France) in 1998,
and kept in laboratory stable conditions (26°C, 90% relative
humidity) since then.
Behavioural experiments
All experiments were performed at 26°C under dimmed red
light. We first conducted a behavioural experiment to assess
how R. flavipes workers and soldiers react to various danger
stimuli. To avoid any effect of social deprivation, we formed
groups (N = 12) of 38 workers and 2 soldiers (natural caste
ratio; see Haverty 1977) that were put in 85mm Petri dishes
lined with moist clean Whatman No. 1 filter paper. The groups
were left to settle for at least 2h prior to testing, and between
two tests. The stimuli set included: (1) light flash (3s, 800lx
intensity, 5500–6000K colour temperature), (2) blow of air,
delivered through a fine straw for 3s, which mimics a breach
into the colony, (3) one crushed worker head and (4) one
crushed soldier head. Crushed heads were prepared by cut-
ting termites at the level of the prothorax and the heads were
crushed on filter paper using a small spatula, allowing the
impregnation of the filter paper by the contents of the frontal
gland (soldier frontal gland is known to produce alarm phero-
mone; Reinhard and Clément 2002). We recorded group activ-
ity without any stimulation as controls for experiments (1) and
(2). For experiments (3) and (4), the controls consisted in the
insertion of a blank filter paper. Pieces of filter paper were
carefully inserted into the Petri dish through a fine slit in the
lid. Each stimulus was tested six times in random order and
on randomly chosen groups (3 stimuli per group in random
order). Behavioural reactions were recorded using a Canon
EOS 6D combined with EF 100mm f/2.8L Macro IS USM
for 3min before stimulation and 6min after the stimulation.

Chemical andvibratory signals used inalarm communication inthetermite Reticulitermes…
1 3
We chose to analyse the speed-of-motion of individuals, to
assess their reaction to our stimuli. Speed-of-motion increases
as workers flee the disturbance source, and soldiers gather
next to it (Reinhard and Clément 2002; Šobotník etal. 2008a,
2010). We tracked two randomly chosen workers and both
soldiers per group for each record. We estimated the group
behaviour 1min before the stimulus introduction, and 1 and
6min after (short-term and long-term responses, respec-
tively), using the Mouse-Tracer Software (see Šobotník etal.
2008a). We then computed the variation of speed-of-motion
for all the stimuli using the difference between the mean speed
before and after the introduction of the stimulus. This method
allowed us to normalize the variance between the different
termite groups. Variations in speed-of-motion for all danger
stimuli were compared to their respective controls.
Chemical analyses
We carried out two experiments to identify the alarm com-
pounds in workers and soldiers of R. flavipes. In the first
experiment, 20 termites were cut at the level of the prothorax
and the anterior parts were successively extracted with 60µl
and 40µl of hexane. Both extractions took place over two
successive nights at 4°C. Both extracts were merged and one
termite equivalent was injected in a 6890N gas chromato-
graph (Agilent, Santa Clara, CA, USA). The most abundant
compounds were identified based on spectral characteristics
and published records (summarized in Šobotník etal. 2010).
In the second experiment, we crushed heads of five individu-
als of each caste in a 1.2mL glass vial with a Pasteur pipette.
The headspace extraction of volatiles was carried out using
SPME fibre holder for manual sampling equipped with a
fused silica fibre coated with 30µm polydimethylsiloxane
(Supelco, Bellefonte, USA). The analytes were desorbed at
220°C in a split/splitless injector of a 5975B quadrupole
mass spectrometer coupled to a gas chromatograph. The
separation was achieved on a DB-5ms capillary column
(30m × 0.25mm, a film thickness of 0.25µm, Agilent) at
a constant flow mode (1mL/min) with helium as a carrier
gas. The column temperature was held at 40°C (1min),
gradually increased at 5°C/min to 200°C, then gradually
increased at 15°C/min to 320°C, and held at 320°C for
3min. The temperatures of the transfer line, ion source and
quadrupole were 280, 230, and 150°C, respectively. The
compounds were ionized at 70eV electrons.
Vibroacoustic experiments
To decipher the vibratory component of R. flavipes behaviour,
we formed ten new groups of termites, using the same ratio we
used in our behavioural experiments. The aim of this experi-
ment was to determine the interaction between a chemical
stimulation and the vibratory component of alarm signalling
in R. flavipes. We crushed one worker and one soldier head on
different pieces of filter paper (following the same protocol as
the behavioural experiment), and used another identical piece
of blank filter paper for control. Due to methodological con-
straints and to avoid the absorption of the termite-produced
vibrations, termite groups were placed into a 85mm Petri dish
without filter paper, and moisture was provided by a piece of
wet cotton attached to the lid. The bottom of the Petri dish was
heavily scratched to allow termites to walk. Our experiments
were carried out in an anechoic room at the Czech Technical
University in Prague under dimmed red light. All experiments
took place on a table hung from the ceiling to avoid any per-
turbations from the experimenters (see Supplementary video
SV1). All experiments were recorded using a SONY DCR-
SR72 camera in night-shot mode fixed above the experimental
arena. These records were only used to link the behaviour of
termite groups to recorded vibrations, and were not used for
behavioural analyses.
Vibratory communication was recorded using accelerom-
eters (Brüel and Kjær type 4507 B 005) glued to the bottom
of Petri dishes. We analysed the recorded vibratory signals
using a Soft dB Tenor recorder (24 bits, sampling frequency
48kHz) and Matlab software (R 2012a; see ESM 1). Prior to
each experiment, we recorded high-resolution videos of both,
disturbed and undisturbed groups of termites, to decipher the
repertoire of vibrations generated by R. flavipes workers and
soldiers. We considered recorded frequencies below 15Hz
as low-frequency vibrations, and frequencies above 15Hz as
high-frequency vibrations. These preliminary tests were also
used to determine the optimal parameters of frequency fil-
ters used for post-processing of vibration records of R. flavi-
pes groups in the described environment. This allowed us to
reduce the background noise.
In these experiments, because termite signals overlap, we
were unable to analyse individual signals. Therefore, we com-
puted the total amount of energy produced by group vibratory
signals after each stimulation. This variable ER, was computed
using the following equation:
in which TA is the evaluation period after disturbance (60s
and 360s, respectively), TB is the evaluation period before
disturbance (60s), and xf(t) is the filtered acceleration signal
(bandpass filter 50–500Hz) and is a function of time (t).
Statistics
We performed Kruskal–Wallis tests and two-by-two post
hoc permutation tests (10,000 permutations) for independent
E
R=10. log
⎡
⎢
⎢
⎣
TB
TA
∫TA
0�
�xf(t)�
�2dt
∫0
−T
B
�
�
xf(t)�
�
2dt
⎤
⎥
⎥
⎦
,

O.Delattre et al.
1 3
samples, and we carried out Friedman tests and two-by-two
post hoc permutation tests (10,000 permutations) for paired
samples. Bonferroni–Holm corrections (Holm 1979) were
applied for multiple comparisons among groups. All sta-
tistical tests were performed with StatXact software (Cytel
Studio, version 9.0.0, 2010).
Results
Behavioural experiments
All groups significantly reacted to all experimental stimuli.
We found significant differences among stimulations for
both soldiers and workers, both 1 and 6min after stimu-
lations (short-term vs. long-term responses, respectively;
Table1).
Workers were sensitive to the blow of air, the crushed
soldier head (CSH) and light flash exposure, but not to the
crushed worker head (CWH). Soldiers revealed similar
responses, but were also sensitive to the crushed worker
head during the first minute after the stimulus introduction
(Fig.1, Fig. S1).
Chemical analyses
Analytical approaches revealed a set of monoterpenes pre-
sent in soldier extracts and SPME, while no candidate com-
pound was detected in workers, irrespective of method used
(Table2). Extracts of workers and soldiers also contained
the cuticular hydrocarbons specific to R. flavipes (see, e.g.
Bagnères etal. 1990; Vauchot etal. 1998; Perdereau etal.
2010).
Vibroacoustic experiments
In response to the stimuli, soldiers and workers displayed
body vibrations that they used to spread alarm within the
groups. Using vibroacoustic preliminary tests in conjunc-
tion with high-resolution video recordings, we observed
that these vibrations were mainly drumming or tremula-
tion. Drumming was produced by abdomen hits against the
substrate, while tremulation was produced by tactile stimu-
lation of nestmates with the head. In a few cases, soldiers
displayed another kind of drumming signal consisting in
powerful hits to the ground with their mandibles.
We found that drumming and tremulation were used in
combination by workers and soldiers, forming thus com-
plex vibrations. These vibrations occurred in two kinds of
bursts (series of hits). The first kind of burst was at a high
frequency of 31 ± 4Hz, while the second kind of burst
occurred at a low frequency of 7.4 ± 1.3Hz. High and
low-frequency vibrations were often performed together,
the high-frequency bursts preceding the low-frequency
bursts (Fig.2, video SV1). This could be defined as the
typical pattern of vibratory behaviour in R. flavipes.
Occasionally, the high- and low-frequency bursts were
performed separately after disturbance. A third kind of
drumming burst was specifically displayed by soldiers,
Table 1 Differences in speed-of-motion between all stimulations after
stimulus introduction for workers and soldiers in R. flavipes
Kruskal–Wallis for multiple comparisons statistic values (H5) and P
values are provided for both castes
Workers Soldiers
Short-term response
(1min) H5 = 46
P < 0.001 H5 = 39.61
P < 0.001
Long-term response
(6min) H5 = 46.01
P < 0.001 H5 = 27.24
P < 0.001
Fig. 1 Change in speed-of-motion of workers (white bars) and sol-
diers (grey bars) in R. flavipes during a 1-min period after exposition
to experimental stimuli in comparison to controls. N = 12 for each
caste and each stimulus. Box plots show the median and 25–75th per-
centiles. Whiskers show all data excluding outliers outside the 10th
and 90th percentiles (circles). Statistical differences are given for
P < 0.05. CWH crushed worker head, CSH crushed soldier head
Table 2 Chemical compounds identified by GC–MS from Reticulit-
ermes flavipes worker and soldier samples (hexane extracts or head-
space SPME)
Ø no volatile compound detected, CHC cuticular hydrocarbons
Extracts SPME
Workers Soldiers Workers Soldiers
CHC α-Pinene Ø α-Pinene
β-Pinene β-Pinene
Limonene Limonene
CHC Unknown compound

Chemical andvibratory signals used inalarm communication inthetermite Reticulitermes…
1 3
and consisted of repeated hits to the substrate with the
mandibles, at an average frequency of 26 ± 2Hz. Unfor-
tunately, this particular behaviour occurred too scarcely
(only 40 series recorded in all experiments combined) to
be analysed in our vibroacoustic work.
Reticulitermes flavipes groups reacted to the alarm
pheromone by producing vibroacoustic signals (Fig. S2).
The intensity of these signals was nearly identical 1 and
6min after the stimulation, with the exception of the
response to CSH, which was lower after 6min (Table3;
Fig.3). The responses to CSH were stronger in the first
minute after the stimulus introduction, while CWH stimu-
lation triggered a long-lasting effect increasing with time
during the timeframe of the experiment (Table4).
Discussion
Alarm communication is an important component of the
defensive strategies of many gregarious, colonial and
social animals, and is used to coordinate defensive activi-
ties. In Reticulitermes flavipes, alarm signals are spread
via tremulations, substrate-borne vibrations and alarm
pheromones. Irrespective of the communication channel,
two modes of alarm transmission can be distinguished in
natural situations: general alarm responses follow strong
disturbance and affect large termite groups, while subtler
specific alarm communication involves a few specimens
reacting to low-level disturbance, such as the encounter
of alien individual into the gallery system (Howse 1965b;
Stuart 1963, 1988).
Here we studied the general alarm responses of R. flavi-
pes, and clearly showed that potential dangers, represented
Fig. 2 Example of vibratory behaviour showing the typical structure
of the vibratory movements of workers and soldiers of Reticulitermes
flavipes. The complex vibrations occurred at two distinct frequencies,
described as high- (31Hz) and low- (7.4Hz) frequency bursts
Table 3 Differences in vibratory behaviour (permutation tests for
paired samples) recorded in termite groups (N = 10) before and after
the introduction of the stimulus
CO control blank paper, CWH crushed worker head, CSH crushed
soldier head
Stimulation Short-term response
(1min)
Long-term
response
(6min)
CO − 0.959
P = 0.359
− 0.5501
P = 0.5977
CWH − 2.067
P = 0.021
− 2.279
P = 0.014
CSH − 1.908
P = 0.006
− 1.574
P = 0.057
Fig. 3 Energy ratio difference in vibrations recorded in groups
(N = 10) before and after the introduction of the stimulus. Box plots
show the median and 25th–75th percentiles. Whiskers show all data
excluding outliers outside the 10th and 90th percentiles (circles). Sta-
tistical differences are shown for *P < 0.05 and **P < 0.01. CO con-
trol blank paper, CWH crushed worker head sample, CSH crushed
soldier head sample
Table 4 Comparison between vibrational responses to crushed ter-
mite heads (N = 10) (permutation tests for paired samples)
CO control blank paper, CWH crushed worker head, CSH crushed
soldier head
Stimulation comparison Short-term response
(1min)
Long-term
response
(6min)
CO vs. CWH − 1.839
P = 0.063
− 2.378
P = 0.027
CO vs. CSH − 1.841
P = 0.047
− 1.579
P = 0.088

O.Delattre et al.
1 3
by air current, light flash, or crushed nestmate heads,
are treated with differing types of alarm responses. The
responses to air currents and crushed soldier heads were
the most pronounced, and triggered immediate increase
of speed-of-motion and quantity of vibroacoustic signals
in workers and soldiers. These behavioural changes were
abrupt and often lasted over 6min.
The observed vibroacoustic signals were made of com-
plex vibratory movements, combining drumming and
tremulations. They were produced by workers and soldiers
repeatedly hitting their abdomen on the substrate. These
examples of vibroacoustic communication combine two
kinds of bursts differing in beat frequencies, as described
in Coptotermes gestroi, another species of Rhinotermitidae
(see Hertel etal. 2011). A duration-dependent effect could
be perceived and CSH samples elicited the strongest reaction
during the first minute after the stimulation.
CSHs were not the only stimuli which could trigger
vibrations, CWH revealed significant effect as well, as evi-
denced by the walking activity of soldiers and vibratory
activity of both soldiers and workers. Our results concur
with previous observations on C. cyphergaster (Termitidae,
Nasutitermitinae) (Cristaldo etal. 2015), in which the CWH
also provoke marked behavioural responses in soldiers. C.
cyphergaster workers possess enlarged mandibular glands
with defensive function (Costa-Leonardo and Shields 1990),
which are possibly the source of chemicals responsible for
the change in behaviour. In contrast, no gland with defensive
role is known in R. flavipes workers, and the source of the
excitement remains unknown.
Soldiers of R. flavipes also used their head and mandibles
to perform powerful hits to the substrate, which produced
a strong substrate-borne vibratory drumming-like signal,
much stronger than the abdominal drumming. The same
way of drumming has also been observed in Archotermop-
sidae (Kirchner etal. 1994), Rhinotermitidae (Hertel etal.
2011) and Macrotermitinae (Termitidae) (Connétable etal.
1999; Hager and Kirchner 2013; Kettler and Leuthold 1995;
Röhrig etal. 1999). The strongest signals are probably used
to warn nestmates and recruit soldiers from deeper inside
the colony.
Our chemical analysis showed high quantity of α- and
β-pinene, and limonene produced by the frontal gland of R.
flavipes soldiers. These compounds form the alarm phero-
mone in R. flavipes, as it has been previously suggested by
several authors (Bagnères etal. 1990; Parton etal. 1981;
Reinhard etal. 2003), although not all compounds might be
necessary for the function, as they were never tested sepa-
rately. The alarm pheromones involve various compounds
classes, such as monoterpenes (also in R. flavipes), sesquit-
erpenes (for review see Šobotník etal. 2010) or quinones
(Delattre etal. 2015), and are known to be released from
the soldier defensive glands, namely the labial glands in
Mastotermitidae and the frontal gland in Rhinotermitidae
and Termitidae (Delattre etal. 2015; Kaib 1990; Kriston
etal. 1977; Pasteels and Bordereau 1998; Reinhard and Clé-
ment 2002; Roisin etal. 1990; Šobotník etal. 2008a; Vrkoč
etal. 1978).
Until recently, alarm positive feedback was only dem-
onstrated either for vibroacoustic (Connétable etal. 1999;
Delattre etal. 2015; Hager and Kirchner 2013; Röhrig
etal. 1999) or chemical signals (Roisin etal. 1990; Vrkoč
etal. 1978). Integrative studies appeared only recently, and
vibroacoustic feedback to chemical alarm has been shown
only in the basal Mastotermes (Mastotermitidae; Delattre
etal. 2015) and the derived Constrictotermes (Termiti-
dae: Nasutitermitinae; Cristaldo etal. 2015). In this study,
we found the third example of vibroacoustic feedback to
chemical alarm, as workers’ and soldiers’ speed-of-motion
increased after exposure to head volatiles. Moreover, as
showed in Connétable etal. (1999), it is likely that termites
spread the alarm further using complex vibratory communi-
cation based on tremulations and drumming, contributing to
the general state of colonial defensive activity (i.e. running
away from the threat).
Our work is the first exhaustive study on the alarm com-
munication strategies in a species of Rhinotermitidae. Both
workers and soldiers of R. flavipes reacted to all stimulations
(light flash, air currents and crushed nestmate heads) with
various degrees of excitement and displaying different vibra-
tory movements. These observations show specialized alarm
communication strategy based on complementary modali-
ties, which could trigger an efficient response according to
the nature and intensity of endangering stimulus. Moreover,
R. flavipes is a pest species in Western USA (Evans 2011;
Evans etal. 2013) and has been introduced to several places
around the world (Bagnères etal. 1990; Smith etal. 2006;
Evans etal. 2013), where it became invasive (originally
described as R. santonensis, and later synonymized with R.
flavipes by Austin etal. 2005). The dominance of R. flavipes
over R. grassei, which has already been observed in the field
(Perdereau etal. 2011), might, at least partially, be explained
by its sophisticated alarm communication strategy.
Acknowledgements This work was supported by the project IGA
A30/17 of the Faculty of Forestry and Wood Sciences, Czech Uni-
versity of Life Sciences, by the project CIGA 20184303 of the Czech
University of Life Sciences Prague, and by the BQR 2014/2015 from
the University Paris 13-SPC.
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