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The labral gland in termites: Evolution and function

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Termites are important contributors to ecosystem functioning. They are highly abundant in tropical and sub-tropical habitats, and represent an important resource for a wide range of predators. Their evolutionary success is driven largely by a life in populous colonies with a complex communication system controlled by a rich set of exocrine glands whose secretions are involved in many aspects of termite life. As many as 20 different exocrine organs are known to occur in termites. Among them, the labral gland has been largely understudied. Here we examine the structure of the labral gland in workers of 28 species and imagoes of 33 species across all termite taxa, and in the Cryptocercus wood roach. The labral gland is present in all species, and comprises two secretory regions located on the ventral side of the labrum and the dorso-apical part of the hypopharynx, respectively. The epithelium of the gland consists of class 1 secretory cells with an abundance of smooth endoplasmic reticulum, and long microvilli with a channel inside, which releases secretion through a modified cuticle. Our observations suggest that the labral gland is involved in defensive communication after encounter with a non-nestmate.
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© 2019 The Linnean Society of London, Biological Journal of the Linnean Society, 2019, 126, 587–597 587
Biological Journal of the Linnean Society, 2019, 126, 587–597. With 4 figures.
The labral gland in termites: evolution and function
VALERIA PALMA-ONETTO1,2, JITKA PFLEGEROVÁ3, RUDY PLARRE4, JIŘÍ SYNEK2,
JOSEF CVAČKA5, DAVID SILLAM-DUSSÈS1, and JAN ŠOBOTNÍK2,*,,
1University Paris 13 - Sorbonne Paris Cité, Laboratory of Experimental and Comparative Ethology, EA
4443, Villetaneuse, France
2Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic
3Institute of Entomology, Biology Centre, Academy of Sciences of the Czech Republic, České Budějovice,
Czech Republic
4Bundesanstalt für Materialforschung und -prüfung, Berlin, Germany
5Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague, Czech
Republic
Received 16 November 2018; revised 12 December 2018; accepted for publication 12 December 2018
Termites are important contributors to ecosystem functioning. They are highly abundant in tropical and sub-tropical
habitats, and represent an important resource for a wide range of predators. Their evolutionary success is driven
largely by a life in populous colonies with a complex communication system controlled by a rich set of exocrine glands
whose secretions are involved in many aspects of termite life. As many as 20 different exocrine organs are known to
occur in termites. Among them, the labral gland has been largely understudied. Here we examine the structure of the
labral gland in workers of 28 species and imagoes of 33 species across all termite taxa, and in the Cryptocercus wood
roach. The labral gland is present in all species, and comprises two secretory regions located on the ventral side of
the labrum and the dorso-apical part of the hypopharynx, respectively. The epithelium of the gland consists of class 1
secretory cells with an abundance of smooth endoplasmic reticulum, and long microvilli with a channel inside, which
releases secretion through a modified cuticle. Our observations suggest that the labral gland is involved in defensive
communication after encounter with a non-nestmate.
ADDITIONAL KEYWORDS: development – evolution – exocrine gland – Isoptera – Termitoidae – ultrastructure.
INTRODUCTION
Termites are among the most important decomposers
of dead plant material and are of prime importance in
both natural and urban areas. Their impact in tropical
regions is immense: they ingest 50–100% of raw plant
biomass in tropical forests (Bignell & Eggleton, 2000).
They probably participated in reducing terrestrial
carbon reserves after their adaptive radiation at the
beginning of the Tertiary (Engel et al., 2009). They also
contribute significantly to the world’s atmospheric
carbon dioxide and methane (Sugimoto et al., 2000).
Termites are often called ecosystem engineers due to
their dramatic impact on terrestrial environments,
including release of nutrients from dead vegetal
matter, soil aeration, transport of tonnes of different
materials per hectare and year, and increase in soil
heterogeneity and net productivity (Jouquet et al.,
2006; Eggleton, 2011; Evans et al., 2011).
The importance of termites is reflected by their
abundance, often exceeding 1000 individuals/m2 in
tropical regions (Eggleton et al., 1996; Dahlsjö et al.,
2014). Termites also represent an important food
source for a wide variety of predators (Deligne et al.,
1981; Redford & Dorea, 1984). Selection pressures
on termites have resulted in an arms race leading
to improved defensive abilities, best expressed in the
specialized caste of defenders, soldiers (Haverty, 1977;
Deligne et al., 1981; Krishna et al., 2013). However,
workers are also important in defence, as they
construct below- or above-ground galleries and nests,
which have a primarily protective function (Eggleton,
2011). Termite colony members, in general, live in a
applyparastyle “fig//caption/p[1]” parastyle “FigCapt”
*Corresponding author. E-mail: sobotnik@fld.czu.cz
These authors contributed equally to the study.
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protected closed system of chambers and galleries,
although the alate imagoes are an exception as they
leave the maternal nest to establish new colonies, and
this is when they are most at-risk, , often being eaten by
non-specialized predators or are later outcompeted by
older colonies (Nutting, 1979). However, the defensive
mechanisms of termites have been almost exclusively
studied in soldiers (for a review see Šobotník et al.,
2010a), while those of imagoes and workers have
largely been neglected (but see e.g. Sands, 1982;
Thorne, 1982; Piskorski et al., 2009; Šobotník et al.,
2012; Bourguignon et al., 2015).
Exocrine glands can have multiple functions,
producing pheromones, defensive chemicals,
antibiotics, lubricants and digestive enzymes
(Chapman, 2013). They are organs of fundamental
importance in all insects, being most abundant and
most diverse in social insects (Billen & Šobotník, 2015).
The complex lives of social insects are also reflected by
plentiful chemical signals produced by as many as 149
different glands described so far. While ants possess 84
exocrine glands producing mostly infochemical signals
(Hölldobler & Wilson, 1990; Billen & Šobotník, 2015),
only 20 exocrine glands have so far been described in
termites. Some termite exocrine glands are present
in all castes, but may be inactive in larval instars
(Šobotník & Hubert, 2003; Šobotník & Weyda, 2003),
while others are limited to only some species and
castes. The exocrine glands produce secretions related
to sexual behaviour occurring in winged imagoes, or to
defence in soldiers, workers and imagoes.
The frontal gland is a defensive organ of prime
importance in termites, occurring in most Neoisoptera
(Stylotermitidae, Rhinotermitidae, Serritermitidae,
Termitidae) soldiers and imagoes (Prestwich &
Collins, 1982; Quennedey, 1984; Šobotník et al., 2004,
2010b; Piskorski et al., 2009; Wu et al. 2018), and
in some workers (Šobotník et al., 2010c). Another
important organ, the labial glands, is universally
present in termites (Noirot, 1969; Billen et al., 1989;
Šobotník & Weyda, 2003). Their function in workers
is connected to feeding (Noirot, 1969; Reinhard et al.,
2002; Fujita et al., 2008) and nest construction (Noirot,
1969; Reinhard et al., 2002), while in all soldiers
and in workers of soldierless species they produce
defensive secretions (Sillam-Dussès et al., 2012).
Workers in general have developed different means
of defence, protecting them during foraging activities
or during invasion of predators into the nest (Deligne
et al., 1981; Prestwich, 1984; Šobotník et al., 2012;
Bourguignon et al., 2015; Poiani & Costa-Leonardo,
2016). The most important contribution of workers to
colony defence is via passive defence, such as gallery
construction and nest fortification (Šobotník et al.,
2010a). Termite workers are often directly engaged in
nest defence (Thorne, 1982; Binder, 1988), and this is
of particular interest with regard to (1) conflicts with
conspecific colonies defended primarily by soldier-
produced toxins due to the presence of specific auto-
detoxification mechanisms (Spanton & Prestwich,
1982), (2) soldierless species in which workers are
considerably more aggressive that soldiered species
(Sands, 1982; Šobotník et al., 2010a), (3) dehiscence
mechanisms when the body wall ruptures and
intestinal contents contaminate opponents (Sands,
1982); and (4) autothysis connected to the release of
toxic or incapacitating compounds from inside the body
(Costa-Leonardo, 2004; Šobotník et al., 2010a, 2012;
Bourguignon et al., 2015; Poiani & Costa-Leonardo,
2016).
The labral gland is an important termite secretory
organ, but has been studied in detail only in soldiers
so far (Deligne et al., 1981; Quennedey, 1984; Šobotník
et al., 2010d; Costa-Leonardo & Haifig, 2014; Palma-
Onetto et al., 2018). It was first reported on the ventral
side of the labrum in Macrotermes bellicosus (Deligne
et al., 1981), later observed on the dorsal side of the
hypopharynx in the same species (Quennedey, 1984),
and finally reported to occur in all termite soldiers
(Palma-Onetto et al., 2018). The gland epithelium
in soldiers consists of class 1 secretory cells in most
representatives, with additional class 3 secretory cells
in a few species (Šobotník et al., 2010d; Costa-Leonardo
& Haifig, 2014; Palma-Onetto et al., 2018). Here, we
describe the occurrence, structure and ultrastructure
of the labral gland in a representative set of termite
workers and imagoes, as well as in the wood roach
Cryptocercus punctulatus.
MATERIAL AND METHODS
Scanning electron microScopy, optical
microScopy and tranSmiSSion electron
microScopy
Observations of the labrum and hypopharynx were
made using optical, scanning electron microscopy
(SEM) and transmission electron microscopy
(TEM). We examined workers (including sub-castes
if present) of 28 species and imagoes of 33 species
representing most extant termite taxa (see Krishna
et al., 2013). We also examined nymphs and female
adults in the cockroach Cryptocercus punctulatus,
member of the sister group to termites (Lo et al., 2000;
Inward et al., 2007). The procedures used for optical
microscopy, TEM and SEM are well established in
our lab, and correspond to those provided in detail
by Šobotník & Weyda (2003) and Palma-Onetto
et al. (2018). Important data are summarized in
Supporting Information Tables S1–S3. We also used
an FEI Helios NanoLab 660 G3 UC scanning electron
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THE LABRAL GLAND IN TERMITES 589
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microscope with focused ion beam milling equipped
for cryo-imaging and correlative light-electron
microscopy.
Behavioural experimentS
Two types of bioassays were performed. In the first
experimental set-up, we ran arena tests in Glossotermes
oculatus and Coptotermes testaceus groups of five
workers and two soldiers, to which we introduced a
single intruder, a worker of a different termite species
or an ant. The behaviour resulting from subsequent
encounters was recorded and specific behavioural
patterns were subsequently analysed. The tests were
performed under dimmed artificial light, and Canon
EOS 60D, 6D or 5D SR cameras combined with Canon
EF 100-mm f/2.8L Macro IS USM lenses were used.
In the second experimental set-up, labral extracts
were prepared by dissecting 60 labra of Prorhinotermes
canalifrons soldiers (four replicates), which were then
extracted in 400 µL of either hexane or methanol (two
repetitions for each solvent), and used in behavioural
tests (repeated six times for each stimulus). These
tests consisted of placing groups of Prorhinotermes
canalifrons (two soldiers and eight workers) in a Petri
dish lined with filter paper split into two sectors: labral
extracts (six labra equivalents in 40 µL of solvent) vs.
control 1 (six legs equivalents in 40 µL of solvent; leg
extracts prepared as for labra) or control 2 (40 µL pure
solvent). The same bioassay was performed six times
using groups of Reticulitermes flavipes consisting of two
soldiers and eight workers, to test for a possible effect
on another termite species. The number of termites on
each sector was recorded using the above-mentioned
equipment 10 min after the introduction of termites
to the Petri dish. The number of termites choosing the
sector treated with labral extracts was compared with
the one in solvent via Student’s t-test (Norusis, 1990). To
identify any preference for a sector, t- Student’s t-tests
were used if the comparison between sectors from the
same Petri dish was normal, and a Mann–Whitney U
test was used if it was not normal (Norusis, 1990).
chemical analySeS
Chemical analyses using samples of 100 labra or 100
legs (as control) extracted in methanol or hexane
were carried out using a 6890N gas chromatograph
(Agilent, Santa Clara, CA, USA) coupled to a 5975B
quadrupole mass spectrometer equipped with an
HP5ms fused silica capillary column (30 m × 0.25 mm,
0.25 µm; Agilent). The carrier gas was helium at 1 mL/
min. The injector was operated in split mode (10:1) at
200 °C; the injected volume was 1 µL. The temperature
programme comprised: 40 °C (2 min), then 8 °C/min
to 200 °C, then 15 °C/min to 320 °C (3 min). Standard
70-eV mass spectra were recorded in the m/z 25–600
range; a 4 min solvent delay was used. Temperatures
of the transfer line, ion source and quadrupole were
280, 230 and 150 °C, respectively. Chemical profiles
of labra and legs extracts were compared to detect
specific compounds from the labral gland.
RESULTS
Scanning electron microScopy
The labrum of a worker or an imago was most often
oval shaped (Fig. 1), broadly attached to the clypeus.
The labrum usually did not differ greatly in size
among species and castes, being c. 2.7 times shorter
than head length (distance between clypeo-frontal
boundary and posterior margin of head), with the
exception of Termes hospes and Microcerotermes sp.
workers, Pseudacanthotermes militaris small workers
and Coptotermes testaceus imagoes, in which the
labrum was about 3.2–5 times shorter than the head
(Tables S1 and S2).
The dorsal side of the labrum was covered by smooth
rectangular plates c. 10 µm in size mixed with a few
hair-like sensillae. The ventral faces of the labrum and
of the hypopharynx consisted of four regions of similar
appearance for workers and imagoes of all studied taxa
(see Fig. 1): (a) a smooth region in the apical zone along
the midline of the labrum, with a wrinkled structure
with numerous pores c. 30–50 nm in diameter (Fig. 1A,
B, D); (b) a basal zone in the midline extending forward
around zone ‘a’, consisting of many irregular hair-like
structures (acanthae based on TEM observations),
ranging in length between 5 and 25 µm; (c) two lines of
sensillae (numerous chemoreceptors usually with four
dendrites and relatively few campaniform sensillae
located predominantly in the basal parts of the sector)
encircling zone ‘b’ (Fig. 1A, B) on the ventral labrum
but missing from the hypopharynx; and (d) lateral
regions, consisting of irregular scales ranging in size
between 2 and 4 µm (Fig. 1).
optical microScopy
The labral gland was found in workers and imagoes
of all studied species. It was located on the ventral
side of the labrum, extending to the dorsal side at
the labrum apex, and in the dorso-apical region of the
hypopharynx (Fig. 2A, B). It appeared as a thickened
epithelium composed of columnar cells (Fig. 2). The
thickness of the secretory epithelium in workers
was in general c. 15–30 µm (on average 17.98 µm).
The epithelium was thinnest in Pseudacanthotermes
militaris small workers (7.80 µm) and thickest
in Mastotermes darwiniensis (29.22 µm) workers
(Table S1). In imagoes, labral gland thickness was
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590 V. PALMA-ONETTO ET AL.
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on average 18.21 µm; the thinnest epithelium was
found in Nasutitermes sp. (8.27 µm) and the thickest
in Neocapritermes araguaia (32.65 µm) (Table S2).
The thickness of the labral gland differed slightly
between sexes in imagoes, but without a clear trend.
For all termite samples, the hypopharyngeal part of
the epithelium was in general significantly thinner,
usually between 8 and 15 µm thick, with the exception
of the ‘lower’ termite workers, in which the thickness
of the secretory epithelium was similar in the labral
and hypopharyngeal portions of the gland.
tranSmiSSion electron microScopy
Secretory cells of the labral gland were always made of
class 1 (according to the classification of Noirot &
Quennedey, 1974), i.e. secretory cells in direct contact
with the body cuticle, through which the secretion
passes. Their ultrastructure was nearly identical in
the labral and hypopharyngeal regions of the labral
gland in all castes and therefore our description is
based on the observation of both parts of the gland.
There was often an abundance of class 3 secretory cells
(Noirot & Quennedey, 1974), i.e. cells separated from
the body wall by epithelial cells, through which they
release their products via (epi)cuticular canals. They
mostly occur at the dorsal face of the labrum, but these
cells never mixed with the labral gland epithelium,
unlike in some soldiers (Palma-Onetto et al., 2018),
and always released their secretion to the dorsal side
of the labrum (Fig. 3A). Class 1 and class 3 secretory
cells were very different, and also easily distinguished
from non-modified epidermal cells (Fig. 3A), which
were much thinner (typically c. 0.5 µm) and contained
virtually no secretory organelles (Fig. S1B).
Labral gland secretory cells were columnar (Fig.
3A–C), and their cytoplasm contained abundant
smooth endoplasmic reticulum (ER), scattered rough
ER, small secretory vesicles, abundant mitochondria,
numerous microtubules orientated predominantly
apico-basally, glycogen granules and sometimes also
myelin figures. While the microtubules are scattered
throughout the secretory cells in most representatives,
they appear grouped into bundles in Glyptotermes
sp. workers. Apical microvilli were well developed
throughout the gland (Fig. 3D), but were longer in the
middle part of the epithelium compared to the margins.
The microvilli were up to 1.3 µm long and about 80 nm
thick, slightly shorter in workers than in imagoes, and
always with a central channel about 30 nm in diameter
Figure 1. Scanning electron micrographs detailing labral gland development. A, ventral side of labrum in Embiratermes
neotenicus female imago. B, ventral side of labrum in Pseudacanthotermes militaris worker. C, dorsal side of the hypopharynx
in Acanthotermes acanthothorax female imago. D, detailed view of region ‘a’ with pores through the epicuticle in labrum of
Microcerotermes sp. female imago. The sectors are abbreviated as follows: a, zone with small porosities located at the apex
of the labrum along the midline; b, zone formed with many irregular hair-like structures located partially around zone ‘a’; c,
two lines of sensillae located around zone ‘b’; d, region with scales of irregular shape located at the labrum margins.
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in termite imagoes and about 40 nm in workers (Fig.
3D; Table S3 and Fig. S1D). Numerous small vesicles
were observed at the microvilli bases (Fig. 3D). These
vesicles were generally electron-lucent when occurring
at the base of microvilli, but sometimes appeared
more electron-dense deeper in the cells, as in males
and females of G. oculatus alate imagoes, in female
alate imagoes of Heterotermes tenuis and in workers
of Thoracotermes sp. Lipid-like droplets were observed
only rarely, but they were more common in Coptotermes
formosanus imagoes, Nasutitermes lujae workers and
Pseudacanthotermes militaris large workers. The
basal parts of the secretory cells differentiated into
invaginations typically about 5 µm deep (up to 12 µm
in workers of Neocapritermes taracua and Coptotermes
formosanus) with frequent formation of pinocytotic
vesicles (Figs 3C, S1C). Free axons were commonly
observed inserted within the basal invaginations.
The basal parts of secretory cells were covered by a
basement membrane (c. 100 nm thick) sometimes
strengthened by clusters of collagen fibres (then up
to 1.5 µm thick). There was no junction between the
neighbouring secretory cells in the basal parts, while
there were always zonulae adherens followed by septate
junctions in the apical parts. The nuclei were elliptical,
located at the cell bases, and usually c. 5 µm long (up
to 7 µm in N. taracua workers and G. oculatus male
alate imagoes). The nuclei contained predominantly
dispersed chromatin with a few aggregates.
The cuticle overlying the labral gland was highly
modified for secretion evacuation, and always thicker
in imagoes than in workers (on average 6.5 and
4 µm, respectively; Fig. 4A). The cuticle was formed
by endocuticle of helicoidal structure, exocuticle and
a thin epicuticle (c. 30 nm thick; Table S3, Fig. 4B).
Modifications to the glandular cuticle were highly
pronounced, especially in the smooth middle part of
the ventral labrum. These modifications included
an increased number of pore canals, which widened
towards the cuticle base (Fig. 4A), and plentiful
epicuticular pores. The cuticle of the hypopharyngeal
portion of the gland was very similar, although the
endocuticle was slightly thicker than in the labrum.
There was no reservoir and the secretion was stored
Figure 2. Sagittal sections of the labral gland. A, head of Termitogeton planus worker. B, labrum and hypopharynx of
Globitermes sulphureus worker. C, labrum of Coptotermes testaceus male imago. D, labrum of Neocapritermes taracua
worker. Note the secretory epithelium of hypopharynx in A and B. Abbreviations: b, brain; cl, clypeus; fg, frontal gland; hy,
hypopharyngeal portion of the labral gland; lb, labium; lg, labral gland; mn, mandible; mm, mandibular muscles; p, pharynx;
spg, subesophageal ganglion.
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592 V. PALMA-ONETTO ET AL.
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only in the space between the secretory epithelium
and the cuticle, or inside the porous cuticle.
The labral gland was also observed in Cryptocercus
punctulatus nymphs and female imagoes. The
epithelium of the labral gland had the same
characteristics as described above, although the
microtubules predominantly occur in large bundles
(Fig. S1F). An important difference was shallower
basal invaginations and shorter microvilli lacking
internal central channels.
Behavioural experimentS
We observed potential use of the labral gland in
soldiers of G. oculatus and Coptotermes testaceus
after encounter with an alien termite or ant worker.
Immediately after such an encounter, the soldiers
changed their behaviour by walking backwards
while rubbing the labrum against the substrate (see
Video S1).
In a second experimental bioassay, the number of
workers and soldiers of Prorhinotermes canalifrons
present at the two sectors did not differ between labral
and leg extracts, using neither methanol (P = 0.869)
nor hexane (P = 0.355) nor pure solvent (P = 0.325
for methanol, P = 0.614 for hexane). Interestingly, the
number of workers and soldiers of R. flavipes avoiding
the sector treated with labral gland extracts was
higher than controls irrespective of the solvent, either
leg extracts (P < 0.0001) or solvent (P < 0.0001).
Figure 3. Ultrastructure of the labral gland in termites. A, middle part of the labrum in Coptotermes testaceus female
imago, showing the labral gland consisting of class 1 secretory cells at the bottom, and class 3 secretory cells occurring at
the dorsal side of the labrum. B, labral gland secretory epithelium in Coptotermes testaceus female imago. C, labral gland
secretory cells in Coptotermes formosanus worker. Note the well-developed invaginations reaching to near cell apices. D,
detailed view of the apex of labral gland secretory cells in Pseudacanthotermes militaris worker showing well-developed
smooth endoplasmic reticulum. Abbreviations: bi, basal invaginations; c, cuticle; c1, class 1 secretory cell; c3, class 3 secretory
cell; h, haemocytes; mv, microvilli; n, nucleus; ser, smooth endoplasmic reticulum; sv, secretory vesicle. Asterisk indicates
mitochondria in the cell cytoplasm.
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chemical analySeS
No specific compounds were detected in the labral
extracts, and the profiles of these extracts and of legs
extracts did not differ to any great extent (data not
shown).
DISCUSSION
The labral gland is an integral part of the labrum in
termites, occurring in soldiers (Palma-Onetto et al.,
2018), workers and imagoes. While its structure is
well known in soldiers (Palma-Onetto et al., 2018),
only anecdotal information about its presence in some
imagoes has previously been published (Křížková et al.,
2014). In the present study, we describe the labral
gland in workers and imagoes of a set of representative
termite species and in nymphs and adults of the wood
roach Cryptocercus punctulatus.
The labrum and the labral gland share the same
characteristics in all species and castes studied so far.
Common features are a higher degree of sclerotization
of the dorsal side of the labrum, which is in general
more pronounced in soldiers, the occurrence of class
3 secretory cells at the dorsal side of labrum but
rarely within the labral gland, and the presence of
the labral gland comprising class 1 secretory cells on
the ventral side of labrum and on the dorsal side of
hypopharynx. The secretory cells are also quite similar
in their ultrastructure, showing well-developed
apical microvilli with a central channel (lacking in
Cryptocercus punctulatus), numerous vesicles of
different electron densities, abundant smooth and
rough ER, cuticle modified for secretion release,
and innervation of the secretory cells through axons
running freely within the basal invaginations (Palma-
Onetto et al., 2018). At the same time, there are also
considerable differences between termite soldiers on
the one hand and workers and imagoes on the other: (1)
the hyaline tip, present in soldiers of many advanced
species, is missing in other castes; (2) the shape of the
labrum is highly variable in soldiers whereas it is almost
identical in all workers and imagoes; and (3) overall
development of microvilli and basal invaginations is
lower in workers and imagoes (Palma-Onetto et al.,
2018). These observations suggest that the labral
gland has the same function in all castes, but plays
a more important function in soldiers. We also cannot
exclude that the labral secretion is used in a different
context by workers and imagoes, as rubbing of labral
gland secretory openings against the substrate was
observed exclusively in soldiers. Although Cryptocercus
punctulatus showed differences in the labral gland
structure in comparison to termites, such as shorter
microvilli devoid of a central channel, the presence of
microtubule bundles was shared particularly between
Cryptocercus punctulatus, and M. darwiniensis and
Hodotermopsis sjoestedti soldiers (Palma-Onetto et al.,
2018), and Glyptotermes sp. workers, suggesting it is a
common feature in basal taxa inherited from cockroach
ancestors.
The hyaline tip, a transparent and extensible
apical part of the labrum, is probably an evolutionary
novelty occurring in some soldiers of Rhinotermitidae
and Termitidae. Our mapping of ancestral characters
(Palma-Onetto et al., 2018) suggests that the hyaline tip
evolved in a common ancestor of Rhinotermitidae and
Termitidae, and was subsequently lost at least in four
independent cases: (1) all soldiers of Nasutitermitinae
in which the whole labrum is highly reduced in size
as well as all other mouth parts; (2 and 3) in snapping
soldiers, represented by two independent lineages,
Figure 4. Cuticle of the labral gland. A, highly modified
cuticle underlying the labral gland in Coptotermes testaceus
male imago. Note the enlarged pore canals. B, detail of the
apical cuticle underlying the labral gland of Coptotermes
testaceus female imago. Note the distinct layers of the
epicuticle. Abbreviations: en, endocuticle; ep, epicuticle; ex,
exocuticle; s, secretion.
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594 V. PALMA-ONETTO ET AL.
© 2019 The Linnean Society of London, Biological Journal of the Linnean Society, 2019, 126, 587–597
Pericapritermes and Neocapritermes, in which the
labrum is highly modified; and (4) in Microcerotermes
for no clear reason apart from the general small size
of the labrum in the soldier caste (Palma-Onetto et al.,
2018). At the same time, workers and imagoes of other
lineages show similarly developed labra without a
hyaline tip, even in taxa with highly modified labra in
soldiers. However, the ultrastructure of secretory cells
is always similar, although the overall size of the labral
gland is much larger in soldiers having a secretory
epithelium approximately twice as thick, apart from
the larger size of the labrum in general (see Palma-
Onetto et al., 2018).
An interesting question is how labral gland
secretion release is controlled. It seems clear that the
release from secretory cells is under neuronal control,
similarly to sternal gland secretion in Mastotermitidae,
Archotermopsidae and Kalotermitidae (Quennedey,
1969, 1975; Quennedey et al., 2008), the nasus gland
of Angularitermes soldiers (Šobotník et al., 2015),
the salivary glands of different insects (Whitehead,
1971; Ali et al., 1993; Ali & Orchard, 1996; Ali, 1997),
including the termite species Kalotermes flavicollis
(Alibert, 1983) and Prorhinotermes simplex (Šobotník
& Weyda, 2003). After release from secretory cells, the
secretion is presumably evacuated from the body by
pressing the labrum (and hypopharynx) against the
substrate, and the pressure is probably controlled
by groups of campaniform sensillae similarly to
trail pheromone release from the sternal gland
(Stuart & Satir, 1968; Quennedey et al., 2008). The
chemoreceptors are clearly more populous within area
c’, but it remains unknown if these receptors are also
involved in the control of secretion release or if they
play a gustatory function.
The labral gland does not form any specific reservoir,
and the secretion is stored only in the space between
the secretory epithelium and the overlying cuticle,
as well as within the cuticle itself. The absence of a
reservoir, a feature characteristic of defensive glands
(Chapman, 2013), excludes a potential defensive
function of the labral gland, in contrast to previous
speculations (see e.g. Deligne et al., 1981 or Quennedey,
1984). The gland also has a very similar structure in
all castes and species, which indicates that it is not
linked to defense. In addition, the high abundance of
smooth ER, an organelle known to produce lipidic and
volatile secretions, typical of pheromone-producing
glands (Percy-Cunningham & MacDonald, 1987;
Nakajima, 1997; Tillman et al., 1999; Alberts et al.,
2002), provides additional evidence for communicative
function rather than strictly defensive function.
We repeatedly observed soldiers wiping the labrum
against the substrate after encountering a threat
(heterospecific termite or an ant worker), and the
observed behaviour (moving backwards combined
with wiping the labrum against the surface) suggests
that the soldiers are warning their nestmates by using
labral gland secretion. Unfortunately, this function
was not proven by our experiments irrespective of
the setting, and only the avoidance of heterospecifics
(which can be considered as potential competitors or
enemies) to labral gland extracts was statistically
significant. However, this avoidance effect can also
result from other compounds dissolved from the labra,
such as cuticular hydrocarbons as species-recognition
cues (Howard & Blomquist, 1982, 2005) or frontal
gland secretion, which inevitably contaminates all
body parts of termite soldiers (Piskorski et al., 2007,
our unpublished observations). Therefore, the function
of the secretion need to be rigorously tested, especially
since we did not detect any labral gland-specific
compounds, probably due to the small quantity of
the secretion linked to the small gland size and the
absence of a reservoir.
CONCLUSION
The labral gland has been thought to have a defensive
function (Deligne et al., 1981; Quennedey, 1984) .
However, Palma-Onetto et al. (2018) suggested based
on gland morphology, structure and ultrastructure
that it may have a communicative function rather
than defensive function. The presence of the labral
gland in other castes and in the closest relative of
termites, the wood roach Cryptocercus punctulatus, as
well as the occurrence of the same basic features of
the gland structure and ultrastructure, reinforce its
alternative function and suggest its essential role in
colony survival and success. Moreover, our behavioural
observations suggest that the labral gland produces
volatiles secreted in response to a threat. A better
understanding of labral gland function in termites and
cockroaches is needed to enhance knowledge of termite
chemical communication behaviour.
ACKNOWLEDGEMENTS
We thank Mirek Hyliš from the Laboratory of Electron
Microscopy (Faculty of Sciences, Charles University in
Prague) for his help and support with SEM and TEM.
We acknowledge the Imaging Methods Core Facility
at BIOCEV, supported by the Czech-BioImaging large
RI projects (LM2015062 and CZ.02.1.01/0.0/0.0/16_0
13/0001775, funded by MEYS CR), for their support
with obtaining high-resolution SEM imaging data
presented in this paper. We are grateful to Thomas
Bourguignon (OIST, Japan) for his help during
species identification, to Aleš Buček (OIST, Japan)
for assistance during recording the behavioural
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THE LABRAL GLAND IN TERMITES 595
© 2019 The Linnean Society of London, Biological Journal of the Linnean Society, 2019, 126, 587–597
experiments, Jean-Luc Durand (LEEC, France) for his
help with statistics, and Anna Jirošová and Jaromír
Hradecký (both CULS, Czech Republic) for their help
and support during the realization of bioassays and
chemical identification. We thank Régis Vigoroux and
other Hydreco members as well as people from Ebogo
II (Cameroon) for their hospitality during fieldwork.
Financial support was provided by the projects IGA
FLD No. A30/17 (Czech University of Life Sciences,
Prague), CIGA No. 20184307 (Czech University of Life
Sciences, Prague) and ‘EVA4.0’, No. CZ.02.1.01/0.0/0.0/
16_019/0000803 financed by OP RDE.
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SUPPORTING INFORMATION
Additional Supporting Information may be found in the online version of this article at the publisher’s web-site.
Figure S1. Ultrastructure of the labral gland. A, non-modified cuticle at the dorsal side of the labrum in
Embiratermes neotenicus male imago. B, non-modified epithelium surrounding the labral gland in Coptotermes
formosanus worker. C, pinocytotic activity at the cell base in the labral gland epithelium in the male imago of
Glossotermes oculatus. Arrows indicate pinocytotic activity at the base of the cell. D, view of the central channels
in the microvilli of Coptotermes testaceus female imago, which allow secretion release from secretory cells. E, detail
of basal part of the labral gland in a worker of Neocapritermes taracua showing free axons located within the
basal invagination. F, large microtubule bundle running through secretory cells in the wood roach Cryptocercus
punctulatus. Abbreviations: a, axon; en, endocuticle; ex, exocuticle; m, mitochondria; mb, microtubule bundle; n,
nucleus; ser, smooth endoplasmic reticulum; sj, septate junction; v, vesicle.
Table S1. List of termite workers used in analyses, with additional information and secretory epithelium
measures. Blank spaces indicate lack of information. All measurements are in micrometres.
Table S2. List of termite imagoes used in our analyses, with additional information and secretory epithelium
measures. Blank spaces indicate lack of information. All measurements are in micrometres.
Table S3. List of termite workers and imagoes used for transmission electron microscopy. Abbreviations: NM,
not modified cuticle; NV, not visible (due to low sample quality). Blank spaces indicate lack of information. All
measurements are in micrometres.
Video S1. Encounter of Glossotermes oculatus with the ant Solenopsis invicta. Note the soldier of G. oculatus
walking backwards while rubbing the labrum against the substrate immediately after the encounter.
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... Many exocrine glands are also known to be involved in communication. Such organs comprise the labral gland, helping to coordinate defensive activities (Palma-Onetto et al., 2018, 2019, the sternal gland, that secretes trail-following pheromones (Bordereau and Pasteels, 2011;Sillam-Duss es, 2010), the labial and frontal glands of soldiers, producing alarm pheromones in several species (Vrko c et al., 1978;Pasteels and Bordereau, 1998;Cristaldo et al., 2014;Delattre et al., 2015), and the imaginal sternal, posterior sternal and tergal glands, known to produce sex pheromones (Bordereau and Pasteels, 2011;Sillam-Duss es et al., 2011). However, the function of most termite glands discovered so far remains hypothetical. ...
... The glands of termites can be classified into two categories according to their distribution among castes and species. The glands present in all taxa and castes are the labral, mandibular, labial and sternal glands (Lambinet, 1959;Noirot, 1969;Sobotník and Hubert, 2003;Sobotník and Weyda, 2003;Palma-Onetto et al., 2018, 2019. All other glands are confined to some groups and/or castes (Billen and Sobotník, 2015), with the exception of the frontal gland, which occurs in most species of Neoisoptera (Deligne et al., 1981;Quennedey, 1984;Sobotník et al., 2010b;Kutalov a et al., 2013;pers. ...
... The recognition was likely chemical, either from pheromones secreted by the termites, perhaps deliberately, cuticular hydrocarbons that had rubbed onto paper from movement, or from faeces deposited on the paper-or a combination of all three. All three of these potential signals differ between termites-e.g., pheromones [48][49][50][51][52], gut bacteria [14,15]-but the exact mechanism is unknown. Given that there are termite species that are inquilines of other termite species, such chemical signals may be attractive (to find hosts) as well as repellent (to avoid more dominant competitors) [53]. ...
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Thesis
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Termites represent a group of eusocial insects that live in colonies containing up to hundreds to millions. They are highly abundant, exceeding in tropics 6,000 individuals per square meter. Due to their abundance, termites represent an important food source for a wide variety of predators. At the same time, termite defensive adaptations allow the colonies to overcome the predator pressures, being extremely evolutionary successful. This achievement is explained by the development of a complex communication system operated by a rich set of exocrine glands. As many as 20 different exocrine organs are known in termites. Some of these organs had received negligible attention being only known by anecdotal observation. One of these was the labral gland. In this study, I examined the structure and ultrastructure of the labrum in soldiers of 28 species, workers of 28 species and imagoes of 33 species across termites’ main representatives, and in the wood roach Cryptocercus. The labral gland was present in all species and castes, and comprises two secretory regions located on the ventral side of the labrum and the dorso–apical part of the hypopharynx, respectively. The epithelium of the gland consisted of class 1 secretory cells, with an addition of class 3 secretory cells in soldiers of few species. A common feature of the secretory cells was the abundance of smooth endoplasmic reticulum (an organelle known to produce lipidic and often volatile secretions), long microvilli with a channel inside, which releases the secretion through a modified cuticle. According to the structure, ultrastructure and behavioural experiments, my results suggest that the labral gland is involved in defensive communication after encounter to an alien. On the other hand, other glands are extensively studied in some castes but have received almost no attention in other castes. It is the case of the frontal gland, an organ without any equivalent among other animals. The frontal gland is well known in soldiers and imagoes but not much was known about it in workers. In order to provide a complete picture of the evolution of this gland in termite workers and consequently in termites, I studied it in 41 additional species across Neoisoptera. The frontal gland of these species was formed by class 1 secretory cells only, and occured as an epithelial without reservoir in all cases. My data suggest that the frontal gland would have caste–specific evolutionary routes, being its ancestral form epithelial with reservoir in soldiers and imagoes, while epithelial thickening in workers. This study was the first to provide a comprehensive picture of the structure of the labral and frontal gland across all termite taxa and castes, providing fundamental information to enhance our understanding about the evolution and social behaviour of Isoptera.
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Termites are eusocial insects currently classified into nine families, of which only Stylotermitidae has never been subjected to any molecular phylogenetic analysis. Stylotermitids present remarkable morphology and have the unique habit of feeding on living trees. We sequenced mitogenomes of five stylotermitid samples from China and Taiwan to reconstruct the phylogenetic position of Stylotermitidae. Our analyses placed Stylotermitidae as the sister group of all remaining Neoisoptera. The systematic position of Stylotermitidae calls for additional studies of their biology, including their developmental pathways and pheromone communication, which have the potential to change our understanding of termite evolution.
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Although the frontal gland has long been known as a prominent defensive device for termite soldiers in many Rhinotermitidae and Termitidae, almost nothing is known about its function in imagoes. In the present study, we show that the frontal gland of imagoes in Prorhinotermes species is well developed at the time of the nuptial flight, and is filled with a complex mixture of sesquiterpene hydrocarbons and nitroalkenes. The sesquiterpene composition varies between Prorhinotermes simplex and Prorhinotermes canalifrons, between geographically distant colonies of P. simplex (Cuba versus Florida), and even between different flights of closely-related subcolonies. The ratio between (E)-1-nitropentadec-1-ene and sesquiterpenes is sex-specific. The volume of secretory cells decreases in functional kings and queens after colony foundation, and the subcellular organization changes into a form resembling unmodified epidermal cells. Dealate reproductives lose the ability for biosynthesis, and their frontal gland is devoid of volatile compounds found in swarming imagoes. The results obtained in the present study clearly show that the frontal gland is only temporarily active at the time of the dispersal flight. The most likely function of this gland is defence by the toxic nitroalkenes. (C) 2009 The Linnean Society of London, Biological Journal of the Linnean Society, 2009, 98, 384-392.
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Suicidal altruism has been reported for some species of eusocial insects, in which the individual dies in defense of the society. The termites of the genus Ruptitermes are known for the suicidal behavior of the workers which liberate a sticky defensive secretion by body bursting. In the present paper it is given a new interpretation of the defense glands of Neotropical Ruptitermes based on the morphological analysis of three species collected at Rio Claro, SP, Brazil. Before the current study, the suicidal defensive behavior was attributed to the dehiscence of the salivary gland reservoirs. The defense or dehiscent glands of Neotropical Ruptitermes are pair structures rounded in shape that are independent of the salivary glands. The dehiscent glands consist of multiple secretory units that are kept together by thin connective tissue. Each secretory unit is composed of one cell generally with one peripheral nucleus and characteristic secretion. The three species studied here present some histological differences in the secretory units, probably related to the chemical composition of the secretion.
Article
Abstract The evolutionary success of termites has been driven largely by a complex communication system operated by a rich set of exocrine glands. As many as 20 different exocrine organs are known in termites. While some of these organs are relatively well known, only anecdotal observations exist for others. One of the exocrine organs that has received negligible attention so far is the labral gland. In this study, we examined the structure and ultrastructure of the labrum in soldiers of 28 termite species. We confirm that the labral gland is present in all termite species, and comprises two secretory regions located on the ventral side of the labrum and the dorso-apical part of the hypopharynx. The labrum of Neoisoptera has a hyaline tip, which was secondarily lost in Nasutitermitinae, Microcerotermes and species with snapping soldiers. The epithelium of the gland generally consists of class 1 secretory cells, with an addition of class 3 secretory cells in some species. A common feature of the secretory cells is the abundance of smooth endoplasmic reticulum, an organelle known to produce lipidic and often volatile secretions. Our observations suggest that the labral gland is involved in communication rather than defence as previously suggested. Our study is the first to provide a comprehensive picture of the structure of the labral gland in soldiers across all termite taxa.
Chapter
Termites have high biomass in many tropical ecosystems and emit the greenhouse gases CO2 and CH4. They are also recognized as ecosystem engineers, mediating decomposition and other aspects of soil function. Therefore, termites may be significant contributors to biogeochemical cycles, notably those of carbon and methane. We review methods of assessing carbon fluxes through termite populations and argue that direct measurements of net CO2 and CH4 emissions from termites in natural settings (in their nests or in the soil) are the best data for scaling-up calculations, if accompanied by accurate estimates of biomass and assemblage feeding-group composition. Actual determinations of gas fluxes from termites, and the attendant computation of regional and global budgets made over the past two decades are reviewed. For CO2, it is concluded that termites contribute up to 2% of the natural efflux from terrestrial sources, a large contribution for a single animal taxon, but small in the global context. For CH4, we note that calculations are still hampered by uncertainties over termite biomass distribution and a general failure to consider local and landscape-level oxidation by methylotrophic microorganisms as a factor mitigating net fluxes. Nevertheless the balance of evidence, including new data on local oxidation, suggests that annual contributions by termites are almost certainly less than 20 Tg, and probably less than 10 Tg (ca. 4% and 2% of global totals from all sources, respectively). Climate changes and land use intensification may cause minor modifications of the overall distribution of termites, but a more serious impact on soil stability and function could result from changes in the balance of feeding groups. The response of termites to changes in the quality and quantity of plant litters is uncertain, but direct effects from elevated atmospheric CO2 are unlikely. Global changes will broadly favour wood- and litter-feeding termites over soil-feeders, but with regional differences and complications arising from patterns of landscape fragmentation and historical factors.