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Molecular Mechanism of the Two-Component Suicidal Weapon of Neocapritermes taracua Old Workers

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In termites, as in many social insects, some individuals specialize in colony defense, developing diverse weaponry. As workers of the termite Neocapritermes taracua (Termitidae: Termitinae) age, their efficiency to perform general tasks decreases, while they accumulate defensive secretions and increase their readiness to fight. This defensive mechanism involves self-sacrifice through body rupture during which an enzyme, stored as blue crystals in dorsal pouches, converts precursors produced by the labial glands into highly toxic compounds. Here, we identify both components of this activated defense system and describe the molecular basis responsible for the toxicity of N. taracua worker autothysis. The blue crystals are formed almost exclusively by a specific protein named BP76. By matching N. taracua transcriptome databases with amino acid sequences, we identified BP76 to be a laccase. Following autothysis, the series of hydroquinone precursors produced by labial glands get mixed with BP76, resulting in the conversion of relatively harmless hydroquinones into toxic benzoquinone analogues. Neocapritermes taracua workers therefore rely on a two-component activated defense system, consisting of two separately stored secretions that can react only after suicidal body rupture, which produces a sticky and toxic cocktail harmful to opponents.
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Article
Molecular Mechanism of the Two-Component Suicidal Weapon
of Neocapritermes taracua Old Workers
Thomas Bourguignon,*
,y,1,2
Jan
Sobotn
ık,*
,y,2
Jana Brabcov
a,
3
David Sillam-Dusse
`s,
4,5
Ale
sBuc
ˇek,
3
Jana Krasulov
a,
3
Blahoslava Vytiskov
a,
2
Zuzana Demianov
a,
6
Michael Mare
s,
3
Yves Roisin,
7
and Heiko Vogel
8
1
School of Biological Sciences, University of Sydney, Sydney, NSW, Australia
2
Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech Republic
3
Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Prague, Czech Republic
4
Institute of Research for Development—Sorbonne Universit
es, iEES-Paris, Bondy, France
5
University Paris 13—Sorbonne Paris Cit
e, LEEC, Villetaneuse, France
6
IMP—the Research Institute of Molecular Pathology, Vienna, Austria
7
Evolutionary Biology and Ecology, Universit
e Libre de Bruxelles (ULB), Brussels, Belgium
8
Department of Entomology, Max Planck Institute for Chemical Ecology, Jena, Germany
y
These authors contributed equally to this work.
*Corresponding author: E-mail: thomas.bourgui@gmail.com; sobotnik@fld.czu.cz.
Associate editor: Nicolas Vidal
Abstract
In termites, as in many social insects, some individuals specialize in colony defense, developing diverse weaponry. As
workers of the termite Neocapritermes taracua (Termitidae: Termitinae) age, their efficiency to perform general tasks
decreases, while they accumulate defensive secretions and increase their readiness to fight. This defensive mechanism
involves self-sacrifice through body rupture during which an enzyme, stored as blue crystals in dorsal pouches, converts
precursors produced by the labial glands into highly toxic compounds. Here, we identify both components of this
activated defense system and describe the molecular basis responsible for the toxicity of N. taracua worker autothysis.
The blue crystals are formed almost exclusively by a specific protein named BP76. By matching N. taracua transcriptome
databases with amino acid sequences, we identified BP76 to be a laccase. Following autothysis, the series of hydroquinone
precursors produced by labial glands get mixed with BP76, resulting in the conversion of relatively harmless hydroqui-
nones into toxic benzoquinone analogues. Neocapritermes taracua workers therefore rely on a two-component activated
defense system, consisting of two separately stored secretions that can react only after suicidal body rupture, which
produces a sticky and toxic cocktail harmful to opponents.
Key words: altruism,colonydefense,termite,Isoptera,laccase.
Introduction
Members of social insect colonies are partitioned into castes
that specialize in colony tasks, such as reproduction, foraging
for food, feeding dependent individuals, or defending the
colony (Wilson 1971). This task partitioning is often associ-
ated with multiple morphological adaptations distributed
among castes. Among the manifold morphological adapta-
tions, the most extreme ones are probably those connected
to colony defense, such as the enlarged mandibles and defen-
sive glands producing a wide array of defensive compounds
(Prestwich 1984;
Sobotn
ık, Jiro
sov
a, et al. 2010).
Social insects colonies can count up to millions of individ-
uals, attracting many predators, and being subjected to in-
tense competition from neighboring colonies (Lepage and
Darlington 2000), making colony defense essential. In the
most complex insect societies, defense is entrusted to sterile
individuals, which often constitute a specialized soldier caste.
Social Hymenoptera defenders have developed sophisticated
strategies, which in extreme cases involve self-destructive pro-
cesses, such as sting autotomy in some bees, wasps, and ants
(Hermann 1971), or autothysis, that is, body rupture to release
defensive substances from specialized glands, in some
Camponotus (Colobopsis)antspecies(Maschwitz and
Maschwitz 1974). Termite defense strategies are comparably
diverse, and although the weaponry is particularly developed
in soldiers, workers may actively take part in colony defense
(Thorne 1982), especially in soldierless species (Sands 1982).
Suicidal defense is common in soldiers of many species that
keep their mandibles locked in the opponent’s wound
(Deligne et al. 1981;Prestwich 1984). Like Camponotus ants,
some termite soldiers or workers can sacrifice themselves, and
perform autothysis to release defensive secretions by body
rupture, or dehiscence, during which the inner organs burst
out of the abdomen. While dehiscence mostly occurs in work-
ers of soldierless termites (Sands 1982), autothysis evolved in-
dependently in soldiers of Serritermitidae (Costa-Leonardo
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and Kitayama 1991;
Sobotn
ık, Bourguignon, et al. 2010),
Globitermes (Bordereau et al. 1997), Dentispicotermes
(
Sobotn
ık, Jiro
sov
a, et al. 2010), and Apilitermes (Deligne
and De Coninck 2006), and in workers of Ruptitermes
(Costa-Leonardo 2004)andN. taracua (all Termitidae)
(
Sobotn
ık et al. 2012,2014).
Termite defensive secretions include a rich set of com-
pounds, terpenoids produced by the frontal gland or ace-
tate-derived quinones produced by labial glands being the
most common (Prestwich 1984;
Sobotn
ık, Jiro
sov
a, et al.
2010). Although these compounds are in general synthesized
de novo (Prestwich et al. 1981), the biosynthetic enzymes are
largely unknown, except for geranylgeranyl diphosphate
synthase involved in diterpene synthesis in Reticulitermes
and Nasutitermes (Hojo et al. 2007,2011). Although proteins
arealsofrequentlyusedastoxinsorvenomsinanimals
(Bettini 1978;Casewell et al. 2013), and despite the presence
of unidentified proteins responsible for stiffening after air ex-
posure in glandular secretions of Mastotermes and
Odontotermes (Moore 1968;Wood et al. 1975), no such
role has been reported in termites so far.
Neocapritermes taracua (Termitidae: Termitinae) nests
and foraging areas are defended by rare soldiers, and by work-
ers that use controlled autothysis, in which the labial gland
secretion is converted into toxic compounds by a highly
abundant protein stored as blue crystals in exterior pouches
(
Sobotn
ık et al. 2012). The “explosive backpacks” are only
ready-to-use in older workers (hereafter called blue workers)
in which the bursting liquid is sticky and toxic to termite
opponents. Young workers (hereafter called white workers)
also have the potential to commit autothysis but do so less
readily and release negligible amounts of toxic secretions
(
Sobotn
ık et al. 2012,2014).
Here,wedescribethemolecularmechanismunderlying
the toxicity of the bursting liquid of old N. taracua workers
(
Sobotn
ık et al. 2012,2014), in which two sets of glands are
involved. The labial glands secrete the precursors that, after
body rupture, react with a blue copper protein produced by
specialized crystal glands. We combined tissue-specific tran-
scriptome sequencing of both white and blue N. taracua
workers with proteomics to identify a candidate laccase
named BP76. Next, we investigated the expression pattern
of BP76 and five additional laccases identified in N. taracua
across different tissues of white and blue workers. Finally, we
identified the compounds produced by the labial glands of
workers and determined the enzymatic activity of BP76 that
converts these compounds into harmful analogues after
autothysis.
Results
Identification of BP76
Blue crystals isolated from dorsal pouches of blue workers
were solubilized and their content separated with denatured
gel electrophoresis (sodium dodecyl sulfate–polyacrylamide
gel electrophoresis [SDS–PAGE]; see supplementary fig. S1,
Supplementary Material online). Coomassie staining of the
gel revealed a single dominant band, named BP76 protein
that forms most of the blue crystals mass (
Sobotn
ık et al.
2012). To enable unambiguous identification of the candidate
protein(s) and subsequently analyze tissue-specific gene ex-
pression levels, we performed NextGen sequencing (RNAseq)
with RNA isolated from 1) body without gut, labial gland, and
crystal gland, 2) labial glands, and 3) crystal glands of white
and blue N. taracua workers. We combined the tissue-specific
data sets to build the de novo transcriptome assembly (TA)
that we used to identify the cDNA sequence of BP76 as well as
related proteins. Three different proteomic methods were
employed to identify BP76, which included data-independent
(Edman sequencing) and data-dependent mass spectrometry
(MS-E and MS/MS) methods. Combining the three different
proteomics approaches resulted in the identification of pep-
tides that covered about half the predicted BP76 protein se-
quence (see supplementary fig. S2,Supplementary Material
online). We subsequently carried out a local BLAST search
against the N. taracua TA and identified the complete cDNA
sequence of BP76 (GenBank accession number: KT945242).
The best NCBI BLAST hit showed that BP76 is 69% similar to a
protein of the termite Zootermopsis nevadensis assigned to L-
ascorbate oxidase (GenBank accession number: KDR11748.1).
The next most similar proteins were laccases of Reticulitermes
flavipes (GenBank accession number: ACX54558.1–
ACX54565.1) and Z. nevadensis (GenBank accession
number: KDR11747.1) that shared slightly more than 50%
similarity with BP76. Further searches against the Conserved
Domain Database (http://www.ncbi.nlm.nih.gov/Structure/
cdd/wrpsb.cgi) identified three cupredoxin domains charac-
teristic of multicopper oxidases. These results confirm that
BP76 is an oxidoreductase and a multicopper protein that we
assigned to insect laccases (see Enzymatic Essays).
Phylogenetic Analysis of Laccases
The TA-contig sequences of the de novo reference assembly
were translated using BLASTx, searched against the NCBI nr
database and functionally annotated by gene ontology and
enzyme commission number analysis. Subsequent screening
for cDNAs encoding laccases resulted in the identification of a
total of six candidate genes,of which we discarded two for a
phylogenetic analysis, as the identified cDNAs encoded for
less than half of the complete proteins. Based on an amino
acid alignment combining laccases from different insect
orders (see supplementary fig. S3,Supplementary Material
online), we then compared the relative positions of the four
N. taracua laccases, using Maximum Likelihood (ML) inferred
phylogenetic analyses. These analyses showed a disjoint dis-
tribution of insect laccases, resulting in at least four separate
clades. Each of the four N. taracua laccases clustered with a
different clade, with all of the critical nodes well supported
(node support values of 80% or above). Neocapritermes tar-
acua candidate laccase BP76 is placed within a group of ter-
mite sequences, including Zootermopsis and Reticulitermes
laccases but excluding other insect laccases (fig. 1).
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FIG.1. Phylogenetic tree of insect laccases reconstructed with ML method. Nodes and tips in blue are the termite cluster to which the laccase BP76
belongs; other Neocapritermes taracua laccases marked in bold. Species names are followed by NCBI GenBank accession numbers. Branch lengths are
proportional to evolutionary distance according to the provided scale. Numbers along branches indicate bootstrap percentage support. Bootstrap
support values less than 50% are not shown.
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Expression of BP76
We profiled the expression of the six laccase genes in N.
taracua by aligning the Illumina 100-bp sequence tags from
theblueandwhiteworkertissuesampleswiththeTA-contig
sequences. Quantitative RNAseq analysis of the transcripts
revealed that most of the laccases (laccases 2, 3, 4, 5) were
expressed at low level, while laccase-1 was expressed at mod-
erate but comparable levels across all tissues sampled. In con-
trast, the candidate laccase BP76 identified at the protein level
displayed variable but clear tissue-specific expression levels.
BP76 is highly expressed in blue workers compared with white
workers, with the highest overall level of expression in the
crystal glands of blue workers, followed by the crystal glands
of white workers (fig. 2). Overall, the expression patterns of
the identified laccases verified a single highly abundant lac-
case (BP76) expressed in crystal glands of blue workers. The
laccase gene expression data obtained by RNAseq was verified
using replicated quantitative real-time reverse transcription
polymerase chain reaction (qRT-PCR) (see supplementary fig.
S4,Supplementary Material online).
Labial Gland Secretions
The dissected labial glands of blue workers contain three
mildly toxic major compounds: the dominant 2-
methyl-hydroquinone, along with 2-ethyl-hydroquinone,
and hydroquinone, while these compounds are not detected
in labial glands of white workers (fig. 3). The bursting liquid of
blue workers contains large amounts of the highly toxic 2-
methyl-p-benzoquinone (along with 2-ethyl-p-benzoquinone
and p-benzoquinone), whereas only traces of the same
compounds were found in the bursting liquid of white work-
ers (fig. 3). In addition, the amounts of p-benzoquinone, 2-
methyl-p-benzoquinone, and 2-ethyl-p-benzoquinone are
low in the labial glands of blue workers but sharply increase
after autothysis. Thus, mildly toxic hydroquinones accumu-
late in the labial glands of blue workers, and are turned into
highly toxic benzoquinones in the bursting liquid following
autothysis, whereas the amounts of hydroquinones and ben-
zoquinones are low in white workers.
Enzymatic Assay
To confirm that BP76, a putative laccase, has phenol oxidase
activity and thus participates in the oxidation of hydroqui-
nones to benzoquinones, we performed a standard functional
test. Solubilized N. taracua blue crystals possess significant
enzymatic activity towards a panel of model phenol oxidase
substrates (table 1). The efficient conversion of p-diphenol
substrates, o-diphenols, 2,6-dimethoxyphenol, and 2,20-azino-
bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) sup-
ported that the tested enzyme exhibits substrate specificity
of laccases (Baldrian 2006). “In gel” zymography analysis con-
firmed that BP76 is the only component of N. taracua blue
crystals responsible for the detected laccase-like activity (see
supplementary fig. S5,Supplementary Material online).
Furthermore, we investigated the sensitivity of BP76 toward
a range of known inhibitors and found effective inhibition by
several selective inhibitors of laccases but also tyrosinases
(table 2). The enzymological analyses demonstrated laccase-
like activity of BP76 and provided functional evidence sup-
porting the proteomic identification of BP76 as a laccase,
although BP76 also depicts enzymological activities similar
to tyrosinase.
Discussion
In this study, we identified and characterized both constitu-
ents of the activated two-component defense system of old
N. taracua workers. Unlike all other termite defensive systems,
the N. taracua worker defense involves two distinct struc-
tures, the crystal glands and the labial glands, whose products
interact after sacrificial autothysis (
Sobotn
ık et al. 2012,2014).
Our results show that the crystal glands secrete BP76, a multi-
copper protein from the laccase family with phenol oxidase
activity (Solomon et al. 1996,2014). The analyses of the labial
glands and bursting liquid allowed tracing conversion of hy-
droquinones into their benzoquinone analogues, which are
well-known for their toxic properties exploited by many ar-
thropods (Bettini 1978;Eisner et al. 2005).
Laccases form a large protein family with representatives in
bacteria, plants, fungi, and animals including insects. They
generally reveal a very broad range of natural substrates,
being able to oxidize diphenols, amino- and methoxy-substi-
tuted phenols, and aromatic diamines (Dittmer and Kanost
2010). In insects, laccases are involved in diverse processes,
ranging from cuticle tanning to detoxification and digestion.
For example, they are a component of the venom of the
parasitoid wasp Pimpla hypochondriaca, probably suppress-
ing immune response of the host (Parkinson et al. 2001,2003),
α
BP76
FIG.2. Heat map showing relative expression levels of the laccase BP76
and five additional laccases in different Neocapritermes taracua tissues.
Abbreviations for individual laccases as well as two control genes are
shown on the left whereas tissues are shown on the top. Two moderate
(EIF4a)tohighly(EF1-) expressed housekeeping genes are shown as
controls to verify equal expression across tissues. The map is based on
log2-transformed RPKM values (blue represents weakly expressed genes,
and red represents strongly expressed genes).
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and are expressed in the salivary glands of the leaf-hopper
Nephottetix cincticeps, in which their function is probably re-
lated to plant compound detoxification (Hattori et al. 2005).
Although laccases were also shown to have digestive func-
tions in R. flavipes (Coy et al. 2010;Scharf et al. 2011), they
generally participate in tanning of soft pro- or endo-cuticle in
many insects (Arakane et al. 2005,2009;Moussian 2010), in-
cluding termites (Reticulitermes speratus:Masuoka et al.
2013). Our phylogenetic analysis showed that the identified
laccases of N. taracua belong to distinct clusters, and that
BP76 is a member of a termite-specific cluster. This cluster
also contains a phenol-oxidizing laccase which was previously
found to be expressed in the salivary glands of termite work-
ers and play a role in lignocellulose digestion (Coy et al. 2010).
The exclusive placement of the N. taracua candidate BP76
within a group of termite sequences, including presumably
duplicated Zootermopsis genes but excluding other insect
laccases, supports a distinct evolutionary history of this ter-
mite-specific group. The acceptance of a wide range of sub-
strates as well as the involvement in a diverse set of functions
in insects likely increases the probability to gain a new func-
tion, for example, after gene duplication events. The main
requirement would then be its expression at the right place
and in adequate amounts, a change which likely results in
FIG.3. GC GC/TOF-MS analyses of (A) labial glands of blue workers; (B) the bursting liquid of blue workers; (C) labial glands of white workers; and (D)
the bursting liquid of white workers.
Table 2. Inhibition of Enzymatic Activity of BP76 by Selective Phenol
Oxidase Inhibitors.
Inhibitor Target
Enzymes
Inhibitor
Concentration (mM)
Remaining
Activity (%)
a
4-Hexylresorcinol 0.5 74.4 0.1
Tyrosinases 1 62.2 0.2
40
Salicylhydroxamic
acid
0.5 54.5 0.1
Tyrosinases 1 44.7 0.5
426.50.2
Cetyltriammonium
bromide
0.5 100 0.5
Laccases 1 60.3 0.2
438.00.6
a
The remaining enzymatic activity of Neocapritermes taracua blue crystals is ex-
pressed relative to the uninhibited control (100%) and was determined using the
activity assay with DMP as substrate. The mean values SD (n=3) are given.
Table 1. Enzymatic Activity of BP76 toward Phenol Oxidase
Substrates.
Substrate Activity (units)
a
ABTS 6.6 0.013
DMP 6.0 0.11
Catechol 72.0 1.21
4-tert-Butylcatechol 11.6 0.49
Hydroquinone (108 1.1) 10
4
Methyl-p-benzoquinone (142 6.0) 10
4
a
One unit of activity corresponds to the formation of 1 mmol of product per minute
by 1 mgofproteinfromNeocapritermes taracua blue crystals. The mean values SD
(n=3) are given.
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little extra costs but sometimes important benefits. The high
level expression of BP76 in crystal glands of old workers fulfills
these requirements.
Although proteins are not common constituents of de-
fensive exudates, some may become sticky after air expo-
sure, and that is why they form defensive secretions in some
termites (Moore 1968;Wood et al. 1975), cockroaches
(Plattner et al. 1972), and other Arthropods (Deslippe
et al. 1996). The same phenomenon was also observed in
N. taracua, in which the blue crystals turn into extremely
sticky material shortly after coming into contact with the
hemolymph, and this feature explains incapacitating prop-
erties of bursting liquid on house fly (
Sobotn
ık et al. 2012).
The crystal gland of N. taracua workers is located just
below the cuticle of the first abdominal tergite (
Sobotn
ık
et al. 2014), suggesting that the production of cuticular
laccases that oxidize quinones was co-opted as a defensive
device. Moreover, this specific position allows BP76 to get
into contact with oxygen from the air which is generally
required as a second substrate for the enzymatic activity
of all laccases. Traces of benzoquinones are also found in
the dissected labial gland samples, because the oxidization
of hydroquinones takes place at air exposure during sample
handling, but the external enzyme BP76 is required for a
quick conversion.
Benzoquinones are reactive compounds produced for
defense against pathogens or predators in many insects
(Bettini 1978;Eisner et al. 2005;Otti et al. 2014), as well as
in the termites, such as Mastotermes and several
Macrotermitinae, in which they are produced in the labial
glands of soldiers (Moore 1968;Wood et al. 1975;Sillam-
Dusse
`s et al. 2012). In comparison, hydroquinones are less
toxic because of their lower electrophilicity resulting in
lowerreactivity(O’Brien 1991). The mode of production
of benzoquinones in N. taracua workers is therefore remark-
able, as mildly toxic hydroquinones are stored inside the
body in anoxic conditions and are converted into benzoqui-
nones only after body rupture by the laccase BP76 (fig. 4).
This two-component mechanism recalls the astounding ap-
paratus of bombardier beetles, which produce a mixture of
hydroquinone and hydrogen peroxide that, when the bee-
tles are disturbed, react and produce a spurt of boiling ben-
zoquinone and water mixture that can severely harm the
opponents (Aneshansley et al. 1969;Arndt et al. 2015).
In N. taracua, the efficiency of the two-component acti-
vated defense system is achieved by two sets of glands,
whose contents need to come in contact by autothysis in
order to allow the activation (fig. 4). If the autothysis is not
triggered, the contents are not mixed and the worker re-
mains safe. This allows the workers to defend against pred-
ators by biting first; only upon imminent danger they can
trigger the two-component system, activating their toxic
weaponry. While the workers will die after autothysis, the
few minutes before death allow them to search for oppo-
nents, contaminating them with toxic benzoquinones. N.
taracua belongs to the most apical family of termites, the
Termitidae (Bourguignon et al. 2015), that evolved the
greatest diversity of defense strategies, and we can as-
sume that the complex two-component suicidal weapon
of N. taracua contributed to its ecological success, making
it an abundant termite species in many forests in Amazonia.
FIG.4. Schematic drawing of the two-component suicidal weapon occurring in Neocapritermes taracua workers.
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Materials and Methods
Termites
Workers were repeatedly extracted from several nests of N.
taracua (Krishna and Araujo 1968) collected in French
Guiana, near Petit Saut dam (N 05040, W 052590), between
2010 and 2013. The nest cores were transported to Prague,
and kept alive in a glass box with moist decayed leaf-litter.
Tissue Dissection and RNA Extraction
We performed RNA extraction on tissue of N. taracua blue
and white workers from three different body parts: 1) whole
worker body without gut, labial glands and crystal glands, (40
termites); 2) labial glands (60 termites), and 3) crystal glands
(100 termites)) stored in TRIzol (Invitrogen) at 80 Cprior
to RNA extraction. The collected tissue material was divided
into three different replicates each, and total RNA was ex-
tracted using standard phenol–chloroform procedure with
TRIzol according to the manufacturer’s protocol (Life
Technologies), followed by digestion of DNA contaminants
with TURBO DNase (Ambion) at 37Cfor1handsubse-
quent purification using the RNAeasy Mini Kit (Qiagen) ac-
cording to the manufacturer’s protocol for RNA cleanup. The
integrity of the RNA was verified using an Agilent 2100
Bioanalyzer and a RNA 6000 Nano Kit (Agilent
Technologies, Palo Alto, CA). The quantity of RNA was de-
termined using a Nanodrop ND-1000 UV/Vis spectrophot-
ometer (Thermo Scientific). For each of the six samples, equal
amounts of total RNA isolated from the three replicates were
pooled for transcriptome sequencing.
Illumina Sequencing, TA, and Annotation
Tissue-specific transcriptome sequencing of the six different
RNA samples was performed with poly(A) +enriched mRNA
fragmented to an average of 150 nt. Sequencing was carried
out by the Max Planck Genome Center Cologne (MPGCC) on
an Illumina HiSeq2500 Genome Analyzer platform using
paired-end (2 100 bp) reads. This yielded approximately
30 million reads for each of the six samples. Quality control
measures, including the filtering of high-quality reads based
on the score given in fastq files, removal of reads containing
primer/adaptor sequences and trimming of read length, were
carried out using CLC Genomics Workbench v6.5 (http://
www.clcbio.com).ThedenovoTAwascarriedoutwiththe
same software, combining all of the six RNAseq samples, and
selecting the presumed optimal consensus transcriptome as
described in (Vogel et al. 2014). The resulting final de novo
reference TA (backbone) of N. taracua contained 96,217 con-
tigs with a N50 contig size of 1,372 bp and a maximum contig
length of 23,644 bp. The transcriptome was annotated using
BLAST, Gene Ontology and InterProScan searches using
BLAST2GO PRO v2.6.1 (www.blast2go.de;G
otz et al. 2008)
as described in Vogel et al. (2014). To identify candidate genes
expressed in the N. taracua crystal glands, we established a
reference set of known or predicted insect-derived laccases
and phenoloxidases using published sequences and searching
our in-house database as well as public databases (NCBI). We
have deposited the complete short read (Illumina HiSeq2500)
data with the following accession numbers: PRJEB11456
(SRA). The complete study can also be directly accessed
here: http://www.ebi.ac.uk/ena/data/view/PRJEB11456.
Cloning and Sequencing of BP76 cDNA
Total RNA isolated from N. taracua blue crystal glands was
transcribed into cDNA using the SuperScript III First-Strand
Kit (Invitrogen) according to the manufacturer’s protocol. To
amplify the cDNA sequence of N. taracua laccase BP76, gene-
specific primers were used which were designed based on the
contigidentiedfromourRNAseqdataandthedesiredpro-
duct amplified by PCR. Positive PCR bands of the correct size
were cut out from the agarose gels, column purified
(Zymogen), ligated into the pCR II TOPO vector
(Invitrogen). Ligations were transformed into Escherichia coli
ELECTROMAX DH5-E electro-competent cells (Invitrogen).
Plasmid minipreparation from bacterial colonies grown in 96
deep-well plates was performed using the 96 robot plasmid
isolation kit (Nextec) on a Tecan Evo Freedom 150 robotic
platform (Tecan). Sequencing of both the 30-and5
0-termini
of as well as with internal primers was carried out on an ABI
3730 xl automatic DNA sequencer (PE Applied Biosystems).
Vector clipping, quality trimming and sequence assembly was
done with the Lasergene software package (DNAStar Inc.).
The full-length cDNA as well as the deduced protein se-
quence of N. taracua laccase BP76 was submitted to
Genbank (Accession Number: KT945242).
Sequencing of BP76 Protein Fragments
Blue crystals were extracted from frozen specimens, solubi-
lized in 20 mM Tris-HCl pH 7.0 containing 0.1M NaCl and
clarified by microfiltration. Aceton-precipitated proteins were
separated on 15% SDS–PAGE gel, electroblotted onto poly-
vinylidene fluoride (PVDF) membrane, and visualized with
Coomassie blue. The BP76 band was cut out of the gel and
blot for mass spectrometric and N-terminal sequencing anal-
ysis, respectively. For internal protein sequencing of BP76, the
solubilized blue crystals were digested with Lys-C (Roche),
resulted fragments separated by SDS–PAGE and analyzed
by Edman sequencing as above using a Procise 494 cLC pro-
tein sequencer (Applied Biosystems). For mass spectrometric
characterization, the BP76 band was digested with trypsin and
analyzed by TripleTOF 5600 system (Sciex, Concord, Canada)
coupled with Ultimate 3000 RSLC nano system (Thermo
Scientific). The top20 MS method was applied. The resulted
MS spectra were searched with Protein Pilot 4.5 (Sciex)
against NCBI protein database (January 2013) limited to tax-
onomy: Insecta. Peptides identified by Edman sequencing and
LC-MS/MS are in supplementary material,Supplementary
Material online.
To enable a precise identification of BP76 utilizing our
transcriptome database, we used an MS-E approach.
Lyophilized proteins from N. taracua blue crystal gland secre-
tions were solubilized in 10 mM Tris (pH 8.0) containing pro-
tease inhibitor cocktail (1 , Pierce). Heat denatured protein
samples were separated on a Criterion 4–12% gradient
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at University of Sydney on February 24, 2016http://mbe.oxfordjournals.org/Downloaded from
polyacrylamide SDS–PAGE gel (BioRad) and stained using
Colloidal Coomassie blue. The single dominant protein
band (see supplementary material,Supplementary Material
online) was excised from the Coomassie-stained gel and tryp-
tic digestion and extraction of tryptic peptides from gel pieces
was carried out as described before (Shevchenko et al. 2006).
For LC-MS, analysis samples were reconstructed in 10 mlaque-
ous 0.1% formic acid.
The samples were analyzed using a nano Acquity nano-
UPLC system on-line connected to a Q-ToF Synapt HDMS
mass spectrometer (Waters, Milford). Desalting of samples
was performed using a Symmetry C18 trap-column
(20 0.18 mm, 5 mm particle size, Waters, Milford) at a
flow rate of 15 ml/min followed by peptide separation on a
nano Acquity C18 analytical column (200 mm 75 mmID,
C18 BEH 15 130 material, 1.7 mm particle size, [Waters,
Milford]). Mass spectrometer settings were as described pre-
viously (Kirsch et al. 2012).LC-MSdatawereacquiredin
positive ESI mode under data-independent acquisition
(MSE) controlled by MassLynx v4.1 software. The collision
energy was set at 4 eV in low energy (MS) scans, and
ramped from 15 to 40 eV in elevated energy (MSE) scans.
The mass range (m/z) for both scans was 300–1,900 and
50–1,700 Da, respectively. The scan time was set at 1.5 s for
both modes of acquisition with an interscan delay of 0.2 s. A
reference compound, human Glu-Fibrinopeptide B
(650 fmol/ml in 0.1% formic acid/acetonitrile [vol/vol, 1:1]),
was infused continuously through a reference sprayer for ex-
ternal calibration.
ProteinLynx Global Server version 2.5.2 (Waters, Milford)
was used for processing of raw files and for database searching.
The continuum LC-MSE data were lock-mass-corrected,
smoothed, background-subtracted, centered, deisotoped, and
charge-state-reduced. Thresholds for low/high energy scan five
ions and peptide intensity were set at 150, 30, and 750 counts,
respectively. Processed data were searched against the
Swissprot database (downloaded on July 27, 2011, from
http://www.uniprot.org/) combined with N. taracua protein
subdatabase constructed from N. taracua transcriptome data-
base by their translation from all six reading frames. Database
searches were performed at 2% false discovery rate, using the
following parameters: minimum number of product ion
matches per peptide (5), minimum number of product ion
matches per protein (7), minimum number of peptide matches
(2), and maximum number of missed tryptic cleavage sites (1).
Sequence Alignment and Phylogenetic Analysis
Amino acid sequences were aligned using MAFFT version
7.017 (E-INS-I parameter set; Katoh et al. 2002) with default
parameters using deduced amino acid sequences from insect
laccase transcripts retrieved from N. taracua andNCBIand
manually inspected for regions of high-quality alignment.
MEGA6 (Tamura et al. 2013) was used to select the best-fit
substitution model for the amino acid alignment. The
unrooted phylogenetic tree was inferred by the ML method
using PhyML (Dereeper et al. 2008) available at LIRMM
(http://www.phylogeny.fr/) and displayed and edited with
FigTree (http://tree.bio.ed.ac.uk/software/figtree). For gene
tree generation using Bayesian inference analysis imple-
mented in Mr. Bayes 3.2.2 (Ronquist and Huelsenbeck
2003), the prior was set for the amino acid models to mix,
thereby allowing jumps between fixed-rate amino acid
models. The Z. nevadensis KDR20236 sequence was used as
outgroup. The Markov Chain Monte Carlo runs were carried
out for 1,000,000 generations. Log likelihood values showed
that equilibrium had been reached after the first 10,000 gen-
erations which were discarded as “burnin.” Two runs were
conducted per data set showing agreement in topology and
likelihood scores. The ML and the Bayesian tree topologies
including their general subfamily relationships were generally
in good agreement.
Digital Gene Expression Analysis
Digital gene expression analysis was carried out by using QSeq
Software (DNAStar Inc.) to remap the Illumina reads from all
six samples onto the reference transcriptome and then
counting the sequences to estimate expression levels, using
previously described parameters for read mapping and nor-
malization (Vogel et al. 2014).Biasesinthesequencedatasets
and different transcript sizes were corrected using the RPKM
algorithm (reads per kilobase of transcript per million
mapped reads) to obtain correct estimates for relative expres-
sion levels. To control for the effect of global normalization
using the RPKM method, we also analyzed a number of highly
conserved housekeeping genes, including several genes
encoding ribosomal proteins (rpl3, rpl5, rpl7a, rps3a, rps5,
rps8, rps18, and rps24), NADH-dh, elongation factor 1alpha
and eukaryotic translation initiation factors 4 and 5. The over-
all expression levels across samples and treatments for these
housekeeping genes was lower than 1.3-fold between samples
(based on log2 transformed RPKM values), indicating they
were not differentially expressed.
Quantitative Real-Time Reverse Transcription PCR
analysis (qRT-PCR)
The differential expression of Laccase-1,-2,-5 and laccase BP76
identified by RNAseq was confirmed by qRT-PCR using pri-
mers designed based on the contig data (supplementary table
S1,Supplementary Material online) using Primer3Plus (http://
primer3plus.com/cgi-bin/dev/primer3plus.cgi). Primers were
tested by generating individual melting curves and by prepar-
ing dilution series for primer efficiency calculations. Primers
with a single peak in the melting curve and an efficiency value
between 0.9 and 1.1 were used in subsequent quantitative
PCR experiments. Reverse transcription and real-time PCR
were carried out on three biological replicates of the different
N. taracua tissue samples using the Verso cDNA synthesis kit
and the Absolute Blue qPCR SYBR Green Mix (ThermoFisher)
on the CFX Connect Real-Time PCR Detection System
(Biorad) according to the manufacturer’s recommendations.
ThedatawerenormalizedagainstActin and RPS8, internal
controls stable across all samples. Relative expression level
differences were calculated based on the CT method.
The resulting values were then used to calculate the relative
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at University of Sydney on February 24, 2016http://mbe.oxfordjournals.org/Downloaded from
expression levels between the White-Body sample (set to 1)
and all other tissue samples relative to the White-Body
sample.
Identification of the Labial Gland Chemicals and
Defensive Secretion
Labial glands of three categories of workers (white, white/blue
intermediate, blue) were dissected from ice-anesthetized in-
dividuals and submerged in methanol (4 ml/individual), ultra-
sonicated for 10 min and extracted at 4C overnight. The
methanol extracts were analyzed by GC-MS (quadrupole
DSQ II, Thermo Scientific) with a nonpolar ZB-5MS column
(30m, id 0.25mm, 0.25mm phase thickness). The tempera-
ture program was from 50C to 120Cat8
C/min rate and
then to 320Cat15
C/min rate. Helium was used as a carrier
gas at a constant flow rate of 1 ml/min.
For the analyses of defensive secretion, 10 blue, 10 white
and 10 intermediate workers were repeatedly forced to ex-
plode by tweezer pressure. The volatiles were extracted from
1.5 ml vials by headspace SPME (yellow SUPELCO SPME fiber,
PDMS, 30 mm, non-bonded) for 5 min. Chemical analyses
were performed using two-dimensional gas chromatography
coupled with mass spectrometric detection (GCxGC/TOF-
MS; LECO, Pegasus 3D). The temperature program for the
primary column (nonpolar ZB5-MS: 30 m, id 0.25 mm,
0.25 mm phase thickness) was 50 C (1 min) to 320 C
(4 min) at 8 C/min; the secondary column (polar RTX-50:
2m, id 0.1mm, 0.1mm phase) was set 10 Chigher.
Methanol extracts of 1 ml were injected, representing about
1/10 of worker equivalent.
The identification of particular compounds was based on a
comparison of their fragmentation patterns with MS library
(NIST MS Search 2.0), and by comparison with commercially
available standards (only hydroquinone and methyl-hydro-
quinone; purchased from Sigma-Aldrich). The calibration
was done using methyl-hydroquinone as an external
standard.
Enzymatic Essay
Phenol oxidase activity was assayed at 25 Cusing2mMof
the following substrates: ABTS, catechol, 4-tert-butylcatechol
(TBC), hydroquinone, and 2,6-dimethoxyphenol (DMP). The
conditions for each substrate were as follow: 1) 2 mM ABTS
("
418
= 36,000 M
1
cm
1
) in 0.1 M acetate buffer, pH 5.0.
Oxidation of ABTS was followed by absorbance increase at
418 nm ("
418
= 36,000 M
1
cm
1
)(Palmieri et al. 1997). 2)
2mM catechol or 2mM 4-tert-butylcatechol (TBC) in 0.1 M
phosphate buffer, pH 7.0. Oxidation was followed by the ab-
sorbance increase at 400 nm ("
400
= 1,450 M
1
cm
1
for
quinone product, "
400
= 1,200 M
1
cm
1
for o-tert-butylqui-
none product) (Lang et al. 2012). 3) 2 mM hydroquinone
("
248
= 22,000 M
1
cm
1
)ormethyl-p-benzoquinone
("
248
= 21,000 M
1
cm
1
) in 50 mM phosphate buffer, pH
5.0. Oxidation was followed by an absorbance increase at
248 nm. 4) 2 mM DMP in McIlvaine’s buffer adjusted to pH
5. Oxidation of DMP was followed by an absorbance increase
at 477 nm ("
477
= 14,800 M
1
cm
1
)(Li et al. 2012). The
reaction mixture consisted of 100 mlofthesubstratewith
5ml of aqueous sample solution (0.15 mgprotein/ml), and
the oxidation of the substrate was monitored without en-
zymes and under atmospheric condition. The oxidation reac-
tion was followed by the absorbance increase at specific
wavelength allowing detection of the newly formed product.
Enzymatic activity was always expressed in international units
per minute, that is the number of mmol product released by
1ml of enzyme solution per minute at 2mM of substrate
concentrations (Lang et al. 2012). Data are expressed as
mean SD (n=3).
We also tested the activity of BP76 using “in gel” zymo-
graphy. Native PAGE was performed using 5% and 8% poly-
acrylamide for the stacking and resolving gels, respectively.
Electrophoresis was carried out at 150 mV at 4C. After elec-
trophoresis the slab gels were subjected to the activity stain-
ing, the gel was first soaked in30ml of 50mM TrisHCl (pH
8.0) for 15 min, and then stained in 50 mM TrisHCl buffer (pH
8.0) containing 0.02 mM DMP and 0.2 mM CuSO
4
at room
temperature for 15 min. The bands of proteins that were as-
sociated with DMP activity were seen as red bands on a white
background. All results were recorded by scanning.
We also tested the effect of laccases and tyrosinase inhib-
itors on BP76 using DMP as substrate. The inhibitors were 4-
hexylresorcinol and salicylhydroxamic acid, dissolved in di-
methyl sulfoxide at a final concentration of 3.3%, and cetyl-
triammonium bromide, dissolved in phosphate buffer at pH
7. First, 0.98 ml of 2 mM DMP substrate was mixed with
0.02 ml of different concentrations of inhibitors. The mixture
was incubated at laboratory temperature for 30 min, then
0.005 ml (0.15 mgprotein/ml) of aqueous sample solution
was added to the mixture and the linear increase in absor-
bance at 477 nm ("
477
= 14,800 M
1
cm
1
)wasmonitored.
Supplementary Material
Supplementary material is available at Molecular Biology and
Evolution online (http://www.mbe.oxfordjournals.org/).
Acknowledgments
The authors are grateful to Marie Zarev
ucka, Zden
ek Voburka
(both IOCB), Miloslav
Sanda (Biochemistry and Molecular
and Cellular Biology, Georgetown University, Washington,
D.C, USA), Natalie Wielsch, and Ales Svato
s (both from the
Max Planck Institute for Chemical Ecology) for technical sup-
port. They also thank P. Cerdan and the staff of the
Laboratoire Environnement HYDRECO of Petit Saut (EDF-
CNEH) for logistic support. Financial support was provided
by the projects CIGA No. 20154314 and IGA No. B03/15
(Czech University of Life Sciences, Prague) and by the
Institute of Organic Chemisty and Biochemistry AS CR v.v.i.
(RVO 61388963). This work was also supported by the Max-
Planck-Gesellschaft. A.B. was partially supported by the
Mobility Fund of Charles University in Prague, Czech
Republic. M.M. was supported by the project InterBioMed
LO1302 from Ministry of Education of the Czech Republic.
T.B. was supported by the University of Sydney through a
postdoctoral fellowship.
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at University of Sydney on February 24, 2016http://mbe.oxfordjournals.org/Downloaded from
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... General Introduction mechanisms to defend conspecifics from chemicals used to attack other termites or ants . More direct defensive strategies include abdomen rupture by dehiscense contaminating the opponent or by autothysis realeasing toxic compounds from inside their bodies Šobotník et al. 2010bBourguignon et al. 2015;Poiani & Costa-Leonard, 2016), and defensive defecation on the enemy . In the same way, workers of soldierless species are known for presenting more aggressiveness compared to other workers (Sands,1972;). ...
... We reconstructed the presence of the hyaline tip using previously published phylogenetic trees (Bourguignon et al., 2015. We carried ancestral state reconstruction with Mesquite (Maddison & Maddison, 2010), on the presence/absence of the hyaline tip, using the Mk1 likelihood model and parsimony analyses. ...
... However, the defensive mechanisms have been almost exclusively studied in soldiers (for a review see Šobotník et al., 2010a), and defensive abilities are with few exceptions (e.g. Thorne, 1982;Bourguignon et al., 2015) completely neglected in other castes. ...
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.
... Preliminary bioassays showed that workers only burst after hard stress, i.e., after various bites from ant enemies, thus showing the existence of a threshold (Bourguignon et al. 2016b). However, the released product causes aggressor discomfort leading to an exaggerated self-grooming, a reaction Mill (1983) attributed to an irritating effect of the secretion. ...
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A new genus, Dissimulitermes Constantini & Cancello gen. nov ., is described from the Neotropical region. The main morphological feature of the new genus is remarkable defensive organs. A new species of Dissimulitermes is described: D. invisibilis Constantini & Cancello, sp. nov . Description, comparisons, measurements, a distribution map, histology of their dehiscent organ, and an illustration of all the fundamental morphological aspects are provided.
... It has been proposed that, as a general rule, evolution of new cell types is contingent upon transcription factors physically interacting in new ways, forming novel 'core-regulatory complexes' that can directly activate loci encoding cell-type functionality (Arendt, 2008;Arendt et al., 2016). Alternatively, different transcription factors may simply be co-expressed in the new cell Box 3. Illuminating gland cell type assembly through single-cell biology Transcriptome sequencing (RNAseq) of biosynthetically active glandular tissue has been used relatively successfully to identify putative genes involved in compound biosynthesis or glandular function (Bourguignon et al., 2015;Buček et al., 2016Buček et al., , 2015Li et al., 2013;Nakaoka et al., 2017;Rork and Renner, 2018;Vogel et al., 2010). However, one caveat with canonical RNAseq is that 'bulk' sequencing of whole gland structures provides only a global view of the transcriptome at the organ level, with no resolution of the transcriptional states of different cells within the gland (e.g. ...
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Across the Metazoa, the emergence of new ecological interactions has been enabled by the repeated evolution of exocrine glands. Specialized glands have arisen recurrently and with great frequency, even in single genera or species, transforming how animals interact with their environment through trophic resource exploitation, pheromonal communication, chemical defense and parental care. The widespread convergent evolution of animal glands implies that exocrine secretory cells are a hotspot of metazoan cell type innovation. Each evolutionary origin of a novel gland involves a process of 'gland cell type assembly': the stitching together of unique biosynthesis pathways; coordinated changes in secretory systems to enable efficient chemical release; and transcriptional deployment of these machineries into cells constituting the gland. This molecular evolutionary process influences what types of compound a given species is capable of secreting, and, consequently, the kinds of ecological interactions that species can display. Here, we discuss what is known about the evolutionary assembly of gland cell types and propose a framework for how it may happen. We posit the existence of 'terminal selector' transcription factors that program gland function via regulatory recruitment of biosynthetic enzymes and secretory proteins. We suggest ancestral enzymes are initially co-opted into the novel gland, fostering pleiotropic conflict that drives enzyme duplication. This process has yielded the observed pattern of modular, gland-specific biosynthesis pathways optimized for manufacturing specific secretions. We anticipate that single-cell technologies and gene editing methods applicable in diverse species will transform the study of animal chemical interactions, revealing how gland cell types are assembled and functionally configured at a molecular level.
... Rupturing can also cause the internal organs to burst out, in this case without toxic secretions, in a process called dehiscence that mostly occurs in soldierless termite workers (Sands, 1982), although it has been found in soldiers of the genera Glossotermes, Serritermes and Apilitermes (Deligne & DeConinck, 2006;Sobotnik et al., 2010a). This tactic differs from others described here, as it is a single-use behaviour, because the worker invariably dies (Sobotnik et al., 2012;Bourguignon et al., 2016). ...
Article
Animal interactions play an important role in understanding ecological processes. The nature and intensity of these interactions can shape the impacts of organisms on their environment. Because ants and termites, with their high biomass and range of ecological functions, have considerable effects on their environment, the interaction between them is important for ecosystem processes. Although the manner in which ants and termites interact is becoming increasingly well studied, there has been no synthesis to date of the available literature. Here we review and synthesise all existing literature on ant–termite interactions. We infer that ant predation on termites is the most important, most widespread, and most studied type of interaction. Predatory ant species can regulate termite populations and subsequently slow down the decomposition of wood, litter and soil organic matter. As a consequence they also affect plant growth and distribution, nutrient cycling and nutrient availability. Although some ant species are specialised termite predators, there is probably a high level of opportunistic predation by generalist ant species, and hence their impact on ecosystem processes that termites are known to provide varies at the species level. The most fruitful future research direction will be to evaluate the impact of ant–termite predation on broader ecosystem processes. To do this it will be necessary to quantify the efficacy both of particular ant species and of ant communities as a whole in regulating termite populations in different biomes. We envisage that this work will require a combination of methods, including DNA barcoding of ant gut contents along with field observations and exclusion experiments. Such a combined approach is necessary for assessing how this interaction influences entire ecosystems.
... Moreover, workers are fully responsible for the nest construction, which represent a passive defense [4]. In some taxa such as Neocapritermes, soldier rate is quite low inside the nest or in foraging sites, thus workers developed suicidal behavior associated with toxic compounds, which may trigger different responses in the enemies and contribute to colony defense [8,9]. Termitidae is the largest family within Isoptera and comprises over 70% of the species [10], including Apicotermitinae, a subfamily in which most species lost the soldier caste. ...
Article
Termite soldiers constitute the defensive frontline of the colonies, despite workers also perform such tasks, especially within the Neotropical Apicotermitinae, in which all species are soldierless. Workers of the genus Ruptitermes display an extreme form of defense, characterized by body rupture and release of a sticky secretion. Previous observations suggested that such behavior may be advantageous against enemies, but the chemical composition of this secretion has been neglected. Here we firstly provide the proteomic profile of the defensive secretion of Ruptitermes reconditus and Ruptitermes pitan workers. Additionally, the mechanisms of action of this behavior was evaluated through different bioassays. A total of 446 proteins were identified in R. reconditus and 391 proteins in R. pitan, which were classified into: toxins, defensins and proteolytic enzymes; sticky components/ alarm communication; proteins related to detoxification processes; proteins involved in folding/conformation and post-translational modifications; housekeeping proteins; and uncharacterized/hypothetical proteins. According to the bioassays, the self-sacrifice is triggered by a physical stimulus, and the defensive secretion may cause immobility and death of the opponents. Assuming that termites are abundant in the tropics and therefore exposed to predators, suicidal behaviors seem to be advantageous, since the loss of an individual benefit the whole colony. Significance Although recent studies have reported the biochemical composition of different weapons in soldiered species of termites, such efforts had not been applied to sordierless taxa up until now. Thus, this is the first report of the defensive mechanisms in soldierless termite species based on proteomic analysis. The diversity of compounds, which included toxin-like and mucin-like proteins, reflect the mechanisms of action of the defensive secretion released by termite workers, which may cause immobility and death of the opponents. Our findings may contribute to the knowledge regarding the development of defensive strategies in termites, especially in groups which lost the soldier caste during the evolution.
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Machadotermes is one of the basal Apicotermitinae genera, living in tropical West Africa. Old observations suggested the presence of a new gland, the intramandibular gland, in Machadotermes soldiers. Here, by combining micro-computed tomography, optical and electron microscopy, we showed that the gland exists in Machadotermes soldiers only as an active exocrine organ, consisting of numerous class III cells (bicellular units made of secretory and canal cells), within which the secretion is produced in rough endoplasmic reticulum, and modified and stored in Golgi apparatus. The final secretion is released out from the body through epicuticular canals running through the mandible cuticle to the exterior. We also studied three other Apicotermitinae, Indotermes, Duplidentitermes, and Jugositermes, in which this gland is absent. We speculate that the secretion of this gland may be used as a general protectant or antimicrobial agent. In addition, we observed that the frontal gland, a specific defensive organ in termites, is absent in Machadotermes soldiers while it is tiny in Indotermes soldiers and small in Duplidentitermes and Jugositermes soldiers. At last, we could also observe in all these species the labral, mandibular and labial glands, other exocrine glands present in all termite species studied so far.
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Salivary glands are omnipresent in termites and occur in all developmental stages and castes. They function to produce, store, and secrete compounds, ranging from a feeding function to defensive mechanisms. Here, we provide a complete morphological overview of the salivary glands in the soldierless species Ruptitermes reconditus and R. xanthochiton, and the first proteomic profile of the salivary glands in a Neotropical Apicotermitinae representative, R. reconditus. Salivary glands from both species were composed of several acini, roughly spherical structures composed of two types of central cells (type I and II) and peripheral parietal cells, as well as transporting ducts and two salivary reservoirs. Central cells were richly supplied with electron-lucent secretory vesicles and rough endoplasmic reticulum, a feature of protein-secreting cells. Parietal cells of Ruptitermes spp. had conspicuous characteristics such as electron-lucent secretory vesicles surrounded by mitochondria and well-developed microvilli. Moreover, different individuals showed variation in the secretory cycle of salivary acini, which may be related to polyethism. Ultrastructural analysis evidenced a high synthesis of secretion and also the occurrence of lysosomes and autophagic structures in central cells. Proteomic analysis of the salivary glands revealed 483 proteins divided into functional groups, highlighting toxins/defensins and compounds related to alarm communication and colony asepsis. Soldierless termites are quite successful, especially due to morphological adaptations of the workers, including unknown modifications of exocrine glands. Thus, according to our morphological and proteomic findings, we discuss the potential roles of the salivary gland secretion in different social aspects of the sampled species.
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Suicidal behavior in termite workers is an extreme defensive strategy, probably a consequence of having a low number of soldiers available in the colony and there being high predation from enemies. We investigated the suicidal mechanism in workers of the Neotropical termite Neocapritermes opacus, which involves salivary gland autothysis followed by body cuticle rupture and the release of a defensive secretion. Autothysis was triggered by a physical stimulus such as a soldier bite that causes the protrusion of the salivary acini, burst reservoirs, and foregut. Histochemical and ultrastructural analyses showed salivary acini composed of peripheral parietal cells and two types of central cells, types I and II. Type I cells are filled with large electron-lucent secretory vesicles, which reacted positively to bromophenol blue and xylidine- Ponceau tests, indicating the occurrence of proteins. Type II cells are elongated and display smaller apical secretory vesicles. Parietal cells present an intracellular canaliculus with dense microvilli and cytoplasm rich in mitochondria and large electron-dense vesicles, which may participate in the self-destructive mechanism.Worker suicidal behavior was previously reported for N. taracua and N. braziliensis. N. opacus is a new species in which a salivary weapon has been developed and factors contributing to this altruistic response are discussed.
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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.
Arthropod venoms have received much attention and have played an important role in folklore and medicine since ancient times. Scorpion envenomation, "tarant­ ism," bee and wasp stings are among those subjects about which most has been speculated and written in the past. In the last 50 years or so, a great number of scientific papers have been devoted to arthropod venoms, but only a few volumes have been designed to collect this rapidly increasing material, and these are not recent. Of late, the chemistry and mode of action of several arthropod venoms have been thoroughly studied, and some of these substances will probably be used as pharmacological tools and also as therapeutic agents. The aim of the present volume is to collect in manual form new information as well as the old notions on arthropod venoms. Even though it was our intention to present a volume on arthropod venoms, and not on venomous arthropods, inevitably we were forced to include information on venom-producing organisms as well. We assumed, in fact, that those scientists for whom the present manual is primarily intended (biochemists, particularly com­ parative biochemists, and pharmacologists) should be familiar with the biologic elements concerning the venom-producing species; which should show them how important it is to operate in close collaboration with biologists specialized in venomous arthropod systematics and biology.
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