Content uploaded by Valeria Palma Onetto
Author content
All content in this area was uploaded by Valeria Palma Onetto on Apr 26, 2022
Content may be subject to copyright.
Content uploaded by Valeria Palma Onetto
Author content
All content in this area was uploaded by Valeria Palma Onetto on Jun 21, 2021
Content may be subject to copyright.
HAL Id: tel-03033808
https://tel.archives-ouvertes.fr/tel-03033808
Submitted on 1 Dec 2020
HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entic research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diusion de documents
scientiques de niveau recherche, publiés ou non,
émanant des établissements d’enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.
Structure, function and evolution of the labral and
frontal glands in termites
Valeria Danae Palma Onetto
To cite this version:
Valeria Danae Palma Onetto. Structure, function and evolution of the labral and frontal glands
in termites. Populations and Evolution [q-bio.PE]. Université Sorbonne Paris Cité, 2019. English.
�NNT : 2019USPCD027�. �tel-03033808�
UNIVERSITÉ PARIS 13, SORBONNE PARIS CITÉ
ECOLE DOCTORALE GALILEÉ
THESE
présentée pour l’obtention du grade de DOCTEUR DE L’UNIVERSITE PARIS 13
Spécialité: Ethologie
Defensive exocrine glands in termites
Présentée par Valeria Palma–Onetto
Sous la direction de: David Sillam–Dussès et Jan Šobotník
Soutenue publiquement le 28 janvier 2019
JURY
Maria Cristina Lorenzi Professeur, Université Paris 13 Présidente du jury
Renate Radek Professeur, Université Libre de Berlin Rapporteur
Yves Roisin Professeur, Université Libre de Bruxelles
Rapporteur
David Sillam–Dussès
Maitre de conférences, Université Paris 13 Directeur de thèse
Jan Šobotník Chargé de Recherche, Czech University of Life Sciences Directeur de thèse
Laboratoire d’Ethologie Expérimentale et Comparée
Structure, function and evolution
of the labral and frontal glands
in termites
2
Structure, function and evolution of the defensive exocrine glands in termites
Structure, function and evolution of the
defensive exocrine glands in termites
Valeria Palma Onetto
A collaboration between the Termites Research Team at the Czech University
of Life Sciences and the Laboratoire d’Ethologie Expérimentale et Comparée
at the Université Paris 13.
3
Structure, function and evolution of the defensive exocrine glands in termites
Dédicace
I dedicate this work to all that people who have been my support during
these hard years. To my friends for hearing my sorrows and laments. To
those colleagues who without knowing me much have taken the time to
speak and support me: Aleš, Cecilia and Rebeca. And, those others who
became closer and provided extense conversations, knowledge and
confidence: Eliska, Katka and Tomáš.
I would like to thanks especially to David, my supervisor, for always
providing nice words, advice, constructive criticism and all the tools I may
have needed during my PhD.
And finally, to the person without who it would have been impossible, to my
girlfriend: Anais. Who got me up in the most difficult moments, heard all my
sorrows, read my e–mails when I was not strong enough to do it by myself,
encouraged me to follow my objectives and gave me the biggest reason to
continue on it.
My parents will never read it, they do not even understand English, but I
still want to say: I am sorry. I am sorry for not have been there in these
years, where things were not easy for you. I am sorry for let my sadness and
pressure overcome my feelings and not had taken the first available flight
when it was needed. I am sorry for letting you alone when you needed
where to hold.
This work is also for you, although it does not replace those moments I have
missed.
4
Contents
Contents
CONTENTS ........................................................................................................................................................4
1. GENERAL INTRODUCTION .............................................................................................................................7
1.1 EUSOCIAL ORGANISMS ......................................................................................................................................... 8
1.2 THE TERMITES .................................................................................................................................................. 11
Phylogeny ..................................................................................................................................................... 13
Economical impact ........................................................................................................................................ 14
Termites’ abundance .................................................................................................................................... 15
1.3 DEFENSE MECHANISMS OF TERMITES .................................................................................................................... 16
The nest ........................................................................................................................................................ 16
The soldiers ................................................................................................................................................... 17
Defensive strategies in other castes ............................................................................................................. 19
1.4 CHEMICAL DEFENSES ......................................................................................................................................... 20
Exocrine glands in termites ........................................................................................................................... 21
1.5 MOTIVATION AND OBJECTIVES OF MY THESIS ......................................................................................................... 24
2. GENERAL METHODS .................................................................................................................................... 26
2.1 ANIMALS OF STUDY ........................................................................................................................................... 27
2.2 HISTOLOGY ..................................................................................................................................................... 27
2.3 MICROSCOPY .................................................................................................................................................. 27
2.4 BEHAVIOURAL TEST ........................................................................................................................................... 28
2.5 OTHERS .......................................................................................................................................................... 28
PAPER 1: THE LABRAL GLAND IN TERMITE SOLDIERS ...................................................................................... 29
INTRODUCTION ...................................................................................................................................................... 32
MATERIAL AND METHODS ....................................................................................................................................... 33
Direct Observations ...................................................................................................................................... 33
Optical microscopy and transmission electron microscopy .......................................................................... 33
Histology ....................................................................................................................................................... 33
Electron Microscopy ...................................................................................................................................... 33
Evolution of the hyaline tip ........................................................................................................................... 34
RESULTS ............................................................................................................................................................... 34
Scanning electron microscopy ...................................................................................................................... 35
Optical microscopy........................................................................................................................................ 36
Transmission electron microscopy ................................................................................................................ 36
DISCUSSION .......................................................................................................................................................... 39
CONCLUSION AND FURTHER HYPOTHESES .................................................................................................................... 42
ACKNOWLEDGEMENTS ............................................................................................................................................ 42
SUPPORTING INFORMATION ..................................................................................................................................... 43
REFERENCES .......................................................................................................................................................... 48
PAPER 2: THE LABRAL GLAND IN TERMITES: EVOLUTION AND FUNCTION ...................................................... 52
INTRODUCTION ...................................................................................................................................................... 55
MATERIAL AND METHODS ....................................................................................................................................... 57
Scanning Electron Microscopy, Optical microscopy and Transmission Electron Microscopy ....................... 57
Behavioural experiments .............................................................................................................................. 57
Chemical analyses ......................................................................................................................................... 58
RESULTS ............................................................................................................................................................... 58
Scanning Electron Microscopy ...................................................................................................................... 58
Optical microscopy........................................................................................................................................ 59
Transmission Electron Microscopy ................................................................................................................ 61
Behavioural experiments .............................................................................................................................. 64
5
Contents
Chemical analyses ......................................................................................................................................... 65
DISCUSSION .......................................................................................................................................................... 65
CONCLUSION ......................................................................................................................................................... 67
ACKNOWLEDGEMENTS ............................................................................................................................................ 68
SUPPLEMENTARY MATERIALS .................................................................................................................................... 69
REFERENCES .......................................................................................................................................................... 74
PAPER 3: THE EVOLUTION OF THE MOST POWERFUL DEFENSIVE ORGAN FOUND IN TERMITES, THE FRONTAL
GLAND, IN NEOISOPTERA ............................................................................................................................... 80
INTRODUCTION ...................................................................................................................................................... 83
MATERIAL AND METHODS ....................................................................................................................................... 85
Termite samples ............................................................................................................................................ 85
Frontal gland occurrence, structure and ultrastructure through optical and electron microscopies ........... 85
Measurements of the gland and its relative size .......................................................................................... 85
Phylogenetic analysis of the frontal gland evolution .................................................................................... 86
RESULTS ............................................................................................................................................................... 86
Common features of the frontal gland in workers ........................................................................................ 86
Systematic survey ......................................................................................................................................... 92
Relative size and volume ............................................................................................................................... 94
Evolution of the frontal gland inferred from its phylogenetic tree ............................................................... 95
DISCUSSION .......................................................................................................................................................... 95
ACKNOWLEDGEMENTS ............................................................................................................................................ 98
REFERENCES .......................................................................................................................................................... 99
TABLES............................................................................................................................................................... 106
PAPER 4: TONSURITERMES, A NEW SOLDIERLESS TERMITE GENUS AND TWO NEW SPECIES FROM SOUTH
AMERICA (BLATTARIA: ISOPTERA: TERMITIDAE: APICOTERMITINAE) ........................................................... 110
INTRODUCTION .................................................................................................................................................... 113
MATERIAL AND METHODS ...................................................................................................................................... 113
TAXONOMY ........................................................................................................................................................ 114
ACKNOWLEDGEMENTS .......................................................................................................................................... 120
REFERENCES ........................................................................................................................................................ 121
TABLES AND FIGURES ............................................................................................................................................ 125
3. GENERAL CONCLUSIONS ........................................................................................................................... 132
4. REFERENCES .............................................................................................................................................. 136
5. DECLARATION ........................................................................................................................................... 153
ABSTRACT ..................................................................................................................................................... 154
APPENDIX ..................................................................................................................................................... 158
PUBLICATIONS OF THE CANDIDATE DURING THE COURSE OF HER PHD ............................................................................. 158
Peer–reviewed journals .............................................................................................................................. 158
POSTER PRESENTATIONS ........................................................................................................................................ 158
International conferences ........................................................................................................................... 158
National conferences .................................................................................................................................. 158
OTHERS ............................................................................................................................................................. 158
7
1. General Introduction
1. General Introduction
“Our whole life is but a greater and longer childhood”
Benjamín Franklin
Angularitermes pinocchio
Photo by Aleš Buček
8
1. General Introduction
1. General Introduction
Social insects represent an important part of our lives. Ants, bees and wasps are
easily recognisable by almost everyone even kids, we can find references about them in the
Holy Bible, and they comprise about the 75 percent of the world's insect biomass (Wilson,
1971). Insects societies have long intrigued and fascinated people, as they also hold a
special place in the biophilia (defined by E.O. Wilson as an innate and genetically
determined affinity of human beings with the natural world), due the parallelism of their lives
with ours. Their colonial life with a central family life, division of labor, communication and the
mutualistic peace interweaves with strife and conflicts, have indisputable similarities with the
achievements and ideals of our own society.
Among all social insects, the one which fits
most closely with humans may be termites, which present among their individuals: parents,
alloparents, builders, soldiers, biochemical–genomic engineers and children. All of these
individuals settled in an extended nuclear family that expand and defend their homes
(Howard & Thorne, 2011). Althought these parallelisms and the fact that termites are one of
the most (if not the most) abundant insects on Earth (overweighting bees and wasps),
termites have received negligected attention in comparison with other social insects.
1.1 Eusocial organisms
Although many animals exhibit social behaviors, such as aggregating in large
numbers at times or parental care, these behaviors do not mean an animal is social.
In fact,
biologists refer to true social animals as eusocial. By definition, eusocial animals share the
following four characteristics: life in groups of the adults, cooperative care of juveniles
(individuals care for brood that is not their own), reproductive division of labor (not all
individuals get to reproduce), and overlap of generations (Wilson, 1971).
The term "eusocial" was introduced for the first time by Suzanne Batra in 1968, who
used it to describe nesting behavior in Halictine bees. She observed colonies which were
founded by a single individual and described the essential cooperative behavior of the bees
and how the activity of one labor division influenced the activity of another. In 1969, Charles
Michenes would expand Batra’s classification with a study aimed to investigate the different
levels of animal sociality and defined by three main characteristics the concept of eusociality:
i) Egg–layers and worker–like individuals among adult females" (division of labor), ii) The
overlap of generations (mother and adult offspring), iii) Cooperative work on the cells of the
bees' honeycomb. But it was not until 1971 when E. O. Wilson extended the terminology to
9
1. General Introduction
include other social organisms which comprehend the following three features:
1) Reproductive division of labor (with or without sterile castes)
2) Overlapping generations
3) Cooperative care of young
Moreover, a crucial evolutionary interrogant has arised with the success of the
eusocial colonies and it is the origin and persistence of a sterile caste in them, whose
existence is the last thing we would expect to be promoted by natural selection and has been
a headache for biologists since Darwin who declared in The Origin of Species the paradox to
be the most important challenge to his theory during the realization of his evolutionary theory.
This solution to this paradox can be approached in many different ways, where the most
influential one is undoubtedly the Hamilton’s inclusive fitness theory (1964). Hamilton
presented a kin selection theory which explains that if a gene promoting altruistic behavior
has copies of itself in others, helping those others survive ensures that the genes will be
passed on. The phenomenon is mathematically described by , where r is the degree of
relatedness between donor and recipient of the altruistic behavior, b the reproductive benefit
to the recipient and c the retroductive cost to the altruist donor.
Table 1. Eusocial animals. Table taken from Plowes, 2010
10
1. General Introduction
When thinking about eusocial animals we may immediately think about insects, but
eusociality has also arisen three different times among some crustaceans that live in
separate colonies (Duffy et al. 2000, Duffy & MacDonald 2010) and two times in mole–rats
(Burda et al. 2000; O’Riain et al. 2008). While in a more controversial view (Gintis, 2012;
Dawkins 2012; Pinker 2016), E. O. Wilson (2012) suggested humans as a eusocial species.
However, the abundance of social animals reaches its peak in the phylum Arthropoda. Inside
this phylum, the order Hymenoptera is the largest and most well–known animal group
eusocial species, although most of them are not eusocial (Ross & Matthews 1991, Nowak et
al. 2010). In fact, the whole concept of eusociality is primarily based on observations on
Hymenopteran taxa, leading to a serie of mismatches when applying to other organisms,
specially the diploid ones (Nowak 2010). In social Hymenoptera, females arise from fertilized
diploid eggs and males arise from unfertilized haploid eggs, a system called haplodiploidy
and which may contribute to kin selection, favoring altruistic behavior in this group (Plowes
2010). Diploid organisms’ sex determination system would not provide especially high
relatedness between some individuals of the group, having all individuals approximately the
same fitness.
Among diploid social organisms, termites are probably the most remarkable group.
We cannot think of an individual in a termite colony as a standard solitary insect. If you
separate it from the colony, it will die (Eggleton 2011). Termites reveal the highest overall
caste diversity (Choe & Crespi 1997; Thorne 1997) and each caste lacks some element that
is present in a solitary insect, forming them all what is call as a “superorganism”. A
superorganism is defined as a social unit of eusocial animals, where division of labour is
highly specialised and where individuals are not able to survive by themselves for extended
periods (Hölldobler & Wilson 2008). And even though they are diploid, they are still eusocial.
Within a single termite colony, you can find individuals at various stages of the termite life
cycle, generations of termites overlap, and there is a constant supply of new adults prepared
to assume responsibility for the colony's care (Nalepa 1994). In termites, two additional
hypotheses have been proposed.
Some theories emerged to explain the evolution of eusociality in termites. One theory
that used to have weight is the existence of relatedness asymmetry inside the colony, mainly
ligated to two mechanisms: (i) The Chromosomal Linkage Hypothesis (Lacy, 1980), which
establishes that much of the termite genome is sex–linked; (ii) cycles of inbreeding and
outbreeding that would increase the relatedness between workers and the parents’ offspring,
favoring their evolution (Bartz 1979). In the first, siblings of the same sex would be related
somewhat above 0.5, but siblings of different sex would have a relatedness less than 0.5
11
1. General Introduction
(Lacy, 1980). Termite workers might then bias their cooperative brood care towards their own
sex. In that way, this hypothesis proposes that workers of a colony would only care for the
offspring of their same sex (Thorne, 1997). However, several studies have undermined both
theories (reviewed in Thorne 1997 and Howard & Thorne 2011). A strongest theory is the
Symbiont Transfer Hypothesis (Cleveland et al. 1934, Nalepa 1984), which points out the
dependence of termites on their symbiotic communities in their guts, which must be
recovered after each molt by interactions with other termites, preventing thus the solitary
way–of–life (Thorne, 1997).
1.2 The termites
Known commonly as “white ants”, termites are eusocial insects, with a broad range of
morphological forms and diets.
Termites are often compared with the social Hymenoptera. Nevertheless, they differ
in their evolutionary origins having big differences in life cycle (Howard & Thorne 2011). In
eusocial Hymenoptera, workers are exclusively female, the males (drones) are haploid and
develop from unfertilised eggs, while females are diploid and develop from fertilised eggs.
On the other hand, termites are diploid individuals in all sexes and castes (Howard & Thorne
2011).
A colony of termites is established by a couple of imagoes, which become the royal
couple (king and queen). They copulate and give birth to immatures individuals, which are
small white, unsclerotised and essentially helpless. Once growing up, these immatures
individuals will become workers, which undertake the most labour within the colony, being
responsible for foraging, constructing, food storage, and brood and nest maintenance
(Eggleton 2011). Some workers can go through further moulting and become soldiers which
defend their colony against predators, or alate imagoes which will fly away from their colony
to pair and establish a new one (Eggleton 2011). This description of caste structure is just a
simplified and basic one, given that some species may have no soldiers, no true workers,
present neotenics or even parthenogenesis (Eggleton 2011, Howard & Thorne 2011,
Bourguignon et al. 2012, Fougeyrollas et al. 2015, Fougeyrollas et al. 2017). However, all
termite’ species have at least one sterile caste that is pre–determined during the immature
stages and follow the three main statements of the eusociality (Boomsma 2009).
All these castes and individuals living inside the colony will conform the animated part
of it, but a colony in fact is conformed also by an inanimate part. The inanimate part of the
colony is the structure built by them, which can be just a few tunnels to huge and
sophisticated structures (Eggleton 2011).
12
1. General Introduction
As well as a sophisticated system of castes and differentiate building strategies,
termites present a highly variable diet. They are detritivores generalists, consuming dead
plants of all decomposition levels (Donovan et al. 2000; Hyodo et al. 2008). Termites rely
primarily upon symbiotic microbes which inhabit predominantly the anterior part of the
hindgut (Eggleton 2011). They can be protozoa, bacteria or flagellate protists which help
termites to digest the cellulose they consume, allowing them to absorb the final products for
their own use (Slaytor, 1992; Ikeda–Ohtsubo and Brune, 2009). Flagellates symbionts are
absent in new individuals, being the workers which pass them to others through proctodeal
trophallaxis. In other words, the immatures are fed by secretions from the anus, which
contain the symbionts and alimentary particles (Ohkuma & Brune 2011). Most evolutionary
advance termites possess cellulase enzymes, therefore they do not count with flagellates but
they rely primarily on bacteria. In these advance termites, the workers fed the immatures
only through stomodeal trophallaxis, method that is also present in older evolutionary
species and consists in feeding from glands located in the thorax (normally the labial glands)
through the mouth (McMahan 1969, Qiu–Ying et al. 2008).
One special case is the symbiosis between the termites and fungi living outside their
body, inside the nest. These termites from the group Macrotermitinae maintain a “garden” of
Termitomyces which is nourished by excrement, then the termites will eat it and their spores
will pass through the intestines until complete a cycle by germinating in the fresh faecal
pellets (Aanen et al. 2002; Mueller and Gerardo, 2002). This fungus farming system allowed
these termites, originally from the rainforest, to colonise the African savannah and other new
environments across Africa and Asia (Roberts et al. 2016).
Feeding preferences of termites are variable, and can present fluctuations between
species, the taxa or even the season (Donovan et al, 2001; Allen et al. 1980). Donovan and
others (2001) classified termites according to the degree of degradation (humification
gradient) of the food they consume, mandibles development and guts structure:
Group I,
feeds on dead wood and grass and have relatively simple guts;
Group II,
feeds on wood,
grass, leaf litter and microepiphytes and have more complex guts;
Group III
feeds on soil–
like material with recognisable plant material in it;
Group IV
feeds on soil–like material with a
high proportion of silica and no recognisable plant material. Bourguignon and others (2011)
have showed later that this classification is merely structural, while the basics split lays
between wood–feeders (lower termites: Groups I and II) and soil–feeders (Higher termites:
Groups III and IV), being these lasts the most advanced evolutionary termites.
13
1. General Introduction
Phylogeny
The phylogeny of termites has been debated for a long time. The most common view
classifies them as the infraorder Isoptera or as the epifamily Termitoidae within the order
Blattodea (cockroaches).
Originally, termites were placed as an order, but in 1934 Cleveland and others have
suggested them to be closely related to wood–feeding cockroaches according to their gut
flagellates. This suggestion became stronger when morphological and phylogenetics studies
supported the closeness between termites and cockroaches (McKittrick 1960; Inward et al.
2007; Eggleton et al. 2007; Legendre et al. 2008; Ware et al. 2008). Termites also share
some behavioural features with their sister group, the cockroaches of the genus
Cryptocercus (Lo et al. 2000, Grimaldi and Engel 2005, Ohkuma et al. 2009). The oldest
unambiguous termite fossils date to the early Cretaceous, predating those of ants and bess
by approximately 35 million years (Thorne et al. 2000, Engel et al. 2007). In the other hand,
the last common ancestor of Cryptocercus and termites lived probably in the Jurassic
(Vrsanky and Aristov 2014, Bourguignon et al. 2014).
About 3,106 species of termites are currently described (Krishna et al. 2013), with
perhaps hundreds more still to be described. They are separated in 9 families which can be
split in two groups: “lower” termites, comprising basal families (Mastotermitidae,
Archotermopsidae, Stolotermitidae, Hodotermitidae, Kalotermitidae, Stylotermitidae,
Serritermitidae and Rhinotermitidae), predominately feeding on wood; and “higher” termites,
harboring the family Termitidae, which consume a wide variety of soft–materials (including
faeces, humus, grass, leaves and roots) (Radek 1999, Engel et al. 2009).
The gut in the
lower termites contains different species of bacteria along with protozoa as symbionts, while
higher termites only have a few species of bacteria with no protozoa (Breznak and Brune
1994).
Higher termites originated 42–54 million years ago in Africa and later dispersed
between the continents at least 24 times in two main periods (Bourguignon et al. 2017). Eight
subfamilies are recognised in Termitidae: Macrotermitinae, Sphaerotermitinae,
Foraminitermitinae, Apicotermitinae, Termitinae, Syntermitinae, Cubitermitinae and
Nasutitermitinae (Krishna et al. 2013). However, this subfamily–level classification is still
unsatisfactory (e.g. see Kambhampati and Eggleton 2000; Inward et al. 2007b), particularly
with respect to the subfamily Termitinae (Inward et al. 2007b). Although termites phylogeny
has been highly debated and mostly disentangled (for review see Eggleton 2001), recent
phylogenies mostly agree on basic pattern of termite phylogenetic tree (Miura et al. 1998, Lo
et al. 2000, Donovan et al. 2000, Thompson et al. 2000, Austin et al. 2004, Inward et al.
2007a,b, Legrendre et al. 2008, Engel et al. 2009, Cameron et al. 2012, Bourguignon et al.
14
1. General Introduction
2015), the tree of Bourguignon and others (2015) being the most accepted and apparently
accurated today (Fig.2).
Figure 1.
Phylogenetic relationships among termites, including their closest relative, the cockroaches of the
genera Cryptocercus. Bourguignon et al. 2015.
Economical impact
Even thought termites are not as well–known as other social insects like
Hymenopterans, most people are aware of termites. They are economic pests, specially in
tropical and subtropical environments where they destroy crops, forests along with wood and
wooden structures of human buildings (Meyer et al. 1999). In fact, they cause a significant
economic loss of about US $ 22 billion to US $ 40 billion annually worldwide (Su 2002, Rust
& Su 2012).
In the USA for example, they cause more economic damage than fire and flood
combined (Eggleton 2011).
Most tropical crops are susceptible to termite attacks worldwide.
Among the most remarkable damage termites can cause are those to eucalyptus (Fonseca
1949, Wood & Pearce 1991, Werner et al. 2008, Faragalla and Al Qhtani 2013), coconuts
and palms (Aisagbonhi 1985, Logan & El–Bakri 1990, Mariau et al. 1992, Tang et al. 2006),
fruit trees (Stansly et al. 2001, Constantino 2002, Ahmed and Qasim 2011, Faragalla and Al
Qhtani 2013, Tomar 2013), sugarcane (Novaretti and Fontes 1998, Ahmed et al. 2007;
Haifig et al. 2008, Alam et al. 2012), rice (Fonseca 1949, Mill 1992, Dario & Villela–Filho
1998, Agunbiade et al. 2009; Oyetunji et al. 2014), maize (Fernandes & Alves 1992, Nkunika
15
1. General Introduction
1994, Mill 1992, Constantino 2002; Faragalla and Al Qhtani 2013), wheat (Ahmed et al.
2004; Pardeshi et al. 2010; Rathour et al. 2014), sorghum (Logan 1991), sunflower (Ashfaq
and Aslam 2001; Sileshi et al. 2009), groundnut (Johnson & Gumel 1981; Johnson et al.
1981, Wood et al. 1987, Wood & Pearce 1991), coffee (Kranz et al. 1981, Cowie & Wood
1989, Neves & Alves 1999), tea (Singha et al. 2011), cotton (Wood et al. 1987), tobacco
(Shah and Shah 2013), pastures (Sands 1973, Cowie and Wood 1989, Mariconi et al. 1994,
Fernandes et al. 1998) and tuber crops (Sands 1973, Tomar 2013).
Apart of their voracity linked to their populous colonies, termites are successful pests
due to their capacity to invade new countries or even continents. Currently, 28 species of
termites are known to be invasive. Most of them are important invasive pests in urban areas,
although 6 of them have colonized natural forests habits (Evans et al. 2013). All these
species share some characteristic in common: they all feed on wood, live and construct their
nests inside of the alimentary source and
easily
produce secondary reproductives (Evans et
al. 2013). Although the economical cost of invasive termite species has not been calculated,
it is known that invasive insects cost a minimum of US$70.0 billion per year globally and the
most expensive insect is purportedly a termite: Coptotermes formosanus, with an estimated
cost higher than US $30.2 billion per year globally (Su 2002; Bradshaw et al. 2016). The
genus Coptotermes is also one of the most spreaded termites’ genera, which along with the
genus Cryptotermes can be found in Africa, Asia, Europe, Oceania and America (Evans et
al. 2013). These two genera plus Heterotermes (presented in Africa, Asia and America)
represent the main invasive group of termites around the world (Evans et al. 2013).
However, termites’ impact is not always negative. They play an important role in the
decomposition of litter on the ground, the regulation of soil structure, soil organic matter and
nutrient cycling, water dynamics, soil erosion, plant growth, restoration of degraded lands,
production of greenhouse gases, and overall biodiversity (see Holt and Lepage 2000,
Jouquet et al.
2011, Bottinelli et al. 2015, Jouquet et al. 2016, Khan et al. 2018, Govorushko
2018, for reviews), including an important role as buffers of ecosystems against climate
change (Bonachela et al. 2015). Termites also possess an economical importance as
alimentary source (Figueirêdo et al. 2015). Forty–three species are known to be used as
food for humans or to feed livestock in Africa, Asia, and North and South America (De
Figueirêdo et al. 2015). However, the economical equivalence of these impacts has not been
determined yet.
Termites’ abundance
Termites are highly abundant in terms of biomass in warm terrestrial ecosystems, where they
may represent 40% to 65% of the overall soil macrofaunal biomass (Loveridge and Moe
16
1. General Introduction
2004). They can exceed 6,000 individuals per square meter in tropics (Lee and Wood 1971,
Eggleton et al. 1996), revealing comparable abundance to another remarkable group: the
ants (Holldöbler & Wilson 1990).
Higher termites are the most abundant group
comprising 83%
of termite genera and about 70% of the species (Krishna et al. 2013), especially the
subfamily Termitinae which can represent 80% of the total termites’ individuals in tropics
(Eggleton et al. 1996).
Due to their abundance, termites represent an important food source
for a wide variety of predators: invertebrate (spiders, scorpions, mites, centipedes, true bugs,
beetles, ants, wasps) and vertebrate (frogs, salamanders, lizards, birds, mammals) (
Redford
& Dorea, 1984)
.
1.3 Defense mechanisms of termites
Termites are vulnerable insects of soft body that have overcome high rates of predation and
competition becoming one of the most ecologically success organisms (Deligne et al. 1981).
They protect themselves through passive and active defence mechanisms, these include: a
cryptic lifestyle characterized by a hidden way of life and the construction of defensive
structures (Korb, 2011), the development of soldiers (Haverty, 1977) and glands that
produce defensive compounds (Prestwich, 1984; Šobotník et al., 2010a).
The nest
Living in a protective nest is a strategy that all social insects share (Howard & Thorne
2011). It promotes the evolution of social cooperation during its construction and defense
(Charnov 1978, Andersson 1984, Alexander et al. 1991, Crespi 1994, Wilson 2008), as well
as by encouraging relatives to stay in close proximity (Hamilton 1978). Their main function is
to protect the colony against enemies and hostile environmental conditions (Noirot &
Darlington 2000, De Visse et al. 2008), but it is also a valuable storage for food reserves
(Myles 1988; Starr 1991; Breed et al. 2004, Korb 2011).
Nests can be: i) fully underground galleries; ii) an epigeal protruding above the soil
surface, which can wind up into very hard mounds of over 8 meters; iii) an arboreal
construction, but always connected to the ground via shelter tubes; iv) a gallery system
inside wooden structures such as logs, stumps and the dead parts of trees, where the colony
develops (Noirot & Darlington 2000). This last is the most primitive way of nesting and
provides a two–fold function, due to the importance of the nest not only for protection but
also as food source (Abe 1987).
Termites build their nests primarily using their faeces, which are relatively inert to
pathogens, are cheap to produce, are a good structural material (Eggleton 2011), and partly
17
1. General Introduction
digested plant matter (arboreal nests) or soil (subterranean and epigeal nests) (Eggleton
2011).
The soldiers
Soldiers are the first truly altruistic caste present in termites (Hare 1937; Thorne et al.
2003). They are highly diverse, the most of all castes, diversifying over time to plentiful
morphs and shapes, which are easily usable to identify genera or even species (Prestwich
1984).
The evolution of a soldier caste represents an autapomorphy of termites (Hare 1937,
Noirot & Pasteels 1987, Roisin & Korb 2011) and is a defining character of termites. In spite of
being ancestral to all extant termite lineages, soldiers are not present in all species, being
secondarily lost in the unrelated genera Anoplotermes, Invasitermes, Orientotermes and
Protohamitermes (Sands 1972, Ahmad 1976; Miller 1984).
The soldiers in a colony have only one function: to defend the colony (Eggleton
2011). They are formed by differentiation of workers through an intermediate presoldier
stage (Noirot 1985, Henderson 1998). It seems probable that soldiers are in the colony to
defend the colony mainly from ants, so their morphology adaptations would be in reponse to
this pressure (Eggleton 2011). Vertebrate predation may also be important, but soldiers
cannot represent a real threat to them and they are generally not killing entire colonies, while
ants do (Leal & Oliveira 1995).
Relative to workers, soldiers have a reduced digestive tract, long and strong legs,
and a highly sclerotised head that usually large along with powerful, highly modified
mandibles (Koshikawa et al. 2002, Eggleton 2011). According to Prestwich et al. (1984),
soldiers mechanical defences can be separated in 9 types, but they can be summarized in 6
main categories:
a) Biting–crushing mandibles (Fig. 3A). Present in most of lower termites (Deligne et al.
1981), they are robust mandibles rich in dentition intended to hurt the opponent by squeezing
or piercing them.
b) Phragmotical head (Fig. 3B). It is a modified highly sclerotized (especially in the rostrum)
head cylindrically shaped with short mandibles, which occurs in some Kalotermitidae
(Deligne et al. 1981). These heads are used as stoppers to plug holes that could be created
during foraging activities or to allow the exit of the alates and thus prevent the entry of
predators into the termite nest.
c) Biting–slashing mandibles (Fig. 3C). In this case, the termites possess slend, straight and
long mandibles with a great angular motion. This mode is frequent in termites and can be
18
1. General Introduction
observed in most Rhinotermitidae, Serritermitidae and Termitidae (Prestwich 1984). The use
of these mandibles is usually coupled with the injection of greasy, irritating, toxic, or viscous
materials into the wound of the enemy.
d) Biting–piercing mandibles. These are slender, inwardly curved mandibles with prominent
marginal teeth (Mill 1982). It is common in some basal Termitinae (e.g. Amitermes),
Syntermitinae (e.g. Armitermes, Rhynchotermes), and major soldiers of higher
rhinotermitines (e.g. Rhinotermes) (Prestwich 1984). As well as for biting–slashing
mandibles, these may be accompanied by chemicals entering the wound, normally from the
frontal gland (Prestwich 1979, Pretwich and Collins 1982).
e) Snapping mandibles. This kind of mandibles is characterized by a long and slender shape
unable to bite, but with the property of releasing energy stored into a single moving
mandible, increasing its kinetic energy imparted at impact, killing or knocking down the
enemy by a powerful strike (Deligne et al. 1981, Prestwich 1984, Seid et al. 2008). Until
recently, snapping mandibles were thought to be present only in some termitines (Deligne et
al. 1981, Prestwich 1981) and have evolved several times independently within this
subfamily Termitinae (Bourguignon et al. 2017). This year, a new genus of snapping termites
has been discovered, it is Roisinitermes, from the Kalotermitidae family (Scheffrahn et al.
2018).
f) Nasute (Fig. 3D). Most evolutionary advanced families of termites have developed a
mandibular regression, where the space in the head which was normally used for the
mandibular muscles is replaced by a huge reservoir for defensive secretions (the frontal
gland) which are ejected through a nasute, entangling and incapacitating smaller enemies,
and causing scratching and cleaning behaviour in larger ones (Prestwich 1984). This
adaptation is characteristic of the Nasutitermitinae subfamily but something similar can be
observed in smaller soldiers of Rhinotermitidae. In these small soldiers, mandibles are
reduced to grabbing or carrying devices, there is no nasute but a labral brush which may
look physically similar, but their defense is in fact accomplished by topical application of
lipophilic contact poisons stored in massive abdominal reservoirs of the frontal gland
(Prestwich 1984).
19
1. General Introduction
Figure 2.
A) Ventral view of the whole body of a Neotermes chilensis soldier, showing its biting–crushing
mandibles. B) Rostrum of the phragmotic head of Cryptotermes cavifrons soldier. C) Dorsal view of the full
head of Heterotermes sp. Note the thin biting–slashing mandibles. D) Dorsal view of the head of
Nasutitermes longinasus large soldier, note the well developed nasute. Photos B and C belong to David
Mora del Pozo. Photo D was taken from Syaukani (2011).
Defensive strategies in other castes
The highest rates of predation against termites occurs when they are realizing
activities out of the nest, such as during the nuptial flight or during foraging activities (Dial &
Vaughan 1987; Lepage 1991, Korb & Salewski 2000, Korb and Schneider 2007). During
foraging activities, as well as in the colony in general, workers outnumber soldiers
considerably (with exception of some Nasutitermitinae species) with proportions which run
from 4: 1 to 400: 1 (Haverty 1977). During the nuptial flight, the termite imagoes leave the
nest and flight for a variable time and then land on the ground to search for a mate (Eggleton
2011). They are bad flyers and most of termites are depraded by invertebrate and vertebrate
predators during these flights, including humans who attract them with lamps and eat them
after removing their wings (Nyakupfuka 2003). In fact, Korb and Schneider (2007) have
determined that the probability of successfully founding of a nest in Cryptotermes secundus
is less than 1%.
Although soldiers are an especially developed defensive caste, workers and imagoes
are not defenseless (Prestwich 1984). Workers have a primary role in passive defense,
building a nest which is the first barrier against predators (Eggleton 2011), but they also
present many other defensive roles. One of these roles is conducting detoxification
20
1. General Introduction
mechanisms to defend conspecifics from chemicals used to attack other termites or ants
(Spanton & Prestwich, 1982). More direct defensive strategies include abdomen rupture by
dehiscense contaminating the opponent (Sands 1982) or by autothysis realeasing toxic
compounds from inside their bodies (Costa–Leonardo, 2004; Šobotník et al. 2010b, 2012;
Bourguignon et al. 2015; Poiani & Costa–Leonard, 2016), and defensive defecation on the
enemy (Prestwich 1984). In the same way, workers of soldierless species are known for
presenting more aggressiveness compared to other workers (Sands,1972; Šobotník et al.
2010a).
Imagoes have also developed several defensive strategies to overcome predation.
Among these, an important one is the existence of synchronous nuptial flights which along
with reducing endogamy (Roisin 1999, Aguilera–Olivares 2015), act as a defensive strategy
increasing the probability of survival by increasing the number of termites flying (Nutting
1969,
Nutting and Haverty 1976,
Thorne 1983, Jones et al. 1988, Bordereau et al. 1991,
Nalepa et al. 2001). Another important defensive mechanism from alate imagoes is the
development of the frontal gland. Indeed, Šobotník and others (2010c) described how wasps
removed the head of Coptotermes testaceus (a termite with a large frontal gland) prior
storing them in the nest, while alates of Anoplotermes s.lat. spp. (a species with tiny frontal
gland) were not.
1.4 Chemical defenses
Exocrine glands are group of cells that produce and secrete substances onto an
epithelial surface by way of a duct or epithelial modification (Young et al. 2013). Insects have
a wide variety of glandular cells and organs which produce a variety of secretions, creating
complex exocrine glandular systems that coordinate
different social interactions or activities,
including foraging, building, mating, defense, and nestmate recognition. If the glands itself may be
not that well–known, their secretions certainly are. Everyone
is familiar with sweat, silk or
venom, all of them results of glandular secretions. Particularly important although not
specially known is their involvement in the production of antibiotics, lubricants, and digestive
enzymes (Billen & Šobotník 2015).
In 1974, Noirot and Quennedey formulated a classification for the exocrine glands,
which has been widely accepted and became universally used. Glandular cells are classified
as: (i) class 1, the cells are adjoined directly to the cuticle which need to be cross to the
release of the secretion; (ii) class 2, the cells are not in direct contact with the cuticle, they
are surrounded by class 1 cells through which the secretion must run before crossing the
cuticle; (iii) class 3, the cells compound units formed by one to several secretory cells
21
1. General Introduction
isolated from the cuticle plus one or two cells that surround a conducting duct that carries the
secretion to the exterior (Fig. 3).
Figure 3.
Classification of insect exocrine glands, based on a rhinotermitid sternal gland. Scheme taken
from Billen and Šobotník (2015), made after Noirot and Quennedey (1974). Abbreviations: C, cuticle; D, duct
cells; G1, secretory cells class 1; G2, secretory cells class 2; G3, secretory cells class 3; S, campaniform
sensilla. The asterisk indicates a subcuticular space.
The life in colonies of social insects is a promoter of exocrine developments, as they are
used extensively to coordinate
different social interactions or activities, including foraging,
building, mating, defense, and nestmate recognition
(Costa–Leonardo & Haifig 2010, Billen
2011).
As many as 149 exocrine glands have been described for social insects so far, from
which 84 can be found in ants, 53 in bees and bumblebees, 49 in wasps and only 20 in
termites (Billen & Šobotník 2015).
Exocrine glands in termites
Termites possess 20 glands spread all over their body, but not necesarilly present in
all castes or species, and they generally consist of epidermal cells of ectodermal origin with
secretory capacities (Blum 1985, Costa–Leonardo & Haifig 2010).
Trail–following and sex pheromones are the most studied exocrine secretions in
termites, followed closely by defensive secretions. Trail–following pheromones are secreted
22
1. General Introduction
to mark the path between the nest and the foraging area. They are used by all termite
species studied so far and are secreted by the
sternal gland
of workers and soldiers, being
their action much stronger in workers (Howard et al. 1976; Sillam–Dussès et al. 2005, 2007,
2009a, 2009b, 2010, 2011, Bordereau et al 2010, Costa–Leonardo & Haifig 2010, Bordereau
& Pasteels 2011).
Sex pheromones are released by imagoes of one sex (usually females) in
order to attract the opposite sex (Pasteels 1972, Bordereau et al. 2010). They are usually
produced by the
tergal glands
, sometimes by the
posterior sternal glands
(both occurring
exclusively in termite imagoes and always involved in mate attraction; Noirot 1969) or
sternal glands
(present in all species, castes and developmental stages) (Bordereau &
Pasteels 2011, Sillam–Dussès et al. 2011). In the case of defensive secretions, they are
known for being mainly produced by the labial and the frontal glands. The
labial glands
(also called salivary glands) are a large paired organ, made of numerous cells arranged in
clumps (called acini) along with paired reservoirs (called water sacs) (Sillam– Dussès et al.
2012), that can be found in all castes and developmental stages of all termite species (Noirot
1969). The defensive function of these glands is restricted to soldiers, while in workers they
are used as food–marking pheromone and as phagostimulant (Noirot 1969, Sillam– Dussès
et al. 2012). On the other side, the
frontal gland
represents a fully defensive organ
incomparable among insects (Noirot, 1969). It is present in almost all imagoes and soldiers
from all species of termites in Neoisoptera clade
(Rhinotermitidae + Serritermitidae + Termitidae) (Deligne et al. 1981, Prestwich 1984,
Šobotník et al. 2010a, 2010c, 2010d, Kutalová et al. 2013). It is always present in
Neoisoptera soldiers and it occurs as a large reservoir, sometimes extending deep into the
abdomen (Rhinotermitidae genera) but normally restricted to the head (all other Neoisoptera
families) (Prestwich 1984, Quennedey 1984). It is present in all Neoisoptera imagoes but
Protermes sp. and Microtermes toumodiensis, as an epithelial thickening (all basal
Neoisoptera groups) or as an epithelial with reservoir (Termitidae except Foraminitermitinae
and Macrotermitinae) (Prestwich 1984, Šobotník et al. 2004, Šobotník et al. 2010c, Kutalová
et al. 2013). This gland is also present in almost all workers from soldierless species, where
it always occurs as an epithelial thickening (Šobotník et al. 2010d). When the gland has
reservoir, it is always accompanied by an opening called “fontanelle”. In those cases where
there is no reservoir, just a modified cuticle allows the secretions to go out of the body.
Frontal gland compounds can be chemicals of diverse nature, but they all have been found
to act as a defensive secretion in soldier, with functions such as: contact poisons, repellents
or irritants, entangling and incapacitating agents, anti–healing compounds, or alarm
pheromones (Piskorski et al. 2007, 2009; Šobotník et al. 2010b). There are only few cases
where the frontal gland is not accompanied by a fontanelle; in these cases, the secretion is
released through autothysis (Deligne & DeConinck 2006, Bordereau et al. 1997, Šobotník et
23
1. General Introduction
al. 2010b). The function of the gland in imagoes with reservoir has been only investigated in
Prorhinotermes simplex and its function seems analogous to soldiers (Piskorski et al., 2007,
2009). The function of the frontal gland when it is present as an epithelial thickening remains
unknown.
There are many other glands in termites, plenty of them presenting unknown or
speculative function. Among them, we can find: the
mandibular gland
, located at the ventral
mandibular condyle and present in all castes and developed stages (Šobotník & Hubert
2003); the
tarsal
glands
, always located on the first and second tarsomere of the leg,
sometimes also on the third tarsomere or the distal part of the tibia and present in most
termite species (Bacchus 1979, Soares & Costa–Leonardo 2002, Šobotník & Weyda 2002);
the
clypeal gland
present at the clypeus of alate imagoes of Rhinotermitidae, Serritermitidae
and Termitidae species (Křížková et al. 2014); the
tegumental glands
described in
Kalotermes and Prorhinotermes neotenics (Sbrenna & Leis 1983, Šobotník et al. 2003); the
lateral thoracic glands
described in 3 Termitidae species (Gonçalves et al. 2010); and the
labral gland
, which had been described in few random observations in soldiers (Deligne et
al. 1981, Quennedey 1984, Šobotník et al. 2010b).
24
1. General Introduction
1.5 Motivation and objectives of my thesis
Termites are fundamental organisms for humans both in their positive and negative
aspects,and learning about their chemical defensive mechanisms provides fundamental
information for a better understanding of their evolution and behaviour. This project was
facilitated through a collaboration between the Termites Research Team, Czech University of
Life Sciences (Czech Republic), and the Laboratoire d’Ethologie Expérimentale et Comparée
(LEEC), Université Paris 13 (France), with the support of a Université Paris 13 doctoral
fellowship.
The presented studies were done under the supervision of David Sillam–Dussès, leader
expert on termite pheromones, whose close collaboration allowed me to learn fundamentals
of termite communication. At the same time, I took the best from collaboration with my co–
supervisor, Jan Šobotník, who is authority in the field of insect exocrine organs, their
structure, function and evolution.
My Ph.D. aimed straight on disentangling the evolutionary processes leading to the
current development of the frontal and labral glands in termites. Three main aims were
raised (corresponding to Chapters 2, 3 and 4). The first aim was:
• To Disentagle the distribution of the labral glands in termite soldiers. This study
represented the first attempt to describe the gland occurrence in a representative set
(28 species) of termite soldiers across all termites (Paper 1). I examined the gland
presence in members of all termite families (except for Stylotermitidae, whose
material is not available) and most of Termitidae subfamilies. The results were
published in the Biological Journal of the Linnean Society (IF: 2.3).
The results of this research were that soldiers from all termite species possess the
labral gland. In addition to personal observations of its occurrence in imagoes as well, these
results suggested that further research should be perform to understand the evolutionary
routes of this gland. Hence, our second aim appeared:
•
To determine the evolution of the labral gland of termites. The study was
carried out among workers and imagoes in a representative set of termite
species and the closest relative, the woodfeeding cockroach Cryptocercus
punctulatus, using the histological procedures (Paper 2). The gained
observations allowed us to describe the evolution of the labral gland across
extant termite taxa. The resulting manuscript has been published in one
leading ecological journal, Biological Journal of the Linnean Society.
The research about the frontal gland in our study presented two main aims:
•
To unravel the evolution of frontal gland in termite workers: I executed
25
1. General Introduction
comparative study of the frontal gland in workers of 37 species across
Neoisoptera representatives using histological procedures, and the gland
secretory activity was evaluated using methods of transmission electron
microscopy in 8 species.
•
To perform a phylogenetic analysis of the frontal gland evolution in Neoisoptera: I
mapped the evolutionary routes leading to the observed diversity of the gland in
soldiers, workers and alate imagoes on a robust phylogenetic tree, which allowed
me to describe the general trends in the gland structure and use in particular
termite taxa.
These two objectives were joined together in a larger manuscript (Paper 3), which I hope
it will be published in a leading biological science journal, such as Proceedings of the Royal
Society London B.
27
2. General Methods
2. General Methods
2.1 Animals of study
For my thesis I used living termite species which were obtained predominantly on the
existing material from my supervisors, but I also realised some necessary field trips (in China,
Ecuador and French Guiana) which were covered by my supervisors. At the same time, my
supervisors already disposed of a set of fixed samples to be used for optical and electron
microscopy, and they also provided me additional material from their field works or through
existing network of their collaborators. The detail of the species and their place of origin can be
found in the supplementary tables of my manuscripts.
2.2 Histology
Histological procedures were done at the laboratory of the Termites Research Team (TRT)
of the Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech
Republic. There, all the equipment for fixation and embedding was available, as well as a
Reichert Ultracut ultramicrotome which I used for sectioning of the samples.
More details about fixative used and fixation methods are provided in the Materials and
Methods of each manuscript presented in this thesis.
2.3 Microscopy
Nikon Ni–E optical microscope equipped with a Nikon DS–Fi1c camera was usually used to
identify presence/absence of the gland. It was available at the TRT in Prague and the software
used for controlling the microscope and for taking and measuring the pictures was Nis–elements
AR.
When it was needed to use Transmission Electron Microscope or Scanning Electron
Microscope, a Jeol 6380 LV scanning electron microscope and a Jeol 1011 transmission electron
microscope were available at the Laboratory of Electron Microscopy of the Faculty of Sciences,
Charles University in Prague, Czech Republic. Mirek Hyliš, the technician in charge of them,
provided me with assistance and collaboration.
28
2. General Methods
2.4 Behavioural test
Behavioural experiments were performed at the Laboratoire d'Ethologie Expérimentale et
Comparée of the Université Paris 13 (France) and at the TRT in Prague. In both cases, they were
carry out in rooms with controlled temperature and humidity.
2.5 Others
Other experiments or details are described in each specific manuscript.
29
Paper 1: The labral gland in termite soldiers
Paper 1: The labral gland in termite soldiers
Valeria Palma–Onetto1, 2, Kristýna Hošková3, Barbora Křížková2, Romana Krejčířová3, Jitka
Pflegerová4, Filipa Bubeníčková3, Rudy Plarre5, Cecilia AL Dahlsjö2, 6, Jiří Synek2, Thomas
Bourguignon2, 7, David Sillam–Dussès1, 8* and Jan Šobotník2*
1 University Paris 13 – Sorbonne Paris Cité, Laboratory of Experimental and Comparative
Ethology, Villetaneuse, France.
2 Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech
Republic.
3 Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences,
Prague, Czech Republic.
4 Institute of Entomology, Biology Centre, Academy of Sciences of the Czech Republic, České
Budějovice, Czech Republic.
5 Bundesanstalt für Materialforschung und –prüfung, Berlin, Germany.
6 Environmental Change Institute, University of Oxford, South Parks Road, Oxford OX1 3QY, UK.
7 Okinawa Institute of Science & Technology Graduate University, Onna–son, Okinawa, Japan.
8 Institute of Research for Development – Sorbonne Universités, Institute of Ecology and
Environmental Sciences of Paris, Bondy, France.
* These authors contributed equally to the study.
Biological Journal of the Linnean Society, Volume 123, Issue 3, 2 March 2018, Pages 535–544,
https://doi.org/10.1093/biolinnean/blx162
Published: 07 February 2018
30
Paper 1: The labral gland in termite soldiers
Résumé
Le succès évolutif des termites repose en grande partie sur un système de communication
complexe géré par un riche ensemble de glandes exocrines. Pas moins de 20 glandes exocrines
différentes sont connues chez les termites. Bien que certaines de ces glandes soient relativement
bien connues, seules des observations anecdotiques existent pour d’autres. La glande labrale est
l’une des glandes exocrines qui n’a retenu jusqu’à présent qu’une attention négligeable. Dans
cette étude, nous avons examiné la structure et l'ultrastructure du labrum chez des soldats de 28
espèces de termites. Nous confirmons que la glande labrale est présente dans toutes les
espèces de termites et comprend deux régions sécrétrices situées sur la face ventrale du labrum
et à la partie dorso–apicale de l'hypopharynx. Le labrum des Neoisoptera a une pointe hyaline,
qui a été ensuite perdue chez les Nasutitermitinae, les Microcerotermes et des espèces à soldats
qui claquent. L'épithélium de la glande est généralement constitué de cellules sécrétrices de
classe 1, avec en plus des cellules sécrétrices de classe 3 chez certaines espèces. Une
caractéristique commune des cellules sécrétrices est l'abondance de réticulum endoplasmique
lisse, un organite connu pour produire des sécrétions lipidiques et souvent volatiles. Nos
observations suggèrent que la glande labrale est impliquée dans la communication plutôt que
dans la défense, comme suggéré précédemment. Notre étude est la première à fournir une
image complète de la structure de la glande labrale chez les soldats parmi tous les taxons de
termites.
Mots–clefs: glande exocrine, hypopharynx, labrum, Termitoidae, ultrastructure, Isoptera
31
Paper 1: The labral gland in termite soldiers
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.
Keywords:
exocrine gland, hypopharynx, labrum, Termitoidae, ultrastructure, Isoptera
32
Paper 1: The labral gland in termite soldiers
Introduction
Termites are an important food resource for a range of animals (Redford & Dorea,
1984), and they compete for resources with other wood– and soil–feeding taxa (Šobotník,
Jirosová & Hanus, 2010a). Termites protect themselves through passive and active defence
mechanisms, including a cryptic lifestyle, the construction of defensive structures (Korb,
2011) and investments into a caste of defenders: the soldiers (Haverty, 1977). While the
primary weapon of termite soldiers is generally their powerful mandibles, glands that produce
defensive compounds are of comparable importance (Prestwich, 1984; Šobot ník et al.,
2010a).
Termites use intricate communication systems, the complexity of which is reflected in
the development of 20 different signal–producing exocrine organs (Billen & Šobotník, 2015).
Four glands are found in most termite species: the frontal gland, the sternal gland, the labial
glands and the mandibular glands. The presence of other exocrine organs is restricted to
specific termite lineages, or to certain castes. The function of these lineage–/caste–specific
glands is not fully understood, apart from the defensive function of the crystal glands in
Neocapritermes taracua workers (Šobotník et al., 2012, 2014; Bourguignon et al., 2016). The
labral gland is one of these poorly known exocrine glands, known only from the soldier caste
of three termite species (Deligne, Quennedey & Blum, 1981; Quennedey, 1984; Šobotník et
al., 2010b; Costa–Leonardo & Haifig, 2014), and from some imagoes (Křížková et al., 2014).
The labral gland was first described on the ventral side of the labrum in Macrotermes
bellicosus (Deligne et al., 1981) and was later found also on the dorsal side of the
hypopharynx in other Macrotermitinae species (Quennedey, 1984). The presence of labral
glands in other taxa is thought to be indicated by a hyaline tip, located on the tip of the
labrum (Deligne et al., 1981). The labral gland of M. bellicosus is composed of class 1
secretory cells only (according to the classification of Noirot & Quennedey, 1974), while
additional class 3 secretory cells have been found in the labral glands of Glossotermes
oculatus and Cornitermes cumulans soldiers (Šobotník et al., 2010b; Costa–Leonardo &
Haifig, 2014). The function of the labral gland has not been studied for any termite species,
and the literature suggests that it produces toxic secretions that impregnate the mandibular
edges (Deligne et al., 1981; Quennedey, 1984). In this paper, we provide the first
comprehensive description of the structure of the labral gland in the soldiers of 28 species,
representatives of the termite tree of life.
33
Paper 1: The labral gland in termite soldiers
Materials and Methods
Direct Observations
Living termites were observed and photographed using Canon EOS 6D and Canon
EOS 5D SR cameras, combined with Canon EF 100 mm f/2.8L Macro IS USM and Canon
MP–E 65 mm f/2.8 lenses, and equipped with the Canon Macro Twin Lite MT–24EX flash.
The photographs were used to compare the shape of the labrum and the presence of a
hyaline tip in termite soldiers.
Optical microscopy and transmission electron microscopy
Soldier labral glands were studied using three different fixatives: fixative with
phosphate buffer (0.2 M, pH 7.2 buffer/formaldehyde 10%/glutaraldehyde 8% = 2 : 1 : 1),
cacodylate buffer (0.2 M, pH 7.3 buffer/glutaraldehyde 8%/distilled water = 2 : 1 : 1) and
standard Bouin’s solution (for details see Supplementary Information, Table S1). For electron
microscopy, soldier heads were cut off and the mandibles were removed to facilitate
sectioning. The mandibles were left intact in the minor soldiers of Rhinotermitinae and in all
Nasutitermitinae. Samples were postfixed using 2% osmium tetroxide, and embedded in
Spurr resin. The samples were cut into 0.5–μm sections using a Reichert Ultracut
ultramicrotome and stained with Azure II for analysis with optical microscopy.
Histology
The samples were dehydrated using a ethanol series, transferred to xylene and embedded in
paraffin. Polymerization was carried out in an oven at 56–58 °C for 2 h. The samples were
cut into sections 5–10 µm thick using Bamed pfm Rotary 3004 M microtome, placed on a
slide coated with eggwhite/glycerol, stained with Mallory’s trichrome stain and then made
clear with xylen. For additional details see Table S1.
Electron Microscopy
We dissected the heads of freshly freeze–killed soldiers, and removed the mandibles,
maxillae and labium. The heads were thereafter dehydrated using an acetone series. The
samples were dried using the critical–point method and glued onto an aluminium holder
using thermoplastic adhesive. The samples were then sputter–coated with gold and
observed using a Jeol 6380 LV scanning electron microscope. The mouthparts of three
species (Embiratermes neotenicus, Coptotermes formosanus and Sphaerotermes
sphaerothorax) were cleaned via argon plasma etching in a sputter coater machine (Bal–Tec
34
Paper 1: The labral gland in termite soldiers
SCD 050).
Ultrastructural features were studied in selected samples (see Table S1) using a Jeol
1011 transmission electron microscope, as described by Šobotník, Weyda & Hanus (2003).
Evolution of the hyaline tip
We reconstructed the presence of the hyaline tip using previously published
phylogenetic trees (Bourguignon et al., 2015, 2017). 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.
Results
The labral gland is a constituent part of the labrum (Fig. 1A, B). The labrum is dorsally
sclerotized, and membranous on the ventral side, with lower sclerotization towards the tip, often
with a transparent inflated apical part termed the ‘hyaline tip’. The hyaline tip appears as a
transparent extensible protrusion of the labrum occurring in many taxa of Rhinotermitidae and
Termitidae (Fig. 1C). The presence of the hyaline tip is variable, depending on species. The
hyaline tip has been lost in several lineages, including the snapping soldiers and all
Nasutitermitinae (Figs 1C, S1).
Figure 1. (A) Sphaerotermes sphaerothorax soldier. Arrow marks the hyaline tip of the labrum. (B) Head
of Neocapritermes taracua soldier. (C) Phylogenetic tree showing the evolution of the hyaline tip in soldier caste
termites. The presence or absence of the hyaline tip is marked by black or white circles, respectively.
35
Paper 1: The labral gland in termite soldiers
Scanning electron microscopy
The ventral facies of the labrum were flexible and appeared wrinkled (Fig. 2A), while
the dorsal facies were more rigid with a sclerotized cuticle. The ventral side of the labrum
generally carried a few tens of sensillae (Fig. 2B), probably acting as contact
chemoreceptors [based on combined scanning (SEM) and transmission electron microscopy
(TEM) evidence, see below], with possible mechanosensitive function (based on striking
similarity to campaniform sensillae). While the dorsal side of the labrum was usually smooth,
the ventral facies of the labrum usually showed borders between the underlying epidermal
cells, which appeared as irregular angular structures between 4 and 6 µm in the largest
dimension. These borders were well delimited in certain parts of the ventral surface of the
labrum, often appearing as ridges or spines extending beyond the cell border. These
features were especially developed in Neotermes cubanus, Glossotermes oculatus,
Neocapritermes taracua, Spinitermes sp. and Labiotermes labralis. The same pattern was
also observed along the midline of the labrum in Prorhinotermes simplex, the basal half of
the labrum in Coptotermes formosanus (Fig. 2A, B) and Sphaerotermes sphaerothorax, and
the basal part of the labrum in Embiratermes neotenicus. In all specimens, the apical and
ventro–lateral part of the labrum possessed numerous pores typically about 30–50 nm in
diameter (Fig. 2C).
Figure 2.
Labral gland development. (A) Micrograph of the ventral side of the labrum of Coptotermes
formosanus; the small rectangle indicates the sector where the micrograph in B was taken. (B) Region with
a group of sensillae (marked with white arrows) in C. formosanus labrum. (C) High–magnification
micrograph of the apical region with epicuticular pores in Sphaerotermes sphaerothorax labrum.
36
Paper 1: The labral gland in termite soldiers
Optical microscopy
The labral gland appeared as a thickened epithelium located on the ventral side of
the labrum, with possible extension to the dorsal side at the labrum apex. An independent
portion of secretory epithelium appeared also on the dorso–apical part of the hypopharynx
(Fig. 3A, B). Labral gland secretions were shown to accumulate in the space between the
secretory epithelium and the overlaying cuticle
with no reservoir.
Figure 3. Sagittal sections of the forehead of
Psammotermes hybostoma medium soldier (A) and
Neocapritermes taracua soldier (B), showing the secretory
epithelium in hypopharynx. Abbreviations: cl, clypeus; hy,
hypopharynx; lb, labium; lg, labral gland.
The labral gland secretory epithelium varied in thickness among species, most
commonly ranging between 20 and 30 µm. The thinnest epithelium was found in
Nasutitermes lujae (2 µm) and the thickest epithelium was found in the large soldiers of
Psammotermes hybostoma (147 µm) (Table S1). Hypopharyngeal thickness varied between
4 and 30 µm. The ultrastructural features were nearly identical between the labral and
hypopharyngeal regions of the labral gland in all species. The shape and overall size of the
labral gland were diverse and not proportional to the size of the labrum. While some labral
glands covered the entire labrum, others covered less than half of the labral ventral area.
Within the four studied species with soldier sub–castes, the thickness of the labral
gland increased with the size of the soldier morph (Table S1).
Transmission electron microscopy
37
Paper 1: The labral gland in termite soldiers
TEM revealed that the labral and hypopharyngeal epithelium were made up of
secretory cells. The ultrastructural features of the secretory cells in the labral and
hypopharyngeal regions of the labral gland were almost identical, and are thus described
together.
The labral gland was predominantly made up of columnar class 1 secretory cells
(according to the classification of Noirot & Quennedey, 1974) that were characterized by an
abundance of smooth endoplasmic reticulum (ER), vesicles of different electron densities,
abundant mitochondria, numerous microtubules orientated apico–basally, glycogen granules,
myelin figures and sparse rough ER mainly located around the nucleus (Fig. 4A–C). The
secretory cells could easily be differentiated from the non–modified cells (Fig. S3A) as the
latter are thinner and lack the characteristics mentioned above. Electron–lucent vesicles
were also relatively common within the cells, although they were rarely observed to be
released (then including the membrane) at the cell apex, while electron–dense granules
were rare. The secretory cell cytoplasm often contained lipid–like droplets (around 1–2 µm in
diameter; Fig. S3B, C) that were located freely in the cytoplasm and particularly abundant in
major soldiers of Dolichorhinotermes longilabius. The droplets in D. longilabius had a foamy
appearance and turned into lucent vesicles that were occasionally excreted at the secretory
cell apex. Junctions between neighbouring class 1 cells were formed by apical zonulae
adherens followed by septate junctions, while the basal parts of the membranes were devoid
of any junctions. Basal invaginations were well developed throughout the gland, and on
average were about 5 µm deep (up to 20 µm in Labiotermes labralis) (Fig. 4A) and showed
frequent pinocytotic activity (Fig. S3D). The nucleus of the class 1 cells was basally located
and elliptic or slightly irregular in shape. The largest dimension of the nucleus was 5 µm
(rarely up to 10 µm) and the nucleus was predominantly filled with dispersed chromatin with
few aggregates. Microvilli were well developed, about 1.5 µm in length (rarely up to 3–4 µm),
approximately 100 nm thick, and always had a central channel about 40 nm in diameter (Figs
4A, S3C, E). The basal invaginations and microvilli of the hypopharyngeal region of the labral
gland were always shorter than those of the labral region. Microvilli were in some cases
longer in the central part of the gland than in the gland margins.
38
Paper 1: The labral gland in termite soldiers
Figure 4.
Ultrastructure of the labral gland in soldiers. (A) Overall development of the labral gland in
Labiotermes labralis. Note the development of the apical microvilli and basal invaginations. (B) Detail of
labral gland secretory cell class 1 cytoplasm in Neocapritermes taracua showing well–developed smooth
endoplasmic reticulum. (C) Detail of labral gland secretory cell class 1 cytoplasm in large soldier of
Dolichorhinotermes longilabius showing a free axon located at the base of the secretory epithelium. (D)
Highly modified cuticle underlying the labral gland in Embiratermes neotenicus. Note enlarged pore canals
ensuring secretion release and the margin of the sensillum. (E) Class 3 secretory cell in Glossotermes
oculatus. Abbreviations: a, axon; c3, class 3 secretory cell; cc, conducting canal; en, endocuticle; ex,
exocuticle; g, glycogen; bi, basal invaginations; m, mitochondria; ms, margin of the sensillum; mt,
microtubule; mv, microvilli; n, nucleus; rer, rough endoplasmic reticulum; ser, smooth endoplasmic reticulum.
The cuticle was in general made up of three layers, the endocuticle of helicoid
structure, exocuticle showing no discernible layers and a thin epicuticle (see Table S1). The
labral gland secretions were stored in the space between the secretory epithelium, the
overlying cuticle and inside the porous cuticle. There was no invaginated reservoir in any of
39
Paper 1: The labral gland in termite soldiers
the studied species. The cuticle showed numerous adaptations for release of the secretion,
and these were more pronounced towards the labral tip (Fig. 2C, 4D, S4A, B). The cuticular
modifications included an increase in the number and width of the pore canals, which
widened towards the cuticle base (Fig. 4D), and the occurrence of epicuticular pores allowing
for the secretion to be evacuated from the body.
Secretory cells were innervated by free axons frequently observed at the base of the
secretory epithelium (Fig. 4C). The singular axons without envelope cells often occurred
among the basal invaginations, and sometimes contained typical electron–dense grains of
neurosecretions. A different kind of neural tissue was represented by groups of sensillae
located along the central line of the labrum, each comprising between two and five sensory
neurons (represented by distal dendrites) and corresponding envelope cells (Figs 4D, S4C).
Apart from the common organelles, large microtubule bundles running through
secretory cells were found in Mastotermes darwiniensis, Hodotermopsis sjoestedti and
Embiratermes neotenicus (Fig. S3D). Additionally, tracheae going through class 1 cells were
found in M. darwiniensis and H. sjoestedti (Fig. S3E). Major soldiers of Dolichorhinotermes
longilabius possessed particularly large amounts of lipid droplets, with electron–dense
granules that dissolved into lucent vesicles. In all studied Nasutitermitinae the labral gland
was relatively underdeveloped, although the cells retained the general characteristics of the
labral gland.
Class 3 secretory cells, when present, commonly occurred on the dorsal side of the
labrum and were generally separated from the secretory epithelium by non–modified
epidermal cells. However, the class 3 cells were in few cases mixed with class 1 cells (Fig.
4E) in Glossotermes oculatus, Termes hospes, and in the minor soldiers of
Dolichorhinotermes longilabius. In Mastotermes darwiniensis, by contrast, the class 3
secretory cells were located adjacent to the class 1 secretory cells.
Class 3 cells did not touch either the apex or the basement membrane of the gland.
Their cytoplasm predominantly contained vesicles of moderate electron density (Fig. 4E), but
also contained rough ER and free ribosomes, Golgi apparatus, mitochondria, microtubules
and rare electron–dense granules. The cells were equipped with porous receiving canals
continuous with a conducting canal approximately 0.4 µm in diameter. The conducting canal
comprised inner (approximately 40 nm thick) and outer (approximately 6 nm thick) epicuticles
(Fig. 4E).
Discussion
The labral gland is an integral part of the labrum, which is a thin lip–like structure that
covers the dorsal side of the pre–oral cavity. The labral gland belongs to the basic body plan
40
Paper 1: The labral gland in termite soldiers
of termites. However, its presence has rarely been investigated. Here we report on its
presence and cytological features in soldiers of 28 species across the termite phylogeny.
The presence of the labral gland in all observed species was unexpected as the gland has
only been reported in soldiers of three termite species previously (Deligne et al., 1981;
Quennedey, 1984; Šobotník et al., 2010b; Costa–Leonardo & Haifig, 2014). The labral gland
was originally recognized as an exocrine organ by Deligne et al. (1981). Quennedey (1984)
described the hypopharyngeal part of the labral gland and suggested that the occurrence of
the hyaline tip proves the presence of the labral gland in termite soldiers. It was only
recently, and following Šobotník et al.’s (2010b) study on the defensive glands in
Glossotermes oculatus, and Costa–Leonardo & Haifig’s (2014) study on the labral gland in
Cornitermes cumulans, that additional data on the labral gland appeared. In addition to the
presence of the labral gland in termite soldiers, it was also recently observed in some
imagoes (Křížková et al., 2014) and certain workers (Palma–Onetto V and Šobotník J, our
unpublished data). These random observations suggest that the labral gland might be
present in all termite castes, pointing to its importance during termite evolution.
The labral gland is split into two secretory regions located in the ventral part of the
labrum and dorso–apical part of hypopharynx, respectively. Although the secretory
epithelium is always thicker in the labral part, the ultrastructure of secretory cells present in
these two secretory regions is virtually identical. We therefore expect that both secretory
regions play the same role, and should thus be treated as a single gland. The nomenclatural
change from ‘labral gland’ to ‘cibarial gland’ proposed by Quennedey (1984), based on gland
development in two regions, is therefore redundant and the original name, well accepted by
the scientific community, should prevail.
The hyaline tip is a traditionally described morphological character. The dorsal side of
the labrum is always sclerotized, while the ventral part is always formed by a lucent
membranous cuticle. However, species may differ in the level of sclerotization of the dorsal
side, especially at the labrum apex. While some soldiers show an unchanged level of labrum
sclerotization (hyaline tip absent), the level of sclerotization often decreases towards the
labrum apex in others (hyaline tip present). All basal taxa primarily lack the hyaline tip, which
evolved in a common ancestor of Rhinotermitidae and Termitidae, and was subsequently
lost at least four times independently: once in Nasutitermitinae, in which the entire labrum is
greatly reduced in size, twice independently in lineages with snapping soldiers,
Pericapritermes and Neocapritermes + Planicapritermes, and once in Microcerotermes.
While the hyaline tip has been shown to disappear in some lineages, the labral gland
was found in all termite families studied here. This suggests that the evolution of snapping
mandibles did not see a loss of the labral gland and that the evolution of mandibles has not
necessarily been accompanied by a reduction or loss of chemical adaptation (Kyjaková et al.
41
Paper 1: The labral gland in termite soldiers
2015).
The cytological features of the labral gland showed many similarities among all
studied species. Additionally, the four species with polymorphic soldiers that we studied
showed that the labral gland volume increased with sub–caste size and was particularly
pronounced in Psammotermes hybostoma.
The common features shared by labral and hypopharyngeal parts of the labral glands
include: (1) abundance of smooth ER, (2) the presence of apical microvilli with a central
channel, (3) well–developed basal invaginations ensuring the intake of precursors from the
haemolymph, and (4) cuticular modifications in the tip of the labral gland allowing gland
secretions to reach the exterior (see also Deligne et al., 1981; Quennedey, 1984; Šobotník et
al., 2010b; Costa–Leonardo & Haifig, 2014). These ultrastructural features are a
conservative account of the characteristics of the two secretory regions in the studied
species, which suggest that the labral gland has the same function among all species. The
labral gland secretion is stored between the secretory epithelium and the overlying cuticle, as
well as within the cuticle itself. Labral secretions from the glandular cells are under neural
control, supposedly from the brain, as singular axons have often been detected at the base
of the secretory epithelium.
The function of the labral gland is probably not defensive due to the absence of a
reservoir, a feature characteristic of defensive glands (Chapman, 2013). Additionally, the
labral gland is present in soldiers of all species, irrespective of their defensive strategies,
including species having soldiers with nasus glands, with snapping mandibles or performing
body rupture. The composition of the labral gland secretion remains unknown despite our
repeated attempts to identify labral gland–specific compounds. This may be due to the small
size of the labral gland and the unknown nature of its secretion. Nevertheless, the high
abundance of a smooth ER suggests that the secretion may have a lipidic and volatile nature
and could be used in communication (Percy–Cunningham & MacDonald, 1987; Nakajima,
1997; Tillman et al., 1999; Alberts et al., 2002).
The presence of specialized receptors on the ventral side of the labrum is likely to aid
in dosage of labral secretions. As all observed receptors contained several dendrites, a
chemosensory function is likely for all species while a mechanoreceptive function remains
hypothetical. The idea that the labral receptors respond to mechanical pressure has a
functional parallel in the sternal gland, secretion releases from which are controlled by
groups of campaniform sensillae (Stuart & Satir, 1968; Quennedey et al., 2008).
Class 3 cells occur frequently on the dorsal side of the labrum and on the sclerotized
body cuticle (Šobotník et al., 2004; Šobotník, Weyda & Hanus, 2005). Class 3 cells may also
occur adjacent to the labral gland secretory epithelium but should not be considered as part
of the labral gland until the two cell classes are combined, as seen in G. oculatus (Šobotník
42
Paper 1: The labral gland in termite soldiers
et al., 2010b), the minor soldiers of D. longilabius (presented here), C. cumulans (Costa–
Leonardo & Haifig, 2014) and T. hospes (presented here). Class 3 cells have not been
observed in the hypopharyngeal part of the labral gland in any of above–mentioned species.
The ultrastructure of the class 3 secretory cells is uniform in termites, irrespective of their
caste (Costa–Leonardo & Shields, 1990; Šobotník et al., 2004) and position in the gland,
such as mandibular (Lambinet, 1959; Cassier, Fain–Maurel & Lebrun, 1977), sternal (Noirot
& Quennedey, 1974; Quennedey et al., 2008), tergal (Ampion & Quennedey, 1981; Šobotník
et al., 2005) and epidermal (Šobotník et al., 2003). The secretory cells are always rich in
rough ER and Golgi apparatus, and contain variable amounts of moderately electron–lucent
vesicles released to the extracellular reservoir (‘end apparatus’), into which the cuticular
canal is inserted. This ultrastructure suggests that rough ER produces proteinaceous water–
soluble secretions that are configured in the Golgi apparatus (Hand & Oliver, 1984) before
being released on the surface of the body cuticle. These secretions may appear as the
uppermost layer of the epicuticles protecting the lower layers from abrasion (Chapman,
2013).
Conclusion and further hypotheses
The labral gland has previously been suggested to be a synapomorphy of Neoisoptera
(Šobotník et al., 2010a). The presence of the labral gland in termite soldiers of all studied
species suggests that the labral gland evolved with the soldier caste where it has remained
an important organ. Moreover, the labral gland has long been thought to primarily have a
defensive function. Gland secretion was thought to be on the mandibles and deposited into
the wound following bite (Deligne et al., 1981; Quennedey, 1984; Šobotník et al., 2010b;
Costa–Leonardo & Haifig, 2014). However, preliminary observations based on the
morphology, structure and ultrastructure of the labral gland suggest that labral gland
secretion has a communicative function.
The presence of a labral gland in soldiers of all termite species suggests that it has a
fundamental role in colony survival and success. Our data suggest that the function of the
labral gland may be related to communication. This hypothesis is supported by personal
observations of soldiers wiping their labrum against the floor after encountering an enemy. A
better understanding of the function of the labral gland in termites is called for to enhance
knowledge of termite defence mechanisms and communication behaviour.
Acknowledgements
Credit for Figure 1B goes to Aleš Buček (OIST, Japan). We thank Mirek Hyliš from
the Laboratory of Electron Microscopy (Faculty of Sciences, Charles University in Prague) for
43
Paper 1: The labral gland in termite soldiers
his help and support with SEM and TEM. We are grateful to Yves Roisin for constructive
criticism of the manuscript. We also thank three anonymous reviewers for their helpful
comments and suggestions. Financial support was provided by the project IGA FLD No.
A13/17 (Czech University of Life Sciences, Prague).
Supporting information
Additional Supporting Information may be found in the online version of this article at
the publisher’s web–site:
Figure S1.
Scanning electron micrograph of the mouth parts of Nasutitermes lujae, with antennae and part
of the maxillary palp removed.
44
Paper 1: The labral gland in termite soldiers
Figure S2.
Labral gland development. Sagital sections of the labrum in: (1) Mastotermes darwiniensis, (2)
Hodotermopsis sjoestedti, (3) Neotermes cubanus small soldier, (4) Neotermes cubanus large soldier, (5)
Dolichorhinotermes longilabius small soldier, (6) Dolichorhinotermes longilabius large soldier, (7)
Prorhinotermes simplex, (8) Psammotermes hybostoma small soldier, (9) Psammotermes hybostoma
medium soldier, (10) Psammotermes hybostoma large soldier, (11) Termitogeton planus, (12) Glossotermes
oculatus, (13) Reticulitermes flavipes, (14) Coptotermes formosanus, (15) Sphaerotermes sphaerothorax,
(16) Pericapritermes sp., (17) Microcerotermes sp., (18) Spinitermes sp., (19) Globitermes globosus small
soldier, (20) Globitermes globosus large soldier, (21) Globitermes sulphureus, (22) Termes hospes, (23)
Inquilinitermes fur, (24) Neocapritermes taracua, (25) Planicapritermes planiceps, (26) Dentispicotermes
brevicarinatus, (27) Labiotermes labralis, (28) Embiratermes neotenicus, (29) Indotermes sp., (30)
Nasutitermes lujae, (31) Constrictotermes cavifrons, (32) Hirtitermes sp., (33) Trinervitermes sp.
45
Paper 1: The labral gland in termite soldiers
Figure S3.
Ultrastructure of the labral gland in soldiers. (A) Non–modified epithelium surrounding the labral
gland in Hirtitermes sp. (B) Labral gland development in Hirtitermes sp. (C) Labral gland development in
Nasutitermes lujae. Note the highly electron–dense vesicles. (D) Pinocytotic activity at the cell base in the
labral epithelium in the large soldier of Dolichorhinotermes longilabius. Arrows indicate the pinocytotic
activity at the base of the cell. (E) View of the central channel present in the microvilli, allowing secretion
release from secretory cells. Abbreviations: en, endocuticle; ex, exocuticle; l, lipid–like droplet; mv, microvilli;
n, nucleus; v, vesicle.
46
Paper 1: The labral gland in termite soldiers
Figure S4.
Ultrastructural features in the labral gland. (A) Highly modified cuticle underlying the labral gland
in Neocapritermes taracua. (B) Detail of apical glandular cuticle at the tip of the labral gland in Nasutitermes
lujae showing epicuticular pores allowing secretion out from the body. (C) Chemoreceptors containing four
or five axons going through the labral epithelium in Hirtitermes sp. (D) Large microtubule bundles running
through secretory cells in Hodotermopsis sjoestedti. (E) Tracheae going through labral gland cells in
Mastotermes darwiniensis. Abbreviations: dd, distal dendrite; dg, electron–dense granule; en, endocuticle;
ep, epicuticle; ex, exocuticle; lv, electron–lucent vesicle; m, mitochondria; mb, microtubule bundle; mv,
microvilli; n, nucleus; s, secretion; ser, smooth endoplasmic reticulum; tr, trachea; v, vesicle.
47
Paper 1: The labral gland in termite soldiers
Table S1.
List of studied termite species, with indication of the fixation buffer used, collection location, species and subcastes (if any), number of repetitions, and
labral and hypopharynx epithelium measures (μm). The last four columns provide detail of the cells analysed by TEM, with indi cation of cell type, thickness of
cuticular layers, smooth ER and presence of axons. Abbreviations: n.a., not applicable; Y, yes.
48
Paper 1: The labral gland in termite soldiers
References
Alberts B, Johnson A, Lewis J, Raff M, Roberts K, and Walter P. 2002.
Molecular Biology
of the cell: the endoplasmic reticulum, 4th edn. New York: Garland Science.
Ampion M, Quennedey A. 1981.
The abdominal epidermal glands of termites and their
phylogenetic significance. In Howse PE, Clément JL, eds. Biosystematics of social insects.
London: Academic Press, 249–261.
Billen J, Šobotník J. 2015.
Insect exocrine glands. Arthropod Structure & Development
44
:
399–400.
Bourguignon T, Lo N, Cameron SL, Šobotník J, Hayashi Y, Shigenobu S, Watanabe D,
Roisin Y, Miura T, Evans TA. 2015.
The evolutionary history of termites as inferred from 66
mitochondrial genomes. Molecular Biology and Evolution
32
: 406–421.
Bourguignon T, Šobotník J, Brabcová J, Sillam–Dussès D, Buček A, Krasulová J,
Vytisková B, Demianová Z, Mareš M, Roisin Y, Vogel H. 2016.
Molecular mechanism of
the two–component suicidal weapon of Neocapritermes taracua old workers. Molecular
Biology and Evolution
33
: 809–819.
Bourguignon T, Lo N, Šobotník J, Ho SY, Iqbal N, Coissac E, Lee M, Jendryka MM,
Sillam–Dussès D, Krížková B, Roisin Y, Evans TA. 2017.
Mitochondrial phylogenomics
resolves the global spread of higher termites, ecosystem engineers of the tropics. Molecular
Biology and Evolution
34
: 589–597.
Cassier P, Fain–Maurel MA, Lebrun D. 1977.
Electron microscopic study of the mandibular
glands of Kalotermes flavicollis fabr. (Isoptera; Calotermitidae). Cell and Tissue Research
182:
327–339.
Chapman RF. 2013.
The insects: structure and function, 5th edn. In Simpson SJ, Douglas
AE, eds. The integument, gas exchange and homeostasis. Cambridge: Cambridge
University Press, 464–496.
Costa–Leonardo AM, Shields KS. 1990.
Morphology of the mandibular glands in workers
of Constrictotermes cyphergaster (Silvestri) (Isoptera: Termitidae). International Journal of
Insect Morphology and Embryology
19
: 61–64.
49
Paper 1: The labral gland in termite soldiers
Costa–Leonardo AM, Haifig I. 2014.
Termite communication during different behavioral
activities. In Witzani G, ed. Biocommunication of animals. Dordrecht: Springer, 161–190.
Deligne J, Quennedey A, Blum MS. 1981.
The enemies and defense mechanisms of
termites. In Hermann HR, ed. Social insects, Vol. 2. New York: Academic Press, 1–76.
Hand AR, Oliver C. 1984.
The role of GERL in the secretory process. In Cantin M, ed. Cell
biology of the secretory process. Basel: Karger Publishers, 148–170.
Haverty MI. 1977
The proportion of soldiers in termite colonies: a list and a bibliography.
Sociobiology
2
: 199–216.
Kyjaková P, Dolejšová K, Krasulová J, Bednárová L, Hadravová R, Pohl R, Hanus R.
2015.
The evolution of symmetrical snapping in termite soldiers need not lead to reduced
chemical defence. Biological Journal of the Linnean Society
115:
818–825.
Korb J. 2011.
Termite mound architecture, from function to construction. In Bignell ED,
Roisin Y, Lo N, eds. Biology of termites: a modern synthesis. Dordrecht: Springer, 349–373.
Křížková B, Bourguignon T, Vytisková B, Šobotník J. 2014.
The clypeal gland: a new
exocrine gland in termite imagoes (Isoptera: Serritermitidae, Rhinotermitidae, Termitidae).
Arthropod Structure & Development
43:
537–542.
Lambinet F. 1959.
La glande mandibulaire du termite à cou jaune (Calotermes flavicollis).
Insectes Soc. 6: 165–17. Maddison WP, Maddison DR. 2010. Mesquite: a modular system
for evolutionary analysis. Version 2.75. Available at:
mesquiteproject.org/mesquite/download/download.html
Nakajima T. 1997.
Cytochrome P450 isoforms and the metabolism of volatile hydrocarbons
of low relative molecular mass. Journal of Occupational Health,
39:
83–91.
Noirot C, Quennedey A. 1974.
Fine structure of insect epidermal glands. Annual Review of
Entomology
19:
61–80.
Percy–Cunningham JE, MacDonald JA. 1987.
Biology and ultrastructure of sex
pheromone–producing glands. In Prestchich GD, Blomquist GJ, eds. Pheromone
biochemistry. London: Academic Press, 27–75.
50
Paper 1: The labral gland in termite soldiers
Prestwich GD. 1984
. Defense mechanisms of termites. Annual Review of Entomology
29
:
201–232.
Quennedey A. 1984.
Morphology and ultrastructure of termite defense glands. In Hermann
HR, ed. Defensive mechanisms in social insects. New York: Praeger, 151–200.
Quennedey A, Sillam–Dussès D, Robert A, Bordereau C. 2008.
The fine structural
organization of sternal glands of pseudergates and workers in termites (Isoptera): a
comparative survey. Arthropod Structure & Development
37:
168–185.
Redford KH, Dorea JG. 1984.
The nutritional value of invertebrates with emphasis on ants
and termites as food for mammals. Journal of Zoology
203:
385–395.
Stuart AM, Satir P. 1968.
Morphological and functional aspects of an insect epidermal
gland. Journal of Cell Biology
36:
527–549.
Šobotník KJ, Weyda F, Hanus R. 2003.
Ultrastructure of epidermal glands in neotenic
reproductives of the termite Prorhinotermes simplex (Isoptera: Rhinotermitidae). Arthropod
Structure & Development
32
: 201–208.
Šobotník J, Weyda F, Hanus R, Kyjaková P, Doubský J. 2004.
Ultrastructure of the frontal
gland in Prorhinotermes simplex (Isoptera: Rhinotermitidae) and quantity of the defensive
substance. European Journal of Entomology
101
: 153–163.
Šobotník J, Weyda F, Hanus R. 2005.
Ultrastructural study of tergal and posterior sternal
glands in Prorhinotermes simplex (Isoptera: Rhinotermitidae). European Journal of
Entomology
102:
81–88.
Šobotník J, Jirosová A, Hanus R. 2010a.
Chemical warfare in termites. Journal of Insect
Physiology
56:
1012–1021.
Šobotník J, Bourguignon T, Hanus R, Weyda F, Roisin Y. 2010b.
Structure and function
of defensive glands in soldiers of Glossotermes oculatus (Isoptera: Serritermitidae).
Biological Journal of the Linnean Society
99
: 839–848.
Šobotník J, Bourguignon T, Hanus R, Demianová Z, Pytelková J, Mareš M, Foltynová
51
Paper 1: The labral gland in termite soldiers
P, Preisler J, Cvačka J, Krasulová J, Roisin Y. 2012.
Explosive backpacks in old termite
workers. Science
337
: 436.
Šobotník J, Kutalová K, Vytisková B, Roisin Y, Bourguignon T. 2014.
Age–dependent
changes in ultrastructure of the defensive glands of Neocapritermes taracua workers
(Isoptera, Termitidae). Arthropod Structure & Development
43
: 205–210.
Tillman JA, Seybold SJ, Jurenka RA, Blomquist GJ. 1999.
Insect pheromones–an
overview of biosynthesis and endocrine regulation. Insect Biochemistry and Molecular
Biology
29
: 481–514.
52
Paper 2: The labral gland in termites: Evolution and function
Paper 2: 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*
1 University Paris 13 – Sorbonne Paris Cité, Laboratory of Experimental and Comparative
Ethology, Villetaneuse, France.
2 Faculty of Forestry and Wood Sciences, Czech University of Life Sciences, Prague, Czech
Republic.
3 Institute of Entomology, Biology Centre, Academy of Sciences of the Czech Republic, České
Budějovice, Czech Republic.
4 Bundesanstalt für Materialforschung und –prüfung, Berlin, Germany.
5 Institute of Organic Chemistry and Biochemistry, Prague, Czech Republic.
* These authors contributed equally to the study.
Biological Journal of the Linnean Society, Volume 126, Issue 3, 28 February 2019, Pages 587–
597, https://doi.org/10.1093/biolinnean/bly212
Published: 02 February 2019
53
Paper 2: The labral gland in termites: Evolution and function
Résumé
Les termites sont des contributeurs importants au fonctionnement de l'écosystème. Ils sont
très abondants dans les habitats tropicaux et subtropicaux et représentent une ressource
importante pour un large éventail de prédateurs. Leur succès évolutif repose en grande
partie sur une vie dans des colonies peuplées avec un système de communication complexe
contrôlé par un riche ensemble de glandes exocrines dont les sécrétions sont impliquées
dans de nombreux aspects de la vie des termites. On sait que jusqu'à 20 organes exocrines
différents sont connus chez les termites. Parmi eux, la glande labrale représente l'un des
organes largement sous–étudiés. Ici, nous avons examiné la structure de la glande labrale
chez des ouvriers de 28 espèces et des imagos de 33 espèces représentants tous les
taxons de termites, ainsi que chez la blatte xylophage Cryptocercus. La glande labrale est
présente chez toutes les espèces et comprend deux régions de sécrétion situées
respectivement sur la face ventrale du labrum et la partie dorso–apicale de l'hypopharynx.
L'épithélium de la glande est constitué de cellules sécrétrices de classe 1 avec une
abondance de réticulum endoplasmique lisse, de longues microvillosités avec un canal à
l'intérieur, qui libèrent les sécrétions à travers une cuticule modifiée. Nos observations
suggèrent que la glande labrale est impliquée dans la communication défensive après la
rencontre avec un étranger.
Mots–clefs: glande
exocrine, Isoptera, Termitoidae, ultrastructure, évolution, développement
54
Paper 2: The labral gland in termites: Evolution and function
Abstract
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 la