Trail-Following Pheromones in the Termite Subfamily Syntermitinae (Blattodea, Termitoidae, Termitidae).

Trail-following behavior is a key to ecological success of termites, allowing them to orient themselves between the nesting and foraging sites. This behavior is controlled by specific trail-following pheromones produced by the abdominal sternal gland occurring in all termite species and developmental stages. Trail-following communication has been studied in a broad spectrum of species, but the "higher" termites (i.e. Termitidae) from the subfamily Syntermitinae remain surprisingly neglected. To fill this gap, we studied the trail-following pheromone in six genera and nine species of Syntermitinae. Our chemical and behavioral experiments showed that (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol is the single component of the pheromone of all the termite species studied, except for Silvestritermes euamignathus. This species produces both (3Z,6Z)-dodeca-3,6-dien-1-ol and neocembrene, but only (3Z,6Z)-dodeca-3,6-dien-1-ol elicits trail-following behavior. Our results indicate the importance of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol, the most widespread communication compound in termites, but also the repeated switches to other common pheromones as exemplified by S. euamignathus.

Molecular phylogeny of parabasalids with emphasis on the order Cristamonadida and its complex morphological evolution.

Parabasalia represents a complex assemblage of species, which recently received extensive reorganization. The newly created order Cristamonadida unites complex hypermastigids belonging to the Lophomonadida like the joeniids, the multinucleate polymonad Calonymphidae, and well-developed trichomonads in the Devescovinidae. All these protists exclusively occur in the guts of termites and related insects. In this study, small subunit rRNA and glyceraldehyde-3-phosphate dehydrogenase genes were identified without cultivation from 14 species in Cristamonadida including previously unstudied genera such as Joenina, Joenia, Joenoides, Macrotrichomonas, Gigantomonas, and Foaina. Despite the great morphological diversity of Cristamonadida, our phylogenetic analyses supported the monophyly of this order. However, almost all the families and subfamilies composing this order are polyphyletic suggesting a complicated morphological evolution. Our analyses also showed that Cristamonadida descends from one lineage of rudimentary trichomonads and that joeniids was basal in this order. Several successive and independent morphological transitions such as the development and reduction of flagellar apparatus and associated cytoskeleton and transition to multinucleated status have likely led to the diversity and complexity of cristamonad lineages.

The phylogeny of termites (Dictyoptera: Isoptera) based on mitochondrial and nuclear markers: Implications for the evolution of the worker and pseudergate castes, and foraging behaviors.

A phylogenetic hypothesis of termite relationships was inferred from DNA sequence data. Seven gene fragments (12S rDNA, 16S rDNA, 18S rDNA, 28S rDNA, cytochrome oxidase I, cytochrome oxidase II and cytochrome b) were sequenced for 40 termite exemplars, representing all termite families and 14 outgroups. Termites were found to be monophyletic with Mastotermes darwiniensis (Mastotermitidae) as sister group to the remainder of the termites. In this remainder, the family Kalotermitidae was sister group to other families. The families Kalotermitidae, Hodotermitidae and Termitidae were retrieved as monophyletic whereas the Termopsidae and Rhinotermitidae appeared paraphyletic. All of these results were very stable and supported with high bootstrap and Bremer values. The evolution of worker caste and foraging behavior were discussed according to the phylogenetic hypothesis. Our analyses suggested that both true workers and pseudergates ("false workers") were the result of at least two different origins. Our data support a traditional hypothesis of foraging behavior, in which the evolutionary transition from a one-piece type to a separate life type occurred through an intermediate behavioral form.

The nasus gland: a new gland in soldiers of Angularitermes (Termitidae, Nasutitermitinae).

Termites have developed many exocrine glands, generally dedicated to defence or communication. Although a few of these glands occur in all termite species, or represent synapomorphies of larger clades, others are morphological innovations of a single species, or a few related species. Here, we describe the nasus gland, a new gland occurring at the base of the nasus of Angularitermes soldiers. The nasus gland is composed of class 1, 2, and 3 secretory cells, a rare combination that is only shared by the sternal and tergal glands of some termites and cockroaches. The ultrastructural observations suggest that the secretion is produced by class 2 and 3 secretory cells, and released mostly by class 3 cells. The base of the nasus has a rough appearance due to numerous pits bearing openings of canals conducting the secretion from class 3 secretory cells to the exterior. We tentatively assign a defensive function to the nasus gland, although further research is needed to confirm this function. Although the gland is described only from species of Angularitermes, other genera of Nasutitermitinae also present a rough nasus base, suggesting the presence of a similar, possibly homologous, gland.

Ultrastructure of posterior sternal glands of Macrotermes annandalei (Silvestri): new members of the sexual glandular set found in termites (Insecta).

In female alates of Macrotermes annandalei, two types of abdominal glands are involved in the secretion of sex pheromone. Tergal glands are found at the anterior margin of tergites 6-10 and posterior sternal glands (PSGs) are located at the anterior margin of sternites 6-7. The cytological features of both types of glands are quite similar. The fine structural organization of PSGs is studied more precisely and described for the first time. The glandular cuticle is pitted with narrow apertures corresponding to the openings of numerous subcuticular pouches. Several Class 3 glandular units open in each pouch. One canal cell and one secretory cell make an individual glandular unit. The canal cell is enlarged apically and is connected with the other canal cells to form a common pouch. Based on the structural features found in these glands, we propose a common secretory process for PSGs and tergal glands. During the physiological maturation of alates inside the nest, secretory vesicles amass in the cytoplasm of secretory cells, while large intercellular spaces collapse the cuticular pouches. At the time of dispersal flight, pouches are filled with the content of secretory vesicles while intercellular spaces are sharply reduced. After calling behavior, no secretion remains in the glands and pouches collapse again, while secretory cells are drastically reduced in size. The structure and the secretory processes of PSGs and tergal glands are compared to those of abdominal sexual glands known in termites.

Sex-pairing pheromones and reproductive isolation in three sympatric Cornitermes species (Isoptera, Termitidae, Syntermitinae).

The species-specificity of pairing has been studied in three sympatric Neotropical termites: Cornitermes bequaerti, Cornitermes cumulans and Cornitermes silvestrii (Termitidae, Syntermitinae). Bioassays showed that sex attraction was highly species-specific between C. bequaerti and C. cumulans but not between C. cumulans and C. silvestrii. The sex-pairing pheromone of the three species is secreted by the tergal glands of female alates. It consists of a common compound (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol. In C. bequaerti, this polyunsaturated alcohol is the only compound of the sex-pairing pheromone, whereas it is associated with the oxygenated sesquiterpene (E)-nerolidol in C. cumulans, and with (E)-nerolidol and (Z)-dodec-3-en-1-ol in C. silvestrii. (3Z,6Z,8E)-Dodeca-3,6,8-trien-1-ol is responsible for sexual attraction, whereas (E)-nerolidol, which is inactive in eliciting attraction of male alates, is responsible for the species-specificity of the attraction. This is the first time that a multicomponent sex-pairing pheromone has been identified in termites. The role of (Z)-dodec-3-en-1-ol present on the surface of the tergal glands of the female alates of C. silvestrii could not be definitively determined, but it is suggested that this compound could be involved in the species-specificity of sex attraction with other sympatric species of Cornitermes. Our study shows that the reproductive isolation in termites is due to a succession of factors, as the chronology of dispersal flights, the species-specificity of sex-pairing pheromones and the species-specific recognition.

Chemical communication in termites: syn-4,6-dimethylundecan-1-ol as trail-following pheromone, syn-4,6-dimethylundecanal and (5E)-2,6,10-trimethylundeca-5,9-dienal as the respective male and female sex pheromones in Hodotermopsis sjoestedti (Isoptera, Archotermopsidae).

The trail-following pheromone and sex pheromones were investigated in the Indomalayan termite Hodotermopsis sjoestedti belonging to the new family Archotermopsidae. Gas chromatography coupled to mass spectrometry (GC-MS) after solid phase microextraction (SPME) of the sternal gland secretion of pseudergates and trail-following bioassays demonstrated that the trail-following pheromone of H. sjoestedti was syn-4,6-dimethylundecan-1-ol, a new chemical structure for termite pheromones. GC-MS after SPME of the sternal gland secretion of alates also allowed the identification of sex-specific compounds. In female alates, the major sex-specific compound was identified as (5E)-2,6,10-trimethylundeca-5,9-dienal, a compound previously identified as the female sex pheromone of the termite Zootermopsis nevadensis. In male alates, the major sex-specific compound was identified as syn-4,6-dimethylundecanal, a homolog of syn-4,6-dimethyldodecanal, which has previously been confirmed as the male sex pheromone of Z. nevadensis. The presence of sex-specific compounds in alates of H. sjoestedti strongly suggests for this termite the presence of sex-specific pairing pheromones which were only known until now in Z. nevadensis. Our results showed therefore a close chemical relationship between the pheromones of the taxa Hodotermopsis and Zootermopsis and, in contrast, a clear difference with the taxa Stolotermes and Porotermes, which is in total agreement with the recent creation of the families Archotermopsidae and Stolotermitidae as a substitute for the former family Termopsidae.

Identification by GC-EAD of the two-component trail-following pheromone of Prorhinotermes simplex (Isoptera, Rhinotermitidae, Prorhinotermitinae).

GC/MS analysis confirmed that neocembrene is the major component of the trail pheromone in the three species of the termite genus Prorhinotermes (P. simplex, P. canalifrons, P. inopinatus). In addition, EAG and GC-EAD experiments with P. simplex strongly suggest that dodecatrienol is a quantitatively minor component but a qualitatively important component of this trail pheromone. Trail-following bioassays confirmed the two-component nature of the trail pheromone. This is the first report of the use of the GC-EAD for the identification of trail pheromone in termites. These original results underline once again the special phylogenetic status of the Prorhinotermitinae among Rhinotermitidae.

The fine structural organization of sternal glands of pseudergates and workers in termites (Isoptera): a comparative survey.

Thirty-nine species belonging to different families of termites are studied to give a comprehensive view of the evolution of the sternal glands. Several modifications occurring at cuticular and cytological levels are described in neuter castes. The outer epicuticle is always pierced by epicuticular pores. In advanced termites the epicuticular filaments greatly increase in number and length creating a thick layer. The pore canals gradually enlarge while the cuticle changes into a lattice structure lining an extracellular space in which the secretion is stored. Two classes of cells are present in basal termites (Mastotermitidae, Hodotermitidae, Termopsidae and Kalotermitidae) but their glandular structures greatly differ between families. A more complex organization with three classes of cells is found in the Serritermitidae and Rhinotermitidae. A regressive evolution occurs in the Termitidae where only two classes of cells are present. A dual nervous control (campaniform sensilla and neurosecretory fibers) is found in lower termites, except for the Hodotermitidae which have mechanosensory bristles. In the other families, neurosecretory fibers are lacking. A comparison with phylogenetic data is given. A more versatile role of sternal glands in neuter castes is hypothesized.

The fine structure of colleterial glands in two cockroaches and three termites, including a detailed study of Cryptocercus punctulatus (Blattaria, Cryptocercidae) and Mastotermes darwiniensis (Isoptera, Mastotermitidae).

The colleterial glands of insects are organs associated with the female genital apparatus. In cockroaches, these glands produce secretions that cover two parallel rows of eggs during oviposition, and in oviparous species, these secretions become the tanned, sculpted, rigid outer casing of the ootheca. The goal of this study was to compare the gross anatomy of the colleterial glands and the ultrastructure of their component tubules in the phylogenetically significant genera Cryptocercus (Blattaria) and Mastotermes (Isoptera). Recent studies indicate that cockroaches in the genus Cryptocercus are the sister group of termites, and Mastotermes is the only termite known to produce a cockroach-like ootheca. One additional oviparous cockroach, Therea, and two additional termites, Zootermopsis and Pseudacanthotermes, were also examined. As in other cockroaches, the colleterial glands of Cryptocercus and Therea are asymmetrical, with a well developed bipartite left gland and a smaller right gland. In the termites Mastotermes, Zootermopsis, and Pseudacanthotermes, the colleterial glands are composed of a well-developed, paired, anterior gland and a small posterior gland; histological staining and cytological evidence suggest that these are homologues of the left and the right colleterial glands of cockroaches, respectively. At the ultrastructural level, colleterial gland tubules are made of cells belonging to a modified class 1 type cell in the cockroaches, in Mastotermes, and in Zootermopsis; the latter lays its eggs singly, without a surrounding ootheca-like structure. In the advanced termite Pseudacanthotermes, the tubules are made of secretory units belonging to the class 3 cell type. This study demonstrates that the cytological characteristics of colleterial glands in basal termites are similar to those of cockroaches, whether the termite secretes an oothecal casing that covers two parallel rows of eggs, as in Mastotermes, or lays its eggs singly, as in Zootermopsis. The function of colleterial glands in non-mastotermitid termites is unknown.

Trail-following pheromones in basal termites, with special reference to Mastotermes darwiniensis.

In the framework of an evolutionary study, trail pheromones have been studied in the most basal extant termite, Mastotermes darwiniensis (Mastotermitidae), and two other basal termites, the Termopsidae Porotermes adamsoni (Porotermitinae) and Stolotermes victoriensis (Stolotermitinae). Although workers of M. darwiniensis do not walk in single file while exploring a new environment under experimental conditions and are unable to follow artificial trails in 'open field' experiments, they do secrete a trail-following pheromone from their sternal glands. This unique behavior might reflect a primitive function of communication of the sternal gland. The major component of the pheromone appears to be the same in the three basal species: the norsesquiterpene alcohol (E)-2,6,10-trimethyl-5,9-undecadien-1-ol. This represents a new chemical category of trail-following pheromones for termites. The quantity of pheromone was estimated as 20 pg/individual in M. darwiniensis, 700 pg/individual in P. adamsoni, and 4 pg/individual in S. victoriensis. The activity threshold was 1 ng/cm in M. darwiniensis and 10 pg/cm in P. adamsoni. In M. darwiniensis, the trail pheromone was secreted by sternal gland 4 and to a lesser degree by sternal gland 3, sternal gland 5 being almost inactive. This study highlighted phylogenetic relationships between the Mastotermitidae and two subfamilies of the Termopsidae, the Porotermitinae and the Stolotermitinae. Furthermore, it indicated a heterogeneity within the Termopsidae, with Porotermitinae and Stolotermitinae on one hand, and Termopsinae on the other. Finally, Mastotermitidae and Termopsidae, with C14 trail pheromones, are clearly separated from the Kalotermitidae, Rhinotermitidae, and Termitidae that secrete C12 or C20 trail pheromones.

Origin and formation of the royal fat body of the higher termite queens.

The formation of the royal fat body was studied by electron microscopy in three species of higher termites (Macrotermes bellicosus, Macrotermes subhyalinus, and Cubitermes fungifaber). The swarming alate imago has a storage fat body typical of most insects. In non-physogastric young queens, during the fasting period, the adipocytes deplete their reserves and then, along with the increased vitellogenesis, acquire protein-synthesizing structures (R.E.R.). During the development of physogastry they progressively specialize in protein synthesis and secretion and undergo many cell divisions. The cytological change is paralleled by a spatial reorganization of the fat body. Observations on the transformation of imaginal adipocytes into royal adipocytes show that the royal fat body is derived from the imaginal fat body and not from the tracheal cells as previously claimed.

Ultrastrucuture of the fat body of the reproductive pair in higher termites.

The fine structure of the fat body of the higher termite king and queen has been studied both in species with (Macrotermes bellicosus, M. subhyalinus) and without (Cubitermes fungifaber) tracheal rosettes. There is a very pronounced sexual dimorphism. The adipocytes of the queen are highly specialized for protein synthesis and secretion; they store only a small quantity of reserves. The adipocytes of the king are not specialized in protein synthesis, but accumulate large amounts of reserve substances. The previously proposed different functions of the termite queen's fat body are discussed; it appears to be mainly concerned with vitellogenesis.

A new C12 alcohol identified as a sex pheromone and a trail-following pheromone in termites: the diene (Z,Z)-dodeca-3,6-dien-1-ol.

The diunsaturated C12 alcohol (Z,Z)-dodeca-3,6-dien-1-ol (dodecadienol) has been characterized by GC-MS and FTIR as a novel releaser pheromone in termites. This alcohol identified in Ancistrotermes pakistanicus (Termitidae, Macrotermitinae) possesses a double pheromonal function which again illustrates the chemical parsimony of termites compared with other social insects. In workers, dodecadienol elicits trail-following at a very low concentration (activity threshold at 0.1 pg/cm of trail); in male alates it induces trail-following at a low concentration (1-10 pg/cm) and sexual attraction at a higher concentration (about 1 ng). Traces of the monounsaturated C12 alcohol (Z)-dodec-3-en-1-ol (dodecenol), known as a trail pheromone of several Macrotermitinae, were also found in the sternal gland extracts of A. pakistanicus, although only dodecadienol was present at the surface of the sternal gland. Workers of A. pakistanicus are not sensitive to dodecenol, but they are as sensitive to dodecatrienol as to dodecadienol. However, in the study area (Vietnam), A. pakistanicus is living in sympatry only with those Macrotermitinae using dodecenol as a trail pheromone, the foraging populations therefore being well isolated through their respective trail pheromones. The presence of three types of unsaturated C12 alcohols as releaser pheromones in the only Macrotermitinae subfamily is discussed, and a possible biosynthetic pathway from linoleic acid is proposed for dodecadienol.