Not only soldiers have weapons: evolution of the frontal gland in imagoes of the termite families Rhinotermitidae and Serritermitidae

Background

The frontal gland is a unique adaptation of advanced termite families. It has been intensively studied in soldiers with respect to its anatomy and chemistry, with numerous novel compounds being discovered within the tremendous richness of identified products. At the same time, the presence of the frontal gland in non-soldier castes received only negligible attention in the past.

Principal Findings

Here, we report on the development of the frontal gland in alate imagoes of 10 genera and 13 species of Rhinotermitidae and Serritermitidae, in order to shed light on the evolution and function of this gland in imagoes. All investigated species possess a frontal gland. In most cases, it is well-developed and equipped with a sac-like reservoir, located in the postero-dorsal part of cranium, but reaching as far as the seventh abdominal segment in some Rhinotermitinae. The only exception is the genus Psammotermes, in which the gland is very small and devoid of the reservoir.

Conclusions

Our direct observations and comparisons with soldiers suggest a defensive role of the gland in imagoes of all studied species. This functional analogy, along with the anatomic homology between the frontal gland in soldiers and imagoes, make it likely that the gland appeared once during the early evolution of rhinotermitid ancestors, and remained as a defensive organ of prime importance in both, soldiers and imagoes.     

The clypeal gland: A new exocrine gland in termite imagoes (Isoptera: Serritermitidae,Rhinotermitidae, Termitidae)

Social insects possess a rich set of exocrine organs producing diverse pheromones and defensive compounds. This is especially true for termite imagoes, which are equipped with several glands producing, among others, sex pheromones and defensive compounds protecting imagoes during the dispersal flight and colony foundation. Here, we describe the clypeal gland, a new termite exocrine organ occurring in the labro-clypeal region of imagoes of most Rhinotermitidae, Serritermitidae and Termitidae species. The clypeal gland of Coptotermes testaceus consists of class 1 (modified epidermal cell) and class 3 (bicellular gland unit) secretory cells. Ultrastructural features suggest that the gland secretes volatile compounds and proteins, probably after starting the reproduction. One peculiar feature of the gland is the presence of multiple secretory canals in a single canal cell, a feature never observed before in other insect glands. Although the function of the gland remains unknown, we hypothesize that it could produce secretion signalling the presence of functional reproductives or their need to be fed.     

Molecular phylogeny of the Rhinotermitidae

Summary. Relationships among genera in the termite family Rhinotermitidae and their relationship to the families Termitidae and Serritermitidae were investigated based on analysis of three mitochondrial genes: COI, COII and 16S rDNA. Maximum Parsimony (MP) bootstrap analysis of each of these genes indicated a low level of phylogenetic incongruence between them, and thus they were combined and analysed by MP and Bayesian analysis. Six main lineages were clearly identified, however relationships among these were not well defined. Tentative support was found for the Rhinotermitid genera Coptotermes, Heterotermes and Reticulitermes being the sister group to the Termitidae, rendering the Rhinotermitidae paraphyletic. The species Serritermes serrifer and Glossotermes oculatus were found to group with strong support, in agreement with the recent transfer of the latter species to the family Serritermitidae based on morphological characteristics. No support was found for the Rhinotermitidae being paraphyletic with respect to the Serritermitidae. A number of disagreements were found between the molecular tree and traditional classifications of genera within subfamilies.Received 20 February 2004; revised 2 April 2004; accepted 19 April 2004.     

Termites and trees: a review of recent advances in termite phylogenetics

Summary: Modern termite phylogenetics is critically reviewed, with an emphasis on tree topologies as phylogenetic hypotheses. Studies have especially concentrated on (1) the position of Isoptera among the Dictyoptera and (2) the family group relationships within the Isoptera. The first of these problems is still controversial; although the weight of evidence now suggests that termites are nested within the cockroaches, thus making "Blattaria" as presently constituted paraphyletic. The exact position of termites within the cockroaches is uncertain, although Cryptocercus is the most plausible sister group.¶Family groups relationships are rather better resolved. Mastotermitidae is now generally accepted to be the most basal termite group. Termopsidae, Hodotermitidae and Kalotermitidae are all basal to (Termitidae + Serritermitidae + Rhinotermitidae), although their relative positions within that part of the tree are disputed. Most recent studies support a sister group relationship for Serritermitidae and (Termitidae + Rhinotermitidae). However, no study has yet unambiguously found the Rhinotermitidae monophyletic. The Termitidae are well established as monophyletic and as the most apical termite family. However, within the Termitidae the monophyly of none of the subfamilies is well established, making subfamily level analyses unreliable.¶A number of problem areas are identified: (1) poor taxon sampling is a universal problem, (2) higher taxonomic groupings are often assumed to be monophyletic a priori without adequate support, (3) datasets are collected from different taxa and character systems without consideration of the overall international effort.     

The sternal glands of termites: segmental pattern,phylogenetic implications

Summary In Mastotermitidae, 3 sternal glands are observed on the 3rd, 4th and 5th abdominal segments. All other families only bear one gland, set on the 4th segment in Termopsidae and Hodotermitidae, and on the 5th in Kalotermitidae, Rhinotermitidae, Serritermitidae and Termitidae. This character may be useful for a phylogenetic analysis.     

Temporal and geographic variations in the morphology and chemical composition of the frontal gland in imagoes of Prorhinotermes species (Isoptera: Rhinotermitidae)

Although the frontal gland has long been known as a prominent defensive device for termite soldiers in many Rhinotermitidae and Termitidae, almost nothing is known about its function in imagoes. In the present study, we show that the frontal gland of imagoes in Prorhinotermes species is well developed at the time of the nuptial flight, and is filled with a complex mixture of sesquiterpene hydrocarbons and nitroalkenes. The sesquiterpene composition varies between Prorhinotermes simplex and Prorhinotermes canalifrons , between geographically distant colonies of P. simplex (Cuba versus Florida), and even between different flights of closely-related subcolonies. The ratio between ( E )-1-nitropentadec-1-ene and sesquiterpenes is sex-specific. The volume of secretory cells decreases in functional kings and queens after colony foundation, and the subcellular organization changes into a form resembling unmodified epidermal cells. Dealate reproductives lose the ability for biosynthesis, and their frontal gland is devoid of volatile compounds found in swarming imagoes. The results obtained in the present study clearly show that the frontal gland is only temporarily active at the time of the dispersal flight. The most likely function of this gland is defence by the toxic nitroalkenes.  © 2009 The Linnean Society of London, Biological Journal of the Linnean Society , 2009, 98 , 384–392.     

Molecular phylogeny of Asian termites (Isoptera) of the families Termitidae and Rhinotermitidae based on mitochondrial COII sequences

The families Termitidae and Rhinotermitidae are the most evolved and diverse groups of the social insects, termites (Order Isoptera), showing elaborated morphology and complex behavior. Molecular phylogeny of termites with the emphasis on these families was examined by Bayesian and maximum-likelihood analyses based on DNA sequence of mitochondrial cytochrome oxidase II (COII) gene of 31 genera sampled in Asia (mainly Thailand and Japan) along with those reported previously. Termitidae was monophyletic and originated from within polyphyletic Rhinotermitidae. Among the four subfamilies of Termitidae, Macrotermitinae was monophyletic suggesting a single common origin of fungus-growing habit characteristic for this subfamily, and was placed in the basal position in the family. A group consisting of other subfamilies Termitinae and Nasutitermitinae, though some important groups were still untouched, was the most apical but neither Termitinae nor Nasutitermitinae formed a monophyletic lineage. It was implied that, as defense systems of the soldier castes, the appearance of snapping mandibles has occurred at a single event, but the development of nasus for chemical secretion has probably not. Our tree provides some evidence concerning contradictions in the previously proposed phylogeny of termites.     

High-level expression of the Geranylgeranyl diphosphate synthase gene in the frontal gland of soldiers in Reticulitermes speratus (Isoptera: Rhinotermitidae)

Defensive strategies of termite soldiers are roughly classified as either mechanical, using mandibles and/or the whole head, or chemical, using frontal gland secretion. Soldiers of the genus Nasutitermes (Termitidae, Nasutitermitinae), which is one of the most derived termite genera, use only chemical defenses, and diterpene defensive secretions were suggested to be synthesized through geranylgeranyl diphosphate (GGPP). On the other hand, soldiers of the genus Reticulitermes (Rhinotermitidae, Heterotermitinae) mainly use mechanical defenses, but also use supplementary chemical defenses involving frontal gland secretions, including diterpene alcohol. In this study, to confirm whether the GGPP is used for diterpene synthesis in a representative of an earlier-branching termite lineage, the GGPP synthase gene (RsGGPPS) was identified in the rhinotermitid Reticulitermes speratus (Kolbe). The relative expression level of RsGGPPS in soldiers was three-fold higher than in workers. Furthermore, RsGGPPS gene expression was detected in epithelial class 1 gland cells around the frontal-gland reservoir. Although GGPP is used for various essential cellular roles in animals, RsGGPPS is suggested to be used not only for these essential roles but also for diterpene synthesis in order to produce defensive secretions. Chemical structures of the diterpene identified from Reticulitermes and Nasutitermes are extremely different from each other, and the two genera are phylogenetically distant from each other. Thus, these two lineages may have independently acquired the abilities of diterpene synthesis from GGPP.     

Generic Keys to the soldier caste of New World Termitidae (Isoptera: Insecta)

Abstract The termite family Termitidae comprises four subfamilies: Apicotermitinae, Macrotermitinae, Termitinae and Nasutitermitinae. Keys are given here to the genera of the Termitinae and Nasutitermitinae found in the New World. The keys rely on morphological features of the soldier caste; for this reason no key is provided to the soldierless apicotermitine genera found in the New World. The Macrotermitinae are absent from the region.     

The leg exocrine system inSerritermes serrifer (Hagen, 1858), phylogenetic implications (Isoptera: Serritermitidae)

Summary The leg exocrine gland was examined in two species of Neotropical termites. Scanning microscopy studies showed a set of pores on the ventral surface of the first and second tarsomeres in all legs ofSerritermes serrifer. InHeterotermes tenuis these pores are present on a sunken plate in all castes. To date, this gland has been observed only in Rhinotermitid species. The presence of leg exocrine gland provides additional evidence supporting a close phylogenetic relationship between the Serritermitidae and Rhinotermitidae.     

Morphology,fine structure,and functional aspects of the labial gland reservoirs of the subterranean termite Reticulitermes santonensis de Feytaud (Isoptera: Rhinotermitidae)

The morphology and fine structure of the labial gland reservoirs in the subterranean termite Reticulitermes santonensis (Isoptera: Rhinotermitidae) was studied by light and transmission electron microscopy. The reservoir wall consists of a single epithelial cell layer and a cuticular intima. The reservoir ducts are formed by a flat epithelial matrix with cuticular ridges lining the duct lumen. Measurements of the ionic concentrations of reservoir fluids and haemolymph show that the osmolality of reservoir fluid ranges from 7 to 28 mosmol kg⁻¹; the haemolymph osmotic pressure was 201 ± 31 mosmol kg⁻. The reservoir lumen is effectively separated from the haemolymph compartment; a net water flow through the reservoir wall could not be induced in physiological experiments. Moreover, typical epithelial structures associated with a fluid transport against an osmotic gradient are lacking. Thus, our fine structural and physiological data support the view that a water transfer from the haemolymph through the reservoir wall into the reservoir lumen does not occur.     

(Z)-dodec-3-en-1-ol, a novel termite trail pheromone identified after solid phase microextraction from Macrotermes annandalei

(Z)-dodec-3-en-1-ol was isolated and identified by GC-MS as the major component of the trail-following pheromone from whole body and sternal gland extracts of workers of the fungus-growing termite, Macrotermes annandalei (Silvestri) (Termitidae, Macrotermitinae). For the first time, this trail pheromone was also identified by using solid phase microextraction from the surface of the secretory sternal gland of workers. Bioassays showed that synthetic dodecenol induced both orientation and recruitment behavioral effects. The activity threshold of (Z)-dodec-3-en-1-ol in eliciting trail-following is similar to that of (3Z,6Z,8E)-dodeca-3,6,8-trien-1-ol in the Rhinotermitidae, but amounts of dodecenol secreted are 100 times higher than those of dodecatrienol. There is about 1 ng of (Z)-dodec-3-en-1-ol per worker. Artificial trails made of synthetic dodecenol are able to compete with natural trails in the field. The activity duration of synthetic (Z)-dodec-3-en-1-ol trails is shorter than that of trails made from whole sternal secretion of workers. Observations showed that (Z)-dodec-3-en-1-ol is probably the only major component of the trail-following pheromone of M. annandalei and that it could be associated with other compounds in a pheromonal blend providing specificity and/or stability to trails.     

THE SPERMATOZOON OF ARTHROPODA: ZOOTERMOPSIS NEVADENSIS AND ISOPTERAN SPERM PHYLOGENY

In this paper, the ultrastructure of the spermatozoon of Zootermopsis nevadensis (Isoptera, Hodotermitidae) and of some Rhinotermitidae and Termitidae is described. Zootermopsis sperm is rod like, aflagellate, immotile, and without an acrosome; it is composed of a filiform nucleus encircled by a monolayered microtubular manchette, and a few mitochondria. This spermatozoon was previously thought to be flagellate, and therefore the most primitive in Isoptera: our present study suggests a new phylogenetical position for Hodotermitidae. All the species of Rhinotermitidae and Termitidae studied by us show a similar spheroidal sperm model, devoid of acrosome, flagellum and manchette at spermatid stage, and are made up of only a round nucleus, two mitochondria and a centriole. This widely distributed model seems to be the more evolved in the order. The nature of sperm evolution in the Isoptera is considered.     

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.     

Evolutionary trend of phylogenetic diversity of nitrogen fixation genes in the gut community of wood-feeding termites

Nitrogen fixation by gut microorganisms is one of the crucial aspects of symbiosis in wood-feeding termites since these termites thrive on a nitrogen-poor diet. In order to understand the evolution of this symbiosis, we analysed the nitrogenase structural gene nifH in the gut microbial communities. In conjunction with the published sequences, we compared approximately 320 putatively functional NifH protein sequences obtained from a total of 19 termite samples that represent all the major branches of their currently proposed phylogeny, and from one species of the cockroach Cryptocercus that shares a common ancestor with termites. Using multivariate techniques for clustering and ordination, a phylogeny of NifH protein sequences was created and plotted variously with host termite families, genera, and species. Close concordance was observed between NifH communities and the host termites at genus level, but family level relationships were not always congruent with accepted termite clade structure. Host groups examined included basal families (Mastotermitidae, Termopsidae, Kalotermitidae, as well as Cryptocercus), the most derived lower termite family Rhinotermitidae, and subfamilies representing the advanced and highly diverse apical family Termitidae (Macrotermitinae, Termitinae, and Nasutitermitinae). This selection encompassed the major nesting and feeding styles recognized in termites, and it was evident that NifH phylogenetic divergence, as well as the occurrence of alternative nitrogenase-type NifH, was to some extent dependent on host lifestyle as well as phylogenetic position.     

Identification of soldier caste-specific protein in the frontal gland of nasute termite Nasutitermes takasagoensis (Isoptera: Termitidae)

The termite soldier is unique because of its defensive task in a colony. In Nasutitermitinae (family Termitidae), soldiers use in their defense frontal glands, which contain various chemical substances. To isolate the gene products related to the chemical defense, we compared the sodium dodecyl sulfate-polyacrylamide gel electrophoresis protein profiles of soldier heads with those of workers of the nasute termite Nasutitermes takasagoensis. We identified a 26-kDa soldier-specific protein (Ntsp1) that exists most abundantly in the dorsal head including the frontal gland. We determined the N-terminal amino acid sequence of Ntsp1, and then cloned the Ntsp1 cDNA by rapid amplification of the cDNA ends-polymerase chain reaction (RACE-PCR). A putative signal peptide was detected upstream of the N-terminus and the Ntsp1 protein showed sequence homologies with known insect secretory carrier proteins, which bind to hydrophobic ligands such as juvenile hormone, suggesting that Ntsp1 belongs to this class of proteins. Northern blot analysis confirmed that the expression level of Ntsp1 was high only in the soldier head. In addition, the localization of Ntsp1 expression was limited in epithelial cells of the frontal gland reservoir, suggesting that this protein binds to some terpenoid(s) preserved in the frontal gland reservoir.     

A Kazal-Type Serine Protease Inhibitor from the Defense Gland Secretion of the Subterranean Termite Coptotermes formosanus Shiraki

Coptotermes formosanus is an imported, subterranean termite species with the largest economic impact in the United States. The frontal glands of the soldier caste termites comprising one third of the body mass, contain a secretion expelled through a foramen in defense. The small molecule composition of the frontal gland secretion is well-characterized, but the proteins remain to be identified. Herein is reported the structure and function of one of several proteins found in the termite defense gland secretion. TFP4 is a 6.9 kDa, non-classical group 1 Kazal-type serine protease inhibitor with activity towards chymotrypsin and elastase, but not trypsin. The 3-dimensional solution structure of TFP4 was solved with nuclear magnetic resonance spectroscopy, and represents the first structure from the taxonomic family, Rhinotermitidae. Based on the structure of TFP4, the protease inhibitor active loop (Cys⁸ to Cys¹⁶) was identified.     

Termites (Isoptera) in forest ecosystems of Cat Tien National Park (Southern Vietnam)

The species composition, abundance and colony sizes of terrestrial termites were studied in five forest habitats of Cat Tien National Park, Southern Vietnam. Twenty-four species belonging to Rhinotermitidae (1 species) and Termitidae (23 species, mostly Macrotermitinae), were found in mounds and in soil samples. The density of inhabited termite nests in different habitats averaged 68 per hectare (range 44–106), most nests belonged to Macrotermes spp. Six mounds of dominant species (Globitermes sulphureus, Microcerotermes burmanicus, Macrotermes carbonarius, M. gilvus, M. malacensis and Hypotermes obscuriceps) were destructively sampled. The number of termites in the nests ranged from 65 000 to 3 150 000 individuals with the biomass ranging from 185 to 2440 g live weight. The abundance of foraging termites in soil and litter averaged 60 ind./m2.The total abundance of Macrotermes species alone could conservatively be estimated as 2.5 million individuals and 20.5 kg live weight per hectare. Four species dominating in the studied habitats (M. carbonarius, M. gilvus, M. malaccensis, and H. obscuriceps) belong to active litter decomposers.     

Revision of the termite family Rhinotermitidae (Isoptera) in New Guinea

Recently, we completed a revision of the Termitidae from New Guinea and neighboring islands, recording a total of 45 species. Here, we revise a second family, the Rhinotermitidae, to progress towards a full picture of the termite diversity in New Guinea. Altogether, 6 genera and 15 species are recorded, among which two species, Coptotermes gambrinus and Parrhinotermes barbatus, are new to science. The genus Heterotermes is reported from New Guinea for the first time, with two species restricted to the southern part of the island. We also provide the first New Guinea records for six species of the genera Coptotermes and Schedorhinotermes. We briefly describe soldiers and imagoes of each species and provide a key based on soldier characters. Finally, we discuss the taxonomic and biogeographical implication of our results. A replacement name, Schedolimulus minutides Bourguignon, is proposed for the termitophilous staphylinid Schedolimulus minutus Bourguignon, to solve a question of secondary homonymy.     

Distinctive fossilised termite nests at Laetoli, Tanzania

A distinctive type of structure found preserved in a volcanic ash layer about 3.7 million years old appears to be a fossilised termite nest. Comparison is made to modern nests of a fungus-growing termite Macrotermes jeanneli (Isoptera, Termitidae, Macrotermitinae). Received: 30 May 2005; revised 27 September 2005; accepted 6 October 2005.