Morphogenesis and gene expression in the soldier-caste differentiation of termites

Summary: Since almost all termite species possess a soldier caste, there must be ubiquitous mechanisms of soldier differentiation throughout isopteran species. In order to reveal the caste differentiation mechanisms, observations during the soldier morphogenesis and identification of soldier specific gene expression are thought to be important. In this article, I summarize research approaches for analyzing caste differentiation in termites, and introduce two of our studies in Hospitalitermes medioflavus (Termitidae) and Hodotermopsis japonica (Termopsidae).¶Colonies of the nasute termite H. medioflavus have soldiers with a frontal projection (nasus) on the head, from which defensive substances are secreted. During soldier differentiation from male minor worker to presoldier, the most dynamic morphogenesis occurs. In the presumptive nasus epithelium of minor workers, a disc-like structure termed "soldier-nasus disc" rapidly develops to form the nasus of presoldiers. This rapid growth is associated with two folding layers of cuticle and epithelium.¶To identify genes specifically expressed in soldiers of the damp wood termite Hodotermopsis japonica, a differential display using RT-PCR was tried, comparing mRNA from the heads of soldiers and pseudergates. An identified gene candidate termed SOL1 was expressed specifically in terminally differentiated mature soldiers, and the product of the gene was suggested to encode a novel protein with a putative signal peptide at the N-terminus. This gene was shown to be expressed in the mandibular glands which actually develop during the soldier differentiation. Thus, these molecular techniques are applicable to reveal the proximate mechanisms of caste determination in termites and other social insects.     

The evolution of symmetrical snapping in termite soldiers need not lead to reduced chemical defence

Termite soldiers use a multitude of defensive strategies, often combining mechanical weapons with defensive chemicals secreted from their frontal gland. In higher termites (Termitidae), both these weapons are situated in the head of the soldiers. Their simultaneous occurrence is supposed to be subjected to energetic and spatial constraints and the resulting trade‐off often leads to a full development of only one of the weapons and reduction of the other. Mandibular snapping represents an extreme and efficient anatomical adaptation, allowing the soldier to knock out arthropod enemies with a violent strike of elongated mandibles. The head of snapping soldiers harbours massive adductor muscles while the frontal gland is reported to be reduced and the chemical defence absent. Here we show that the symmetrical snapping soldiers of the Neotropical termite Cavitermes tuberosus possess a well‐developed frontal gland situated in the frontal projection on their head. The gland produces a blend of unbranched alkenes and alkadienes combined with three diterpene hydrocarbons, derived from a novel bicyclic hydrocarbon skeleton. We characterized the molecular structure of the most abundant of these diterpenes and proposed for it a trivial name, cavitene. We conclude that the evolution of the mandibular snapping has not necessarily been accompanied by the reduction or loss of chemical defensive adaptations and that the two defensive modes are not mutually incompatible. © 2015 The Linnean Society of London, Biological Journal of the Linnean Society, 2015, ●● , ●●–●●.     

Structure and function of defensive glands in soldiers of Glossotermes oculatus (Isoptera: Serritermitidae)

The soldier caste represents the most conspicuous realization of termite eusociality, characterized by an extreme anatomical, behavioural, and physiological specialization. Numerous strategies have evolved in soldiers, including extreme adaptations such as self‐sacrifice by autothysis. In the present study, we investigated the structure and function of defensive glands in Glossotermes oculatus soldiers aiming to understand their use in combat. Three glands are involved in defence: labral, frontal, and labial glands. Mandibles are used to bite the enemy, whereas the secretions of labral and labial glands are discharged into the wound. A striking characteristic of G. oculatus is the lack of the frontal pore; the secretion of the frontal gland is discharged by a rupture of the body wall. We hypothesized that this self‐sacrifice is an efficient way of blocking a gallery under attack. A similar development of the frontal gland occurs in Serritermes serrifer, which supports the close relationship between the two genera inferred from morphological and genetic analyses. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 99 , 839–848.     

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.     

Soldier morphogenesis in a nasute termite: discovery of a disc-like structure forming a soldier nasus

Nasute termites belonging to the subfamily Nasutitermitinae, have a soldier caste that possesses a frontal projection (nasus) on the head, from which defensive substances are secreted. In the course of caste differentiation of the processional nasute termite Hospitalitermes medioflavus, the most dynamic morphogenesis occurs in the stage of moulting from male minor worker to presoldier (the stage preceding the soldier stage). We examined the presumptive nasus epithelium in minor workers and determined that the nasus develops rapidly just prior to the moulting to presoldiers. The rapid growth is associated with two folding layers of cuticle and epithelium, which we termed the soldier-nasus disc, and resembles the imaginal discs found in holometabolous insects.     

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.     

Soldier‐biased gene expression in a subterranean termite implies functional specialization of the defensive caste

In a termite colony, reproduction is typically monopolized by a small number of sexuals that are supported by reproductively altruistic soldiers and workers. We expect caste differentiation to be associated with clear‐cut differences in gene expression, and for these differences to reflect caste function and development. Here, we use RNA‐Sequencing to compare the gene expression profiles of sexual nymphs and two non‐reproductive helper castes (i.e., workers and soldiers) of the Eastern subterranean termite Reticulitermes flavipes. We found that of n = 93 genes that are strictly expressed as a function of caste, a majority (78%) show a soldier‐specific pattern. This conspicuous soldier‐bias in genome‐wide expression suggests that this defensively specialized caste is functionally well‐differentiated from both the reproductive and the other non‐reproductive caste of this species, despite a shared developmental program with workers. Gene ontology analysis supports the notion of functional specialization by soldiers, as soldier‐biased gene sets are enriched for novel biological processes. Whether this pattern reflects ancient or more recent bouts of selection for caste novelty at the gene‐regulatory level is not known, but because soldiers are sterile and thus have no direct fitness, any selection for novelty must have been mediated indirectly, through reproducing relatives.     

Relationships between frontal‐gland formation and mandibular modification during JH III‐induced presoldier differentiation in the termite Reticulitermes speratus (Isoptera: Rhinotermitidae)

Termite soldiers engage in colony defense and they possess weapons in order to attack enemies. Defensive strategies vary among species depending on the morphology of the soldier's weapons. Both the frontal gland and elongated mandibles are formed during soldier differentiation of Reticulitermes speratus workers, which is associated with an increase in juvenile hormone (JH) titer. It was shown that the amount of JH applied and coexistence with soldiers affected mandibular elongation of the induced presoldiers. To determine the relationship between applied JH concentration and frontal‐gland development of R. speratus, mandibular modification and frontal‐gland formation of presoldiers induced by 20, 40 and 80 µg JH III in the presence or absence of soldiers were observed. Both presoldier differentiation rates and their mandibular lengths were affected by the presence of soldiers in treatments with 20 and 40 µg JH III. Especially in the treatment with 20 µg JH III, frontal‐pore malformation was observed in some presoldiers. All presoldiers with a malformed frontal pore had almost the same sized mandibles, compared with those of stationarily molted workers. Interestingly, sections of these presoldiers showed that frontal‐gland invagination occurred incompletely in comparison with presoldiers with relatively elongated mandibles. Consequently, frontal‐gland formation might be affected by JH titer (applied and endogenous JH), and individuals with a malformed frontal pore were probably differentiated by JH titers only slightly above the threshold for presoldier differentiation.     

Comparative Study of the Labial Gland Secretion in Termites (Isoptera)

Labial glands are present in all castes and developmental stages of all termite species. In workers, their secretion contains a food-marking pheromone and digestive enzymes, while soldier secretion plays a defensive role. However, these functions were studied only in a limited set of species, and do not allow drawing general conclusions. Hence, we have investigated the chemical composition of the labial gland extracts from soldiers and workers in 15 termite species belonging to 6 families using an integrative approach based on proteomic and small-molecule profiling. We confirmed the presence of hydroquinone and cellulase in the labial glands of workers, and we identified new toxic compounds in soldiers and workers of several species. Our results highlight the dual role of labial gland secretion, i.e. the defensive role in soldiers and workers of several termite species, and the digestive function in workers.     

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.     

Control of Distal-less expression in the Drosophila appendages by functional 3' enhancers

The expression of the Hox gene Distal-less (Dll) directs the development of appendages in a wide variety of animals. In Drosophila, its expression is subjected to a complex developmental control. In the present work we have studied a 17 kb genomic region in the Dll locus which lies downstream of the coding sequence and found control elements of primary functional importance for the expression of Dll in the leg and in other tissues. Of particular interest is a control element, which we have called LP, which drives expression of Dll in the leg primordium from early embryonic development, and whose deletion causes severe truncation and malformation of the adult leg. This is the first Dll enhancer for which, in addition to the ability to drive expression of a reporter, a role can be demonstrated in the expression of the endogenous Dll gene and in the development of the leg. In addition, our results suggest that some enhancers, contrary to the widely accepted notion, may require a specific 5′ or 3′ position with respect to the transcribed region.     

X‐ray computed tomography reveals that intraspecific competition promotes soldier differentiation in a one‐piece nesting termite

Investment in soldier production in eusocial lineages involves a trade‐off between maintenance costs and defense benefits. Termites are eusocial insects that live in colonies organized into three castes: primary reproductives, soldiers, and workers or pseudergates. Neotermes chilensis (Blanchard) (Isoptera: Kalotermitidae) is a one‐piece nesting termite that nests and forages in a single piece of wood. Two scenarios may be of importance in a defense context of one‐piece nesting termites: during swarms, when colonies may be invaded by winged termites (alates) in search of a place to found a new colony, and when colonies of conspecifics are present within the same substrate. It was hypothesized that the ratio of soldiers to non‐soldiers would be higher at the onset of the swarming period and in substrates bearing more than one termite colony. A method based on X‐ray computed tomography (CT) was developed to study gallery connectivity in colonies of N. chilensis and caste composition within colonies. Computed tomography allowed the digital reconstruction of the galleries within the substrate, even when they belonged to different colonies, and was effective in distinguishing termites from substrate, and soldiers from reproductives and pseudergates. Using CT, the ratio of soldiers to non‐soldiers was shown to be highest in colonies within multicolonial scapes (i.e., neighboring colonies were present in the same substrate) during the swarming season, thus supporting our initial hypotheses. These results constitute a unique example of induced defenses arising from intraspecific interactions in termites.     

Parallel evolution of termite‐egg mimicry by sclerotium‐forming fungi in distant termite groups

Among the great diversity of insect–fungus associations, fungal mimicry of termite eggs is a particularly fascinating consequence of evolution. Along with their eggs, Reticulitermes termites often harbour sclerotia of the fungus Fibularhizoctonia sp., called ‘termite balls’, giving the fungus competitor‐free habitat within termite nests. The fungus has evolved sophisticated morphological and chemical camouflage to mimic termite eggs. To date, this striking insect–fungus association has been found in eight temperate termite species, but is restricted to the lower termite genera Reticulitermes and Coptotermes. Here, we report the discovery of a novel type of termite ball (‘Z‐type’) in the subtropical termite, Nasutitermes takasagoensis. Phylogenetic analysis indicated that the Z‐type termite ball is an undescribed Trechisporoid fungus, Trechispora sp., that is phylogenetically distant from Fibularhizoctonia, indicating two independent origins of termite‐egg mimicry in sclerotium‐forming fungi. Egg protection bioassays using dummy eggs revealed that Reticulitermes speratus and N. takasagoensis differ in egg‐size preference. A comparative study of termite ball size and egg‐size preference of host termites showed that both fungi evolved a termite ball size that optimized the acceptance of termite balls as a unit investment. Termite‐egg mimicry by these fungi offers a model case of parallel evolution. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 100 , 531–537.     

Proximate factors influencing soldier development in the basal termite <Emphasis Type="Italic">Cryptotermes secundus</Emphasis> (Hill)

Summary Regulation of soldier development in termites at the endocrinological and social scale was intensively studied in the 60s and 70s, but conclusive results are still scanty. In termites, the presence of soldiers inhibits further soldier differentiation, but the mechanism is not well understood. Secretions from the frontal glands seem to be important, but dry- and dampwood termites do not have frontal glands. We investigated the influence of precocene I, a JH-antagonist, and of soldier head extracts on the development of soldiers in the drywood termite, Cryptotermes secundus (Hill). Soldierless colonies produced fewer soldiers when treated with precocene I or soldier head extract than control colonies. Furthermore, colonies treated with precocene I had produced worker-nymph intercastes, last stage nymphs and alates by the end of the experiment (after six months) which were not present in control colonies. Hence, both treatments inhibited soldier development while precocene I simultaneously promoted the development of adult traits, probably by reducing (directly or indirectly) the JH level. The inhibition effect of the soldier head extract indicates that at least in termites without a frontal gland, other sources of material in the head have a regulatory function.     

Chemical Identification and Ethological Function of Soldier-Specific Secretion in Japanese Subterranean Termite Reticulitermes speratus (Rhinotermitidae)

We identified the soldier-specific compounds in the Japanese subterranean termite, Reticulitermes speratus, to clarify their ethological roles. Silica gel column chromatography separated one major soldier-specific compound in the hexane fraction accounting for 70–80% of the total amount of the fraction, while cuticular hydrocarbons constituted the rest. We identified the compound as β-selinene by gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy. Comparative GC analyses of the major exocrine glands detected the compound in the soldier’s frontal gland. Both soldiers and workers made aggregation to the hexane fraction, as well as to the crushed heads and head extract of the soldiers. They did not aggregate to cuticular hydrocarbons, making it likely that β-selinene was the aggregation pheromone in this species. The opportunistic predator of this termite, Lasius japonicus, was also attracted to the compounds. The ant workers, therefore, would use the termite aggregation pheromone as a kairomone for hunting them.     

Worker aggression of ant Lasius japonicus enhanced by termite soldier–specific secretion as an alarm pheromone of Reticulitermes speratus

The subterranean termite Reticulitermes speratus usually nests in rotten wood trunks, which may also be occupied by the Japanese garden ant Lasius japonicus. Few battles were observed between them under ordinary circumstances because they inhabit separate nesting sites. However, once the termite nesting sites were artificially broken, the ant workers invaded and hunted the termites, although the termite soldiers fought against the ants. This study aims to confirm intra‐ and inter‐specific chemical interactions between the termite and ant. Solid phase microextraction–gas chromatograph (SPME‐GC) analyses revealed that R. speratus soldiers secreted caste‐specific sesquiterpene hydrocarbon when they were irritated. Both the hexane extract of the soldiers and its hydrocarbon fraction, as well as the crushed soldier bodies, attracted the soldiers but dispersed the workers when presented on the trails. We also confirmed that the soldier chemicals enhanced aggressiveness of L. japonicus, which rushed around the odor sources and hunted any termites that were present. These findings suggest that: (i) the soldier–specific secretion might serve as an alarm pheromone in termite chemical communication, in which components recruit soldiers and also warn the other colony members away; and (ii) termite communication is eavesdropped on by L. japonicus workers to locate and hunt the termites.     

Age-dependent changes in ultrastructure of the defensive glands of Neocapritermes taracua workers (Isoptera,Termitidae)

Protection against predators and competitors is one of the main concerns of termite colonies, which developed a specialised defensive caste, the soldiers. However, soldiers are rare or even missing in several lineages of termites, while workers often develop new defence strategies especially in soil-feeding species. Here, we describe the morphology and ultrastructure of the autothysis-associated glands of Neocapritermes taracua workers and report their age-related changes in structure. The defensive glands of N. taracua workers consist of a pair of labial and a pair of crystal glands, whose secretions mix together through autothysis. Autothysis always occurs at the line of weakness connecting the anterior parts of the crystal-bearing pouches. The crystal glands consist of groups of bicellular secretory units (secretory and corresponding canal cells) which secrete the blue crystal material into external pouches. Their secretory activity is maximal in the middle of worker life, and is considerably lower in very young and old workers. The labial glands are composed of two types of secretory cells: the central and the parietal cells. While the central cells are developed similarly to other termites and secrete proteinaceous secretion into labial gland ducts, the parietal cells develop proteinaceous granules which may eventually bud off the cells. The secretory function of parietal cells is so far unique to N. taracua and differs from other termite species in which they are only responsible of water uptake by acini. The defensive device of N. taracua is truly exceptional as it involves a new gland and a previously undescribed function for parietal cells, being a remarkable example of evolution of morphological innovation.     

Cell migration within the embryonic limb primordium of Drosophila as revealed by a novel fluorescence method to visualize mRNA and protein

 We report a new technique using fluorescent probes to detect a mRNA and a protein simultaneously in the Drosophila embryo. For in situ hybridization, 3-hydroxy-N-2′-biphenyl-2-naphthalenecarboxamide phosphate ester (HNPP)/Fast Red TR was used as a fluorescent substrate for alkaline phosphatase. It was possible to compare protein and mRNA expression on a cell by cell basis with a laser scanning confocal microscope. We applied this technique to analyse the dynamics of Distal-less (Dll) enhancer activity in the thoracic limb primordium in the early Drosophila embryo. We stained embryos bearing the Dll early enhancer (Dll-304) fused to the Escherichia coli lacZ gene. LacZ mRNA was detectable in the ventral region of the limb primordium, and β-galactosidase protein in the dorsal region. In the middle, both mRNA and protein were detectable. These results suggest that the Dll enhancer is activated in the ventral region of the limb primordium and that Dll-positive cells migrate from a ventral position to a dorsal one within a single limb primordium. Received: 7 April 1997 / Accepted: 15 May 1997