Socio-environmental and endocrine influences on developmental and caste-regulatory gene expression in the eusocial termite <Emphasis Type="Italic">Reticulitermes flavipes</Emphasis>

Background  

Strict regulation of caste differentiation, at the molecular level, is thought to be important to maintain social structure in insect societies. Previously, a number of extrinsic and intrinsic factors have been shown to influence caste composition in termite colonies. One important factor is the influence of nestmates; in particular, soldier termites are known to inhibit hormone-dependent worker-to-soldier differentiation. However, soldier influences on nestmates at the molecular level are virtually unknown. Here, to test the hypothesis that soldiers can influence nestmate gene expression, we investigated the impact of four treatments on whole-body gene expression in totipotent Reticulitermes flavipes workers: (i) juvenile hormone III (JHIII; a morphogenetic hormone), (ii) soldier head extracts (SHE), (iii) JHIII+SHE, and (iv) live soldiers.     

Knockdown of the juvenile hormone receptor gene inhibits soldier-specific morphogenesis in the damp-wood termite Zootermopsis nevadensis (Isoptera: Archotermopsidae)

The Methoprene-tolerant (Met) protein has been established as a juvenile hormone (JH) receptor. Knockdown of the Met gene caused precocious metamorphosis and suppression of ovarian development. However, the function of Met in caste development of social insects is unclear. In termites, JH acts as a central factor for caste development, especially for soldier differentiation, which involves two molts from workers via a presoldier stage. Increased JH titer in workers is needed for the presoldier molt, and the high JH titer is maintained throughout the presoldier period. Although presoldiers have the fundamental morphological features of soldiers, the nature of the cuticle is completely different from that of soldiers. We expected that JH signals via Met are involved in soldier-specific morphogenesis of the head and mandibles during soldier differentiation, especially in the presoldier period, in natural conditions. To test this hypothesis, we focused on soldier differentiation in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Met homolog (ZnMet) expression in heads increased just after the presoldier molt. This high expression was reduced by ZnMet double stranded (dsRNA) injection before the presoldier molt. Although this treatment did not cause any morphological changes in presoldiers, it caused strong effects on soldiers, their mandibles being significantly shorter and head capsules smaller than those of control soldiers. Injection of ZnMet dsRNA throughout the presoldier stage did not affect the formation of soldier morphology, including cuticle formation. These results suggested that the rapid increase in ZnMet expression and subsequent activation of JH signaling just after the presoldier molt are needed for the formation of soldier-specific weapons. Therefore, besides its established role in insect metamorphosis, the JH receptor signaling also underlies soldier development in termites.     

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.     

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.     

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.     

Flight muscle breakdown in the soldier caste of the gall-inducing thrips species, Kladothrips intermedius Bagnall

Within the gall-inducing thrips of Australia, genus Kladothrips, is a single origin of a soldier caste. A subsequent radiation has led to at least seven social species, and two species that are likely to have independently lost the soldier caste. Both losses of soldiers are connected to a shift in the insects’ host plant. A third inferred host shift is correlated with life history changes in a species with soldiers, K. intermedius, which might suggest this defensive caste may be in transition. The soldiers of this species have variable wing lengths that overlap with that of the flight-capable dispersers. Our study was designed to assess the possibility that some soldiers in this species retain the ability to disperse by wing. A morphological assessment shows that a proportion of soldiers have body dimensions and wing lengths that fall within the range of dispersers, with males more so than females. However, longer wing length in soldiers (sample of primarily females) did not correspond with a disperser like walking behaviour. Furthermore, histological sections suggest that wing muscles of soldiers are deteriorated or absent, which is similar to what was observed in their foundress mothers. The presence of long wings did not correspond with being flight capable in this caste.     

Association between caste and genotype in the termite Mastotermes darwiniensis Froggatt (Isoptera: Mastotermitidae)

Abstract Termite workers and soldiers differ markedly in their morphology and behaviour. We sought evidence for genetic influences on caste determination in the giant northern termite, Mastotermes darwiniensis , by investigating if workers and soldiers from the same colony differed genetically. The genotypes of 795 termites from 11 distinct colonies were assayed at six polymorphic microsatellite loci. We found that the multilocus genotypes of workers and soldiers from 8 of the 11 colonies did not differ significantly. Thus, the majority of the data provided no evidence for a genetic association with caste in workers and soldiers of M. darwiniensis . However, the genotype frequencies of workers and soldiers from three colonies differed, suggesting that genotype is occasionally associated with caste in this species. The genetic differentiation of castes within these colonies could reflect differences in the propensities of termites with distinct genotypes to develop into particular castes and provide a selective advantage to colonies headed by multiple reproductives.     

Evolution of eusociality and the soldier caste in termites: a validation of the intrinsic benefit hypothesis

In termites the evolution of reproductive altruism is not based on a particularly high relatedness between nestmates. For the evolution and maintenance of the ancestral sterile soldier caste, the benefits generated by the soldiers' presence must compensate the loss of the soldiers' reproductive potential. To study the impact of soldiers on colony's fitness, we manipulated the proportion of soldiers to nonsoldiers in colonies of the dry-wood termite Cryptotermes secundus.'Soldier-less' colonies were obtained by removing soldiers and inhibiting their development with an extract of soldier heads. The colonies were set up for 1 year in experimental nests in the field. 'Soldier-less' colonies produced fewer soldiers. The reduction of soldiers neither affected colony survival nor helper growth, but fewer dispersing sexuals were produced in 'soldier-less' than in control colonies. This confirms what was only supposed so far, that in termites soldiers are maintained for their intrinsic benefit to cost ratio.     

Morphometric changes during soldier differentiation of the damp-wood termite Hodotermopsis japonica (Isoptera, Termopsidae)

Summary. To clarify the allometric development of body parts accompanying soldier differentiation in termites, we measured 16 body parts of soldiers, presoldiers, pseudergates (workers), nymphs and larvae of the damp-wood termite Hodotermopsis japonica. Principal component analysis (PCA) was performed using these parameters, which revealed that differentiation into soldiers differed distinctly from development into adult (reproductive) individuals. In particular, the anterior body parts enlarged during development of soldiers. Similarly, elongation of the apical portion of both mandibles was noted during soldier differentiation. X-ray analysis of mandibles revealed sclerotization of the soldier mandibles after differentiation into terminal soldiers. These morphological changes during soldier differentiation are associated with changes in their roles within the colony. Through soldier differentiation, the morphology of this caste of termite becomes functionally suited for attacking predators, and unsuitable for feeding on wood using their mandibles. Based on these data, we suggest that there must be some morphogenetic factors leading caste specific morphology such as soldier mandibles.     

Preliminary studies on the control of caste formation in the harvester termiteHodotermes mossambicus (Hagen)

The control of caste formation in the harvester termiteHodotermes mossambicus (Hagen) was investigated.Normal laboratory colonies contained a primary reproductive pair, between 6.59 and 24.98% workers, 0.39 and 3.45% soldiers and 74.64 and 92.06% larvae. Supplementary reproductives did not develop in colonies deprived of their king and queen. Continuous removal of soldiers stimulated soldier production. We have suggested that a pheromone produced by the soldiers plays a major role in controlling the soldier population of a colony. A similar phermone system appreared to control the production of workers.Evidence was also obtained that the primary reproductive pair influenced the production of workers and soldiers.     

Endogenous synchronization of the chemical signature of Reticulitermes (Isoptera: Rhinotermitidae) worker termites

Termites of the genus Reticulitermes are characteristic of temperate regions. Their colonies comprise various castes, the most numerous being that of workers which can develop into soldiers or secondary reproductives (neotenics). Each caste has a mixture of hydrocarbons (HCs) on the cuticle forming a chemical signature. The primary aim of this study was to compare the changes in the chemical signature of a population of worker termites fed on paper with juvenile hormone to differentiate them into soldiers with a control population of termites fed only on paper or wood for one month. Gas chromatography was used to analyze the cuticular profiles of Reticulitermes flavipes termites to determine whether they changed, and, if so, when and how. The data collected over one month showed that the workers fed with JH did not differentiate into soldiers but that there were progressive changes in the hydrocarbon profile independent of the treatment. These results indicate that the differentiation of the chemical signature of the worker caste is a dynamic process, depending only on time and not on colony membership, confirming that, for these termites, this signature has a lesser role in colony membership than caste membership, unlike the chemical signatures of other social insects. The temporal process of this cuticular change is also associated with a change in the alkene/methyl-branched alkane ratio.     

Sexual difference in juvenile-hormone titer in workers leads to sex-biased soldier differentiation in termites

In termites, the soldier caste, with its specialized defensive morphology, is one of the most important characteristics for sociality. Most of the basal termite species have both male and female soldiers, and the soldier sex ratio is almost equal or only slightly biased. However, in the apical lineages (especially family Termitidae), there are many species that have soldiers with strongly biased sex ratio. Generally in termites, since high juvenile hormone (JH) titer is required for soldier differentiation from a worker via a presoldier stage, it was hypothesized that the biased soldier-sex ratio was caused by differences in JH sensitivity and/or JH titer between male and female workers. Therefore, we focused on the presoldier differentiation and the worker JH titer in species with only male soldiers (Nasutitermes takasagoensis) and with both male and female soldiers (Reticulitermes speratus) in natural conditions. In the former species, there are four types of workers; male minor, male medium, female medium and female major workers, and presoldiers differentiate from male minor workers. First, we tried to artificially induce presoldiers from male and female workers. In N. takasagoensis, the presoldier differentiation rate and mortality was significantly higher in male minor workers. Morphological analyses showed that both male and female induced presoldiers possessed normal soldier-specific morphologies. It was suggested that female workers, from which soldiers do not differentiate under natural conditions, also maintained the physiological and developmental potential for soldier differentiation. In R. speratus, however, no differences were observed in solder differentiation rate and mortality between male and female workers. Second, the JH titers of each sex/type of workers were quantified by high performance liquid chromatography–mass spectrometry in two different seasons (April and December). The results showed that, in N. takasagoensis, JH titer in male minor workers was consistently higher than those in other worker types. In R. speratus, in contrast, there were no significant differences in JH titers between male and female workers. These results suggested that, in N. takasagoensis, male minor workers maintain JH titers at a high level throughout a year, and this may cause the male-biased presoldier differentiation.     

Soldier-specific modification of the mandibular motor neurons in termites

Social insects exhibit a variety of caste-specific behavioral tendencies that constitute the basis of division of labor within the colony. In termites, the soldier caste display distinctive defense behaviors, such as aggressively attacking enemies with well-developed mandibles, while the other castes retreat into the colony without exhibiting any aggressive response. It is thus likely that some form of soldier-specific neuronal modification exists in termites. In this study, the authors compared the brain (cerebral ganglion) and the suboesophageal ganglion (SOG) of soldiers and pseudergates (workers) in the damp-wood termite, Hodotermopsis sjostedti. The size of the SOG was significantly larger in soldiers than in pseudergates, but no difference in brain size was apparent between castes. Furthermore, mandibular nerves were thicker in soldiers than in pseudergates. Retrograde staining revealed that the somata sizes of the mandibular motor neurons (MdMNs) in soldiers were more than twice as large as those of pseudergates. The enlargement of MdMNs was also observed in individuals treated with a juvenile hormone analogue (JHA), indicating that MdMNs become enlarged in response to juvenile hormone (JH) action during soldier differentiation. This enlargement is likely to have two functions: a behavioral function in which soldier termites will be able to defend more effectively through relatively faster and stronger mandibular movements, and a developmental function that associates with the development of soldier-specific mandibular muscle morphogenesis in termite head. The soldier-specific enlargement of mandibular motor neurons was observed in all examined species in five termite families that have different mechanisms of defense, suggesting that such neuronal modification was already present in the common ancestor of termites and is significant for soldier function.     

Winged presoldiers induced by a juvenile hormone analog in Zootermopsis nevadensis: implications for plasticity and evolution of caste differentiation in termites

To elucidate the switching mechanism of caste differentiation in termites and to examine the possible induction of soldier-reproductive intercastes experimentally, we investigated the effects of juvenile hormone on the morphologies of soldier caste by applying a juvenile hormone analog (JHA) to nymphs of the damp-wood termite Zootermopsis nevadensis (Isoptera : Termopsidae). JHA treatment for about 2 weeks induced a variety of intermediate castes, showing both alate and soldier morphological features. The principal component analysis (PCA) of those morphological characters showed that those intercastes were a deviation from the developmental line into alates to soldier differentiation, which is known to be triggered by juvenile hormone. Detailed morphological examination of the compound eyes, wing joint, and mandibles showed that those intercastes expressed soldier features, although they had started to develop alate characteristics. The morphology of the resultant intercastes seemed to be determined by the nymphal stage, at which JHA treatment was applied. The induced intercastes with exaggerated soldier-specific characteristics (e.g., mandibles) repressed alate-specific characteristics (e.g., wings), namely, the alate and soldier morphological characteristics in induced intercastes show opposite responses against the application of JHA. On the other hand, ovarian development was not suppressed by the JHA application, even in the soldier-like individuals. Naturally differentiated presoldiers also possessed developed ovarioles, although ovaries of mature soldiers were degenerated. Our results suggest that the juvenile hormone plays complicated roles in the expression of caste morphologies and ovarian development in termites.     

Antifungal activity in thrips soldiers suggests a dual role for this caste

The social insect soldier is perhaps the most widely known caste, because it often exhibits spectacular weapons, such as highly enlarged jaws or reinforced appendages, which are used to defend the colony against enemies ranging in size from wasps to anteaters. We examined the function of the enlarged forelimbs of soldiers (both male and female) of the eusocial, gall-inhabiting insect Kladothrips intermedius, and discovered that they have little impact on their ability to repel the specialized invading thrips Koptothrips species. While the efficacy of the enlarged forelimb appears equivocal, we show that soldiers secrete strong antifungal compounds capable of controlling the specialized insect fungal pathogen, Cordyceps bassiana. Our data suggest that these thrips soldiers have evolved in response to selection by both macro- and micro-organisms. While it is unknown whether specialized fungal pathogens have been major selective agents in the evolution of the soldier caste in general, they were probably present when sociality first evolved and may have been the primordial enemies of social insects.     

Soldier presence suppresses presoldier differentiation through a rapid decrease of JH in the termite Reticulitermes speratus

The regulation of caste differentiation is essential to insect eusociality. Termite soldiers are sterile and cannot eat by themselves because they have specialized mouth morphology. Almost all termite species have a soldier caste, and the soldier ratio per colony is maintained at a low level, probably by elaborate regulatory mechanisms. Although the soldier presence is considered to negatively affect soldier differentiation in all examined species, the detailed mechanism remains unclear. Presoldier differentiation can be induced artificially by juvenile hormone (JH) application to workers, showing that JH is a key factor underlying the regulation of soldier differentiation. In this study, to elucidate physiological changes in workers because of the soldier presence during the molt into presoldiers, JH III applications and JH titer quantifications were carried out in the rhinotermitid termite Reticulitermes speratus. Firstly, the effects of soldier presence before the molt into presoldiers induced by JH III application to workers were investigated. The rates of presoldier molt induced by the treatments with soldiers were significantly lower than those without soldiers. Secondly, worker JH titers in the presence or absence of soldiers were quantified by LC-MS on day 0, 5, 10, and 15 after JH application. Results indicated that the worker JH titers (endogenous + applied JH III) in the presence of soldiers were significantly lower than those without soldiers on day 5 after the JH treatment. On days 10 and 15, such soldier effects were not observed. Finally, the effective duration of soldier presence after the JH application was elucidated. A 4 day period of co-existence with soldiers suppressed presoldier differentiation, suggesting that the soldier presence rapidly decreased the JH titer in other colony members (i.e., workers), resulting in the inhibition of presoldier production.     

Group size effect on worker juvenile hormone titers and soldier differentiation in Formosan subterranean termite

In a finite environment, population growth can lead to crowding, increased densities and stress. Termites live in highly organized societies and densities can increase astronomically as colonies grow. However, little is known about juvenile hormone (JH) changes in these insects as numbers increase, despite the fact that JH is a critical caste regulator in this insect. Using Formosan subterranean termites as a model, we evaluated minimum group size requirements for soldier differentiation and the effect of density (50, 100, 500, and 1000 individuals/experimental unit) on worker JH titers either with or without initial soldiers being present. The minimum group size investigation indicated that groups initiated with 5 workers were insufficient for soldier differentiation. Soldiers were produced in groups initiated with 10 or more workers. As density increased from 50 to 1000 individuals per experimental unit, worker JH levels were elevated. Presence of soldiers lessened the effect of density on rising worker JH titers, indicating that soldiers have the ability to down-regulate worker JH. The study provides direct evidence of a population density effect on JH in eusocial insects and sheds light on understanding of the regulatory mechanisms associated with termite soldier caste differentiation.