Impact of a juvenile hormone analogue on the anatomy and the frontal gland secretion of Prorhinotermes simplex (Isoptera: Rhinotermitidae)

In termites, juvenile hormone plays a key role in soldier differentiation. To better understand the evolutionary origin of the soldiers, we studied the external and inner morphology of pseudergate-soldier intercastes and neotenic-soldier intercastes formed artificially by the application of juvenile hormone analogue in Prorhinotermes simplex. A majority of these intercastes had a soldier phenotype, whereas the inner anatomy had an intermediary form between two castes or a form specific to intercastes. Our experiments showed that traits of neotenics and soldiers can be shared by the same individuals, although such individuals do not exist naturally in P. simplex, and they have not been reported in other species but in some Termopsidae. Our results reinforce the hypothesis that soldiers may have emerged from soldier neotenics during the evolution of termites.     

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.     

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.     

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.     

Developmental regulation of caste-specific characters in social-insect polyphenism

Phenotypes of organisms are not determined completely genetically, but vary according to environmental factors (phenotypic plasticity). Some organisms express several discrete adaptive phenotypes (polyphenism). Social insects possess a few types of individuals (castes) in their colonies, to which specific tasks are allocated. Here, I review studies on caste polyphenism in ants and termites, in terms of the developmental mechanisms of caste-specific characters, such as alate wings and soldier mandibles. In ants, the developmental fate of caste is probably determined by the pattern-formation genes in the early stage of postembryonic development, but apoptotic degeneration occurs in the wing primordia of future workers. As apoptotic wing degeneration has been observed in two phylogenetically distant groups of ants, this phenomenon is suggested to be conserved in many ant species. On the other hand, all termite species possess distinct sterile soldiers with specific morphologies suitable for defense. Recent studies using molecular techniques isolated genes related to soldier differentiation and analyzed the expression profiles of those genes in order to understand the mechanism of caste differentiation and the link between molecular and social evolution. In this review, I focus on these studies, in terms of the alteration of body plan in response to environmental signals, and discuss the evolutionary process of the interaction between ontogeny and environment.     

SOLDIER-REPRODUCTIVE INTERCASTES IN A SPECIES OF TUMULITERMES (ISOPTERA: TERMITIDAE)

i
Two aberrant specimens in a collection of a species of Tumulitermes are described. These specimens are basically soldier like in appearance, but show a number of reproductive caste characters in such structures as the antennae, head capsule and mandibles, and in the presence of wing rudiments. They are evidently soldier-reproductive intercastes, which are quite uncommon in the higher termites.     

Response variability of juvenile hormone and juvenile hormone analogs in workers of the Formosan subterranean termite (Coptotermes formosanus Shiraki)

Juvenile hormone (JH) or juvenile hormone analog (JHA) can induce soldier formation in termites. However, different studies have yielded inconsistent results on the effects of JHA on soldier production in Coptotermes formosanus Shiraki. Using filter paper as the testing substrate, the effects of JH III, pyriproxyfen and methoprene on the intact filter paper left, worker mortality and presoldier formation were tested on six colonies. Our results showed that pyriproxyfen and methoprene were more repellent than JH III. No significant difference in toxicity was observed among methoprene, pyriproxyfen and JH III. JH III and pyriproxyfen did not cause workers to differentiate into presoldiers, but methoprene can induce formation of presoldiers. Colony response variability to methoprene was observed. The confirmation of the effects of JH/JHA on C. formosanus establishes the foundation for molecular studies of soldier differentiation in this species.     

Juvenile hormone titers and caste differentiation in the damp-wood termite Hodotermopsis sjostedti (Isoptera, Termopsidae)

Termites are social insects, presenting morphologically distinct castes, performing specific tasks in the colony. The developmental processes underlying caste differentiation are mainly controlled by juvenile hormone (JH). Although many fragmentary data support this fact, there was no comparative work on JH titers during the caste differentiation processes. In this study, JH titer variation was investigated using a liquid chromatography-mass spectrometry (LC-MS) quantification method in all castes of the Japanese damp-wood termite Hodotermopsis sjostedti, especially focusing on the soldier caste differentiation pathway, which was induced by treatment with a JH analog. Hemolymph JH titers fluctuated between 20 and 720pg/microl. A peak of JH was observed during molting events for the pseudergate stationary molt and presoldier differentiation, but this peak was absent prior to the imaginal molt. Soldier caste differentiation was generally associated with high JH titers and nymph to alate differentiation with low JH titers. However, JH titer rose in females during alate maturation, probably in relation to vitellogenesis. In comparison, JH titer was surprisingly low in neotenics. On the basis of these results in both natural and artificial conditions, the current model for JH action on termite caste differentiation is discussed and re-appraised.     

Effects of juvenile hormone III on Reticulitermes flavipes: changes in hemolymph protein composition and gene expression

Termites express polyphenism during caste differentiation that is mostly undefined at the molecular level. Using the eastern subterranean termite, Reticulitermes flavipes Kollar, we wanted (1) to test juvenile hormone (JH) model assays for their ability to induce detectable molecular changes in worker termites and (2) to investigate hemolymph proteins and their corresponding genes during JH-induced soldier caste differentiation. Our results illustrate pronounced changes in two hemolymph proteins after JH treatment, as well as differences among several caste phenotypes. Significant increases in the expression of four genes encoding hemolymph proteins, including two vitellogenins and two hexamerins, were observed after JH exposure. These findings are the first to demonstrate such protein and gene expression changes during termite caste differentiation. These results also validate the utility of JH model assays for inducing detectable molecular changes in worker termites that have begun presoldier differentiation.     

Agonistic Behavior of the Termite <Emphasis Type="Italic">Prorhinotermes canalifrons</Emphasis> (Isoptera: Rhinotermitidae)

While intercolonial and interspecific agonism has been broadly documented in termites, detailed studies of individual behavior and its differentiation among castes are scarce. We studied the responses of non-reproductive castes of Prorhinotermes canalifrons (pseudergates, presoldiers, soldiers of different age and artificial intercastes) to conspecific and heterospecific aliens. Each caste displayed a specific behavioral repertoire. Soldiers, but to a lesser extent pseudergates as well, are responsible for colony defense. The behavioral repertoire of the soldier appears on the second day after exuviation and becomes complete on the third day, well before its defensive secretion is present. The behavior of artificial intercastes induced by a JH analog is intermediate between that of pseudergates and soldiers. Reactions to heterospecific aliens were much quicker and more violent than reactions to conspecifics, which suggests that individuals experience difficulties in identifying conspecific aliens.     

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.     

Soldier-like intercastes in the rotten-wood termite Hodotermopsis sjostedti (Isoptera: Termopsidae)

All termite species possess a distinct sterile-soldier caste in their colonies, although reproductive soldiers, with soldier characteristics and reproductive ability, have been reported from several species of the family Termopsidae. Such intercastes have been considered the primitive-soldier caste, and based on this many researchers have discussed the evolutionary origin of termite soldiers. We investigated whether such soldier-reproductive intercastes also exist in the Japanese rotten-wood termite Hodotermopsis sjostedti. Abnormal individuals with soldier-like characteristics were found and designated as soldier-like intercastes, which appeared to have both soldier and reproductive characteristics. Based on our morphometric analyses and histological examinations, we suggest that the developmental origin of this intercaste is a pseudergate, nymph or sixth-instar larva. In addition, the intercaste was found to have relatively well-developed gonads, although mature oocytes and spermatozoa were not found in female ovaries and male seminal vesicles, respectively. We conclude that the soldier-like intercaste of H. sjostedti does not have reproductive ability, which is different from all other known soldier-like intercastes in Termopsidae.     

Caste differentiation responses of two sympatric <Emphasis Type="Italic">Reticulitermes</Emphasis> termite species to juvenile hormone homologs and synthetic juvenoids in two laboratory assays

Summary We report on our investigations comparing three juvenile hormone (JH) homologs and two synthetic juvenoids to induce caste differentiation in laboratory colonies of Reticulitermes flavipes and R. tibialis. Two laboratory assays were evaluated as model systems for inducing caste differentiation: (1) shorter-term dish assays on groups of 20 individuals and (2) longer-term feeding assays on groups of 500 individuals. Each assay possessed attributes that can be considered advantageous under certain conditions. Specifically, dish assays were most suitable for presoldier and soldier induction, while jar assays provided for the induction of nymphs, presoldiers, soldiers, neotenic reproductives, and intercastes. Differences in response to the JH homologs and synthetic juvenoids were noted between species, suggesting differences in JH physiology may exist between R. flavipes and R. tibialis. Substantial morpholo-gical impacts were noted in association with some treatments, including (1) juvenoid-induced mandibular mal-formation in presoldiers, (2) JH II-induced abdominal elongation in R. flavipes soldiers and workers (associated with a presence of internal reproductive anatomy that is consistent with what would be expected to occur in pseudergates), and (3) JH II-induced soldier-nymph intercastes in R. tibialis that were able to further molt into soldier-alate intercastes. Findings are discussed in relation to the potential differences in JH-related physiology between R. flavipes and R. tibialis, and the use of model systems to induce rare castes and intercastes for molecular investigations of caste differentiation.     

The homolog of Ciboulot in the termite (<Emphasis Type="Italic">Hodotermopsis sjostedti</Emphasis>): a multimeric β-thymosin involved in soldier-specific morphogenesis

Background  

Caste differentiation in social insects is a type of polyphenism that enables division of labor among members of a colony. This elaborate social integration has attracted broad interest, although little is known about its regulatory mechanisms, especially in Isoptera (termites). In this study, we analyzed soldier differentiation in the damp-wood termite Hodotermopsis sjostedti, focusing on a possible effector gene for caste development. The gene for an actin-binding protein, HsjCib, which shows a high level of expression in developing mandibles during soldier differentiation, is characterized in detail.     

Histological analysis of fat body development and molting events during soldier differentiation in the damp-wood termite, Hodotermopsis sjostedti (Isoptera, Termopsidae)

The caste system of termites is well defined, with a high degree of polyphenism among colony members. Polyphenic caste characteristics are hormonally regulated, and juvenile hormone (JH) is particularly involved in caste determination, as is the case with many other social insects. In the present study, soldier differentiation in the damp-wood termite, Hodotermopsis sjostedti, was induced by treatment with a JH analog (pyriproxyfen) in order to establish the chronology of tissular modifications appearing in response to the hormone.The fat body is involved in the physiological events that prepare the insect for the molting transition. The development of the fat body started within three days after hormonal treatment, and it filled the entire abdominal cavity for about four days prior to the molt to presoldier, maintaining this state until the next molt to soldier. Fat body development was accompanied by the accumulation of protein granules in the cytoplasm, but these granules disappeared during the few days preceding the molt to presoldier. The timing of consumption of these storage proteins corresponded to the window of epidermal growth, which was conspicuous about 14 days after hormonal treatment, and synthesis of the new cuticle, which was initiated 10 days after treatment. We summarize the chronology of the histological events under hormonal control.     

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.     

The influence of juvenile hormone analogue on soldier differentiation in the higher termite,Macrotermes michaelseni*

ABSTRACT. The influence of topically-applied or vapour-administered juvenile hormone analogue (ZR-515, methoprene (JHA)), on soldier differentiation in a higher termite species, Macrotermes michaelseni (Sjostedt) (Isoptera; Macro-termitinae) was examined. It was found that even male third larvae, which under natural conditions develop exclusively into major workers, could be induced by JHA to differentiate into presoldiers. Intermediate forms between workers and soldiers were produced from some of the treated individuals. Administered in vapour form, JHA induced a higher proportion of presoldiers and a lower proportion of intermediate forms than it did when administered topically. The rate of presoldier formation was found to be JHA-dose-dependent. Biometric analysis showed that male presoldiers have the largest head capsule size, but the smallest mandibular index. The possible mechanism of soldier induction is discussed.     

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.     

Screening of genes expressed in developing mandibles during soldier differentiation in the termite Hodotermopsis sjostedti

We investigated the morphological changes accompanying soldier differentiation in the damp-wood termite Hodotermopsis sjostedti. Genes expressed in the developing mandibles, which undergo the most remarkable morphological changes during soldier differentiation, were screened using fluorescent differential display. Database searches for sequence similarities were conducted and the relative expression levels were then quantified by real-time polymerase chain reaction. Among the identified candidate genes, 12 genes were upregulated during soldier differentiation. These included genes for cuticle proteins, nucleic acid binding proteins, ribosomal proteins and actin-binding protein, which were inferred to be involved in caste-specific morphogenesis in termites.