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.     

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.     

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.     

Competence of Macrotermes michaelseni (Isoptera: Macrotermitinae) larvae to differentiate into soldiers under the influence of juvenile hormone analogue (ZR-515, methoprene)

It has been shown that only third instar larvae of Macrotermes michaelseni have the competence to differentiate into presoldiers under the influence of juvenile hormone analogue (JHA). The timing of events leading to presoldier formation was independent of JHA dose above the threshold. Further studies with homogeneous groups of third instar larvae of different ages showed that only larvae of a certain age (0–6 days) could respond to topically applied JHA to produce presoldiers and intercastes (intermediate forms). Older larvae did not respond, hence, 0–6 days interval is the competence period for presoldier differentiation in this species. It seems also that the corpora allata of those individuals which differentiate into presoldiers become activated during the competence period, possibly by the parents or other means.     

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.     

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.     

A juvenile hormone analogue with potential for termite control: laboratory tests with Reticulitermes santonensis, Reticulitermes flaviceps and Coptotermes formosanus (Isopt., Rhinotermitidae)

Laboratory experiments with three species of termites were conducted to study the efficacy of a carbamate derivative of 2-(4-hydroxybenzyl)-1-cyclohexanone (W-328). The compound has been found to be highly active causing differentiation of termite workers into presoldiers, soldiers and/or soldier-intercastes. Differentiation of excessive soldiers induced by juvenile hormone analogues (JHAs) may disrupt the social homeostasis and consequently cause death of the whole termite colony. W-328 induced differentiation into soldiers in 41–100% of individuals when applied in range of 50–500 ppm to a feeding substrate of wood in force-feeding and choice bioassays with groups of Reticulitermes santonensis Feytaud and R. flaviceps (Oshima). Efficacy of W-328 was also confirmed in choice bioassays with isolated groups of workers and soldiers and with incipient colonies of Coptotermes formosanus Shiraki. A high proportion of JHA-induced differentiation into soldier caste was accompanied by increased mortality. Termites accept a matrix treated with W-328 quite well. Significant feeding deterrence of the compound was observed only at higher concentrations: 500 ppm in some tests with Reticulitermes spp. and 5000 ppm in colonies of C. formosanus . Adequate persistence of W-328 in corrugated cardboard (a potential feeding substrate for baits) was confirmed by bioassay with R. santonensis and by chemical analysis. The residue levels of the compound, applied at 500 ppm concentration, after 27 days of conditioning remain high enough to result in significant soldier differentiation.     

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.     

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.     

Caste development systems of the Neotropical termite <Emphasis Type="Italic">Constrictotermes cyphergaster</Emphasis> (Isoptera,Termitidae)

The caste developmental system of the Neotropical termite Constrictotermes cyphergaster was investigated in 12 natural colonies from the semi-arid Caatinga region of northeastern Brazil. Twelve measurements were taken of different body parts of the larvae, workers, presoldiers, soldiers, nymphs, and alates for morphometric analyses, and principal component analyses (PCA) were performed based on these parameters. The nymphal line comprised one larval instar, followed by five nymphal instars, and alates, in agreement with the developmental pattern described for Termitidae species. The apterous line included two larval instars, followed by workers, presoldiers, and soldiers. Both workers and soldiers were found to be monomorphic and composed only of male specimens, with only one instar phase each. C. cyphergaster, like most of the Nasutitermitinae, has only male soldiers. The presence of male workers, with only one instar, suggests a simplified system of differentiation in the apterous line of C. cyphergaster. These patterns, which had not previously been described for ‘full nasute’ Nasutitermitinae, are similar to the differentiation patterns of the ‘mandibulate nasute’ Cornitermes walkeri.     

Development of non-reproductive castes in the neotropical termite generaCornitermes,Embiratermes andRhynchotermes (Isoptera,Nasutitermitinae)

Summary The developmental pattern of the neuter castes was studied in the mandibulate nasute generaCornitermes, Embiratermes andRhynchotermes. InCornitermes walkeri, all the workers and soldiers are male. There are two larval and a single worker instar. Workers can molt into presoldiers. InEmbiratermes chagresi andRhynchotermes perarmatus, both sexes are present among the neuters. A slight sexual dimorphism (males > females) is discernible among both larval instars and among workers ofE. chagresi; female workers can molt into presoldiers. InR. perarmatus, the sexual dimorphism is conspicuous from the first larval instar on. Male larvae go through two instars, then give rise to workers, which do not molt. InR. perarmatus, there is no worker stage in females, but a third larval instar, preceding the presoldier. Hypotheses are proposed as to the evolution of these caste patterns, attempting to conciliate present knowledge of Nasutitermitinae phylogeny and known evolutionary trends affecting termite caste patterns, according to the species' ecology.Research Associate: National Fund for Scientific Research (Belgium).     

Influence of group size and composition on soldier differentiation in female final larval instars of a higher termite, Macrotermes michaelseni

ABSTRACT. Experiments with Macrotermes michaelseni (Sjöstedt), involving artificial groups of different sizes of final larval instar females, showed that the proportion of presoldiers produced in each group decreased exponentially with increase in the size of the colonies. This relationship was not smooth, however, but stepwise. Increasing the number of workers in a group with larvae improved the chance of the larvae differentiating into presoldiers, although the normal proportion of soldiers for the species was not achieved under these conditions, even at maximum crowding. The inhibitory effect of soldiers in stopping larvae differentiating into more soldiers was established, and evidence obtained for a non-volatile or low volatility pheromone. It is postulated that dominance by larvae which have acquired 'competence' over others, allows them to suppress these others from differentiating into presoldiers through the pheromone acting on specific behavioural elements.     

Correlation between larval weights,endocrine gland activities and competence period during differentiation of workers and soldiers in Macrotermes michaelseni (Isoptera:Termitidae)

Correlations were established between corpora allata and prothoracic gland activities, changes in fresh weights and competence period for soldier formation in a higher termite species, Macrotermes michaelseni. There was a time-course differential increase in fresh weights observed in pairs of female third (final)-instar larvae of the same age previously adopted by a pair of female reproductives from incipient colonies. A bimodal distribution of these larvae according to weights was evident from the first day of the instar. Heavier larvae were associated with higher corpora allata and prothoracic gland volumes (activities), and differentiation into presoldiers, while lighter larvae had considerably less-active corpora allata and prothoracic glands and developed into workers. The peak in the activities of these glands in both cases marked the end of the competence period.The chain of events that may precede caste differentiation is discussed briefly and the significance of the findings outlined.     

Physical contact between soldier and worker is essential in soldier self-regulation of Coptotermes formosanus (Isoptera, Rhinotermitidae)

In a termite society, the soldier proportion is regulated at a species-specific level with a seasonal fluctuation. A feedback mechanism in soldier proportion regulation is well recognized, while the proximate means by which the presence of soldiers inhibits the new soldier formation from workers remains mostly elusive. In the present study, some possible means for this inhibition were tested with the Formosan subterranean termite, Coptotermes formosanus Shiraki. The results showed that neither soldier-originated volatiles nor non-volatile chemicals without physical contact were responsible for the inhibition of new soldier formation. The essentiality of physical contact between workers and soldiers in soldier regulation was established. However, by physical contacts, as the soldier-replacement experiment showed, workers could not individually recognize or “count” soldiers to maintain a set soldier proportion. The possible feedback mechanisms for soldier regulation are discussed.     

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.     

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.     

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.     

Alarm Reaction of European Reticulitermes Termites to Soldier Head Capsule Volatiles (Isoptera, Rhinotermitidae)

The alarm behavior of worker and soldier castes of four European subterranean termite species of the genus Reticulitermes (R. santonensis, R. lucifugus, R. grassei, R. banyulensis) is described. In petri dish bioassays we presented filter papers with squashed soldier heads as an odor source to trigger a alarm reaction. Untreated filter papers were used as controls. The behavioral patterns were alike for all four species. In the control situation (no alarm), only occasional workers and few soldiers were seen walking in the petri dish. When the odor source was presented, a general state of alertness was observed, showing as higher activity and presence, zigzag running, antennation of nestmates, jittering and jerking, mandible snapping, head-banging, and attraction to the odor source. Workers were attracted within a few seconds (6.9–13.4 s); soldiers followed much later (18.5–64.6 s). Both workers and soldiers inspected the odor source only briefly (workers for 8.7–12.8 s, soldiers for 10.0–19.5 s), with the exception of R. santonensis, where soldiers stayed an average of 209.8 s. The number of individuals, of 50 workers and 3 soldiers, near the odor source averaged 6.0–9.2 workers and 1.2–2.3 soldiers. The presence of workers is crucial: without workers, a significant alarm reaction in soldiers could not be induced. The roles of soldier and worker castes of Reticulitermes species within alarm communication and the origin and nature of possible alarm signals are discussed.     

Developmental arrest induced by juvenile hormone in larvae of Bombyx mori

Injection of the juvenile hormone analog (JHA) methoprene into day 3, fifthinstar larvae of Bombyx mori induced developmental arrest. Feeding activity declined, and the larvae remained as larvae for more than 2 weeks, after which they died. After JHA injection, the hemolymph ecdysteroid titer was low, and the prothoracic glands were almost inactive for 7 days. During this period, prothoracic glands were stimulated by prothoracicotropic hormone (PTTH) in vitro, indicating that JHA did not inhibit the competence of the glands to respond to PTTH. When brain-corpora cardiaca-corpora allata complexes were removed from intact fifth-instar larvae on day 4, the prothoracic glands became autonomously active and produced enough ecdysone for pupation. When PTTH injections were given to larvae previously injected with JHA (7 days before), the larvae recovered feeding activity, purged their guts, and pupated. Injections of 20-hydroxyecdysone into larvae that had been injected with JHA 7 days earlier induced larval molting. These results suggest that JHA affects both the brain and the prothoracic gland.