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.     

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.     

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.     

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.     

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).     

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.     

Caste morphology and development in Termitogeton nr. planus (Insecta,Isoptera, Rhinotermitidae)

The termite family Rhinotermitidae displays a wide diversity in its patterns of social organization in castes. The genus Termitogeton probably branched off early in the evolution of this family. We studied the developmental pathways of a putative undescribed species from New Guinea, Termitogeton nr planus. The development begins with two white inactive instars (larvae), the second of which possesses small wing buds. These are followed by a relatively homogenous group of active immature stages (pseudergates) among which a biometric study revealed the presence of four instars. The first of these instars possesses wing buds that regress at subsequent molts. The external morphology of older instars resembles that of higher termite workers. Older pseudergates can differentiate into presoldiers and then into soldiers, but they are also able to molt into a unique stage with long wing pads (nymph), preceding the imago. Colony maturity can be reached with about 10(3) individuals. The sex ratio is near 1:1 in all castes except soldiers, among which females are more numerous than males. The caste pattern of T. nr. planus is reminiscent of those of the Kalotermitidae and Termopsidae, and of Prorhinotermes among the Rhinotermitidae. None of these taxa possess a true worker caste, permanently diverted from imaginal development: social tasks are done by unspecialized immatures that retain a full array of developmental options, including that of proceeding to the imago. The most remarkable trait of T. nr. planus is the presence in all second-instar individuals of wing buds that later regress to reappear in the single nymphal stage. We suggest that the traditional definitions of the terms larvae, nymphs, and pseudergates should be revised because they cannot be satisfactorily applied to the castes of Termitogeton.     

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.     

Soldier determination in Pheidole bicarinata: Inhibition by adult soldiers

In the ant Pheidole bicarinata, adult soldiers suppress the induction of soldier developments in larvae that have been treated with methoprene, as well as in acetone-treated controls. Induction of soldier development is inhibited in methoprene-treated larvae when the adult soldiers are present as nurses, when they are present in the colony but minor workers serve as nurses, and when they are held in a minor worker-permeable cage to prevent them from contacting larvae. Suppression of soldier determination can be overridden by high doses of methoprene applied during the juvenile hormone-sensitive period for soldier determination. The degree of suppression increases with increasing contact between larvae and adult soldiers. The most likely mechanism of inhibition is a contact soldier pheromone. On the basis of our results, we expand our model of soldier determination to accommodate the fact that the threshold titre of juvenile hormone necessary to induce soldier development changes in the presence of soldiers. A threshold that is sensitive to the presence of adult soldiers provides a mechanism whereby the worker caste ratio in colonies can be regulated.     

Termite soldier differentiation in incipient colonies is related to parental proctodeal trophallactic behavior

Termite soldiers represent a peculiar caste among social insects in terms of their specific defensive roles. Numbers of soldiers are relatively low in a mature colony, and it is impossible to identify the individuals that will differentiate into soldiers. If it were possible to specify these individuals prior to soldier differentiation, it would facilitate a better understanding of the regulatory mechanisms of soldier differentiation under natural condition. Here we analyzed soldier differentiation in incipient colonies of Zootermopsis nevadensis, in which only a single soldier develops via a presoldier stage, and is stable during early colony ontogeny. We observed that the oldest third instar differentiated into a presoldier within about eight days from its appearance. Caste differentiation, however, was not strictly determined on an individual basis. The oldest third instars never differentiated into presoldiers if primary reproductives were removed soon after their appearance. Behavioral observations of primary reproductives and their offspring prior to presoldier differentiation, showed that primary reproductives transferred proctodeal materials to the oldest third instar significantly more frequently than to other larva. A high juvenile hormone (JH) titer is required for the soldier differentiation, and we suggest that the JH itself or some nutrients/factors increasing larval JH titer may be transferred to the oldest third instar via a parental proctodeal fluid.     

Gene expression changes in the tyrosine metabolic pathway regulate caste-specific cuticular pigmentation of termites

In social insects, all castes have characteristic phenotypes suitable for their own tasks and to engage in social behavior. The acquisition of caste-specific phenotypes was a key event in the course of social insect evolution. However, understanding of the genetic basis and the developmental mechanisms that produce these phenotypes is still very limited. In particular, termites normally possess more than two castes with specific phenotypes (i.e. workers, soldiers, and reproductives), but proximate developmental mechanisms are far from being fully understood. In this study, we focused on the pigmentation of the cuticle as a model trait for caste-specific phenotypes, during the molts of each caste; workers, soldiers, presoldiers (intermediate stage of soldiers), and alates (primary reproductives) in Zootermopsis nevadensis. Expression patterns of cuticular tanning genes (members of the tyrosine metabolic pathway) were different among each molt, and high expression levels of several “key genes” were observed during each caste differentiation. For the differentiation of castes with well-tanned cuticles (i.e. soldiers and alates), all focal genes except DDC in the former were highly expressed. On the other hand, high expression levels of yellow and aaNAT were observed during worker and presoldier molts, respectively, but most other genes in the pathway were expressed at low levels. RNA interference (RNAi) of these key genes affected caste-specific cuticular pigmentation, leading to soldiers with yellowish-white heads and pigmented mandibular tips, presoldiers with partly pigmented head cuticles, and alates with the yellow head capsules. These results suggest that the pigmentation of caste-specific cuticles is achieved by the regulation of gene expression in the tyrosine metabolic pathway.     

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.     

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.     

Elimination of sexual brood in the Argentine antLinepithema humile: queen effect and brood recognition

Mated queens of the antLinepithema humile (Iridomyrmex humilis Mayr) introduced into dequeened colony fragments rearing sexual brood elicited worker aggression resulting in queen larvae being bitten and eliminated. By contrast, male larvae were spared. Regarding queen brood, killing mainly concerned small and medium sized larvae. A large proportion of the large larvae escaped extermination, and prepupae and pupae were spared. These data suggest that workers were able to discriminate sex, caste and age of the brood. That a queen pheromone may be involved was shown by experiments using whole or cut corpses that were either rinsed or not rinsed in pentane. The pheromone eliciting worker aggressive behaviour was shown to act over a short distance, suggesting that it is somewhat volatile. Similarities and differences between this new queen pheromone and other known queen pheromones acting on queen production or worker attraction are discussed as well as the origin of the signals underlying the recognition of the larval classes.     

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.     

Response to orphaning in two Neotropical termites: shape Armitermes euamignathus and shape Embiratermes festivellus

In this paper we examine the potential of the termites Armitermes euamignathus Silvestri: 1901 and Embiratermes festivellus (Silvestri, 1901) (Isoptera, Termitidae, Nasutitermitinae) to produce neotenics experimentally. Three nests of the mound-building termite A. euamignathus, from the Brazilian cerrado, had their primary queens removed in August 1994. After 12 months, only one mound survived; it had a normal appearance. In this healthy, orphaned colony we found the primary king, six physogastric nymphoid female replacement reproductives, two ergatoid female replacement reproductives, 46 nymphs, several presoldiers, soldiers, workers, larvae and many eggs. These data show that neotenics in A. euamignathus may originate from both workers and nymphs, but nymphoids are produced in larger numbers. The biometric study of nymphs and nymphoids suggests that these brachypterous neotenics were derived from third instar nymphs after a single moult or from four instar nymphs after a reduction of wing bud length. A piece of an E. festivellus nest with some third instar nymphs, soldiers and workers was kept under laboratory conditions. After 12 months, the whole experimental subcolony was examined and appeared to contain two pigmented nymphoid females, two pigmented nymphoid males, only one larva, seven nymphs of the same instar, 148 workers, five soldiers and many eggs. These results also indicate the capacity of the termite E. festivellus to produce nymphoid neotenics. These neotenic females were laying eggs, but they were not physogastric after a year, unlike some nymphoids of the same species collected from natural colonies.     

Morphology,development and evolutionary significance of the working stages in the caste system of Prorhinotermes (Insecta,Isoptera)

Summary The existence of a worker caste in Prorhinotermes inopinatus was determined with the help of biometrical analyses and morphological observations. No dichotomy between alate and worker lines was detected among the young larval instars. Mandible sclerotization and wear show that the larvae become active by the fourth instar in mature colonies, and by the third in incipient ones. Prothoracic finger-shaped expansions characterize the most dependent larval instars. The antennal structure is the best feature for distinguishing late instars. The developmental schema is very flexible: the late larvae of stages 5 to 8 (at least) can give rise to wing-padded nymphs, to the next larval instar, to presoldiers, or most likely to neotenic reproductives as well; the wing-padded individuals (nymphs) normally moult direct to alates, but they can also undergo a regression of their wing pads and revert to worker-like individuals or differentiate into soldiers or neotenics. Slight variations in alate size and antennal morphology result from their origin in different larval instars. Whole-colony censuses revealed that the major part of the colony's work force is composed of pseudergates, i.e. late larvae that do not follow the most direct pathway to the alate. The pseudergates of Prohinotermes do not constitute a distinct worker caste irreversibly diverted from the alate developmental pathway. For this reason the caste system of Prorhinotermes is analogous to that of the Kalotermitidae and Termopsidae and much more primitive than those of the other Rhinotermitidae so far studied, Reticulitermes, Schedorhinotermes and Coptotermes.Contribution No. 139 from King Léopold III Biological Station, Laing Island, Papua New GuineaSenior research assistant of the National Fund for Scientific Research, Belgium     

Energetics and development of incipient colonies of the harvester termite, <Emphasis Type="Italic">Trinervitermes trinervoides</Emphasis> (Sjöstedt) (Termitidae,Nasutitermitinae)

Energy dynamics within incipient colonies of the southern African mound dwelling harvester termite Trinervitermes trinervoides (Sj?stedt) (Termitidae: Nasutitermitinae) were investigated. Development of incipient colonies established from field collected alates and changes in energy content of reproductives and brood were studied over a 25 week period in the laboratory. Colonies were then transferred into the field to study the established of the first mound. An initial four-week egg-laying cycle started within a week of pairing. Hatching occurred six weeks later. Two lines emerged from first instar undifferentiated larvae. Small larvae moulted first into minor presoldiers and then minor soldiers. Large larvae developed into major workers. Foraging began once second instar major workers had differentiated 20 weeks after colony foundation. First instar major workers did not forage but there is evidence of pre-foraging utilisation of soil humus. The epigeous mound was produced after three years. The energy available to maintain incipient termite colonies until the first workers start foraging is crucial to their survival. The royal pair of T. trinervoides does not feed during the rearing of the first brood, the fate of which depends largely on the reproductives accumulated energy reserves. The males and females expended 84.54 and 83.07% respectively of their total energy during this period. Once foraging started, the energy content of the royal pair increased and a second egg-laying cycle began. Received 25 July 2007; revised 19 March and 22 May 2008; accepted 4 September 2008.