Incipient Colonies Research Articles

A lipocalin protein, Neural Lazarillo, is key to social interactions that promote termite soldier differentiation.

Social communication among castes is a crucial component of insect societies. However, the genes involved in soldier determination through the regulation of inter-individual interactions are largely unknown. In an incipient colony of the damp-wood termite Zootermopsis nevadensis, the first larva to develop into a third instar always differentiates into a soldier via frequent trophallactic feeding from the reproductives. Here, by performing RNA-seq analysis of third instar larvae, a homologue of Neural Lazarillo (named ZnNLaz1) was found to be the most differentially expressed gene in these soldier-destined larvae, compared with worker-destined larvae. This gene encodes a lipocalin protein related to the transport of small hydrophobic molecules. RNAi-induced knockdown of ZnNLaz1 significantly inhibited trophallactic interactions with the queen and decreased the soldier differentiation rates. This protein is localized in the gut, particularly in the internal wall, of soldier-destined larvae, suggesting that it is involved in the integration of social signals from the queen through frequent trophallactic behaviours. Based on molecular phylogenetic analysis, we suggest that a novel function of termite NLaz1 has contributed to social evolution from the cockroach ancestors of termites. These results indicated that a high larval NLaz1 expression is crucial for soldier determination through social communication in termites.

Asssesment of Initial Colony Founding by Swarming Reproductives of the Western Drywood Termite, Incisitermes minor

Colony founding activities in drywood termite is difficult to assess, since the colony forage and nest inside the wood. Non-destructive observation using X-ray computed tomography was used to evaluate nest-gallery establishment and colony breeding of the incipient colonies. The infested timbers were marked by year of infestation and were left on its original position to allow termite colonies develop in natural environment. Selected timbers were subjected for X-ray scanning analysis in Kyushu National Museum, Japan, in which colony founding from 5 royal pairs in the first year development was monitored. The CT data of the first six months colony founding indicated two different outcome in colony founding; two colonies distributed the energy to breed and slowed down in foraging simultaneously and other three concentrated in foraging. The CT scan after one year showed that only one royal pair showed no breeding activity, while the other four had been observed to have 2 – 5 new colony members. Colony extraction also confirmed that colony size in the first and second year varied from 0 – 5 new colony members. The new colony members were mostly in larval instars, sometimes with the presence of one small soldier. Colony founding through nuptial flight is a risky process, as 59% of the observed chambers were failed colony founding, resulted from the dead of a royal pair or being abandoned (empty chamber).

TGFβ signaling related genes are involved in hormonal mediation during termite soldier differentiation.

A working knowledge of the proximate factors intrinsic to sterile caste differentiation is necessary to understand the evolution of eusocial insects. Genomic and transcriptomic analyses in social hymenopteran insects have resulted in the hypothesis that sterile castes are generated by the novel function of co-opted or recruited universal gene networks found in solitary ancestors. However, transcriptome analysis during caste differentiation has not been tested in termites, and evolutionary processes associated with acquiring the caste are still unknown. Termites possess the soldier caste, which is regarded as the first acquired permanently sterile caste in the taxon. In this study, we performed a comparative transcriptome analysis in termite heads during 3 molting processes, i.e., worker, presoldier and soldier molts, under natural conditions in an incipient colony of the damp-wood termite Zootermopsis nevadensis. Although similar expression patterns were observed during each molting process, more than 50 genes were shown to be highly expressed before the presoldier (intermediate stage of soldier) molt. We then performed RNA interference (RNAi) of the candidate 13 genes, including transcription factors and uncharacterized protein genes, during presoldier differentiation induced by juvenile hormone (JH) analog treatment. Presoldiers induced after RNAi of two genes related to TGFβ (Transforming growth factor beta) signaling were extremely unusual and possessed soldier-like phenotypes. These individuals also displayed aggressive behaviors similar to natural soldiers when confronted with Formica ants as hypothetical enemies. These presoldiers never molted into the next instar, presumably due to the decreased expression levels of the molting hormone (20-hydroxyecdysone; 20E) signaling genes. These results suggest that TGFβ signaling was acquired for the novel function of regulating between JH and 20E signaling during soldier differentiation in termites.

Transmission of fungal partners to incipient Cecropia-tree ant colonies.

Ascomycete fungi in the nests of ants inhabiting plants (= myrmecophytes) are very often cultivated by the ants in small patches and used as food source. Where these fungi come from is not known yet. Two scenarios of fungus recruitment are possible: (1) random infection through spores or hyphal fragments from the environment, or (2) transmission from mother to daughter colonies by the foundress queen. It is also not known at which stage of the colony life cycle fungiculture is initiated, and whether the- symbiont fungi serve as food for the ant queen. To clarify these questions, we investigated four Azteca ant species inhabiting three different Cecropia species (C. insignis, C. obtusifolia, and C. peltata). We analysed an rRNA gene fragment from 52 fungal patches produced by founding queens and compared them with those from established Azteca colonies (n = 54). The infrabuccal pockets of winged queens were dissected to investigate whether young queens carry fungi from their mother colony. Additionally, 15N labelling experiments were done to verify whether the queen feeds on the patches until she is nourished by her first worker offspring. We infer from the results that the fungi cultivated in hollow plant structures are transferred from the parental colony of the young queen. First, fungal genotypes/OTU diversity was not significantly different between foundress queen patches and established colonies, and second, hyphal parts were discovered in the infrabuccal pockets of female alates. We could show that fungiculture already starts before queens lay their eggs, and that the queens do not feed on fungal patch material but feed it to the larvae. Our findings suggest that fungiculture may be crucial for successful colony founding of arboreal ants in the tropics.

No evidence for social immunity in co-founding queen associations

Ant queens often associate to found new colonies, yet the benefits of this behaviour remain unclear. A major hypothesis is that queens founding in groups are protected by social immunity and can better resist disease than solitary queens, due to mutual grooming, sharing of antimicrobials, or higher genetic diversity among their workers. We tested this hypothesis by manipulating the number of queens in incipient colonies of Lasius niger and measuring their resistance to the fungal entomopathogen Metarhizium brunneum. We found no evidence for social immunity in associations of founding queens. First, co-founding queens engaged in self-grooming, but performed very little allo-grooming or trophallaxis. Second, co-founding queens did not exhibit higher pathogen resistance than solitary queens, and their respective workers did not differ in disease resistance. Finally, queens founding in groups increased their investment in a component of individual immunity, as expected if they do not benefit from social immunity but respond to a higher risk of disease. Overall, our results provide no evidence that joint colony founding by L. niger queens increases their ability to resist fungal pathogens.

Irreversible transfer of brood care duties and insights into the burden of caregiving in incipient subterranean termite colonies

1. In incipient termite colonies, biparental brood care rapidly shifts towards alloparental brood care. This transition was suggested to recapitulate the evolutionary trajectory from subsocial wood roach ancestors to eusociality in termites.2. Incipient colonies of the subterranean termite Coptotermes gestroi (Wasmann) were investigated to determine if this transition was irreversible and if the burden of brood care on the first alloparents could be measured. To do so, the initial size of the work force necessary for an incipient colony to survive once the brood care became alloparental was determined.3. The results of the study show that within 5 months after foundation, brood care duties were fully transferred to workers and the primary reproductives became irreversibly dependent on these workers for survival, reproduction, and colony growth.4. Once the brood care became strictly alloparental, the presence of a single worker was enough to maintain the survival of the king and queen, confirming that ‘reversed parental care’ was also achieved. However, major brood loss and suppressed egg‐laying activity from the queen was observed, suggesting that the burden of brood care was too high for a single worker to absorb. Therefore, once brood care has shifted to alloparents, a critical number of workers is necessary to prevent brood loss and initiate colony growth.5. As the initial cost of brood care is rapidly absorbed with colony growth in termites, the performance in brood care of the first few alloparents in a subsocial wood roach ancestor may have contributed to the emergence of eusociality in this clade.

Termite’s royal cradle: does colony foundation success differ between two subterranean species?

Colony foundation modes play a key role in social insect societies as they strongly impact colony survival. In subterranean termites, we do not yet know which factors most influence the early stages of colony foundation since studies on the topic are scarce. In the present study, we examined how variation in life-history traits can influence colony foundation success in two European termite species: Reticulitermes grassei, an indigenous, and R. flavipes, an invasive. We conducted an experiment in which we observed the development of colonies founded by 165 royal pairs on a weekly basis over a 6-month period. Several key results emerged: (1) survival rates of royal pairs were lower in R. grassei; (2) R. grassei pairs laid fewer eggs; and (3) all castes contained fewer individuals in R. grassei colonies, but (4) their timing of appearance are similar for both species. Overall, R. grassei pairs seemed less robust and produced fewer offspring. To our knowledge, our study is the first to describe the foundation of R. grassei incipient colonies in detail over a 6-month period. We also discovered new information about R. flavipes’ colony foundation. The data suggest that the two species have different levels of colony foundation success, resulting in differences in colony development. This finding could help explain the invasiveness of R. flavipes. Our study contribute to our understanding of the spread of two termite pest species and shed light on critical steps in the colony foundation process.

Role of accelerated developmental pathway and limited nurturing capacity on soldier developmental instability in subterranean termite incipient colonies

In the subterranean termite Coptotermes gestroi (Wasmann), soldiers developing in incipient colonies display strong fluctuating asymmetry when compared with soldiers developing in mature colonies. This strong asymmetry may arise from two different types of stress factors on individuals. First, the accelerated development of nanitic (small) soldiers may impose a direct physiological stress as Coptotermes soldiers produced in incipient colonies have two less molting events than soldiers produced in mature colonies. Second, the environmental conditions in incipient colonies present a major constraint with limited access to resources and small numbers of workers to care for the developing brood. In this study, 459 soldiers from 73 incipient colonies (6-month-old) displaying only nanitic soldiers were investigated in a range of nurturing capacity scenarios. Nanitic soldiers developing in incipient colonies with high nurturing capacity displayed more symmetrical traits than the ones developing in colonies with low nurturing capacity. In addition, the first soldiers to emerge in the colony were the most asymmetrical individuals, showing that as the conditions improve rapidly with the growth of the colony, newly produced nanitic soldiers manifest the lessening of stress in normalization of their morphology. However, the nurturing capacity of the colony only partially explained the developmental instability variability, implying that the accelerated development of nanitic soldiers remains an important stress factor in incipient colonies, in comparison with soldiers developing in mature colonies with two additional molts and homeostatic environmental conditions.

Soldiers are differentiated from male larval stages in incipient colonies of Nasutitermes takasagoensis (Isoptera: Termitidae)

In phylogenetically ancestral taxa of termites (the so-called lower termites), at least one soldier emerges and is maintained longitudinally in each incipient colony. However, in apical taxa (the so-called higher termites), the developmental pathway and regulation of soldiers in incipient colonies currently remain unknown. We therefore examined soldier and worker development in incipient colonies of higher termites (Nasutitermes takasagoensis Shiraki). Developmental stages and castes were successfully discriminated by head width in incipient colonies 4 months after colony foundation. Furthermore, differences were observed in the number of bristles on antennae between first- and second-instar larvae. In N. takasagoensis, there was more than one soldier in each incipient colony 4 months after its foundation. Presoldiers in the incipient colonies were differentiated from an earlier instar (male second-instar larvae), whereas, in mature colonies, they were differentiated from male third instars (= minor workers). The developmental period of the former (7 days) was markedly shorter than that of the latter (14 days). All female second-instar larvae molted into workers. The developmental processes shown here are useful for obtaining a clearer understanding of the mechanisms of soldier/worker differentiation in higher termites.

Potential Hybridization between Two Invasive Termite Species, Coptotermes formosanus and C. gestroi (Isoptera: Rhinotermitidae), and Its Biological and Economic Implications.

The Asian subterranean termite, Coptotermes gestroi, is a tropical species but has increasingly been collected from the subtropics in recent years, making it sympatric to the Formosan subterranean termite, C. formosanus in at least three areas, Taiwan, Hawaii, and Florida. Simultaneous flights by these two species were observed since 2013 in South Florida, during which interspecies tandems were observed. Laboratory mating of C. formosanus and C. gestroi alates produced hybrid incipient colonies of larger population size. Studies are underway to examine the presence in the field of hybrid colonies in sympatric areas of Taiwan and Florida. Other biological characteristics of C. formosanus × C. gestroi hybrids being studied include temperature tolerance and preference, colony growth rate, wood-consumption rate, and reproductive fertility. This current research aims to determine the potential establishment of a hybrid termite population in south Florida and Taiwan. It investigates the risk of introgressive hybridization in field populations, with an emphasis on its potential ecological, evolutionary, and economic consequences.

Ant community and habitat limit colony establishment by the fire ant, Solenopsis invicta

Summary Hypotheses of community assembly include limitation through habitat physical attributes, as well as competition among species. Such hypotheses must be resolved through experimental tests. Previous experiments have shown that: (i) fire ants of the monogyne social form occur mostly in highly disturbed habitat where they do not compete with mature colonies of co‐occurring ants; (ii) in native pine forests of northern Florida, habitat disturbance favours fire ants while simultaneously reducing native ants; (iii) fire ants thrive in these disturbances but do not persist as these become less disturbed over time; and finally, (iv) newly mated, dispersing/colony‐founding fire ant queens settle preferentially in such disturbed sites. We now show that by choosing disturbed sites, newly mated, monogyne fire ant queens greatly increase their chances of successful colony establishment. Experimental plots were created in the native ground cover of a north Florida pine forest with all combinations of tilling, shading or reduction of the native ant community. Newly mated fire ant queens, incipient colonies and small colonies were planted in these plots. Only five of 980 (0·5%) newly mated queen nests survived after 120 days, and only five of 400 incipient colonies (1·3%) survived after 30 days. All survivors were in plots with tilling and/or native ant reduction. Extrapolation indicated that 0·04% of newly mated queens and 0·1% of incipient colonies were likely to have survived at 1 year. In contrast, planting small colonies resulted in much higher rates of survival – in plots with native ant reduction, fire ants increased on baits throughout the year but decreased in unreduced control plots. Fifteen months after planting 108 colonies, 21 mounds (19%) were found in the ant‐reduced plots, but <2% of 108 colonies survived in the control plots. Taken together, these results show that by landing in disturbed habitat with its reduced native ant population, newly mated fire ants queens increase their chances of successful colony establishment. In contrast to much of the previous literature, our results suggest that ant community assembly proceeds primarily by queen habitat choice and secondarily by filtering and competition. A lay summary is available for this article.

Studies on the development of captive termite colonies

AbstractDifferent authors have tried in the past to develop captive colonies of termites in their laboratories, starting with capturing of alate pairs and confining them in Petri dishes or small boxes to facilitate mating, then transferring incipient colonies into progressively larger containers. Twenty-three termite species of the genera Coptotermes, Cortaritermes, Cryptotermes, Hodotermes, Macrotermes, Mastotermes, Microtermes, Odontotermes, Pseudacanthotermes, Reticulitermes, Trinervitermes, and Zootermopsis have been explored following this method in 33 reported studies. However, in most of the attempts, incipient colonies did not grow beyond the population of a few hundred insects and tended to die off in a few months. In this paper, we report the efforts made to develop colonies of Hypotermes obscuriceps, Macrotermes convulsionarius, Microcerotermes cameroni, Odontotermes brunneus, Pericapritermes sp., and Trinervitermes biformis found in the study area. Several strategies were attempted to develop ...

Male same-sex pairing as an adaptive strategy for future reproduction in termites

A wide variety of animals display same-sex behaviours, including courtship, copulation and pairing. However, these behaviours create a paradox, as selection seemingly acts on maladaptive traits, and they have often been regarded as cases of mistaken identity, especially in invertebrates. We show that termite males show nest establishment and pairing formation that usually occur in monogamous colony foundation and demonstrate how this contributes to their fitness. We found that pairs of male dealates stopped searching for females and established nests without females, although single males rarely ceased searching for mates. Males in these male–male pairings had much higher survival than single males. Our colony fusion experiment showed that a male in a surviving same-sex pair can replace a male in an incipient colony and produce offspring. A mathematical model demonstrated that the observed strategy of establishing a male–male pairing instead of searching for females is advantageous when the risk of predation is high, even when colony fusion is very rare. These results indicate that, under certain ecological conditions, a cooperative same-sex pairing with a potential rival for reproduction can be adaptive. Our study implies the existence of various possibilities for explaining the adaptive significance of same-sex sexual behaviours.

Distribution and origin of intraspecific social variation in the California harvester ant Pogonomyrmex californicus

The California seed harvester ant Pogonomyrmex californicus exhibits striking differences in queen behavior and colony organization throughout its range. Most populations show the likely ancestral condition of single-queen colonies (monogyny). However, in one known population, cooperation between queens during colony founding (pleometrosis) occurs, and is suspected to persist to the mature colony stage (primary polygyny). To better understand this variation in social phenotype, we estimate relatedness among co-foundresses, measure the frequency of polygyny in adult colonies across three focal populations, and assess population genetic structure between populations. We additionally measure queen mating frequency (polyandry) across focal populations. With few possible exceptions, queens within pleometrotic associations are unrelated. We demonstrate a correlation between pleometrosis in incipient colonies and polygyny in adult colonies at the population level. Nuclear microsatellites and mtDNA barcoding indicate significant structure between focal populations. Queens from all three focal populations engage in relatively high levels of polyandry. These findings, when examined with previous data demonstrating that pleometrosis leads to polygynous colonies in the laboratory, strongly suggest that the same occurs in nature. However, the possibility of additional queen adoption in adult colonies (secondary polygyny) is not excluded. Thus, polygynous P. californicus colonies potentially represent long-term associations of unrelated family groups, containing multiple unrelated queens and their mixed-paternity offspring. Due to its pronounced variation in social behavior, P. californicus provides a unique opportunity to examine the evolutionary history and genetic underpinnings of cooperation among non-relatives, a strategy of intense interest in behavioral ecology.

Is the initial nest depth adapted to favorable conditions for the incipient colony in leaf-cutting ants?

The nest dug by the leaf-cutter ant queen of the genus Atta is a vertical tunnel interconnected to a small chamber to hold its offspring and the symbiotic fungus. The depth of the initial chamber of the Atta sexdens rubropilosa ranges from 10 to 30 cm below the soil surface. This available information, could it be that the ideal initial nest depth is adapted to favorable conditions for the initial colony? We hypothesized this depth can provide a minimum temperature range with almost constant temperature, leading to the development of symbiotic fungus and brood yet to emerge. To test this hypothesis, laboratory experiments were carried out and the soil temperature was measured at different depths. The colony development at different temperatures was studied in the laboratory and the brood production (number of eggs, larvae, pupae and adults) was measured until the first workers emergency. Additionally, lipid content and the survival of queens at different temperatures were determined. Our results show an suitable temperature range (ranging from 24.82±3.14°C to 24.11±1.30°C) at a depth of 5 to 25 cm from the ground, an ideal brood development at temperatures of 24 and 28 °C, and consequently a reduction in lipid content of the queens at high temperatures, without affecting their survival in the trial period. These results indicate that the depth of the initial chamber excavated by the queen is suitable for the success of the incipient colony.

Dopamine regulates termite soldier differentiation through trophallactic behaviours.

Caste polyphenism in social insects is regulated by social interactions among colony members. Trophallaxis is one of the most frequently observed interactions, but no studies have been conducted identifying the intrinsic factors involved in this behaviour and caste differentiation. Dopamine (DA) has multiple roles in the modulation of behaviours and physiology, and it produces species-specific behaviours in animals. Here, to verify the role of DA in termite soldier differentiation, we focused on the first soldier in an incipient colony of Zootermopsis nevadensis, which always differentiates from the oldest 3rd instar (No. 1 larva) via a presoldier. First, brain DA levels of the No. 1 larva at day 3 after its appearance were significantly higher than day 0. Second, DA synthesis gene expression levels were extraordinarily high in the No. 1 larva at day 0–1 after appearance. Finally, injection of a DA receptor antagonist into the No. 1 larva resulted in the inhibition of presoldier differentiation. Behavioural observations of the antagonist or control-injected larvae suggested that brain DA and signalling activity regulate the frequencies of trophallaxis from reproductives and presoldier differentiation. Because trophallaxis is a social behaviour frequently observed in natural conditions, the role of DA should be investigated in other social insects with frequent trophallactic and allogrooming behaviour.

Impact of imidacloprid on new queens of imported fire ants, Solenopsis invicta (Hymenoptera: Formicidae).

Neonicotinoid insecticides are commonly used in managing pest insects, including the imported fire ant, Solenopsis invicta Buren. There is increasing evidence that neonicotinoid insecticides at sublethal concentrations have profound effects on social insects. However, the sublethal effect of neonicotinoids on S. invicta has never been investigated. In this study, the newly mated queens were fed with water containing 0.01 or 0.25 μg/ml imidacloprid. Imidacloprid at both concentrations did not cause any increase in queen mortality during the founding stage; however, it significantly reduced queens’ brood tending ability. In the 0.25 μg/ml imidacloprid treatment, the time to larval emergence was significantly delayed and no pupae or adult workers were produced. This study provides clear evidence that imidacloprid at sublethal concentrations has a significant detrimental impact on S. invicta queens and the development of incipient colonies.

Expressions of juvenile hormone biosynthetic genes during presoldier differentiation in the incipient colony of Zootermopsis nevadensis (Isoptera: Archotermopsidae)

Soldier differentiations require high juvenile hormone (JH) titers in workers and two molts via a presoldier stage. However, it is still unclear when the endogenous JH titer increases in workers, because identifying an individual before it undergoes the presoldier molt is quite difficult in natural conditions. In an incipient colony of Zootermopsis nevadensis Hagen, the oldest 3rd instar larva (Νo. 1 larva) always differentiates into a presoldier and the second 3rd instar larva (Νo. 2 larva) molts into a 4th instar larva. Consequently, by focusing on the ontogeny of the Νo. 1 larva, the timing of the increase in the JH titer can be determined. Here, we compared the expression levels of JH biosynthetic genes in the heads of Νo. 1 and Νo. 2 larvae. Most genes involved in JH biosynthesis were found in a genome database, and two candidate JH epoxidase (CYP15A1) genes were identified. Expression levels of JH acid methyltransferase (JHAMT) and one candidate CYP15A1, probably involved in the final steps of the JH biosynthesis, were specifically high in Νo. 1 larvae at 2–3 and 4–5 days after the appearance, respectively. Expression patterns of these genes may be used for the prediction of endogenous JH titer changes during presoldier differentiation.

Animal personality in a foundation species drives community divergence and collapse in the wild.

Despite thousands of papers on the topic, precious few of the studies on animal personality have considered the role of personality in shaping community-level processes. Here, we test the effect of individual variation on the long-term trajectories of biological communities, from initiation to their demise. The spider Anelosimus studiosus builds webs that serve as habitat for >50 species of spider, which together construct a species-rich silken reef. This species also exhibits a temporally consistent behavioural polymorphism where females exhibit either an aggressive or docile phenotype (personality). In this study, we established incipient colonies of either two docile or two aggressive females and then tracked community succession and persistence over 7 years in the field. In particular, we noted the community compositions associated with colony extinction/collapse events, which are common in this species. The community composition of webs founded by different phenotypes diverged rapidly in their early successional stages. However, this period of divergence was ephemeral and all communities eventually converged on a similar composition; communities founded by aggressive females merely took longer to reach it. This secondary stage of community convergence was itself short-lived and independent of founders' personality; all communities collapsed within a year of achieving it. Experimentally imposing this fatal climax composition on colonies caused 80% of communities to collapse within a year, suggesting that this climax composition actually causes the collapse of the community. Community collapse was characterized by a complete die-off of the foundation species and the dispersal of all other spiders. Thus, the behavioural traits of the founding, foundational individuals of these communities dictate their path of succession and longevity in the wild.

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.