Reproductive schedule and factors affecting soldier production in the eusocial bamboo aphid Pseudoregma bambucicola (Homoptera, Aphididae)

Summary: The reproductive characteristics of the soldier-producing aphid Pseudoregma bambucicola were studied in Kagoshima, Southern Japan, to know the factors affecting soldier production of eusocial aphids. The soldier proportion in aphid colonies was highest from October to November. In some large colonies, soldiers were observed in all seasons except in July when colony size was relatively small. Multiple regression analysis showed that the colony size was a principal factor affecting soldier proportion throughout a year. Other social or environmental factors such as aphid composition, host plant conditions and predator abundance were not always significant. Rearing experiments revealed that large colonies (̿,000 individuals) produced soldiers in almost all seasons while small colonies (<1,000) never produced any soldiers. The caste-production schedule of adult females was examined in the field. When solitary females produced both castes, they usually produced normal nymphs first and then soldiers. Females from large colonies tended to produce more soldiers in the earlier period of their lifetime, whereas females from newly established small colonies produced no or only a few soldiers at later times. The average number of soldiers and normal nymphs produced consecutively by a single female was >10 and >20, respectively. Because they have a small number of ovarioles (<15 on average), females should alter caste production within the same ovarioles according to changes in environmental conditions. Artificial removal or introduction of predators and reduction of colony size did not affect soldier production over two successive generations, revealing maternal effects on soldier production. Females cannot shift caste production quickly in response to changes in predator abundance and colony size. This is probably due to early developmental determination of castes within the mother's body.     

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

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

Soldier‐biased gene expression in a subterranean termite implies functional specialization of the defensive caste

In a termite colony, reproduction is typically monopolized by a small number of sexuals that are supported by reproductively altruistic soldiers and workers. We expect caste differentiation to be associated with clear‐cut differences in gene expression, and for these differences to reflect caste function and development. Here, we use RNA‐Sequencing to compare the gene expression profiles of sexual nymphs and two non‐reproductive helper castes (i.e., workers and soldiers) of the Eastern subterranean termite Reticulitermes flavipes. We found that of n = 93 genes that are strictly expressed as a function of caste, a majority (78%) show a soldier‐specific pattern. This conspicuous soldier‐bias in genome‐wide expression suggests that this defensively specialized caste is functionally well‐differentiated from both the reproductive and the other non‐reproductive caste of this species, despite a shared developmental program with workers. Gene ontology analysis supports the notion of functional specialization by soldiers, as soldier‐biased gene sets are enriched for novel biological processes. Whether this pattern reflects ancient or more recent bouts of selection for caste novelty at the gene‐regulatory level is not known, but because soldiers are sterile and thus have no direct fitness, any selection for novelty must have been mediated indirectly, through reproducing relatives.     

X‐ray computed tomography reveals that intraspecific competition promotes soldier differentiation in a one‐piece nesting termite

Investment in soldier production in eusocial lineages involves a trade‐off between maintenance costs and defense benefits. Termites are eusocial insects that live in colonies organized into three castes: primary reproductives, soldiers, and workers or pseudergates. Neotermes chilensis (Blanchard) (Isoptera: Kalotermitidae) is a one‐piece nesting termite that nests and forages in a single piece of wood. Two scenarios may be of importance in a defense context of one‐piece nesting termites: during swarms, when colonies may be invaded by winged termites (alates) in search of a place to found a new colony, and when colonies of conspecifics are present within the same substrate. It was hypothesized that the ratio of soldiers to non‐soldiers would be higher at the onset of the swarming period and in substrates bearing more than one termite colony. A method based on X‐ray computed tomography (CT) was developed to study gallery connectivity in colonies of N. chilensis and caste composition within colonies. Computed tomography allowed the digital reconstruction of the galleries within the substrate, even when they belonged to different colonies, and was effective in distinguishing termites from substrate, and soldiers from reproductives and pseudergates. Using CT, the ratio of soldiers to non‐soldiers was shown to be highest in colonies within multicolonial scapes (i.e., neighboring colonies were present in the same substrate) during the swarming season, thus supporting our initial hypotheses. These results constitute a unique example of induced defenses arising from intraspecific interactions in termites.     

尖唇散白蚁工蚁、兵蚁和繁殖蚁精子发生的比较研究

为探讨白蚁非生殖品级和生殖品级生殖细胞发育差异,采用组织学染色技术对尖唇散白蚁Reticulitermes aculabialis Tsaiet Hwang繁殖蚁、工蚁和兵蚁的精巢发育以及精子发生进行了显微观察和比较研究。结果发现3个品级间精巢发育的程度差异很大,三者精巢切面面积相对大小之比为:繁殖蚁∶工蚁=1.7∶1;繁殖蚁∶兵蚁=29.3∶1;工蚁∶兵蚁=17.1∶1。繁殖蚁和工蚁精巢管内有精子的形成,工蚁和繁殖蚁精子的发生都经历了精原细胞、初级精母细胞、次级精母细胞、精细胞和精子时期,但工蚁有大量次级精母细胞呈细胞凋亡状态。兵蚁生殖细胞发育仅有精原细胞、初级精母细胞、次级精母细胞,没有精细胞和精子产生。工蚁的生殖细胞显著小于同一时期繁殖蚁的生殖细胞,兵蚁的各时期生殖细胞均极显著小于繁殖蚁同一时期的生殖细胞。研究表明各品级之间生殖功能分化与生殖细胞发育有直接关系,工蚁有转化为补充繁殖蚁和兵蚁的能力;而兵蚁由于精巢极度退化不能产生精细胞和精子,因此是非生殖品级分化的终极形式,不具有转化成补充繁殖蚁或其它品级的能力。     

A microsatellite-based test of the <Emphasis Type="Italic">Reticulitermes speratus</Emphasis> genetic caste determination model in <Emphasis Type="Italic">Coptotermes lacteus</Emphasis>

Coptotermes lacteus, a termite found in eastern Australia, is a central-site nester that constructs mounds up to 2 m in height. Mature colonies typically contain the primary king and queen, and up to a million or more worker and soldier offspring. Once a year, male and female nymphs are produced in approximately equal numbers and develop into new alates. Experimental removal of the queen in the field and subsequent mating of the king with a replacement queen, results in an unusual phenomenon whereby the production of female nymphs completely ceases, while the production of male nymphs, and workers of both sexes, continues. The proximate cause of this nymph male-bias is yet to be discovered. The production of male nymphs and workers of both sexes in de-queened C. lacteus colonies is equivalent to offspring production patterns in laboratory crosses of male nymphoid (nymph-derived) with female ergatoid (worker-derived) replacement reproductives in the related species Reticulitermes speratus. An X-linked genetically influenced caste determination (GCD) mechanism has been proposed to account for such offspring patterns in R. speratus. We examined microsatellite genotypes in C. lacteus to test a prediction arising from the R. speratus GCD model: that nymphs should result from reproduction by neotenics, rather than the primary pair. In five of six colonies examined, genotypes indicated that all workers and nymphs were derived from a single reproductive pair. In three of these cases, the primary queen was also located and examined; her genotype matched that inferred from worker and nymph genotypes. These results suggest that the GCD model proposed for R. speratus does not apply to C. lacteus, at least under field conditions. The male-nymph bias following queen removal therefore remains an unresolved issue in C. lacteus.     

Density-dependent induction and suppression of soldier differentiation in an aphid social system

We investigated the mechanism underlying the control of soldier production in colonies of a social aphid, Tuberaphis styraci, which has a sterile soldier caste in the second instar. High aphid density was shown to induce soldier production in T. styraci. Analysis of natural colonies revealed that the soldier proportion tended to increase with aphid density but reached a plateau. Artificial diet experiments identified a similar plateau of soldier proportion under high-density conditions. In order to gain insights into the controlling mechanism of soldier production, the effect of soldiers on reproducing adult aphids was examined using the artificial diet system. It was experimentally demonstrated that soldier production was suppressed by coexisting soldiers, whereas coexisting non-soldiers facilitated soldier production. These results suggested that caste ratio in the colony of T. styraci is controlled by positive and negative feedbacks consisting of density-dependent induction and suppression of soldier differentiation.     

Proximate factors influencing soldier development in the basal termite <Emphasis Type="Italic">Cryptotermes secundus</Emphasis> (Hill)

Summary Regulation of soldier development in termites at the endocrinological and social scale was intensively studied in the 60s and 70s, but conclusive results are still scanty. In termites, the presence of soldiers inhibits further soldier differentiation, but the mechanism is not well understood. Secretions from the frontal glands seem to be important, but dry- and dampwood termites do not have frontal glands. We investigated the influence of precocene I, a JH-antagonist, and of soldier head extracts on the development of soldiers in the drywood termite, Cryptotermes secundus (Hill). Soldierless colonies produced fewer soldiers when treated with precocene I or soldier head extract than control colonies. Furthermore, colonies treated with precocene I had produced worker-nymph intercastes, last stage nymphs and alates by the end of the experiment (after six months) which were not present in control colonies. Hence, both treatments inhibited soldier development while precocene I simultaneously promoted the development of adult traits, probably by reducing (directly or indirectly) the JH level. The inhibition effect of the soldier head extract indicates that at least in termites without a frontal gland, other sources of material in the head have a regulatory function.     

Social structure and the defensive role of soldiers in a eusocial bamboo aphid,Pseudoregma bambucicola (Homoptera: Aphididae): A test of the defence-optimization hypothesis

A complete understanding of the evolution of sociality in aphids requires a detailed knowledge of the patterns of soldier investment in their ecology. The eusocial bamboo aphidPseudoregma bambucicola has a morphologically specialized first-instar soldier caste. The proportion of soldiers was positively correlated with colony size. Within a colony, soldiers were evenly distributed among subcolonies; within each subcolony, however, their distribution was biased toward peripheries which were exposed to many predators. Field experiments introducing natural enemies such asEupeodes confrater (Diptera: Syrphidae) andSynonycha grandis (Coleoptera: Coccinellidae) revealed that the survival rate of these predators was negatively correlated with the density of soldiers, suggesting that soldiers can more or less defend their colonies by killing or removing a range of natural enemies. Observations suggest that large mature colonies attract more predators than newly established small colonies and that, within a colony, the predators attack each subcolony regardless of its position on bamboo shoots. This implies the presence of a positive correlation between colony size and predation risk. Thus, the investment in soldiers seems to reflect the attacking pattern of predators within a colony. These results agree with the defence-optimization hypothesis in soldier investment ofP. bambucicola colonies.     

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.     

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.     

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.     

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.     

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.     

The ontogeny of soldiers in Prorhinotermes simplex (Isoptera, Rhinotermitidae)

The ontogeny of the soldier caste in Prorhinotermes simplex is studied with emphasis on the instar origin of soldiers, the duration of the presoldier stage, and changes in external anatomy during the soldier morphogenesis. The soldiers of different instar origin are compared morphometrically and by analysis of the soldier defensive secretion. The soldiers were found to develop from larvae of the second up to the eighth instar, via a short (13–17 days) presoldier stage. The early soldier instars were found exclusively in incipient colonies while the mature colony contains late instar soldiers only. The abrupt change in the external anatomy occurs in two steps. During the larva-presoldier moult, the head increases in length but only slightly in width. During the presoldier-soldier moult, both the length and width of the head increase markedly and the typical cordate shape is attained; the relative size of the pronotum increases considerably. The long falcate mandibles develop mainly during the larva-presoldier moult. One antennal segment is added during these two moults. With increasing instar age of soldiers a relative increase of the pronotum and the head size in its posterior region was observed. No functional differences in external anatomy were found among the six soldier instars. The composition of the frontal gland secretion is similar in the six soldier instars, an extraordinarily high amount of a defensive substance, (E)-1-nitropentadecene, was detected together with (Z,E)-α-farnesene, presumably an alarm substance. The strategy of soldier production is discussed. We conclude that the observed patterns of soldier production in P. simplex correspond well to its ecological strategy of an advanced single-site nester able to migrate and form foraging groups. Received 16 August 2005; revised 21 November and 6 February 2006; accepted 16 February 2006.     

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.     

Social aphids use their antennae to perceive density cue for soldier production

In social insects, local interactions among colony members facilitate information transfer, and allow the whole colony to regulate division of labor and task allocation in an integrated and coordinated manner. In particular, regulation of caste differentiation in response to external cues is important for sustaining social insect colonies. The social aphid Tuberaphis styraci exhibits a caste polyphenism, producing second‐instar soldiers and non‐soldiers. Previous studies using an artificial diet rearing system identified high aphid density as a crucial cue for soldier production, which acts on embryos in the maternal body and newborn first‐instar nymphs to induce soldier differentiation. While direct contact stimuli from live non‐soldiers were suggested to mediate the density effect, how the aphids perceive the stimuli has been unknown. Here we investigated how antennal removal of adult females affects the soldier production in T. styraci. Under a high density condition, intact females produced the highest percentage of soldiers, females deprived of both antennae produced the lowest percentage of soldiers, and females deprived of one antenna exhibited an intermediate percentage of soldiers. Scanning electron microscopic observations of the aphids revealed the existence of sensory organs for chemoreception and tactile sensation on the antennae of the mother aphids. These results indicate that T. styraci females use their antennae to perceive soldier‐inducing density cue, suggesting that maternal perception of density cue is involved in regulation over caste phenotype of their offspring.     

Density triggers soldier production in a social aphid

For evolution and maintenance of the social system of insect colonies, investment in the sterile caste should be adequately controlled in response to environmental cues. Recent developments using artificial diet rearing techniques have revealed an underlying mechanism of caste control in a gall-forming aphid, Tuberaphis styraci, which has a soldier caste in the second instar. Statistical analyses of field-collected galls detected a significant positive correlation between aphid density and soldier proportion in the natural colonies of T. styraci. Artificial diet experiments showed that soldiers are produced under crowded conditions. Detailed experiments demonstrated that soldiers are produced in a density-dependent manner rather than in a colony size-dependent manner. From these results, it was concluded that aphid density is the crucial cue that triggers soldier production in T. styraci. This study provides, to our knowledge, the first experimental demonstration of an environmental factor involved in aphid soldier differentiation.