Comparative analysis of caste differentiation during embryogenesis of social aphids whose soldier castes evolved independently

Summary. We investigated the morphological characters of normal nymphs, soldier nymphs and developing embryos of a social aphid, Colophina arma, which has a sterile soldier caste in the first instar. Morphometric analysis revealed that normal nymphs and soldier nymphs were clearly distinguishable on the basis of several morphological characters. At late embryonic stages, normal embryos and soldier embryos were also distinguishable morphologically. The younger the embryonic stages, the smaller the morphological differences between them. In young embryos of less than 600 m in body length, normal embryos and soldier embryos were no more distinguishable, suggesting that the onset of soldier differentiation occurs at an early embryonic stage. Through the embryonic development, morphological differentiation of soldier caste proceeded gradually: forelegs and midlegs were exaggerated, and growth of mouthpart was suppressed, which resulted in the soldier morphology specialized for attacking behavior. On the basis of these results, developmental aspects in soldier differentiation of C. arma were compared with those of Pseudoregma bambucicola, a social aphid with a first instar soldier caste of independent evolutionary origin. Ecological and evolutionary relevance of the differences between the two social aphids was discussed.Received 30 June 2004; revised 20 October 2004; accepted 9 November 2004.     

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.     

Soldier differentiation during embryogenesis of a social aphid, Pseudoregma bambucicola

To understand the developmental process of aphid soldier differentiation, we investigated the morphological characters of normal nymphs, soldier nymphs and developing embryos of Pseudoregma bambucicola. Results of morphometric analyses showed that normal and soldier nymphs formed discrete clusters on the basis of several morphological characters, although a small number of intermediate individuals, termed ‘intercaste nymphs’, were present. In late embryonic stages, normal and soldier embryos were morphologically distinguishable. The earlier the embryonic stage, the smaller the morphological differences between them. In early embryos less than 1000 µm in length, normal and soldier embryos were not morphologically distinguishable, suggesting that the onset of soldier differentiation occurs at an early embryonic stage. Throughout embryonic development, morphological differentiation of the soldier caste proceeded gradually. Notably, several morphological characters of soldiers grew remarkably upon larviposition. Observation of embryonic leg cuticle revealed a characteristic folding structure, indicating that some morphological traits of the soldier are exaggerated upon larviposition through expansion of the folded cuticle. We suggest that morphological differentiation of the soldier caste in P. bambucicola comprises two phases: gradual growth during embryogenesis and rapid growth upon larviposition.     

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.     

Altruistic defenders in a gall-forming aphid of the tribe Hormaphidini (Homoptera,Aphididae, Hormaphidinae) on its primary host

Summary. We found defensive behavior in the aphid Hamamelistes miyabei on its primary host plant, Hamamelis japonica, where it forms a spiny gall. Introduction of moth caterpillars into the galls elicited attacking behavior of aphid nymphs with their stylet. Although older nymphs sometimes attacked, first-instar nymphs were the main defenders. Immature and mature galls contained a large proportion of first-instar nymphs. Open galls still contained first-instar nymphs, but the proportion was remarkably smaller. In immature and mature galls, particularly, the molting rate of first-instar nymphs was significantly lower than that of older instars. These data suggest that the defensive strategy of H. miyabei is such that 1) molting of first-instar nymphs is suppressed, 2) the duration of the first instar is prolonged, 3) the proportion of defender nymphs in the gall is elevated, and 4) consequently the colony in the gall is effectively defended against predators. No morphological differences were found either between attacking and non-attacking first-instar nymphs or between molting and non-molting first-instar nymphs. Some first-instar nymphs in open galls had the next instar cuticle developing inside the body. These data suggest that first-instar nymphs of H. miyabei are monomorphic defenders, and that at least some of them are able to develop and reproduce. In addition to the attacking behavior, first-instar nymphs of H. miyabei performed characteristic behaviors such as gall cleaning and hindleg waving. This is the first time that altruistic defenders are described in the primary host generation of an aphid from the tribe Hormaphidini.     

Secondary host generation of the gall aphid Cerataphis jamuritsu (Homoptera)

Colonies of a Cerataphis species with well‐developed horns were found on the rattan Calamus quinquesstinervis in southern Taiwan. The morphology of first instar nymphs from the colonies accorded well with the morphology of first instar nymphs laid by alates of Cerataphis jamuritsu from galls on Styrax suberifolia, indicating that the rattan aphids are the secondary host generation of C. jamuritsu. Although the aphid colonies were attended by ants, the sharp horns of the first instar nymphs suggest that they might attack predators.     

The proximate cue of density-dependent soldier production in a social aphid

Tuberaphis styraci is a social aphid that produces 2nd instar soldiers with morphological, behavioral and reproductive division. High aphid density was shown to induce soldier production in T. styraci, although direct cue of soldier induction associated with high density has been unknown. In order to identify the proximate environmental cue underlying the density-dependent soldier production, a series of experiments was conducted using an artificial diet rearing technique. When adult aphids were reared with live normal nymphs, live soldiers, dead normal nymphs, shed skins, honeydew globules and excreted wax, only live normal nymphs effectively induced soldier production. In order to gain insights into the nature of soldier-inducing cue associated with normal aphids, we performed artificial diet experiments using partitioned and non-partitioned chambers, in which direct contact between aphids was either inhibited or allowed. Induction of soldiers was observed only when direct contact was allowed. Therefore, it was shown that the soldier-inducing cue is neither volatile in the air nor diffusible through the diet, but is transmitted between normal non-soldier aphids via direct contact. On the basis of these results, we suggest that the soldier-inducing cue might be physical stimulus combined with non-volatile surface chemicals whose properties differ between normal aphids and soldiers.     

Formation of long‐lasting galls by overwintered nymphs in the Japanese aphid Quadrartus yoshinomiyai (Hemiptera: Aphididae: Hormaphidinae)

This paper examines the life history of a generation of galls created by the aphid Quadrartus yoshinomiyai (Hormaphidinae: Nipponaphidini) on its primary host plant, Distylium racemosum. First‐instar fundatrix nymphs of Q. yoshinomiyai initiated galls on stems of developing shoots in early April and incipient enclosed galls were found from later the same month. The galls lasted for up to 14 months, during which they grew to maturity, opened in early or mid‐April of the following year and dried up by the end of June. First‐instar fundatrix nymphs were found on winter buds, indicating that they hatched from eggs in autumn and overwintered as nymphs. These results suggest that Q. yoshinomiyai has a three‐year life cycle.     

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.     

Defenders in the North American aphidPemphigus obesinymphae

Summary Gall-inhabiting individuals of the aphidPemphigus obesinymphae act as defenders, protecting other colony members against attack by dipteran and neuropteran larvae that are the primary predators of this species. As first instar nymphs, the progeny of the fundatrix patrol surfaces of galls and adjoining leaves. These first instar nymphs attack potential predators by mounting and grasping them and inserting their stylets. This defensive behavior, which is not exhibited by nymphs in later instars, appears to be effective in reducing predation. The fundatrix typically produces defenders throughout the extended gall-inhabiting phase, and her progeny delay development beyond the defensive first instar stage. By August, galls contain an average of 101 defenders. Early death of the fundatrix reduces the number of defenders in the gall and advances maturation of defenders into winged migrants, which otherwise mature in September and October. InPemphigus, defensive behavior by first instar nymphs appears to have evolved in the context of several types of derived life cycle, each involving an extended gall-inhabiting phase.     

Clonal mixing in the soldier-producing aphid Pemphigus spyrothecae (Hemiptera: Aphididae)

Illuminating the genetic relationships within soldier-producing aphid colonies is an essential element of any attempt to explain the evolution of the altruistic soldier caste. Pemphigus spyrothecae is a soldier-producing aphid that induces galls on the leaf petioles of its host (trees of the genus Populus). At least a quarter of the aphids within the clonally produced gall population are morphologically and behaviourally distinct first-instar soldiers that defend the gall population from predation. Using field trapping and microsatellites, we investigated the degree of clonal mixing within natural gall populations. Field trapping in the UK showed that all the migrants of P. spyrothecae and of two other Pemphigus species were wingless first-instar soldiers. The average degree of mixing estimated from trapping P. spyrothecae migrants was 0.68% (range = 0-15%). Microsatellite genotyping of 277 aphids from 13 galls collected in Italy revealed an average mixing level of 10.4% (range = 0-59%). Six galls contained more than one clone (range = 2-5 clones). Non-kin aphids were not restricted to the soldier caste but were evenly distributed across instars. An additional gall, from which 527 occupants were genotyped, contained 12 non-kin aphids distributed among nine clones, showing that clonal diversity can be high even when mixing is very low. These observations suggest that although soldiers migrate regularly and can moult and reproduce within foreign galls, clonal mixing in this species is generally low and is unlikely to provide a barrier to the evolution of investment by the aphid clones in an altruistic soldier caste.     

Defense by a few first-instar nymphs in the closed gall ofdinipponaphis autumna (homoptera, aphididae, hormaphidinae)

The defensive behavior of the aphidDinipponaphis autumna, which forms small, completely closed galls on leaves ofDistylium racemosum, was observed. In mature galls, in addition to tens of mature or nearly mature aphids of the 3rd generation, there remained 3 – 17 1st-instar nymphs of the same generation, which had well-sclerotized legs and the antennae with developed setae. Despite their minority, these 1st-instar nymphs clung to experimentally introduced insect larvae and stung them with their stylets. Fourth-instar wingpadded nymphs, the majority of the 3rd generation at the experiment, also attacked the introduced larvae, but they were readily spilt from the larvae. Four out of 205 1st-instar nymphs remaining in mature galls had the next instar cuticle developing inside, indicating that they are not destined to be sterile.     

Shift in life-history strategy from reproduction to defense with colony age in the galling aphidHemipodaphis persimilis producing defensive first-instar larvae

Colony defense in some aphids is performed by sterile soldiers but in others by monomorphic larvae of a specific instar stage. This paper, focusing on a galling aphid Hemipodaphis persimilis with monomorphic defensive first instars, examined the mechanism by which the proportion of defenders is regulated in the colonies. Demographic analyses showed that the ratios of first instars (defenders) were kept constantly high (58% on average) from mid June to late September. High proportions of first instars could be explained by consistently high birth rates (birth rate hypothesis) or by a prolonged duration of the first instar stage (instar span hypothesis). With the progress of colony age, the mature-embryo content of apterous adults, used as an index of the birth rate, decreased and the proportions of advanced instars increased. These results did not support the birth rate hypothesis. By contrast, calculation of a newly proposed index, the molting rate, showed that the duration of the first-instar stage was short in incipient galls but became longer with colony age. The duration of other instar stages was always kept short. These results corroborate the instar span hypothesis and suggest that the prolongation of the first-instar stage is an adaptive mechanism by which the defender ratio is kept high in mature colonies where the birth rate is declining. The frequency of aggressive behavior in first instars increased from incipient to mature galls. Seasonal changes in the instar span and aggressiveness of first instars suggest that in H. persimilis colonies there is a strategic shift from the reproductive to defensive phase with colony age.     

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.     

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.     

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.     

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.     

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     

Effects of a juvenile hormone analog, pyriproxyfen, on the apterous form of Lipaphis erysimi

Effects of a juvenile hormone analog, pyriproxyfen, on various developmental stadia of the apterous form of the turnip aphid, Lipaphis erysimi (Kaltenbach), were bioassayed at three concentrations (50, 100, and 150 mg [AI]/l) in the laboratory. Supernumerary-molted nymphs with 1-3 extra molts were found when the first three nymphal instars were treated, and all these nymphs died prematurely. In contrast, all treated fourth instar nymphs molted normally to adults. The longevity of all pyriproxyfen-treated L. erysimi nymphs and adults and the fecundity of treated adults were reduced by 50%. Higher concentrations of pyriproxyfen (100 and 150 mg [AI]/l) caused sterility in adults from treated fourth instars; whereas the lowest concentration (50 mg [AI]/l) did not cause any adult sterility. A majority of adults from treated fourth instars and treated adults produced normal nymphs, and some adults produced dead nymphs with normal appendages or dead and deformed nymphs without any appendages. Normal nymphs produced by the adults from treated adults or treated at fourth instars developed more slowly from first instar to adult than the water-treated nymphs, and all adults in the new generation apparently reproduced normally. The potential role of pyriproxyfen in a vegetable aphid IPM program was discussed.