Ecdysteroid production during caste differentiation in larvae of the ant, Plagiolepis pygmaea

ABSTRACT. Variations in ecdysteroids were measured by radio-immunoassay in worker-biased and queen-biased larvae of the ant Plagiolepis pygmaea Latr. (Hymenoptera, Formicidae) during late larval development, i.e. from the end of winter diapause up to the prepupal period. At the end of diapause, larvae are bipotential and, depending on culture conditions, can become either queens or workers. Ecdysteroid profiles revealed that there are striking differences between the two castes: worker larvae showed high titres during their development, queen larvae had low titres over the same period.     

Postembryonic development of the wing imaginal discs in the female wingless bagworm moth Eumeta variegata (Lepidoptera,Psychidae)

The process of wing disc development and degeneration in the bagworm moth Eumeta variegata was investigated histologically. Morphological differences between two sexes first appear in the penultimate (eighth) larval instar. In the male, wing discs proliferate rapidly in the penultimate larval instar and continue proliferating; a conspicuous peripodial epithelium forms in the last (ninth) larval instar. The hemopoietic organs break down in this stage and disappear completely by the prepupal stage. In the female, in contrast, the wing discs remain as in the previous (seventh) instar, without proliferation of cells inside. No peripodial epithelium forms in the penultimate instar or later. Hemopoietic organs are still attached to the wing discs in the last larval instar and the entire wing discs transform into a plain, thick epidermis in the prepupal period. It is suggested that the hemopoietic organs may prevent the wing discs from developing in E. variegata.     

Female-specific wing degeneration is triggered by ecdysteroid in cultures of wing discs from the bagworm moth, Eumeta variegata (Insecta: Lepidoptera, Psychidae)

Female adults of the bagworm moth, Eumeta variegata, are completely wingless; by contrast, the male adults have functional wings. Sex-specific differences in the development of wing discs appear to arise during the 8th (penultimate) larval instar. We have previously found that the wing discs of female E. variegata terminate development and disappear during the prepupal period, whereas the wing discs of males continue to develop fully into adult wings. We have investigated the effects of ecdysteroid (20-hydroxyecdysone, 20E) when cultured with larval wing discs, which are normally attached to the larval integument of both male and female larvae. Male wing discs cultured with 20E undergo a remarkable transformation: the discs undergo apolysis and then differentiation. Female wing discs cultured with 20E also undergo apolysis; however, the disc cells enter apoptosis. We have observed condensed chromatin, fragmented nuclei, and secondary lysosomes in the epithelial cells of these female discs. This report establishes that the reduction of female wing discs arises through apoptotic events triggered by ecdysteroid in vitro.     

Hormonally regulated expression of arginine kinase in Drosophila melanogaster

Summary Arginine kinase (AK) is present throughout the life cycle of Drosophila melanogaster, but there is a sharp, transient peak of AK activity during the prepupal period and a second period of elevated activity at the time of eclosion of the adult. Imaginal discs show the greatest increase in AK activity at the prepupal stage of those tissues assayed. The prepupal peak is not seen when the temperature-sensitive ecdysoneless mutant ecd-1 is shifted to 29° C at mid-third instar larval stage. The peak in activity reappears when ecd-1 is either shifted back to 20° C after 60 h at 29° C or is fed 20-hydroxyecdysone. At the restrictive temperature, imaginal discs from ecd-1 larvae progressively lose AK activity, whereas discs from 20-hydroxyecdysone-fed larvae have a marked increase in AK activity at stage P3 of the prepupal period. These data suggest that the prepupal peak is regulated by the hormone 20-hydroxyecdysone.     

我国大陆红火蚁不同等级和虫态过冷却点的测定

了解我国红火蚁Solenopsis invicta Buren的耐低温能力并为其风险分析提供科学依据,本文对采自我国4个地点红火蚁蚁群中不同等级和虫态的过冷却点,以及不同季节和室内不同饥饿程度的工蚁过冷却点进行了测定。结果表明：蚁群中不同等级和虫态的过冷却点差异显著。工蚁成虫的过冷却点（－11.8±0.4℃）显著低于有翅雌蚁（－8.1±0.6℃）及蚁后（－7.9±0.5℃）,工蚁蛹和幼虫的过冷却点分别为－13.6±0.3℃和－12.1±0.4℃,蚁后蛹和幼虫的分别为－13.3±0.3℃和－12.2±0.3℃。工蚁蛹的过冷却点均显著低于其成虫和幼虫。蚁后蛹与其幼虫的过冷却点没有显著差异,但与其成虫的差异显著。同一未成熟期不同等级之间的也没有显著差异;不同季节的工蚁过冷却点有显著差异,其中以春季的最高（－4.3±0.2℃）,夏季（－6.6±0.2℃）、秋季（－9.4±0.1℃）的次之,冬季的（－12.0±0.3℃）最低;深圳、珠海、广州和陆川4个地理种群的工蚁过冷却点在春、秋和冬季无显著差异,但夏季陆川种群的过冷却点显著低于其他3个种群的（P<0.05）。饥饿可使工蚁的过冷却点明显升高,但大工蚁的过冷却点较小工蚁升高的幅度更大,饥饿15 d后的大工蚁比饥饿前的过冷却点升高了7.0℃,而小工蚁仅升高了4.9℃。这些结果为评价红火蚁在我国潜在的传播风险提供了理论依据。     

The composition of larval food and the significance of exocrine secretions in the bumblebee Bombus terrestris

Summary: This paper describes a study on the relation between the composition of larval food and the development of female castes in bumblebees. The first aim was to evaluate the significance of glandular secretions in the larval diet as a possible factor involved in larval feeding and caste differentiation. Small amounts of proteinaceous secretions were found to be added during the ingestion of sucrose but not while discharging food to the larvae. It is discussed that these secretions are digestive in function rather than food additives that would possibly play a role in the process of caste differentiation.¶Secondly, a comparative analysis was made of the general composition of food samples obtained from larvae of different castes and ages and from various periods in the social development of the colony. No significant differences in the total amount of pollen, sucrose and protein were detected between the castes or different age groups. Unlike honeybee workers, individual bumblebee workers did not change the composition of the diet they supplied to the brood in relation to their own age, nor to the social development of the colony. These findings suggest that all larvae receive the same nourishment during their total development and indicate that differences in development between queen larvae and worker larvae are neither caused by variations nor by a qualitative modification of their food with respect to the amount of pollen, protein and carbohydrates.     

Soldier determination in Pheidole bicarinata: Effect of methoprene on caste and size within castes

Topical application of methoprene to final-instar larvae of the ant Pheidole bicarinata can induce soldier development. Soldier induction takes place if methoprene levels are above a soldier-determining threshold during a critical period of juvenile hormone-sensitivity that occurs during about days 4–6 of the final instar. Furthermore, the amount of exogenous methoprene applied affects the timing of metamorphosis and the adult size in both the minor worker and soldier castes. When larvae that receive methoprene treatment become minor workers these are always larger than acetone-treated controls. In larvae that become soldiers, growth and timing of metamorphosis vary with the dose of methoprene, but in a more complex way. A high dose of methoprene produces a metamorphic delay and large soldiers. However, the lowest effective dose for soldier induction produces early metamorphosis and small adults. On the basis of these results, we have expanded our model of a mechanism by which juvenile hormone could control determination of worker castes in Pheidole bicarinata.     

Apoptotic wing degeneration and formation of an altruism-regulating glandular appendage (gemma) in the ponerine ant <Emphasis Type="Italic">Diacamma</Emphasis> sp. from Japan (Hymenoptera,Formicidae, Ponerinae)

We here show an example of morphological novelties, which have evolved from insect wings into the specific structures controlling social behaviour in an ant species. Most ant colonies consist of winged queen(s) and wingless workers. In the queenless ponerine ant Diacamma sp. from Japan, however, all female workers have a pair of small thoracic appendages, called gemmae, which are homologous to the forewings and acts as an organ regulating altruism expression. Most workers, whose gemmae are clipped off by other colony members, become nonreproductive helpers, while only a single individual with complete gemmae becomes functionally reproductive. We examined histologically the development of gemmae, and compared it with that of functional wings in males. Female larvae had well-developed wing discs for both fore- and hindwings. At pupation, however, the wing discs started to evaginate and later degenerate. The hindwing discs completely degenerated, while the degeneration of forewing discs was incomplete, leading to the formation of gemmae. The degeneration process involved apoptotic cell death as confirmed by TUNEL assay. In addition, glandular cells differentiated from the epithelial cells of the forewing buds after completion of pupation. The mechanism of developmental transition from wing to gemma can be regarded as an evolutionary gain of new function, which can be seen in insect appendages and vertebrate limbs.Edited by P. Simpson     

A mechanical signal biases caste development in a social wasp

Understanding the proximate mechanisms of caste development in eusocial taxa can reveal how social species evolved from solitary ancestors. In Polistes wasps, the current paradigm holds that differential amounts of nutrition during the larval stage cause the divergence of worker and gyne (potential queen) castes. But nutrition level alone cannot explain how the first few females to be produced in a colony develop rapidly yet have small body sizes and worker phenotypes. Here, we provide evidence that a mechanical signal biases caste toward a worker phenotype. In Polistes fuscatus, the signal takes the form of antennal drumming (AD), wherein a female trills her antennae synchronously on the rims of nest cells while feeding prey-liquid to larvae. The frequency of AD occurrence is high early in the colony cycle, when larvae destined to become workers are being reared, and low late in the cycle, when gynes are being reared. Subjecting gyne-destined brood to simulated AD-frequency vibrations caused them to emerge as adults with reduced fat stores, a worker trait. This suggests that AD influences the larval developmental trajectory by inhibiting a physiological element that is necessary to trigger diapause, a gyne trait.     

Task-specific distribution of the two worker castes in extranidal activities inMacrotermes bellicosus (Smeathman): Observation of behaviour during food acquisition

Summary Caste-specific division of labour can be observed in extranidal activities inMacrotermes bellicosus. Food is acquired through a sequence of tasks shared between the two worker castes. During the exploration phase, a network of subterranean galleries is built outwards from the nest, almost exclusively by minor workers. The frequency of excursions made by major workers during this building period is low, but increases significantly when food is needed by the colony. After a food-source has been discovered, it is hardly exploited by the minor workers and no recruitment occurs. They continue to construct galleries, while individuals which have contact with the food show local fidelity in their building activities within the gallery nearest to the food. After a single sporadic major worker accidentally comes across a food-source, the ratio of major to minor workers continually increases. New major workers aim for the food, bite off pieces and may transfer them to the minor workers. In general, only major workers are recruited after food has been discovered, and they then orientate towards the food-source more distinctly than the minor workers.     

Ultrabithorax gene expression in Drosophila imaginal discs and larval nervous system

Using a monoclonal antibody and image-processing procedures, the patterns of expression of the Ultrabithorax (Ubx) gene product have been characterized in Drosophila larvae. As reported previously, the metathoracic imaginal discs stain most intensely with anti-Ubx, with some mesothoracic and no prothoracic expression detectable. In the metathoracic discs, the greatest modulation in anti-Ubx staining is along the proximodistal axis. Ubx is generally expressed at higher levels in the posterior regions of metathoracic discs, although relatively high anterior expression is found in some areas. Expression in the mature wing disc is confined to the squamous peripodial membrane cells; in younger wings, Ubx expression fills the posterior half of the peripodial side of the disc. The mesothoracic leg stains with a pattern that is qualitatively similar (but not identical) to that of the metathoracic leg; Ubx is expressed in some anterior regions of the mesothoracic leg, in parasegment 4. Double staining with anti-Ubx and anti-engrailed reveals that discontinuities in Ubx expression that have been suggested to correspond to compartment borders do not coincide with the compartment boundaries in some cases. In the larval ventral ganglion, Ubx expression is greatest in parasegments 5 and 6, as in the embryonic nervous system.     

Growth and patterning are evolutionarily dissociated in the vestigial wing discs of workers of the red imported fire ant, Solenopsis invicta

Over the last decade, it has become clear that organismal form is largely determined by developmental and evolutionary changes in the growth and pattern formation of tissues. Yet, there is little known about how these two integrated processes respond to environmental cues or how they evolve relative to one another. Here, we present the discovery of vestigial wing imaginal discs in worker larvae of the red imported fire ant, Solenopsis invicta. These vestigial wing discs are present in all worker larvae, which is uncommon for a species with a large worker size distribution. Furthermore, the growth trajectory of these vestigial discs is distinct from all of the ant species examined to date because they grow at a rate slower than the leg discs. We predicted that the growth trajectory of the vestigial wing discs would be mirrored by evolutionary changes in their patterning. We tested this prediction by examining the expression of three patterning genes, extradenticle, ultrabithorax, and engrailed, known to underlie the wing polyphenism in ants. Surprisingly, the expression patterns of these three genes in the vestigial wing discs was the same as those found in ant species with different worker size distributions and wing disc growth than fire ants. We conclude that growth and patterning are evolutionarily dissociated in the vestigial wing discs of S. invicta because patterning in these discs is conserved, whereas their growth trajectories are not. The evolutionary dissociation of growth and patterning may be an important feature of gene networks that underlie polyphenic traits.     

The time during which beta-ecdysone is required for the differentiation in vitro and in situ of wing imaginal discs of Drosophila melanogaster.

The time during which β-ecdysone is required for the apolysis and imaginal differentiation of wing discs of Drosophila both in vitro and in situ has been examined, and it is concluded that β-ecdysone is required as a sustained stimulus rather than as a trigger for differentiation. These results are compared with the requirement for β-ecdysone for the puffing of salivary gland polytene chromosomes during the prepupal stage (Richards, G. P., 1976, Develop. Biol.48, 191–195). It is suggested that imaginal discs and larval salivary glands require different exposures to β-ecdysone to fulfill their developmental commitments and that the drop in β-ecdysone titer during the early prepupal stage, which is necessary for the subsequent puffing of the polytene chromosomes, plays little or no part in imaginal disc differentiation.     

Defective gap-junctional communication associated with imaginal disc overgrowth and degeneration caused by mutations of the dco gene in Drosophila

The lethal(3)discs overgrown (dco) locus of Drosophila melanogaster, located on the third chromosome at cytogenetic position 100A5,6-100B1,2, is necessary for normal development and growth control in the imaginal discs of the larva. Three recessive lethal alleles (dco2, dco3, and dco18) in heteroallelic combinations and one allele (dco3) when homozygous cause the imaginal discs to continue to grow beyond the normal disc-intrinsic limit during an extended larval period. Some degeneration also occurs in the overgrowing discs. The discs overgrow even when transplanted early in their development into wild-type hosts, whereas normal discs stop growth at about the normal final size under such conditions, indicating that the overgrowth is a disc-autonomous effect of the mutations. During overgrowth the imaginal discs retain their single-layered epithelial structure except near regions of degeneration, and they differentiate into disc-appropriate but abnormal adult structures when transplanted into wild-type larval hosts. When the mutant larvae are reared under certain conditions a small percentage develop to the pharate adult stage, and these animals show a characteristic syndrome of abnormalities including swollen leg segments with many extra bristles, small or missing eyes, duplicated antennae and palpi, and separated vesicles of cuticle. A fourth recessive lethal allele (dcole88), when homozygous or in heteroallelic combination with the overgrowth alleles, causes the imaginal discs to degenerate, producing a "discless" phenotype. Gap junction-mediated communication was assayed by observing the intercellular transfer of injected fluorescein complexon (dye coupling). Dye coupling in the imaginal discs of the dco genotypes that cause overgrowth was dramatically reduced at 4 days after egg laying (AEL) compared with wild-type controls. Coupling was more normal although still significantly reduced at 7-8 and 12-14 days AEL. In c43hs1, another disc overgrowth mutant, the imaginal disc cells also showed very reduced dye coupling at 4 days and incomplete coupling at 9 days. In contrast, discs from wild-type larvae, two other imaginal disc overgrowth mutants, and a cell death mutant showed extensive dye coupling at all stages tested. Electron microscopic morphometry revealed a reduction in gap-junction length per unit lateral plasma membrane length in dco3/dco18 and c43hs1 wing discs, although not in dco2/dco3, compared with wild-type wing discs. The results suggest that gap-junctional cell communication may be involved in the cell interactions that limit cell proliferation in vivo.     

Insights into the dynamics of hind leg development in honey bee (Apis mellifera L.) queen and worker larvae - A morphology/differential gene expression analysis

Phenotypic plasticity is a hallmark of the caste systems of social insects, expressed in their life history and morphological traits. These are best studied in bees. In their co-evolution with angiosperm plants, the females of corbiculate bees have acquired a specialized structure on their hind legs for collecting pollen. In the highly eusocial bees (Apini and Meliponini), this structure is however only present in workers and absent in queens. By means of histological sections and cell proliferation analysis we followed the developmental dynamics of the hind legs of queens and workers in the fourth and fifth larval instars. In parallel, we generated subtractive cDNA libraries for hind leg discs of queen and worker larvae by means of a Representational Difference Analysis (RDA). From the total of 135 unique sequences we selected 19 for RT-qPCR analysis, where six of these were confirmed as differing significantly in their expression between the two castes in the larval spinning stage. The development of complex structures such as the bees’ hind legs, requires diverse patterning mechanisms and signaling modules, as indicated by the set of differentially expressed genes related with cell adhesion and signaling pathways.     

Ergatoid queen development in the ant Myrmecina nipponica: modular and heterochronic regulation of caste differentiation

Caste polyphenism in social insects provides us with excellent opportunities to examine the plasticity and robustness underlying developmental pathways. Several ant species have evolved unusual castes showing intermediate morphologies between alate queens and wingless workers. In some low-temperature habitats, the ant Myrmecina nipponica produces such intermediate reproductives (i.e. ergatoids), which can mate and store sperm but cannot fly. To gain insight into the developmental and evolutionary aspects associated with ergatoid production, we conducted morphological and histological examinations of the post-embryonic development of compound eyes, gonads and wings during the process of caste differentiation. In compound eyes, both the queen-worker and ergatoid-worker differences were already recognized at the third larval instar. In gonads, queen-worker differentiation began at the larval stage, and ergatoid-worker differentiation began between the prepupal and pupal stages. Wing development in ergatoids was generally similar to that in workers throughout post-embryonic development. Our results showed that the developmental rate and timing of differentiation in body parts differed among castes and among body parts. These differences suggest that the rearrangement of modular body parts by heterochronic developmental regulation is responsible for the origination of novel castes, which are considered to be adaptations to specific ecological niches.     

Macroevolutionary integration of phenotypes within and across ant worker castes

Phenotypic traits are often integrated into evolutionary modules: sets of organismal parts that evolve together. In social insect colonies, the concepts of integration and modularity apply to sets of traits both within and among functionally and phenotypically differentiated castes. On macroevolutionary timescales, patterns of integration and modularity within and across castes can be clues to the selective and ecological factors shaping their evolution and diversification. We develop a set of hypotheses describing contrasting patterns of worker integration and apply this framework in a broad (246 species) comparative analysis of major and minor worker evolution in the hyperdiverse ant genus Pheidole. Using geometric morphometrics in a phylogenetic framework, we inferred fast and tightly integrated evolution of mesosoma shape between major and minor workers, but slower and more independent evolution of head shape between the two worker castes. Thus, Pheidole workers are evolving as a mixture of intracaste and intercaste integration and rate heterogeneity. The decoupling of homologous traits across worker castes may represent an important process facilitating the rise of social complexity.     

Reciprocal transplantation of wing discs between a wing deficient mutant (fl) and wild type of the silkworm, Bombyx mori

The wingless mutant flügellos ( fl ) of the silkworm lacks all four wings. Although wing discs of the fl seem to develop normally until the fourth larval instar, wing morphogenesis stops after the fourth larval ecdysis, probably caused by aberrant expression of an unidentified factor, referred to as fl . To characterize factor fl , the wing discs dissected from the wild-type (WT) and fl larvae were transplanted into other larvae and developmental changes of the discs were examined. When the wing disc from a WT larva was transplanted into another WT larva and allowed to grow until emergence, a small wing appeared that was covered with scales. Thus, the transplanted wing discs can develop autonomously, form scales and evert from adult skin. The WT wing discs transplanted into the fl larvae also developed at a high rate. However, the fl wing discs transplanted into the WT larvae did not develop during the larval to pupal developmental stages. These data suggest that the fl gene product (factor fl) works in the wing disc cells during wing morphogenesis. Its function cannot be complemented by hemolymph in the WT larva. It is also implied that the level of humoral factors and hormones required for wing morphogenesis are normally maintained in the fl larva.     

Testing homology with morphology, development and gene expression: sex-specific thoracic appendages of the ant Diacamma

Females of the ants belonging to the queenless genus Diacamma have a pair of unique tiny thoracic appendages, called "gemmae," located on the mesothoracic segment. They are covered with sensory hairs, filled with exocrine glands and are involved in the behavioral regulation of reproduction. We report here a morphological, developmental, and genetic study of the development of the gemmae. Both male and female larvae have dorsal mesothoracic discs, although differing in shape and fate. In Diacamma ceylonense, we show that, contrary to butterflies, these discs specify parts of the adult thorax in addition to wing tissues, as in Drosophila. We have cloned and studied the expression of wingless (wg) and scalloped (sd), two genes known to play a critical role in wing morphogenesis in Drosophila. In the fly's mesothoracic dorsal disc, sd is specifically expressed in the wing pouch. In Diacamma, we show that sd is also expressed in male dorsal thoracic discs, whereas its expression was undetectable in females. From this result and observations of shape and growth of cultured isolated discs, we suggest that gemmae originate from a more ventral part of the dorsal disc than the wing pouch and discuss the pro and cons of gemma/wing homology.     

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).