Cell death and wing reduction during the metamorphosis of sex-specific flightless morphs in winter geometrid moths.

Winter geometrid moths show striking sexual dimorphism by having female-specific flightless morphs. The evolutionary grades of wing reduction in winter geometrid moths vary and range from having short wings, vestigial wings, to being wingless. Although the ontogenetic processes underlying the development of the wingless or short-wing morphs in Lepidoptera has been well studied, the mechanisms underlying the development of vestigial wing morphs in winter geometrid moths during metamorphosis are poorly understood. In the winter geometrid moth Sebastosema bubonaria Warren, 1896, the males have functional wings, but the females have vestigial wings. Here, we studied the ontogenetic processes affecting wing reduction in the winter geometrid moth S. bubonaria using light microscopy and transmission electron microscopy, and compared the ontogenetic process of wing reduction in this species with that in another species of the wingless-female winter moth that we investigated previously. Our results showed that, in the vestigial-wing morphs, the loss of pupal wing epithelium was terminated in the middle of the wing degeneration process, whereas in the wingless morph, the pupal wing epithelium disappeared almost completely and the final appearance of the wings differed slightly among flightless morphs. We propose that the extent and location of cell death in the pupal wing play an important role in the various patterns of reduced wings that are observed in flightless moths.

Early cellular development induced by ecdysteroid in sex-specific wing degeneration of the wingless female winter moth.

The winter moth, Nyssiodes lefuarius, exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that the steroid hormone 20-hydroxyecdysone (20E) triggered massive programmed cell death (PCD) only in the female pupal wing epithelium; however, when and how early sexual trait development of the pupal wings is initiated during pupal-adult metamorphosis remains obscure. To clarify the detailed morphological changes and mechanisms underlying early sexual trait development and cell death, we examined the effects of 20E on early ultrastructural and histological changes in the pupal wing epithelium of both sexes. Before the onset of adult differentiation, no morphological differences were observed in the epithelial cells of both sexes at an ultrastructural level. When 5.4 µg of 20E was injected into pupae of both sexes at 15 days after the onset of pupation, retraction of the wing epithelium from the pupal cuticle was initiated at day 2 after 20E injection in both sexes. Although overt degeneration of wing tissue was not still obvious, apoptotic body-like structures and auto-phagosomes were visible at day 3 after 20E injection in females, whereas development of scale precursor cells started on day 4 after injection in males. Our results suggest that (1) the injection of 20E induced sexually dimorphic changes in the pattern of organelle distribution in wing epithelial cells, and (2) abnormally shaped mitochondria in the cytoplasm of the female wing epithelium might be involved in the PCD that occurs during wing tissue degeneration.

Discovery of wing imaginal discs in the penultimate instar of the lacewing Mallada desjardinsi (Insecta: Neuroptera: Chrysopidae) with histological notes on postembryonic imaginal disc development.

Holometabolous insects are alternatively named "Endopterygota" because, in the larvae of many taxa, the wing primordia in the lateral regions of the meso- and metathoracic segments form more or less invaginated structures called wing imaginal discs. Holometabolous insects exhibit differential developmental timing of the wing during ontogeny. The condition in which wing growth is deferred until the end of larval life has been considered ancestral, whereas early disc formation has been recognized as the derived condition. Even though wing disc development in holometabolous insects has been studied extensively in select groups, many questions remain about the development of the wing imaginal disc in the orders Raphidioptera, Megaloptera, Neuroptera, and Mecoptera. To clarify whether the wing imaginal disc of Neuroptera is typical of the derived condition, we examined the ontogeny of the wing imaginal discs in the lacewing Mallada desjardinsi histologically. Using both light microscopy and transmission electron microscopy, we were able to recognize wing imaginal discs in the penultimate larval instar (prefinal larval instar) of this species. To date, neuropteran insects have been characterized as having late-forming wing imaginal discs. However, our findings show that the developmental pattern of the wing imaginal discs within the Neuroptera represents a more derived pattern of development in the Holometabola than was assumed previously.

Morphological and histological examination of short-wing formation in the winter moth Protalcis concinnata (Insecta: Lepidoptera, Geometridae).

Winter geometrid moths exhibit sexual dimorphism in wing length and female-specific flightlessness. Female-specific flightlessness in insects is an interesting phenomenon in terms of sexual dimorphism and reproductive biology. In the winter geometrid moth, Protalcis concinnata (Wileman), adult females have short wings and adult males have fully developed wings. Although the developmental process for wing reduction in Lepidoptera is well studied, little is known about the morphology and the developmental pattern of short-winged flightless morphs in Lepidoptera. To clarify the precise mechanisms and developmental processes that produce short-winged morphs, we performed morphological and histological investigations of adult and pupal wing development in the winter geometrid moth P. concinnata. Our findings showed that (a) wing development in both sexes is similar until larval-pupal metamorphosis, (b) the shape of the sexually dimorphic wings is determined by the position of the bordering lacuna (BL), (c) the BL is positioned farther inward in females than in males, and (d) after the short pupal diapause period, the female pupal wing epithelium degenerates to approximately two-thirds its original size due to cell death. We propose that this developmental pattern is a previously unrecognized process among flightless Lepidoptera.

Morphology of the tentorium in the ant genus Lasius Fabricius (Hymenoptera: Formicidae).

The tentorium is the internal skeleton of the head capsule of insects. Several studies have shown that the structure of the tentorium is an important factor not only for the morphology and systematics but also for the phylogeny and evolution. In ants, however, only three studies have reported tentorial morphology so far. We reveal the fundamental structure of the tentorium of the genus Lasius (Hymenoptera, Formicidae) and its minor variation among six species of the genus. Based on the results, we give new terminologies of the organ, presenting a schematic diagram of the tentorium. We clarify muscle attachment to the tentorium by constructing a three-dimension image of the tentorium and muscles. We then verify the attachment areas of the antennal muscles and maxillary adductor muscles on the tentorium. The results show that the muscular attachment areas are broader than previously thought. Our study indicates that the key to understanding the evolution of the tentorium is its functional morphology, in relation to the attachments of the muscles originating from the tentorium within the head capsule. This is the first report of the three-dimensional images of the ant tentorium and its attached muscles. The intra- and inter-specific variations of the tentorium is also reported for the first time.

Asymmetric larval head and mandibles of Hydrophilus acuminatus (Insecta: Coleoptera, Hydrophilidae): Fine structure and embryonic development.

The larvae of a water scavenger beetle, Hydrophilus acuminatus, have strongly asymmetric mandibles; the right one is long and slender, whereas the left one is short and stout. The fine structure and embryonic development of the head capsule and mandibles of this species were examined using light and scanning electron microscopy, and asymmetries in shape were detected in these structures applying an elliptic Fourier analysis. The larval mandibles are asymmetric in the following aspects: whole length, the number, structure and arrangement of retinacula (inner teeth), and size and shape of both the molar and incisor regions. The larval head is also asymmetric; the left half of the head capsule is larger than the right, and the left adductor muscle of the mandible is much thicker than the right. The origin and developmental process of asymmetric mandibles were traced in developing embryos whose developmental period is about 270 h and divided into 10 stages. Mandibular asymmetries are produced by the cumulative effects of six stepwise modifications that occur from about 36% of the total developmental time onward. The significance of these modifications was discussed with respect to the functional advantages of asymmetries and the phylogeny of members of the Hydrophilidae.

Evolution of female-specific wingless forms in bagworm moths.

The evolution of winglessness in insects has been typically interpreted as a consequence of developmental and other adaptations to various environments that are secondarily derived from a winged morph. Several species of bagworm moths (Insecta: Lepidoptera, Psychidae) exhibit a case-dwelling larval life style along with one of the most extreme cases of sexual dimorphism: wingless female adults. While the developmental process that led to these wingless females is well known, the origins and evolutionary transitions are not yet understood. To examine the evolutionary patterns of wing reduction in bagworm females, we reconstruct the molecular phylogeny of over 30 Asian species based on both mitochondrial (cytochrome c oxidase subunit I) and nuclear (28S rRNA) DNA sequences. Under a parsimonious assumption, the molecular phylogeny implies that: (i) the evolutionary wing reduction towards wingless females consisted of two steps: (Step I) from functional wings to vestigial wings (nonfunctional) and (Step II) from vestigial wings to the most specialized vermiform adults (lacking wings and legs); and (ii) vermiform morphs evolved independently at least twice. Based on the results of our study, we suggest that the evolutionary changes in the developmental system are essential for the establishment of different wingless forms in insects.

Ecdysteroid-induced programmed cell death is essential for sex-specific wing degeneration of the wingless-female winter moth.

The winter moth, Nyssiodes lefuarius, has a unique life history in that adults appear during early spring after a long pupal diapause from summer to winter. The moth exhibits striking sexual dimorphism in wing form; males have functional wings of normal size, whereas females lack wings. We previously found that cell death of the pupal epithelium of females appears to display condensed chromatin within phagocytes. To provide additional detailed data for interpreting the role of cell death, we performed light microscopy, transmission electron microscopy, and TUNEL assay. We consequently detected two modes of cell death, i.e., dying cells showed both DNA fragmentation derived from epithelial nuclei and autophagic vacuole formation. To elucidate the switching mechanism of sex-specific wing degeneration in females of N. lefuarius, we tested the effects of the steroid hormone 20-hydroxyecdysone (20E) on pupal diapause termination and wing morphogenesis in both sexes. When 20E (5.4 µg) was injected into both sexes within 2 days of pupation, wing degeneration started 4 days after 20E injection in females, whereas wing morphogenesis and scale formation started 6 days after 20E injection in males. We discuss two important findings: (1) degeneration of the pupal wing epithelium of females was not only due to apoptosis and phagocytotic activation but also to autophagy and epithelial cell shrinkage; and (2) 20E terminated the summer diapause of pupae, and triggered selective programmed cell death only of the female-pupal wing epithelium in the wingless female winter moth.

Fine-tuned bee-flower coevolutionary state hidden within multiple pollination interactions.

Relationships between flowers and pollinators are generally considered cases of mutualism since both agents gain benefits. Fine-tuned adaptations are usually found in the form of strict one-to-one coevolution between species. Many insect pollinators are, however, considered generalists, visiting numerous kinds of flowers, and many flower species (angiosperms) are also considered generalists, visited by many insect pollinators. We here describe a fine-tuned coevolutionary state of a flower-visiting bee that collects both nectar and pollen from an early spring flower visited by multiple pollinators. Detailed morphology of the bee proboscis is shown to be finely adjusted to the floral morphology and nectar production of the flower. Behavioral observations also confirm the precision of this mutualism. Our results suggest that a fine-tuned one-to-one coevolutionary state between a flower species and a pollinator species might be common, but frequently overlooked, in multiple flower-pollinator interactions.

In vitro effects of juvenile hormone analog on wing disc morphogenesis under ecdysteroid treatment in the female-wingless bagworm moth Eumeta variegata (Insecta: Lepidoptera, Psychidae).

Female adults of the bagworm moth, Eumeta variegata, lack wings completely, whereas male adults of this species have functional wings. We previously found that ecdysteroid induces apoptotic events in the female wing rudiment of E. variegata in vitro, whereas the male wing discs cultured with 20-hydroxyecdysone (20E) underwent apolysis and then cell differentiation. To investigate whether juvenile hormone (JH) in involved in sex-specific cellular response to ecdysteroid during wing development between sexes of E. variegata, we tested the effects of juvenile hormone analog (JHA), methoprene, and 20E on wing disc morphogenesis between sexes in vitro. Using transmission electron microscopy (TEM), we found that both higher concentration of JHA (5 µg/ml) and 20E (1 µg/ml) addition induced cell death (apoptosis) in the male wing discs but not induced cell death in the female wing rudiments in vitro in E. variegata. These culture experiments clearly detected the differential responses of wing discs to JHA under ecdysteroid treatment between sexes. We propose two important hypotheses: (1) JH is not significantly involved in the suppression of the female wing rudiment morphogenesis under 20E treatment, (2) female wing rudiment has lost the ability for cell proliferation in response to the stimulus of 20E.

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.

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 caused by ecdysteroid in the Tussock Moth, Orgyia recens: hormonal and developmental regulation of sexual dimorphism.

Females of the tussock moth Orgyia recens have vestigial wings, whereas the males have normal wings. During early pupal development, female wings degenerate drastically compared with those of males. To examine whether ecdysteroid is involved in this sex-specific wing development, we cultured pupal wings just after pupation with ecdysteroid (20-hydroxyecdysone, 20E). In the presence of 20E, the female wings degenerated to about one-fifth their original size. In contrast, the male wings cultured with 20E showed only peripheral degeneration just outside the bordering lacuna, as in other butterflies and moths. TUNEL analysis showed that apoptotic signals were induced by 20E over the entire region of female wings, but only in the peripheral region of male wings. Semi-thin sections of the wings cultured with ecdysteroid showed that phagocytotic hemocytes were observed abundantly throughout the female wings, but in only peripheral regions of male wings. These observations indicate that both apoptotic events and phagocytotic activation are triggered by ecdysteroid, in sex-specific and region-specific manners.