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.     

Modulation of the ecdysteroid-induced cell death by juvenile hormone during pupal wing development of Lepidoptera

Females of the tussock moth Orgyia recens have only vestigial wings, whereas the males have normal wings. We previously found that ecdysteroid induces both apoptotic events and phagocytotic activation in sex-specific and region-specific manners. To investigate whether different responses to ecdysteroid are controlled at the receptor level, we cloned ecdysteroid receptor isoforms, EcR-A and EcR-B1, in O. recens. In both male and female wings, EcR-A signal was detected in the distal region of the bordering lacuna (BL), whereas EcR-B1 signal was detected in the proximal region of the BL. The similar expression patterns of both EcR isoforms suggested that molecules other than EcR should be involved in different ecdysteroid responses between male and female of O. recens. We next tested juvenile hormone (JH) effects on pupal wing morphogenesis in O. recens. Interestingly, both JH and 20E addition induced wing degeneration not only in females but also in males. In addition, higher concentration of JH pre-treatment of the pupal wings of the silkworm, Bombyx mori, also caused wing degeneration under ecdysteroid treatment. These results indicate that JH modulates the ecdysteroid action to induce the cell death on pupal wings, generally in Lepidoptera.     

Ecdysteroid-induced programmed cell death and cell proliferation during pupal wing development of the silkworm, Bombyx mori

The wing margin of adult wings of Lepidoptera is defined by the position of a "bordering lacuna"(BL). During adult wing development, cell proliferation and scale formation proximal to this lacuna and programmed cell death distal to the lacuna are generally observed. To determine the effect of 20-hydroxyecdysone (20E) on these events, we cultured the silkworm pupal wings with or without 20E and analyzed regional specificity for cell death by the TUNEL method and cell proliferation by 5-bromodeoxyuridine labeling. Programmed cell death was induced by 20E after 5 days of culture and was detected only in the region distal to BL. Cell proliferation after 1 day of culture and scale formation after 5 days of culture were also inducible by 20E and detected in the region proximal to BL. These results suggest that two types of pupal wing cells, which are divided by the position of the BL, respond to ecdysteroid in different manners. Higher concentrations of 20E (more than 1,000 ng/ml) repressed the scale formation, while such repression could not be observed in the peripheral cell death even with 5,000 ng/ml 20E. The ecdysteroid may work both as a trigger to make the wing margin and scales and as a developmental timer to arrange these cellular responses.     

Morphological comparison of pupal wing cuticle patterns in butterflies

Butterfly wing color-patterns are determined in the prospective wing tissues during the late larval and early pupal stages. To study the cellular differentiation process of wings, morphological knowledge on pupal wings is prerequisite. Here we systematically examined morphological patterns of the pupal wing cuticular surface in a wide variety of nymphalid butterflies in relation to adult color-patterns. Several kinds of pupal wing patterns corresponding to particular adult color-pattern elements were widely observed in many species. Especially noteworthy were the pupal "focal" spots corresponding to the adult border ocelli system, which were detected in many species of Nymphalinae, Apaturinae, Argynninae, Satyrinae, and Danainae. Striped patterns on the pupal wing cuticle seen in some species of Limenitinae, Ariadnae, and Marpesiinae directly corresponded to those of the adult wings. In Vanessa cardui, eyespot-like pattern elements were tentatively produced during development in the wing tissue underneath the pupal spots and subsequently erased, suggesting a mechanism for producing novel color-patterns in the course of development and evolution. The pupal focal spots reasonably correlated with the adult eyespots in size in Precis orithya and Ypthima argus. We physically damaged the pupal focal spots and their corresponding cells underneath in these species, which abolished or inhibited the formation of the adult eyespots. Taken together, our results clarified that pupal cuticle patterns were often indicative of the adult color-patterns and apparently reflect molecular activity of organizing centers for the adult color-pattern formation at least in nymphalid butterflies.     

Programmed cell death at the periphery of the pupal wing of the butterfly,Pieris rapae

The outline of the adult wing of lepidopteran insects (butterflies and moths) emerges as a result of disappearance of a group of cells at the periphery of the pupal wing. Histological observation of the pupal wing of Pieris rapae showed that, just after apolysis of the wing epithelium from the pupal cuticle, there occurs a rapid and localized decrease of the number of cells at the periphery of the wing. This decrease occurs through cell death, which lasts 1–1.5 days at 20°C. Dying cells lose contact with the neighbouring cells and show condensation of chromatin and cytoplasm. They then appear to be phagocytosed by neighbouring epithelial cells or discharged through the basal surface of the epithelium into the lumen within the wing and taken up by phagocytes. Fragmentation of DNA in the nuclei was detected in the dead cells or their debris. These results indicate that programmed cell death in the lepidopteran wing proceeds through a mechanism closely similar to that of apoptosis in the vertebrate.     

Bursicon signaling mutations separate the epithelial-mesenchymal transition from programmed cell death during Drosophila melanogaster wing maturation

Following eclosion from the pupal case, wings of the immature adult fly unfold and expand to present a flat wing blade. During expansion the epithelia, which earlier produced the wing cuticle, delaminate from the cuticle, and the epithelial cells undergo an epithelial–mesenchymal transition (EMT). The resulting fibroblast-like cells then initiate a programmed cell death, produce an extracellular matrix that bonds dorsal and ventral wing cuticles, and exit the wing. Mutants that block wing expansion cause persistence of intact epithelia within the unexpanded wing. However, the normal progression of chromatin condensation and fragmentation accompanying programmed cell death in these cells proceeds with an approximately normal time course. These observations establish that the Bursicon/Rickets signaling pathway is necessary for both wing expansion and initiation of the EMT that leads to removal of the epithelial cells from the wing. They demonstrate that a different signal can be used to activate programmed cell death and show that two distinct genetic programs are in progress in these cells during wing maturation.     

Localized cell death in Drosophila imaginal wing disc epithelium caused by the mutation apterous-blot

Drosophila melanogaster carrying the mutation apterous-blot have blistered wings. Trypan blue stains a patch of dead cells localized to the wing pouch of imaginal discs and the same area shows acid phosphatase (AcPase) activity suggesting that the cell death is lysosomal. Autophagic vacuoles and other secondary lysosomes show AcPase activity within the disc epithelium and enzyme activity is found in fragments of dead cells which have been extruded basally. The cell death, although extensive and confined to the presumptive wing region, does not result in loss of adult structures.     

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.     

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.     

Ultrastructure of cell death in gamma- or X-irradiated imaginal wing discs of Drosophila

When Drosophila larvae were irradiated with 1300-1500 R of gamma rays both apoptotic and necrotic cell death were observed in imaginal wing discs. The ultrastructure of cell death by apoptosis was characterized by fragmentation of dead cells into highly condensed, membrane-bound particles. The ultrastructure of cell death by necrosis was characterized by cell lysis and organelle degeneration. Marked contrast was also seen in the distribution of the two types of cell death: apoptosis was universal in irradiated discs and affected widely distributed single cells, or small groups of cells, whereas necrosis formed lesions by afflicting large numbers of contiguous cells. It was noted that even where there were large lesions in the epithelial cell layer, which is the primary component of imaginal discs, the basement membrane associated with this epithelium always remained intact. Lesions could be identified in freshly extirpated discs by staining with trypan blue and were found in 50-70% of irradiated discs (depending on the larval age at the time of irradiation). Lesions were seen in all regions of the wing disc and varied greatly in size. In spite of extensive necrotic cell death wing discs developed into normal adult wings. Regenerative growth in this case would appear to require significant reorganization of cells. Implications of this for the appropriate interpretation of clonal analysis are discussed.     

Butterfly Wings Are Three-Dimensional: Pupal Cuticle Focal Spots and Their Associated Structures in Junonia Butterflies

Butterfly wing color patterns often contain eyespots, which are developmentally determined at the late larval and early pupal stages by organizing activities of focal cells that can later form eyespot foci. In the pupal stage, the focal position of a future eyespot is often marked by a focal spot, one of the pupal cuticle spots, on the pupal surface. Here, we examined the possible relationships of the pupal focal spots with the underneath pupal wing tissues and with the adult wing eyespots using Junonia butterflies. Large pupal focal spots were found in two species with large adult eyespots, J. orithya and J. almana, whereas only small pupal focal spots were found in a species with small adult eyespots, J. hedonia. The size of five pupal focal spots on a single wing was correlated with the size of the corresponding adult eyespots in J. orithya. A pupal focal spot was a three-dimensional bulge of cuticle surface, and the underside of the major pupal focal spot exhibited a hollowed cuticle in a pupal case. Cross sections of a pupal wing revealed that the cuticle layer shows a curvature at a focal spot, and a positional correlation was observed between the cuticle layer thickness and its corresponding cell layer thickness. Adult major eyespots of J. orithya and J. almana exhibited surface elevations and depressions that approximately correspond to the coloration within an eyespot. Our results suggest that a pupal focal spot is produced by the organizing activity of focal cells underneath the focal spot. Probably because the focal cell layer immediately underneath a focal spot is thicker than that of its surrounding areas, eyespots of adult butterfly wings are three-dimensionally constructed. The color-height relationship in adult eyespots might have an implication in the developmental signaling for determining the eyespot color patterns.     

Color pattern formation on the wing of a butterfly Pieris rapae. 2. Color determination and scale development

It has been shown that microcautery on the prospective apical black region of the early pupal forewing of a butterfly, Pieris rapae , causes alteration of the scale color on the adult wing and a delay in histogenesis of the pupal wing. From these results, it has been assumed that the developmental delay of scale cells in the pupal wing alters their developmental fate and the hypothesis that different color fates of scales are determined by differences in the developmental timetables between scale cells is proposed. In this study, we attempted to find the developmental timetables of individual scales expressing specific color to test this hypothesis. It was found that the holes on the upper surface of a scale become larger as they develop and the hole sizes of scales in the white region are always larger than in the black region on the same wings either during pupal period or after eclosion. This suggests that the scale hole size is a good index that reflects developmental rate of the scale and a difference in the hole size between adult scales is attributed to a difference in the developmental timetables when their ancestral scale precursor cells were in the pupal period. A comparison of the hole sizes between adult scales in different color regions suggested that normal white scales were in a more advanced state than were the black ones but white scales induced by microcautery were in a less advanced state than black ones on the same wing. This supports our hypothesis.     

Effect of different larval rearing temperatures on the productivity (Ro) and morphology of the malaria vector Anopheles superpictus Grassi (Diptera: Culicidae) using geometric morphometrics

Temperature affects both the biology and morphology of mosquito vectors. Geometric morphometrics is a useful new tool for capturing and analyzing differences in shape and size in many morphological parameters, including wings. We have used this technique for capturing the differences in the wings of the malaria vector Anopheles superpictus, using cohorts reared at six different constant temperatures (15°, 20°, 25°, 27°, 30°, and 35° C) and also searched for potential correlations with the life tables of the species. We studied wing shape in both male and female adults, using 22 landmarks on the wing in relation to ecological parameters, including the development rate. The ecological zero was calculated as 9.93° C and the thermal constant as 296.34 day‐degrees. The rearing temperature affects egg, larval, and pupal development and also the total time from egg to adult. As rearing temperatures increased, longevity decreased in both sexes. In An. superpictus, R_o value and productivity correlated with the statistically significant gradual deformations in the wing shape related to size in both sexes. These deformations directly linked to differences in immature rearing temperatures. Analysis using PCA and UPGMA phenograms showed that although wings of females became narrower dorsoventrally as the temperature increased, they became broader in males. Comparisons of the wing landmarks indicated the medial part of the wing was most affected by larval rearing temperatures, showing relatively more deformations. Algorithmic values of the life tables were determined in correlation with the results of geometric morphometrics. Comparisons of centroid sizes in the cohorts showed that overall wing size became smaller in both sexes in response to higher rearing temperatures.     

Effect of heat shock,pretreatment and hsp70 copy number on wing development in Drosophila melanogaster

Naturally occurring heat shock (HS) during pupation induces abnormal wing development in Drosophila; we examined factors affecting the severity of this induction. The proportion of HS-surviving adults with abnormal wings varied with HS duration and intensity, and with the pupal age or stage at HS administration. Pretreatment (PT), mild hyperthermia delivered before HS, usually protected development against HS. Gradual heating resembling natural thermal regimes also protected wing development against thermal disruption. Because of the roles of the wings in flight and courtship and in view of natural thermal regimes that Drosophila experience, both HS-induction of wing abnormalities and its abatement by PT may have marked effects on Drosophila fitness in nature. Because PT is associated with expression of heat-inducible molecular chaperones such as Hsp70 in Drosophila, we compared thermal disruption of wing development among hsp70 mutants as well as among strains naturally varying in Hsp70 levels. Contrary to expectations, lines or strains with increased Hsp70 levels were no more resistant to HS-disruption of wing development than counterparts with lower Hsp70 levels. In fact, wing development was more resistant to HS in hsp70 deletion strains than control strains. We suggest that, while high Hsp70 levels may aid cells in surviving hyperthermia, high levels may also overly stimulate or inhibit numerous signalling pathways involved in cell proliferation, maturation and programmed death, resulting in developmental failure.     

Live Cell Imaging of Butterfly Pupal and Larval Wings In Vivo

Butterfly wing color patterns are determined during the late larval and early pupal stages. Characterization of wing epithelial cells at these stages is thus critical to understand how wing structures, including color patterns, are determined. Previously, we successfully recorded real-time in vivo images of developing butterfly wings over time at the tissue level. In this study, we employed similar in vivo fluorescent imaging techniques to visualize developing wing epithelial cells in the late larval and early pupal stages 1 hour post-pupation. Both larval and pupal epithelial cells were rich in mitochondria and intracellular networks of endoplasmic reticulum, suggesting high metabolic activities, likely in preparation for cellular division, polyploidization, and differentiation. Larval epithelial cells in the wing imaginal disk were relatively large horizontally and tightly packed, whereas pupal epithelial cells were smaller and relatively loosely packed. Furthermore, larval cells were flat, whereas pupal cells were vertically elongated as deep as 130 μm. In pupal cells, many endosome-like or autophagosome-like structures were present in the cellular periphery down to approximately 10 μm in depth, and extensive epidermal feet or filopodia-like processes were observed a few micrometers deep from the cellular surface. Cells were clustered or bundled from approximately 50 μm in depth to deeper levels. From 60 μm to 80 μm in depth, horizontal connections between these clusters were observed. The prospective eyespot and marginal focus areas were resistant to fluorescent dyes, likely because of their non-flat cone-like structures with a relatively thick cuticle. These in vivo images provide important information with which to understand processes of epithelial cell differentiation and color pattern determination in butterfly wings.     

Uric acid metabolism during pupal-adult development of Pieris brassicae

Uric acid metabolism has been investigated during the pupal and adult stages of Pieris brassicae. Uric acid and its main metabolite, allantoic acid, have been quantified in various organs (fat body, gut, wings) during development, in order to determine synthesis, degradation, and transport phenomena. Both labelling experiments (using 2-¹⁴C uric acid, guanine, and guanosine) and enzymatic studies (xanthine dehydrogenase, guanine deaminase, and uricase) were performed.Labelled uric acid, when injected into a young pupa, accumulates preferentially into the fat body, and its degradation leads to an increase in allantoic acid, which is found chiefly in imaginal structures (wings, heads, body wall). Since uricase is present only in low levels through the pupal stage, only a small fraction of uric acid is metabolized.In the developing pharate adult, uric acid is transported via the haemolymph from fat body to the wings and gut. Male wings accumulate more uric acid than female wings. At emergence, a large amount of uric acid and most of the allantoic acid are excreted into the meconium, but not together; uric acid is excreted into the so-called ‘meconium 1’ containing ommochromes, whereas its metabolite is eliminated only after wing expansion into ‘meconium 2’, a colourless fluid. Shortly before emergence, the fat body recovers its ability to synthesize uric acid, a fraction of which is excreted within ‘meconium 1’.During adult life, the synthesis of uric acid occurs in the fat body and ovaries, where it is especially abundant. Ageing organs (wings, heads, testes) accumulate it markedly. A small fraction is excreted together with allantoic acid by the butterfly.Purine catabolism pathways have been investigated, showing that in guanine derivatives, the freebase state of guanine leads quickly to uric acid (and its metabolites), whereas ¹⁴C-guanosine may be transformed into nucleotide and incorporated efficiently into wing pteridines when it is injected at the time of adult pigmentation.Another purine derivative, identified as adenosine, has been shown to accumulate in male fat body just before adult emergence. Its amount increases during the first days of emerged adult life, and it corresponds to an alternative pathway of purine catabolism. Its absence in females is related to development of the ovaries.     

Color pattern formation on the wing of the butterfly Pieris rapae. 1. Cautery induced alteration of scale color and delay of arrangement formation

Experimental approaches to color pattern formation of lepidopteran insects have been made exclusively by analyzing pattern alterations in adult wings induced by operations. We microcauterized the presumptive black region of the dorsal forewing of the butterfly Pieris rapae and analyzed not only the resultant color pattern in the adult wing but also the cell behavior in the pupal wing epidermis around the injury. Cautery induced color alterations were as follows: (i) cautery up to 49.5 h after pupation resulted in white regions appearing within the black region while later cauteries induced larger white regions; (ii) cautery between 50 and 59.5 h resulted in the white regions induced by the cauteries being dramatically decreased; (iii) cautery after 60 h resulted in white regions that had almost disappeared. The examination of the cell behavior in the pupal wing epidermis after cauteries showed that the row formation of scale precursor cells was delayed. This delayed area varied with the time of cautery, in the same manner as that in the induced white area in the adult wing ((i) – (iii) above). The relationship between scale color alteration and the developmental delay of the scale row formation is discussed.     

Morphology of wing mechanoreceptors involved in the wing retraction reflex in the pteropod mollusc Clione limacina

Tactile stimulation of the wings (parapodia) of actively swimming Clione limacina results in inhibition of swimming and retraction of the wings. Electrophysiological evidence suggests that wing mechanoreceptors have central cell bodies and wide innervation fields in the ipsilateral wing. Scanning electron microscopy of expanded wings reveals ciliary cone processes arranged in a pattern that is similar to the electrophysiologically‐determined innervation fields of wing mechanoreceptors. Transmission electron microscopy suggests that the ciliary cone structures are terminal processes of neuron‐like cells. Three‐dimensional reconstructions of serially‐sectioned terminal processes indicate that cell bodies are not found in the wing epithelium or immediately under the epithelium, further supporting the notion that the wing mechanoreceptors have central cell bodies.