Real-Time In Vivo Imaging of Butterfly Wing Development: Revealing the Cellular Dynamics of the Pupal Wing Tissue

Butterfly wings are covered with regularly arranged single-colored scales that are formed at the pupal stage. Understanding pupal wing development is therefore crucial to understand wing color pattern formation. Here, we successfully employed real-time in vivo imaging techniques to observe pupal hindwing development over time in the blue pansy butterfly, Junonia orithya. A transparent sheet of epithelial cells that were not yet regularly arranged was observed immediately after pupation. Bright-field imaging and autofluorescent imaging revealed free-moving hemocytes and tracheal branches of a crinoid-like structure underneath the epithelium. The wing tissue gradually became gray-white, epithelial cells were arranged regularly, and hemocytes disappeared, except in the bordering lacuna, after which scales grew. The dynamics of the epithelial cells and scale growth were also confirmed by fluorescent imaging. Fluorescent in vivo staining further revealed that these cells harbored many mitochondria at the surface of the epithelium. Organizing centers for the border symmetry system were apparent immediately after pupation, exhibiting a relatively dark optical character following treatment with fluorescent dyes, as well as in autofluorescent images. The wing tissue exhibited slow and low-frequency contraction pulses with a cycle of approximately 10 to 20 minutes, mainly occurring at 2 to 3 days postpupation. The pulses gradually became slower and weaker and eventually stopped. The wing tissue area became larger after contraction, which also coincided with an increase in the autofluorescence intensity that might have been caused by scale growth. Examination of the pattern of color development revealed that the black pigment was first deposited in patches in the central areas of an eyespot black ring and a parafocal element. These results of live in vivo imaging that covered wide wing area for a long time can serve as a foundation for studying the cellular dynamics of living wing tissues in butterflies.

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title: Elements of butterfly wing patterns.
abstract: The color patterns on the wings of butterflies are unique among animal color patterns in that the elements that make up the overall pattern are individuated. Unlike the spots and stripes of vertebrate color patterns, the elements of butterfly wing patterns have identities that can be traced from species to species, and typically across genera and families. Because of this identity it is possible to recognize homologies among pattern elements and to study their evolution and diversification. Individuated pattern elements evolved from non-individuated precursors by compartmentalization of the wing into areas that became developmentally autonomous with respect to color pattern formation. Developmental compartmentalization led to the evolution of serially repeated elements and the emergence of serial homology. In these compartments, serial homologues were able to acquire site-specific developmental regulation and this, in turn, allowed them to diverge morphologically. Compartmentalization of the wing also reduced the developmental correlation among pattern elements. The release from this developmental constraint, we believe, enabled the great evolutionary radiation of butterfly wing patterns. During pattern evolution, the same set of individual pattern elements is arranged in novel ways to produce species-specific patterns, including such adaptations as mimicry and camouflage.

title: Color Pattern Analysis of Nymphalid Butterfly Wings: Revision of the Nymphalid Groundplan
abstract: To better understand the developmental mechanisms of color pattern variation in butterfly wings, it is important to construct an accurate representation of pattern elements, known as the “nymphalid groundplan”. However, some aspects of the current groundplan remain elusive. Here, I examined wing-wide elemental patterns of various nymphalid butterflies and confirmed that wing-wide color patterns are composed of the border, central, and basal symmetry systems. The central and basal symmetry systems can express circular patterns resembling eyespots, indicating that these systems have developmental mechanisms similar to those of the border symmetry system. The wing root band commonly occurs as a distinct symmetry system independent from the basal symmetry system. In addition, the marginal and submarginal bands are likely generated as a single system, referred to as the “marginal band system”. Background spaces between two symmetry systems are sometimes light in coloration and can produce white bands, contributing significantly to color pattern diversity. When an element is enlarged with a pale central area, a visually similar (yet developmentally distinct) white band is produced. Based on the symmetric relationships of elements, I propose that both the central and border symmetry systems are comprised of “core elements” (the discal spot and the border ocelli, respectively) and a pair of “paracore elements” (the distal and proximal bands and the parafocal elements, respectively). Both core and paracore elements can be doubled, or outlined. Developmentally, this system configuration is consistent with the induction model, but not with the concentration gradient model for positional information.

title: Scale Arrangement Pattern in a Lepidopteran Wing. 1. Periodic Cellular Pattern in the Pupal Wing of Pieris rapae
abstract: In most species of lepidopteran insects, anteroposterior rows formed by scales are arranged at regular intervals in the adult wing; within each row two kinds of scales are alternately arranged. To investigate the cellular basis for the scale arrangement pattern, we examined cell arrangement in the epidermal monolayer of the pupal wing of a small white cabbage butterfly, Pieris rapae, by scanning electron microscopy and light microscopy.

title: Pattern formation on lepidopteran wings: determination of an eyespot.
abstract: Color patterns on lepidopteran wings are believed to be organized around a series of hypothetical foci. Foci presumably serve as sources of positional information, directing synthesis of appropriate pigments in their vicinity. Species-specific color patterns arise by variations in the number of foci on the wing and variations in the rules by which positional information is interpreted. The present paper provides experimental evidence for the existence of a focus that determines the large eyespot on the forewing of the Buckeye butterfly,Precis coenia. Cautery of a small group of about 300 cells at the center of the presumptive eyespot, early during wing development, can completely inhibit development of the eyespot. The same group of cells can be transplanted to another region of the wing and induce ring-shaped pigment patterns in the host tissue around the graft. These findings show that a focus is a physiological entity. Color pattern induction is an intrinsic property of a small group of cells. Presumably these cells produce a factor that affects the production of enzymes involved in pigment synthesis.

title: Cautery-induced colour patterns in Precis coenia (Lepidoptera: Nymphalidae).
abstract: Cautery of the dorsal hindwing in the butterfly, Precis coenia, induces the formation of a concentric colour pattern around the site of injury. The induced pattern is identical in pigmentation to the eyespots that normally develop on this wing surface. This response to cautery also occurs, though much less dramatically, on the ventral forewing. In addition to the peculiar response to cautery, the dorsal hindwing of Precis also develops a series of unique pattern aberrations in response to coldshock. These consist of irregular elongation of the anterior eyespot along the proximodistal axis of the wing. In the most dramatic aberrations the eyespot field covers the entire anterior half of the wing surface. An analysis is presented that attempts to reconcile the effects of cautery on the Precis hindwing with the very different morphological effects of cautery on the colour pattern of Ephestia kühniella, described by Kühn & Von Engelhardt. Computer simulations reveal that the finding presented in this paper, as well as the classical work on Ephestia, can both be explained by assuming that the site of cautery becomes a sink for one of the morphogens involved in colour pattern determination. The experimental findings furthermore indicate that minor perturbations of the wing epidermis can evoke the physiological conditions that attend normal eyespot determination. It is shown that this interpretation also helps to explain the unusual pattern modifications following coldshock.

title: The development of eyespot patterns on butterfly wings : morphogen sources or sinks?
abstract: We have studied the development of the eyespot colour pattern on the adult dorsal forewing of the nymphalid butterflies, Bicyclus safitza and B. anynana, by cauterising the presumptive eyespot centres (the foci) on the pupal wing. The effects on pattern depended on age at cautery. Early focal cautery (at 1–12 hours after pupation) usually reduced or eliminated the eyespot, while cautery at a non-focal site usually had no effect. These results resemble those of a previous study on another species but, in addition, we find that a later cautery (at 12–24 hours) had the converse effect of generating pattern, so that focal cautery enlarged the anterior eyespot (but usually not the large posterior eyespot) and non-focal cautery induced a new ectopic eyespot. The effects of cautery on patterning are more extensive by an order of magnitude than the cell death which is caused, so implicating a long-range mechanism, such as a morphogen gradient, in eyespot development. The focus clearly acts to establish the normal eyespot pattern, but a simple source/diffusion model is not supported by the response to late cautery. We suggest two alternative forms of gradient model in which late damage can mimic and augment the action of a focus. In the Source/Threshold model, the focus is a morphogen source, and cautery can remove the focus but also transiently lowers the response threshold in surrounding cells. In the Sink model, the focus generates the gradient by removing morphogen, and cautery can eliminate the focus but it also causes a transient destruction or leakage of morphogen. These models can explain most features of the results of cautery.

title: Eyespot development on butterfly wings: the epidermal response to damage.
abstract: Eyespot colour patterns decorate the wings of many butterfly species. The eyespot is specified in the early pupal epidermis by signals from a central "focus," and it has been suggested that the focus is the source of a diffusible morphogen gradient. We show that ectopic eyespots can be induced in nonfocal positions throughout the distal, but not the proximal, wing epidermis of Bicyclus anynana by mild epidermal damage inflicted at 12-18 hr (into a 6- to 7-day pupal period). Damage may lower, locally and transiently, the threshold for response to morphogen. Here, we have tested two predictions of the gradient model. As predicted, mild damage close to the focus (parafocal) locally extends the eyespot, making it markedly asymmetrical, even after early operations (1 or 6 hr), when remote epidermis is non-responsive. Early parafocal operations also have an unexpected result, however, reducing the extent of the eyespot in other directions, perhaps through a long-range "wound effect" on the signaling activity of the focal cells. The model also correctly predicts that increasing the severity of a nonfocal operation will prolong the transient damage and hence give ectopic eyespots after early (1- or 6-hr) operations. We do not, however, find the expected size increase in ectopics induced by the later (12 or 18 hr) severe damage. Similarly, we demonstrate the predicted effect of early (1 or 6 hr) mild damage in increasing the response to a subsequent (18 hr) operation at the same position. The gradient model is therefore supported by most aspects of eyespot induction in response to epidermal damage.

title: Single locus affects embryonic segment polarity and multiple aspects of an adult evolutionary novelty
abstract: BackgroundThe characterization of the molecular changes that underlie the origin and diversification of morphological novelties is a key challenge in evolutionary developmental biology. The evolution of such traits is thought to rely largely on co-option of a toolkit of conserved developmental genes that typically perform multiple functions. Mutations that affect both a universal developmental process and the formation of a novelty might shed light onto the genetics of traits not represented in model systems. Here we describe three pleiotropic mutations with large effects on a novel trait, butterfly eyespots, and on a conserved stage of embryogenesis, segment polarity.ResultsWe show that three mutations affecting eyespot size and/or colour composition in Bicyclus anynana butterflies occurred in the same locus, and that two of them are embryonic recessive lethal. Using surgical manipulations and analysis of gene expression patterns in developing wings, we demonstrate that the effects on eyespot morphology are due to changes in the epidermal response component of eyespot induction. Our analysis of morphology and of gene expression in mutant embryos shows that they have a typical segment polarity phenotype, consistent with the mutant locus encoding a negative regulator of Wingless signalling.ConclusionsThis study characterizes the segregation and developmental effects of alleles at a single locus that controls the morphology of a lineage-specific trait (butterfly eyespots) and a conserved process (embryonic segment polarity and, specifically, the regulation of Wingless signalling). Because no gene with such function was found in the orthologous, highly syntenic genomic regions of two other lepidopterans, we hypothesize that our locus is a yet undescribed, possibly lineage-specific, negative regulator of the conserved Wnt/Wg pathway. Moreover, the fact that this locus interferes with multiple aspects of eyespot morphology and maps to a genomic region containing key wing pattern loci in different other butterfly species suggests it might correspond to a 'hotspot' locus in the diversification of this novel trait.

title: Tungstate-Induced Color-Pattern Modifications of Butterfly Wings are Independent of Stress Response and Ecdysteroid Effect
abstract: Systemic injections of sodium tungstate, a protein-tyrosine phosphatase (PTPase) inhibitor, to pupae immediately after pupation have been shown to efficiently produce characteristic color-pattern modifications on the wings of many species of butterflies. Here we demonstrated that the tungstate-induced modification pattern was entirely different from other chemically-induced ones in a species of nymphalid butterfly Junonia (Precis) orithya. In this species, the systemic injections of tungstate produced characteristic expansion of black area and shrinkage of white area together with the move of parafocal elements toward the wing base. Overall, pattern boundaries became obscure. In contrast, an entirely different modification pattern, overall darkening of wings, was observed by the injections of stress-inducing chemicals, thapsigargin, ionomycin, or geldanamycin, to pupae under the rearing conditions for the adult summer form. On the ventral wings, this darkening was due to an increase of the proportion of peppered dark scales, which was reminiscent of the natural fall form of this species. Under the same rearing conditions, the injections of ecdysteroid, which is a well-known hormone being responsible for the seasonal polyphenism of nymphalid butterflies, yielded overall expansion of orange area especially around eyespots. Taken together, we conclude that the tungstate-induced modifications are clearly distinguishable from those of stress response and ecdysteroid effect. This conclusion then suggests that the putative PTPase signaling pathway that is sensitive to tungstate uniquely contributes to the wing-wide color-pattern development in butterflies.

title: Physiological characterization of the cold-shock-induced humoral factor for wing color-pattern changes in butterflies.
abstract: Butterfly wing color patterns can be modified by the application of temperature shock to pupae immediately after pupation, which has been attributed to a cold-shock-induced humoral factor called cold-shock hormone (CSH). Here, we physiologically characterized CSH and pharmacological action of tungstate, using a nymphalid butterfly Junonia orithya. We first showed that the precise patterns of modification were dependent on the time-point of the cold-shock treatment after pupation, and confirmed that the modification properties induced in a cold-shocked pupa were able to be transferred to another pupa in a parabiosis experiment. Cold-shock application after removal of the head and prothorax together still produced modified wings, excluding major involvement of the brain-retrocerebral neuroendocrine complex. Furthermore, tungstate injection induced modifications even in individuals whose head and prothorax were removed. Importantly, transplantation of tracheae isolated from cold-shocked pupae induced modifications in the recipient wings. We identified a chemical peak in hemolymph of the cold-shocked individuals using HPLC, which corresponded to dopamine, and demonstrated that dopamine and its related biogenic amines have ability to induce small color-pattern changes. Taken together, the present study suggests that CSH is likely to be secreted from trachea-associated endocrine cells upon cold-shock treatment and that tungstate may change color patterns via its direct action on wings.

title: Larval temperature experience determines sensitivity to cold-shock-induced wing color pattern changes in the blue pansy butterfly Junonia orithya
abstract: The butterfly wing color patterns are unique to a species but are modified in response to cold-shock and tungstate treatments at the pupal stage, producing characteristic temperature–shock (TS) phenotypes that are distinct from the color patterns of seasonal polyphenism. In this study, we examined the efficiency of cold-shock and tungstate treatments for color pattern modifications at the pupal stage in relation to larval rearing conditions for the fall or summer morph using the blue pansy butterfly Junonia orithya. We found that larvae reared under the low-temperature condition that induces the fall morph exhibited hardiness against the color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. When larvae were fed an artificial diet containing tungstate under the high-temperature condition that induces the summer morph, they were still vulnerable to color pattern changes imposed by cold-shock or tungstate treatment at the pupal stage. Furthermore, larvae reared under the high-temperature condition were subjected to cold-shock or tungstate treatments at the pupal stage. In addition to the expected TS-type changes, these individuals exhibited a reduced number of eyespots in adults, which is a feature of the fall morph. These results suggest that the temperature condition experienced by the larvae, but not their consumption of tungstate, determines the sensitivity of the wing imaginal discs to cold-shock and tungstate treatments at the pupal stage.

title: Reversed type of color-pattern modifications of butterfly wings: a physiological mechanism of wing-wide color-pattern determination.
abstract: Application of cold shock or tungstate to butterfly pupae produces a unique color-pattern modification type on the adult wings, in which the color-pattern elements are dislocated toward the reduced focal elements. This modification-inducing activity has been primarily attributed to the putative cold-shock hormone (CSH) that is secreted into the hemolymph upon cold shock. Here, using a species of nymphalid butterfly Junonia almana, a new "reversed" type of the color-pattern modifications of butterfly wings was obtained by the application of heat shock or thapsigargin, a calcium-ATPase inhibitor, in which most elements were dislocated away from the enlarged focal elements. This result suggests that the endocrine secretion of CSH is sensitive to a wide range of temperature shocks, which then affects the cellular interpretation of the wing-wide positional information that is emitted from the focal locations. Ecdysteroid contributes to the wing-wide patterning primarily independently from CSH, but these two systems negatively interact with each other, probably in the intracellular signaling pathways.

title: Structural analysis of eyespots: dynamics of morphogenic signals that govern elemental positions in butterfly wings
abstract: BackgroundTo explain eyespot colour-pattern determination in butterfly wings, the induction model has been discussed based on colour-pattern analyses of various butterfly eyespots. However, a detailed structural analysis of eyespots that can serve as a foundation for future studies is still lacking. In this study, fundamental structural rules related to butterfly eyespots are proposed, and the induction model is elaborated in terms of the possible dynamics of morphogenic signals involved in the development of eyespots and parafocal elements (PFEs) based on colour-pattern analysis of the nymphalid butterfly Junonia almana.ResultsIn a well-developed eyespot, the inner black core ring is much wider than the outer black ring; this is termed the inside-wide rule. It appears that signals are wider near the focus of the eyespot and become narrower as they expand. Although fundamental signal dynamics are likely to be based on a reaction-diffusion mechanism, they were described well mathematically as a type of simple uniformly decelerated motion in which signals associated with the outer and inner black rings of eyespots and PFEs are released at different time points, durations, intervals, and initial velocities into a two-dimensional field of fundamentally uniform or graded resistance; this produces eyespots and PFEs that are diverse in size and structure. The inside-wide rule, eyespot distortion, structural differences between small and large eyespots, and structural changes in eyespots and PFEs in response to physiological treatments were explained well using mathematical simulations. Natural colour patterns and previous experimental findings that are not easily explained by the conventional gradient model were also explained reasonably well by the formal mathematical simulations performed in this study.ConclusionsIn a mode free from speculative molecular interactions, the present study clarifies fundamental structural rules related to butterfly eyespots, delineates a theoretical basis for the induction model, and proposes a mathematically simple mode of long-range signalling that may reflect developmental mechanisms associated with butterfly eyespots.

title: Morphological Comparison of Pupal Wing Cuticle Patterns in Butterflies
abstract: 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.

title: Local Pharmacological Effects of Tungstate on the Color-Pattern Determination of Butterfly Wings: A Possible Relationship Between the Eyespot and Parafocal Element
abstract: Butterfly wing color patterns can be changed by the application of a temperature shock or pharmacological agents such as tungstate, producing a distinctive type of elemental modification called the TS (temperature shock) type. Heterochronic uncoupling between the signaling and reception steps during the color-pattern determination process has been proposed as a mechanism for TS-type changes. As an extension of this hypothesis, both the parafocal element (PFE) and the eyespot in the same wing compartment are considered to be determined by morphogenic signal(s) emitted from the same eyespot focus. However, these models need to be examined with additional experimental data. Furthermore, there is controversy as to whether the action of tungstate on wing color patterns is direct or indirect. Using a species of nymphalid butterfly (Junonia orithya), we have devised a simple method for the local application of pharmacological agents directly on developing wings of pupae. Local tungstate application resulted in reduced eyespots and circular dislocated PFEs in the eyespot-less compartments only on the treated wing, demonstrating that tungstate directly induces color-pattern changes on wings. We further examined the eyespot-PFE relationship in normal and cold-shocked individuals, showing that an eyespot can be superimposed on a PFE and vice versa, probably depending on the timing of their fate determination. Taken together, we propose a two-morphogen model for the normal color-pattern determination, in which the morphogenic signals for the eyespot and PFE are different from each other despite their identical origin. This two-morphogen model is compatible with the heterochronic uncoupling model for TS-type changes.

title: Color‐pattern analysis of parafocal elements in butterfly wings
abstract: Butterfly wing color‐patterns are composed of anatomically identifiable pattern elements, whose positional relations are idealized in the “nymphalid groundplan”. But the status of the parafocal element and submarginal band (which are collectively called “the peripheral elements” for convenience) in this groundplan has been ambiguous. Herein, I descriptively analyze homologous color‐patterns of the parafocal elements in the representative nymphalid butterflies. Cases are presented where the distal and proximal peripheral elements are symmetric on the eyespot focal axis, they are continuous around eyespots, and a given parafocal element shows a concentric pattern. Multi‐compartmental comparison of the consecutive parafocal elements on a given wing surface indicates that the relative locations of the parafocal elements can correlate with the eyespot size and that a peripheral part of an eyespot can be “disconnected” from its focal part. These cases suggest the fundamental difficulty in unequivocally distinguishing the parafocal element from the eyespot at least in some nymphalid species. Taken together, it appears that the parafocal element is developmentally regulated by the organizing activity of the prospective eyespot focus, and thus they are to be considered as a part of the border symmetry system. However, the parafocal element also seems to be organized by its own focus that acts as the secondary organizing center at least in some species. These conclusions based on the natural color‐patterns are in good agreement with those based on the physiologically‐induced color‐pattern changes.

title: Generation of Butterfly Wing Eyespot Patterns: A Model for Morphological Determination of Eyespot and Parafocal Element
abstract: The determination of color patterns of butterfly wing eyespots has been explained by the morphogen concentration gradient model. The induction model has been proposed recently as a more realistic alternative, in which the eyespot-specifying signal does not depend entirely on focal activity. However, this model requires further elaboration and supporting evidence to be validated. Here, I examined various color patterns of nymphalid butterflies to propose the mechanics of the induction model. Based on cases in which an eyespot light ring is identical to the background in color, I propose that eyespots are fundamentally composed of dark rings and non-dark “background” spaces between them. In the induction model, the dark-ring-inducing signal that is released from a prospective eyespot focus (the primary organizing center) as a slow-moving wave effects both selfenhancement and peripheral induction of the dark-ring-inhibitory signal at the secondary organizing centers, resulting in an eyespot that has alternate dark and light rings. Moreover, there are cases in which an unseen “imaginary light ring” surrounds an eyespot proper and in which PFEs are integrated into the eyespot. It appears that PFEs constitute a periodic continuum of eyespot dark rings; thus, a background space between the eyespot and a PFE is mechanistically equivalent to eyespot light rings. The eyespot dark-ring-inducing signals and PFE-inducing signal are likely to be identical in quality, but released at different times from the same organizing center. Computer simulations based on the reaction-diffusion system support the feasibility of the induction model.

title: Pattern formation and eyespot determination in butterfly wings.
abstract: Butterfly wings display pattern elements of many types and colors. To identify the molecular processes underlying the generation of these patterns, several butterfly cognates of Drosophila appendage patterning genes have been cloned and their expression patterns have been analyzed. Butterfly wing patterns are organized by two spatial coordinate systems. One system specifies positional information with respect to the entire wing field and is conserved between fruit flies and butterflies. A second system, superimposed on the general system and involving several of the same genes, operates within each wing subdivision to elaborate discrete pattern elements. Eyespots, which form from discrete developmental organizers, are marked by Distal-less gene expression. These circular pattern elements appear to be generated by a process similar to, and perhaps evolved from, proximodistal pattern formation in insect appendages.

title: Development, plasticity and evolution of butterfly eyespot patterns
abstract: The developmental and genetic bases for the formation, plasticity and diversity of eyespot patterns in butterflies are examined. Eyespot pattern mutants, regulatory gene expression, and transplants of the eyespot developmental organizer demonstrate that eyespot position, number, size and colour are determined progressively in a developmental pathway largely uncoupled from those regulating other wing-pattern elements and body structures. Species comparisons and selection experiments suggest that the evolution of eyespot patterns can occur rapidly through modulation of different stages of this pathway, and requires only single, or very few, changes in regulatory genes.

title: Contribution of Distal-less to quantitative variation in butterfly eyespots
abstract: The colour patterns decorating butterfly wings provide ideal material to study the reciprocal interactions between evolution and development. They are visually compelling products of selection, often with a clear adaptive value, and are amenable to a detailed developmental characterization. Research on wing-pattern evolution and development has focused on the eyespots of the tropical butterfly Bicyclus anynana. There is quantitative variation for several features of eyespot morphology but the actual genes contributing to such variation are unknown. On the other hand, studies of gene expression patterns in wing primordia have implicated different developmental pathways in eyespot formation. To link these two sets of information we need to identify which genes within the implicated pathways contribute to the quantitative variation accessible to natural selection. Here we begin to bridge this gap by demonstrating linkage between DNA polymorphisms in the candidate gene Distal-less (Dll) and eyespot size in B. anynana.

title: Butterfly Wing Pattern Evolution Is Associated with Changes in a Notch/Distal-less Temporal Pattern Formation Process
abstract: In butterflies there is a class of "intervein" wing patterns that have lines of symmetry halfway between wing veins. These patterns occur in a range of shapes, including eyespots, ellipses, and midlines, and were proposed to have evolved through developmental shifts along a midline-to-eyespot continuum. Here we show that Notch (N) upregulation, followed by activation of the transcription factor Distal-less (Dll), is an early event in the development of eyespot and intervein midline patterns across multiple species of butterflies. A relationship between eyespot phenotype and N and Dll expression is demonstrated in a loss-of-eyespot mutant in which N and Dll expression is reduced at missing eyespot sites. A phylogenetic comparison of expression time series from eight moth and butterfly species suggests that intervein N and Dll patterns are a derived characteristic of the butterfly lineage. Furthermore, prior to eyespot determination in eyespot-bearing butterflies, N and Dll are transiently expressed in a pattern that resembles ancestral intervein midline patterns. In this study we establish N upregulation as the earliest known event in eyespot determination, demonstrate gene expression associated with intervein midline color patterns, and provide molecular evidence that wing patterns evolved through addition to and truncation of a conserved midline-to-eyespot pattern formation sequence.

title: Comparative insights into questions of lepidopteran wing pattern homology
abstract: BackgroundButterfly and moth eyespots can share a similar appearance, involving multiple concentric rings of colored scales, but usually occuring in non-homologous positions on the wing. Within the butterflies, on the other hand, spots that share the same homologous position may not share the concentric ring structure; and, in butterfly species that have eyespots with concentric rings, ectopic eyespots with a similar ring structure can be induced by means of a simple epidermal wound. The extent to which all these eyespots, natural or induced, share similar genes and developmental mechanisms is investigated here by means of protein in-situ localizations in selected butterfly and moth species. In addition to looking at some of the transcription factors previously identified as being involved in eyespot formation, we also tested the involvement of candidate genes from the Wingless and TGF-β signaling pathways as putative morphogens for eyespot development.ResultsSaturniid moth and nymphalid butterfly eyespots with concentric rings of color express at least two transcription factors, Distal-less and Engrailed, in the center of the future pattern. Nymphalid eyespots centers also express the ligand Wingless and an activated signal transducer, a phosphorylated Smad protein, but neither these proteins nor the previous two proteins are found in pierid spot centers, which consist of a single patch of color. Both butterfly wing patterns, however, express a third transcription factor, Spalt, a portion of whose expression domain maps to the black scales on the adult wing. Wounding a nymphalid wing, on the other hand, leads to upregulation of Distal-less, engrailed and spalt in subsets of cells around the wounding site, mimicking concentric eyespot development.ConclusionWingless and TGF-β ligands are both candidate morphogens involved in nymphalid butterfly eyespot formation. These eyespots, as well as saturniid moth eyespots with concentric circles, share two genes that are associated with the differentiation of the signaling cells in nymphalid eyespots. This commonality suggests that they may be produced via the same developmental mechanism despite their non-homologous location. By contrast, pierid butterfly spots of a single color share some of the same genes but appear to be produced by a different mechanism. Eyespots with concentric rings may have co-opted a wound healing genetic network during their evolution.

title: Evolutionary history of the recruitment of conserved developmental genes in association to the formation and diversification of a novel trait
abstract: BackgroundThe origin and modification of novel traits are important aspects of biological diversification. Studies combining concepts and approaches of developmental genetics and evolutionary biology have uncovered many examples of the recruitment, or co-option, of genes conserved across lineages for the formation of novel, lineage-restricted traits. However, little is known about the evolutionary history of the recruitment of those genes, and of the relationship between them -for example, whether the co-option involves whole or parts of existing networks, or whether it occurs by redeployment of individual genes with de novo rewiring. We use a model novel trait, color pattern elements on butterfly wings called eyespots, to explore these questions. Eyespots have greatly diversified under natural and sexual selection, and their formation involves genetic circuitries shared across insects.ResultsWe investigated the evolutionary history of the recruitment and co-recruitment of four conserved transcription regulators to the larval wing disc region where circular pattern elements develop. The co-localization of Antennapedia, Notch, Distal-less, and Spalt with presumptive (eye)spot organizers was examined in 13 butterfly species, providing the largest comparative dataset available for the system. We found variation between families, between subfamilies, and between tribes. Phylogenetic reconstructions by parsimony and maximum likelihood methods revealed an unambiguous evolutionary history only for Antennapedia, with a resolved single origin of eyespot-associated expression, and many homoplastic events for Notch, Distal-less, and Spalt. The flexibility in the (co-)recruitment of the targeted genes includes cases where different gene combinations are associated with morphologically similar eyespots, as well as cases where identical protein combinations are associated with very different phenotypes.ConclusionsThe evolutionary history of gene (co-)recruitment is consistent with both divergence from a recruited putative ancestral network, and with independent co-option of individual genes. The diversity in the combinations of genes expressed in association with eyespot formation does not parallel diversity in characteristics of the adult phenotype. We discuss these results in the context of inferring homology. Our study underscores the importance of widening the representation of phylogenetic, morphological, and genetic diversity in order to establish general principles about the mechanisms behind the evolution of novel traits.

title: Species‐specific color‐pattern modifications of butterfly wings
abstract: We have previously shown that the systemic injection of sodium tungstate, a general protein‐tyrosine phosphatase (PTPase) inhibitor, efficiently produces characteristic color‐pattern modifications on the wings of the Painted Lady butterfly, Vanessa cardui. By using this method in the present study, we analyzed modification patterns of six species of Japanese butterflies. Whereas in Vanessa indica the black spots on the forewings reduced in size in response to the treatment, in Lycaena phlaeas the morphologically similar black spots enlarged in size. However, the metallic blue spots on the forewings of V. indica did enlarge in size, showing different behavior even within a single wing surface. The response patterns of Ypthima argus differed markedly from those of other species in that ectopic color‐pattern elements were created. Colias erate showed minor modifications that coincidentally resembled the natural color‐pattern of a closely related species, Colias palaeno. Through a comprehensive literature search, we confirmed the existence of naturally occurring aberrant color patterns with close similarities to the experimentally induced phenocopies in each of the modified species. Our results point out the possibility that a hypothetical transduction pathway with a PTPase for the scale‐cell differentiation globally coordinates the wing‐wide color‐pattern development in butterflies.

title: Physiologically induced color-pattern changes in butterfly wings: mechanistic and evolutionary implications.
abstract: A mechanistic understanding of the butterfly wing color-pattern determination can be facilitated by experimental pattern changes. Here I review physiologically induced color-pattern changes in nymphalid butterflies and their mechanistic and evolutionary implications. A type of color-pattern change can be elicited by elemental changes in size and position throughout the wing, as suggested by the nymphalid groundplan. These changes of pattern elements are bi-directional and bi-sided dislocation toward or away from eyespot foci and in both proximal and distal sides of the foci. The peripheral elements are dislocated even in the eyespot-less compartments. Anterior spots are more severely modified, suggesting the existence of an anterior-posterior gradient. In one species, eyespots are transformed into white spots with remnant-like orange scales, and such patterns emerge even at the eyespot-less "imaginary" foci. A series of these color-pattern modifications probably reveal "snap-shots" of a dynamic morphogenic signal due to heterochronic uncoupling between the signaling and reception steps. The conventional gradient model can be revised to account for these observed color-pattern changes.

title: Color-Pattern Evolution in Response to Environmental Stress in Butterflies
abstract: It is generally accepted that butterfly wing color-patterns have ecological and behavioral functions that evolved through natural selection. However, particular wing color-patterns may be produced physiologically in response to environmental stress, and they may lack significant function. These patterns would represent an extreme expression of phenotypic plasticity and can eventually be fixed genetically in a population. Here, three such cases in butterflies are concisely reviewed, and their possible mechanisms of genetic assimilation are discussed. First, a certain modified color-pattern of Vanessa indica induced by temperature treatments resembles the natural color-patterns of its closely related species of the genus Vanessa (sensu stricto). Second, a different type of color-pattern modification can be induced in Vanessa cardui as a result of a general stress response. This modified pattern is very similar to the natural color-pattern of its sister species Vanessa kershawi. Third, a field observation was reported, together with experimental support, to show that the color-pattern diversity of a regional population of Zizeeria maha increased at the northern range margin of this species in response to temperature stress. In these three cases, modified color-patterns are unlikely to have significant functions, and these cases suggest that phenotypic plasticity plays an important role in butterfly wing color-pattern evolution. A neutral or non-functional trait can be assimilated genetically if it is linked, like a parasitic trait, with another functional trait. In addition, it is possible that environmental stress causes epigenetic modifications of genes related to color-patterns and that their transgenerational inheritance facilitates the process of genetic assimilation of a neutral or non-functional trait.

title: The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. II. Scale‐forming and socket‐forming cells
abstract: In wings of the giant silkmoth, Hyalophora cecropia, scale‐forming and socket‐forming cells are first observed on day four of pharate adult development. Scale‐forming cells appear synthetically active when they are first observed, for their basal region is filled with huge stacks of polyribosome‐studed lamellate endoplasmic reticulum, numerous Golgi complexes containing secretory vesicles and many elongated mitochondria. During later development, some of these organelles diminish in number. Neck and scale regions are predominantly filled with longitudinally oriented microtubules and microfibril bundles, suggesting that their function is one of transport rather than synthesis. The scales originate as finger‐like projections of the cell body. They subsequently elongate, flatten out and deposit a cuticle which has a surface differing somewhat from that in other lepidopterans. It consists of longitudinal ridges (1.8‐2.4 μ apart), transverse ribs (0.6‐1.0 μ apart) and microribs (0.10‐0.13 μ apart).

title: Evidence for Notch-mediated lateral inhibition in organizing butterfly wing scales
abstract: Here I present gene expression data that implicate a Notch-mediated lateral inhibition process in the spatial organization of butterfly wing scales. During early pupal development the receptor molecule Notch is expressed in a grid-like pattern in the wing epithelium, resulting in parallel rows of uniformly spaced cells with low Notch expression. Previous work has shown that these low-Notch cells express a homolog of the achaete-scute proneural transcription factors and develop into scales. All of these observations are consistent with the Drosophila model of Notch-mediated bristle determination and support the hypothesis that bristles and scales share an underlying patterning mechanism.

title: Butterfly wings shaped by a molecular cookie cutter: evolutionary radiation of lepidopteran wing shapes associated with a derived Cut/wingless wing margin boundary system
abstract: Butterflies and moths show a remarkable diversity of specialized wing shapes, yet little is known about the molecular basis of wing shape determination. To learn more about this process we examined the expression of dorsoventral (DV) boundary candidate genes in developing wings of several species of Lepidoptera. We found that the transcription factor Cut and mRNA for the signaling molecule wingless (wg) are strongly co‐expressed in a discrete zone around the larval wing disc margin. Surprisingly, the expression boundary of Cut and wg clearly presages complex future adult wing shapes, including the hindwing tails of swallowtail butterflies, very early in final‐instar wing disc development. During pupal wing development the cells in this zone undergo apoptosis, thereby defining the actual margin of the adult wing. Comparison with gene expression in beetle and fly wings suggests that this delineation of a topologically independent boundary running parallel to the DV boundary is a derived feature of Lepidoptera. We propose that the developmental decoupling of wing margin determination and DV boundary formation was a major developmental innovation that facilitated the radiation of specialized wing shapes in moths and butterflies.

title: Positional dependence of scale size and shape in butterfly wings: wing-wide phenotypic coordination of color-pattern elements and background.
abstract: Butterfly wing color-patterns are a phenotypically coordinated array of scales whose color is determined as cellular interpretation outputs for morphogenic signals. Here we investigated distribution patterns of scale shape and size in relation to position and coloration on the hindwings of a nymphalid butterfly Junonia orithya. Most scales had a smooth edge but scales at and near the natural and ectopic eyespot foci and in the postbasal area were jagged. Scale size decreased regularly from the postbasal to distal areas, and eyespots occasionally had larger scales than the background. Reasonable correlations were obtained between the eyespot size and focal scale size in females. Histological and real-time individual observations of the color-pattern developmental sequence showed that the background brown and blue colors expanded from the postbasal to distal areas independently from the color-pattern elements such as eyespots. These data suggest that morphogenic signals for coloration directly or indirectly influence the scale shape and size and that the blue "background" is organized by a long-range signal from an unidentified organizing center in J. orithya.

title: Morphometric analysis of nymphalid butterfly wings: number, size and arrangement of scales, and their implications for tissue‐size determination
abstract: Animal body size and tissue size depend on genetic and environmental factors, but the precise mechanisms of how tissue size is determined in proportion to body size remain unknown. Here we focused on wings from three nymphalid butterflies, Junonia orithya (Linnaeus), Vanessa cardui (Linnaeus) and Danaus chrysippus (Linnaeus) (Lepidoptera: Nymphalidae), to examine the contributions of the number and size of scales to macroscopic structures, represented by wing compartments, and to investigate the positional dependence of scale size, density and arrangement. The whole wing area and wing compartment area exhibited a high correlation in all three species. Similarly, the wing compartment area and the blue or orange area showed a high correlation in three species, indicating isometric relationships among wings of different sizes. However, only in J. orithya, the blue area was highly correlated with the number of constituent scales and, to a lesser extent, with scale size. In contrast, reasonable correlations were obtained between the blue or orange area and the number of rows in all three species. These results suggest that variations of the background area accompany changes in the number of scales through changes in the number of rows. In a background region of the compartment, scale size gradually decreased and scale density increased from the proximal to the distal side in all three species. Our findings suggest that butterfly wing tissue size is determined primarily by the number of scale cells and secondarily by the size change of scale cells before or during the period of row arrangement.

title: Localization of ecdysone receptor protein during colour pattern formation in wings of the butterfly Precis coenia (Lepidoptera: Nymphalidae) and co-expression with Distal-less protein
abstract: Butterfly wing colour patterns are determined during late larval and early pupal development, a part of metamorphosis controlled by ecdysteroid hormones via their nuclear hormone receptors. We have sequenced a fragment of the common regions of the ecdysone receptor (EcR) from the butterflies Precis coenia and Bicyclus anynana and found high identities (83.5% to 100%) to EcR from a moth, Manduca sexta. In P. coenia, we sequenced a putative EcR-B1 isoform with 80.4% identity with the A/B-region of the M. sexta-EcR-B1. Consequently, we used antibodies generated against MsEcR-B1 to localise EcR protein during wing development of P. coenia. Nuclear staining of EcR was observed in different cell types during the course of colour pattern formation. Major observations are as follows: EcR is expressed in cell nuclei corresponding to wing lacunae and prospective veins. EcR is expressed early in pupal wing development in "focal" cells which are thought to release determining signals in a process leading to eyespot formation. Scale forming cells differentiate first and show EcR signal in the eyespot foci and most of the wing sheet, but not in areas corresponding to prospective eyespots. In the eyespots, 20–24 h after pupation, EcR expression seems to play a role in formation of scale rows preceding a later expression (28 h) in scale-forming cells. The results demonstrate that EcR is locally expressed in correlation to all major events of wing development and colour pattern formation. In particular, EcR expression patterns in prospective eyespots show that these are special pattern elements which are specified in concert with other factors of colour pattern formation such as the transcription factor Distal-less. In eyespot foci, Distal-less is expressed simultaneously with EcR, but clearly precedes EcR expression in eyespot scale-forming cells. This indicates a potential interaction between "short-range" signalling systems and "long-range" hormonal systems.

title: Eyespot colour pattern determination by serial induction in fish: Mechanistic convergence with butterfly eyespots
abstract: Vertebrate and invertebrate colour pattern determination mechanisms are considered distinct; recently, however, both fish and butterfly colour patterns have been partly explained by reaction-diffusion mechanisms. Here, we show that multi-coloured eyespots of the spotted mandarin fish, which are reminiscent of butterfly eyespots, are determined by the serial induction of colour patterns. The morphological characterisation of eyespots indicates a sequence of colour pattern development and dynamic interactions between eyespots. A substantial part of an eyespot can be surgically removed and is then reconstructed by regeneration. Strikingly, ectopic patterns are induced by damage at a background (eyespotless) area, but focal damage did not change the eyespot size. Early stages of damage repair were accompanied by calcium oscillations. These results demonstrate that fish eyespots are determined by serial induction, which is likely based on a reaction-diffusion mechanism. These findings suggest mechanistic similarities between the fish and butterfly systems.

title: Development of polyploidy of scale-building cells in the wings of Manduca sexta.
abstract: The developing wings of butterflies and moths are composed of two epithelial monolayers. Each epithelial sheet is made up of two kinds of cells, diploid cells that make up the epidermal surface and body of the wing, and large polyploid cells that become the scale-building cells whose cytoplasmic projections develop into the scales that will cover the adult wing and bear the pigment pattern. We studied the development of polyploidization of the scale-building cells during the pupal stage of the tobacco hornworm moth, Manduca sexta. The endomitotic divisions of the presumptive scale-building cells and the mitotic divisions of the diploid epithelial cells begin on day 3 of the pupal stage and continue until day 7. We show that scales of different colors and positions on the wing differ in size, and that the size of the scale is proportional to the ploidy of the scale-building cell. Scale-building cells are arranged in irregular rows and within each row there is an alternation of ploidy levels, with the lower ploidy cells giving rise to the underscales and the higher ploidy cells giving rise to the cover scales that carry the color pattern. Along the wing there is a proximo-distal decreasing gradient of average ploidy and scale size. Scale-building cells of high ploidy are surrounded by fewer epidermal cells than those of low ploidy. This inverse relationship is known as Henke's compensation principle, which posits that the number of endomitoses of a pre-polyploid cell and the number of mitotic divisions of its diploid daughter cell add up to a constant. We show that the inverse relationship fits the predictions of the compensation principle and does not fit constraints imposed by packing density, and we discuss mechanisms that could give rise to the inverse relationship.

title: Color-Pattern Analysis of Eyespots in Butterfly Wings: A Critical Examination of Morphogen Gradient Models
abstract: Butterfly wing color patterns consist of many color-pattern elements such as eyespots. It is believed that eyespot patterns are determined by a concentration gradient of a single morphogen species released by diffusion from the prospective eyespot focus in conjunction with multiple thresholds in signal-receiving cells. As alternatives to this single-morphogen model, more flexible multiple-morphogen model and induction model can be proposed. However, the relevance of these conceptual models to actual eyespots has not been examined systematically. Here, representative eyespots from nymphalid butterflies were analyzed morphologically to determine if they are consistent with these models. Measurement of ring widths of serial eyespots from a single wing surface showed that the proportion of each ring in an eyespot is quite different among homologous rings of serial eyespots of different sizes. In asymmetric eyespots, each ring is distorted to varying degrees. In extreme cases, only a portion of rings is expressed remotely from the focus. Similarly, there are many eyespots where only certain rings are deleted, added, or expanded. In an unusual case, the central area of an eyespot is composed of multiple “miniature eyespots,” but the overall macroscopic eyespot structure is maintained. These results indicate that each eyespot ring has independence and flexibility to a certain degree, which is less consistent with the single-morphogen model. Considering a “periodic eyespot”, which has repeats of a set of rings, damage-induced eyespots in mutants, and a scale-size distribution pattern in an eyespot, the induction model is the least incompatible with the actual eyespot diversity.

title: Patterning and growth control by membrane-tethered Wingless
abstract: Wnts are evolutionarily conserved secreted signalling proteins that, in various developmental contexts, spread from their site of synthesis to form a gradient and activate target-gene expression at a distance. However, the requirement for Wnts to spread has never been directly tested. Here we used genome engineering to replace the endogenous wingless gene, which encodes the main Drosophila Wnt, with one that expresses a membrane-tethered form of the protein. Surprisingly, the resulting flies were viable and produced normally patterned appendages of nearly the right size, albeit with a delay. We show that, in the prospective wing, prolonged wingless transcription followed by memory of earlier signalling allows persistent expression of relevant target genes. We suggest therefore that the spread of Wingless is dispensable for patterning and growth even though it probably contributes to increasing cell proliferation.

title: Baculovirus-mediated gene transfer in butterfly wings in vivo: an efficient expression system with an anti-gp64 antibody
abstract: BackgroundCandidate genes for color pattern formation in butterfly wings have been known based on gene expression patterns since the 1990s, but their functions remain elusive due to a lack of a functional assay. Several methods of transferring and expressing a foreign gene in butterfly wings have been reported, but they have suffered from low success rates or low expression levels. Here, we developed a simple, practical method to efficiently deliver and express a foreign gene using baculovirus-mediated gene transfer in butterfly wings in vivo.ResultsA recombinant baculovirus containing a gene for green fluorescent protein (GFP) was injected into pupae of the blue pansy butterfly Junonia orithya (Nymphalidae). GFP fluorescence was detected in the pupal wings and other body parts of the injected individuals three to five days post-injection at various degrees of fluorescence. We obtained a high GFP expression rate at relatively high virus titers, but it was associated with pupal death before color pattern formation in wings. To reduce the high mortality rate caused by the baculovirus treatment, we administered an anti-gp64 antibody, which was raised against baculovirus coat protein gp64, to infected pupae after the baculovirus injection. This treatment greatly reduced the mortality rate of the infected pupae. GFP fluorescence was observed in pupal and adult wings and other body parts of the antibody-treated individuals at various degrees of fluorescence. Importantly, we obtained completely developed wings with a normal color pattern, in which fluorescent signals originated directly from scales or the basal membrane after the removal of scales. GFP fluorescence in wing tissues spatially coincided with anti-GFP antibody staining, confirming that the fluorescent signals originated from the expressed GFP molecules.ConclusionsOur baculovirus-mediated gene transfer system with an anti-gp64 antibody is reasonably efficient, and it can be an invaluable tool to transfer, express, and functionally examine foreign genes in butterfly wings and also in other non-model insect systems.