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.     

Color-pattern analysis of eyespots in butterfly wings: a critical examination of morphogen gradient models

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.     

Reversed type of color-pattern modifications of butterfly wings: a physiological mechanism of wing-wide color-pattern determination

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.     

Tungstate-induced color-pattern modifications of butterfly wings are independent of stress response and ecdysteroid effect

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.     

Positional dependence of scale size and shape in butterfly wings: Wing-wide phenotypic coordination of color-pattern elements and background

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.     

Color pattern analysis of nymphalid butterfly wings: revision of the nymphalid groundplan

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.     

Stress-induced color-pattern modifications and evolution of the Painted Lady butterflies Vanessa cardui and Vanessa kershawi

It has been proposed that phenotypic plasticity and genetic assimilation through natural selection partly determine the direction of divergent selection that eventually results in speciation. To elucidate a process of butterfly color-pattern evolution and speciation in the light of this hypothesis, morphological and physiological differences between a pair of sister species, the Painted Lady butterfly Vanessa cardui and the Australian Painted Lady butterfly Vanessa kershawi, were investigated. Ten different traits of wing color-pattern were indicated, most of which concerned the darker coloration of V. kershawi, with the notable exception of the blue foci at the center of the black focal elements only in V. kershawi. Differences in behavior and life history between the two species appeared to be minimal, but importantly, V. kershawi tends to prefer a "stressful" arid environment. The experimental treatment of pupae of V. cardui either by low temperature or by injection of thapsigargin, a stress-inducing chemical, readily produced individuals with the darker coloration and the blue foci as a result of a general stress response. These stress-induced color-pattern modifications were considered to be the revelation of phenotypic plasticity in V. cardui. Taken together, I propose that the ancestral species of V. kershawi had similar phenotypic plasticity. Natural selection exploited this plasticity and shaped the present V. kershawi as an independent species, whose specific color-pattern traits are by-products of this adaptation process.     

Species-specific color-pattern modifications of butterfly wings

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.     

An analysis of the phenotypic effects of certain colour pattern genes in Heliconius (Lepidoptera: Nymphalidae)

The colour patterns of Heliconius butterflies are built up from an array of serially homologous pattern elements known as the nymphalid groundplan. An analysis of the phenotypic effects of ten genetic loci from H. melpomene and H. cydno reveals that each alters the expression either of a single element of the groundplan or of an entire row of serially homologous elements. Five of the ten loci affect the size (or presence/absence) of specific pattern elements, two affect the colour in which a pattern element is expressed, two affect pattern-inducing activity of the wing veins, and one appears to affect an overall threshold for pattern determination. Three of the ten loci have identical effects on homologues of the fore- and hindwing. We show that most of the apparently large and qualitative phenotypic effects of these genes can be readily explained by relatively small and quantitative changes in the dimensions or positions of specific pattern elements.     

Quantitative genetics of butterfly wing color patterns

Developmental processes exert their influence on the evolution of complex morphologies through the genetic correlations they engender between traits. Butterfly wing color patterns provide a model system to examine this connection between development and evolution. In butterflies, the nymphalid groundplan is a framework used to decompose complex wing patterns into their component pattern elements. The first goal of this work has been to determine whether the components of the nymphalid groundplan are the products of independent developmental processes. To test this hypothesis, the genetic correlation matrices for two species of butterflies, Precis coenia and Precis evarete, were estimated for 27 wing pattern characters. The second purpose was to test the hypothesis that the differentiation of serial homologs lowers their genetic correlations. The “eyespots” found serially repeated across the fore- and hindwing and on the dorsal and ventral wing surfaces provided an opportunity to test this hypothesis. The genetic correlation matrices of both species were very similar. The pattern of genetic correlation measured between the different types of pattern elements and between the homologous repeats of a pattern element supported the first hypothesis of developmental independence among the elements of the groundplan. The correlation pattern among the differentiated serial homologs was similarly found to support the second hypothesis: pairs of eyespots that had differentiated had lower genetic correlations than pairs that were similar in morphology. The implications of this study are twofold: First, the apparent developmental independence among the distinct elements of wing pattern has facilitated the vast diversification in morphology found in butterflies. Second, the lower genetic correlations betweendifferentiated homologs demonstrates that developmental constraints can in fact be broken. The extent to which genetic correlations readily change, however, remains unknown. © 1994 Wiley-Liss, Inc.     

Wnt signaling underlies evolution and development of the butterfly wing pattern symmetry systems

Most butterfly wing patterns are proposed to be derived from a set of conserved pattern elements known as symmetry systems. Symmetry systems are so-named because they are often associated with parallel color stripes mirrored around linear organizing centers that run between the anterior and posterior wing margins. Even though the symmetry systems are the most prominent and diverse wing pattern elements, their study has been confounded by a lack of knowledge regarding the molecular basis of their development, as well as the difficulty of drawing pattern homologies across species with highly derived wing patterns. Here we present the first molecular characterization of symmetry system development by showing that WntA expression is consistently associated with the major basal, discal, central, and external symmetry system patterns of nymphalid butterflies. Pharmacological manipulations of signaling gradients using heparin and dextran sulfate showed that pattern organizing centers correspond precisely with WntA, wingless, Wnt6, and Wnt10 expression patterns, thus suggesting a role for Wnt signaling in color pattern induction. Importantly, this model is supported by recent genetic and population genomic work identifying WntA as the causative locus underlying wing pattern variation within several butterfly species. By comparing the expression of WntA between nymphalid butterflies representing a range of prototypical symmetry systems, slightly deviated symmetry systems, and highly derived wing patterns, we were able to infer symmetry system homologies in several challenging cases. Our work illustrates how highly divergent morphologies can be derived from modifications to a common ground plan across both micro- and macro-evolutionary time scales.     

The significance of wing pattern diversity in the Lycaenidae: mate discrimination by two recently diverged species

Closely related species of lycaenid butterflies are determinable, in part, by subtle differences in wing pattern. We found that female wing patterns can act as an effective mate‐recognition signal in some populations of two recently diverged species. In field experiments, we observed that males from a Lycaeides idas population and an alpine population of L. melissa preferentially initiate courtship with conspecific females. A morphometric study indicated that at least two wing pattern elements were important for distinguishing the two species: hindwing spots and orange crescent‐shaped pattern elements called aurorae. We deceived male L. idas into initiating courtship with computer generated paper models of heterospecific females when these pattern elements were manipulated, indicating that the wing pattern elements that define the diversity of this group can be effective mate recognition signals.     

Physiological characterization of the cold-shock-induced humoral factor for wing color-pattern changes in butterflies

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.     

Color-pattern modifications of butterfly wings induced by transfusion and oxyanions

The color-pattern determination of butterfly wings was studied, focusing on the cold-shock-induced color-pattern modifications of a species of butterfly, Vanessa (Cynthia) cardui (Lepidoptera: Nymphalidae). It was shown that the modification property could be transferred to the noncold-shocked individuals by the transfusion of hemolymph taken from the cold-shocked individuals, suggesting the existence of an unknown diffusible factor or hormone, induced or activated by the cold shock. The involvement of a receptor tyrosine kinase for the color-pattern modifications was tested by the simple application of some oxyanions such as sodium tungstate, sodium molybdate, and molybdic acid to pupae, since these oxyanions have been known to up-regulate the process of phosphorylation via receptor tyrosine kinases in general. It was shown that they could modify the wing color-pattern in a way very similar to the cold shock. Moreover, the topical applications of sodium tungstate or molybdic acid induced large ectopic black spots on the treated pupal wings. Among the treatment methods, the sodium tungstate treatment was by far more effective than the cold shock treatment itself. Taken together, these data suggest that an unknown cold-shock hormone activates the process of phosphorylation via a receptor tyrosine kinase necessary for the color-pattern development.     

THE EVOLUTIONARY GENETICS AND DEVELOPMENTAL BASIS OF WING PATTERN VARIATION IN THE BUTTERFLY BICYCLUS ANYNANA

We have studied interactions between developmental processes and genetic variation for the eyespot color pattern on the adult dorsal forewing of the nymphalid butterfly, Bicyclus anynana. Truncation selection was applied in both an upward and a downward direction to the size of a single eyespot consisting of rings with wing scales of differing color pigments. High heritabilities resulted in rapid responses to selection yielding divergent lines with very large or very small eyespots. Strong correlated responses occurred in most of the other eyespots on each wing surface. The cells at the center of a presumptive eyespot (the “focus”) act in the early pupal stage to establish the adult wing pattern. The developmental fate of the scale cells within an eyespot is specified by the “signaling” properties of the focus and the “response” thresholds of the epidermis. The individual eyespots can be envisaged as developmental homologues. Grafting experiments performed with the eyespot foci of the selected lines showed that additive genetic variance exists for both the response and, in particular, the signaling components of the developmental system. The results are discussed in the context of how constraints on the evolution of this wing pattern may be related to the developmental organization.     

Correlations between scale structure and pigmentation in butterfly wings

SUMMARY We examined the correlation between color and structure of wing scales in the nymphalid butterflies Bicyclus anynana and Heliconius melpomene . All scales in B. anynana are rather similar in comparison to the clear structural differences of differently pigmented scales in H. melpomene . Where scale structural differences in H. melpomene are qualitative, they seem to be quantitative in B. anynana . There is a "gradient" in the density of some structural elements, the cross ribs, in the scales of B. anynana : black, gold, and brown scales show progressively lower cross rib density within an individual. There is, however, high individual variation in the absolute cross rib densities (i.e., scales with a particular color and cross rib density in one individual may have a different color but similar density in another individual). By ectopically inducing color pattern during early pupal development, we examined whether a scale's color and its microstructure could be uncoupled. The effect of these manipulations appears to be different in B. anynana and H. melpomene . In Bicyclus , "black" scales induced by wing damage at an ectopic location normally containing brown scales acquire both an intermediate structure and color between that of brown and normal black scales. In Heliconius , however, intermediate colors or scale structure were never observed, and scales with an altered color (due to damage) always have the same structure as normal scales with that color. The results are discussed on the basis of gene expression patterns, variability in rates of scale development and pigment, and scale sclerotization pathways.     

Elements of butterfly wing patterns

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.     

Mutants highlight the modular control of butterfly eyespot patterns

SUMMARY The eyespots on butterfly wings are thought to be serially homologous pattern elements. Yet eyespots differ greatly in number, shape, color, and size, within and among species. To what extent do these serially homologues have separate developmental identities, upon which selection acts to create diversity? We examined x‐ray–induced mutations for the eyespots of the nymphalid butterfly Bicyclus anynana that highlight the modular control of these serially homologous wing pattern elements. These mutations reduce or eliminate individual eyespots, or groups of eyespots, with no further effect on the wing color pattern. The collection of mutants highlights a greater potential developmental repertoire than that observed across the genus Bicyclus. We studied in detail one such mutation, of codominant effect, that causes the elimination of two adjacent eyespots on the ventral hindwing. By analyzing the expression of genes known to be involved in eyespot formation, we found an alteration in the differentiation of the “organizing” cells at the eyespot's center. No such cells differentiate in the wing subdivisions lacking the two eyespots in the mutants. We propose several developmental models, based on wing compartmentalization in Drosophila, that provide the first framework for thinking about the molecular evolution of butterfly wing pattern modularity.