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.