Wing patterning and coloration
The dorsal wings of male S. charonda display a contrasting pattern created by a dark brown margin and cream to orange-yellow spots that surround a UV/blue-reflecting central area, which furthermore contains bright white spots (Fig. 1a, b). Upon oblique illumination with an angle of incidence of ~ 45° (Fig. 1c), the blue reflection of the central area (Fig. 1b, #1) vanishes, which demonstrates its iridescence. The reflectance spectra of the blue wing parts show a narrow band (bandwidth ~ 50 nm) peaking at ~ 400 nm (Fig. 1b, g, #1). Not surprisingly, the centrally located white spots show a high reflectance throughout the whole wavelength range, but an enhanced UV/blue reflectance peak also exists (Fig. 1b, g, #2). The orange-yellow patches adjacent to the central blue area have a broadband reflectance spectrum (Fig. 1b, g, #3), while the reflectance of the red spots of the dorsal hindwings is more restricted to longer wavelengths (Fig. 1b, h, #4). The reflectance of the brown margins is low throughout the whole wavelength range and slightly increases with increasing wavelength, characteristic of melanin-pigmented tissue; however, the minor peak in the UV spectrum suggests a structural contribution (Fig. 1b, h, #5).
The pattern on the ventral wings resembles that on the dorsal wings, but the strong blue coloration of the dorsal wings is absent. The ventral hindwings display a greenish shimmer, suggesting a structural basis (Fig. 1e, h, #6). The dark brown margins and the orange-yellow, red and white spots appear to be colocalized on both wing sides, which is confirmed when applying transillumination (Fig. 1f). This finding demonstrates that the white areas are transparent, i.e., unpigmented. The orange-yellow spots on the fore- and hindwings are also rather clear in transmitted light, which indicates that pigment absorption in the visible wavelength range is minor. The red spots on the hindwings are red in both epi- and transillumination, which means that absorption in the visible wavelength range is considerable.
To investigate the light-absorbing pigments more closely, we measured the absorbance spectra of the pigments extracted from the yellow and red spots (Fig. 1i, #3, 4). The obtained spectra show a high absorbance in the wavelength ranges where the reflectance is low (Fig. 1g, h, #3, 4). This finding indicates the prominent presence of 3-OH-kynurenine and ommochrome pigment in the yellow and red scales, respectively. The central and marginal wing areas are dark and light brown, respectively, or equivalently, the melanin pigment concentration gradually decreases from the butterfly’s body towards the periphery of its wings.
Electron microscopy of the scale types
The scales are not only colored by pigments, but their structure is certainly important as well (Fig. 2). Scanning electron micrographs show that the scales of both the blue area of the dorsal wing and the white spots have tightly arranged, parallel ridges (Fig. 2, #1, 2). The ridges of the orange-yellow and red scales are spaced wider than the abovementioned scales, and thus together with the crossribs, they frame rather large windows (Fig. 2, #3–5). The scales of the greenish ventral wing area are particularly widely spaced (Fig. 2, #6).
The transmission electron microscopy results corroborate the different scale architectures. In all the scales, the lower lamina is a thin plate. The blue (Fig. 2a-c, #1) and white (Fig. 2d-f, #2) scales have an upper lamina consisting of tall ridges with characteristic multilayer-forming folds (Fig. 2c, f). The blue scales have pillars below the ridge structures with high electron density, which indicates a substantial amount of melanin (Fig. 2c), quite in contrast with the white scales, which appear to be unpigmented (Fig. 2f). The other scales have the standard structure of butterfly wing scales, where the upper lamina is a loose network of parallel ridges connected by crossribs resting with pillars on the lower lamina.
Optics of isolated scales
The anatomy allows a convenient interpretation of the results of imaging scatterometry and reflectance spectrophotometry that we performed on individual wing scales. We therefore glued the scales to a glass pipette so that the abwing (upper) and adwing (lower) sides of the same scale could be investigated (Fig. 3). Figure 3a shows the abwing side of a scale taken from the blue dorsal wing area. With epi-illumination microscopy, the scale area that reflects blue incident light back into the microscope objective is limited to a band, which occurs because the scale is rather curved and not flat. The scale area outside the blue band is black (Fig. 3a) because the incident light is effectively absorbed by the melanin of the scale ridges and trabeculae.
Very similar to Morpho wing scales, the blue reflections are created by the ridges, which consist of a stack of lamellae. Hence, we expected a scatterogram similar to those of Morpho scales [24]. Indeed, the abwing (upper) side of the blue scale produces a scatterogram with a distinct blue stripe and a fainter stripe (Fig. 3c). The two stripes in the scatterogram are due to the ridge lamellae of the upper lamina and the thin film of the lower lamina, and the stripes are spatially segregated because the ridge lamellae are inclined with respect to the lower lamina, similar to Morpho wing scales [8]. Similar to Morpho scales, the abwing reflectance spectrum, measured with a microspectrophotometer, is a narrow band (peak at ~ 400 nm, Fig. 3e). The blue scale’s adwing side has a greenish color and is rather wrinkled (Fig. 3b). As a result, its scatterogram shows a distinctly distributed spot (Fig. 3d). The adwing side has a broadband reflectance spectrum; its peak at 570 nm and shape resemble that of a chitinous thin film with a thickness of 275 nm (Fig. 3e). The color as well as the adwing reflectance spectrum of different blue scales somewhat varies, which is in accordance with the TEM anatomy, yielding a spread in lower lamina thickness of 210–310 nm.
The anatomy showed that a scale from a white spot (Fig. 3f) closely resembles a blue scale with multilayered ridges (Fig. 2c, f). In fact, the white and blue scales feature identical blue bands, demonstrating similar scale curvatures. However, the white scale’s area outside the blue band is whitish because an appreciable fraction of the incident light is backscattered, clearly because of the absence of strongly absorbing melanin pigment. The scatterogram of the abwing side of the white scale (Fig. 3h) closely resembles the blue scale’s abwing scatterogram (Fig. 3c), but there is a background signal due to diffuse scattering by the unpigmented scale. Accordingly, the abwing reflectance spectrum, peaking again at approximately 400 nm, has a substantial background signal. The adwing side of the white scale is blue-purplish (Fig. 3g), and due to its wrinkles, the scatterogram is again slightly spread out (Fig. 3i). As the reflectance spectrum measured locally from the adwing side resembles the abwing spectrum, the spectrum is presumably the result of a lower lamina with a thickness of 190 nm (Fig. 3j). As with the blue scales, the anatomy showed that the lower lamina of the white scales also has a variable thickness, 185–310 nm, which results in a variable adwing reflectance spectrum and color (see Fig. 3g).
An orange-yellow scale of the dorsal forewing (Fig. 3k) has a strongly blue-colored adwing side (Fig. 3l). The scatterogram of the abwing side shows a diffuse pattern due to scattering by the upper lamina in a wide angular space (Fig. 3m). The adwing scatterogram shows a distinct spot because of the locally flat lower lamina, acting as a thin-film reflector (Fig. 3n). The reflectance spectrum of the adwing side indicates that the local thickness of the lower lamina is 380 nm (Fig. 3o). The TEM anatomy of several orange-yellow scales of the dorsal forewing yielded a thickness range of 300–385 nm. The abwing reflectance spectrum, which has peaks and valleys corresponding to those in the adwing spectrum, can be understood as partly the result of blue/purple light reflected by the thin-film lower lamina and yellow light reflected by the pigmented upper lamina (Fig. 3o).
Somewhat surprisingly, a rather similar orange-yellow scale from the ventral forewing (Fig. 3p) features at the adwing side in the proximal area a quite different, strongly yellow metallic reflection (Fig. 3q). The abwing scatterogram shows a clear line due to ridge diffraction plus a diffuse background, representing widespread scattering by the scale structures (Fig. 3r). The adwing scatterogram is dotlike (Fig. 3s) due to the locally rather flat lower lamina (Fig. 3q), which behaves like a thin-film reflector with a thickness of 300 nm (Fig. 3t). The ab- and adwing reflectance spectra are similar, indicating the presence of only a minor amount of pigment.
The lattice of scales on the wing
With the gained insight into the optics of single scales, we can understand the coloration of the butterfly’s wings when covered by the intact lattice of scales. Epi-illumination microscopy of a transition area with blue and white scales on the dorsal forewing demonstrates that both scale types have the same blue band but on a black or white background, respectively (Fig. 4a). When rotating the wing around an axis parallel to the blue bands, the transition area shifts over the scales. In natural conditions, the scales’ curvature is presumably used to radiate the blue color into a large spatial angle. The melanin pigment of the black-blue scales effectively absorbs scattered stray light, creating a contrasting background for the reflection of the blue color (see also Fig. 3e). As the white-blue scales lack pigmentation, part of the incident light is scattered and reflected by the scales as well as by the underlying wing substrate, which thus creates a white background (Fig. 4a). This phenomenon ultimately results in white spots inside the blue areas of the dorsal forewings (Fig. 1b).
A rather different coloration system is encountered in the orange-yellow spots of the dorsal forewing. Figure 4b-d shows an area where part of the scales is removed, revealing the wing substrate. With approximately normal illumination (and normal observation, i.e., perpendicular to the wing), applying linear polarized light and using a parallel analyzer, the wing substrate reflection is white-yellowish, but surprisingly, the scales show a mixture of blue-purplish colors (Fig. 4b). The latter clearly emerges from the thin-film lower lamina (Fig. 3l). With the analyzer in the crossed position, both the specular reflections from the wing substrate and the thin-film reflections of the scales’ lower lamina are extinguished, and the remaining depolarized reflected light has an orange-yellow color (Fig. 4c). A similar color emerges from the scales (but not the wing substrate) when illuminating the same area from an oblique (~ 45°) direction (Fig. 4d). With oblique illumination, only a small fraction of the incident light will be able to enter the scale windows, while a major part of the applied light will hit the scattering ridges and crossribs. As they contain the short-wavelength-absorbing yellow pigment, the color seen with wide-angular, natural illumination is principally determined by the pigmentation.
Close-up inspection of the red scales shows that they behave very similar to the orange-yellow scales. Here, a red pigment causes the overall red color, but with normal illumination, an additional blue flare also exists, which emerges from the scales’ lower lamina, and the same phenomenon can be observed in the brown, melanized scales of the wing margin (not shown; however, see the UV hump in the reflectance spectrum of Fig. 1h, #5).
Coloring unpigmented scales by thin-film interference
Similar to the scales in Fig. 3p (#7), the scales on the ventral hindwing have large windows (Fig. 2q, r, #6), and therefore, not only normally but also obliquely incident light will largely pass the ridges and crossribs and thus will subsequently reach the lower lamina unhampered upon reaching the lower lamina. This light will be partly reflected, depending on the thickness of the lower lamina. The lower lamina of the ventral hindwing scales is again quite wrinkled, although epi-illumination still produces a distinct color due to light interference in the thin film (Fig. 5a). Applying transmitted light at an isolated scale on a microscope slide may suggest the presence of melanin (Fig. 5b), but in immersion oil, the scales become almost invisible, meaning that the pigmentation is negligible (Fig. 5c). The thin-film characteristics of the lower lamina of an unpigmented scale therefore play a principal role in the scale coloration.
A local set of ventral wing scales observed in situ on the wing appears to be somewhat variably colored, indicating that the thickness of the lower lamina is not constant (Fig. 5d). Indeed, reflectance spectra measured from a number of adjacent scales vary slightly (Fig. 5e), similar to the reflectance spectra of chitinous thin films with thicknesses varying between 260 and 300 nm (Fig. 5f). The experimentally obtained spectra have a background signal, whereas the theoretical spectra show zero reflectance at specific wavelengths. The background signal is partly due to scattering contributions by the ridges and crossribs, but additional in situ back-scattering from the wing substrate is also important.