Colour formation on the wings of the butterfly Hypolimnas salmacis by scale stacking

The butterfly genus Hypolimnas features iridescent blue colouration in some areas of its dorsal wings. Here, we analyse the mechanisms responsible for such colouration on the dorsal wings of Hypolimnas salmacis and experimentally demonstrate that the lower thin lamina in the white cover scales causes the blue iridescence. This outcome contradicts other studies reporting that the radiant blue in Hypolimnas butterflies is caused by complex ridge-lamellar architectures in the upper lamina of the cover scales. Our comprehensive optical study supported by numerical calculation however shows that scale stacking primarily induces the observed colour appearance of Hypolimnas salmacis.


Results
Optical appearance of scales on the wing. The dorsal side of the Hypolimnas salmacis wings features dark brown or black, white, and blue regions. On the ventral side, white regions mimic the dorsal side while everywhere else the wings are covered by light brown scales. The basal region is almost black near the thorax for both forewing and hindwing. The outer margin of the whole wing is mostly brown and covered with blue and white patches in the middle. A stereo-microscopy image of the Hypolimnas salmacis wing is shown in Fig. 1b. Interestingly, the blue colouration is visible only in diffused illumination, suggesting that the origin of colouration is purely physical. Moreover, tilting the wing shifts the blue colour to purple-violet (Fig. 1c) that demonstrates the iridescent property of the Hypolimnas salmacis wing. As in most other Lepidoptera species, both white and black/ brown areas are uniformly covered with cover and ground scales 4 .
As clearly visible in Fig. 1b, some of the cover scales in the blue areas are not completely blue. While examining thoroughly scales in blue areas, we observed that white cover scales stacked on brown ground scales appear blue. And, the same cover scales look white when lying on top of white ground scales. To further confirm our observation, we picked a single white cover and a single brown ground scale from the blue region. We manually overlapped the white cover and brown ground scales whereupon the blue colouration appears as a result of the stacking (see Fig. 1d). The cover scale appeared white when the bottom brown scale was absent. Hence, the formation of the blue colour on the Hypolimnas salmacis wings occurs due to scale stacking when a white scale is on top of a brown one.
Electron microscopy of scales. Creating blue colour out of white and brown does not accord with conventional colour blending or filtering mechanism. To understand this effect, we first analysed the scales by electron microscopy ( Fig. 2a-d). The blue areas consist of stacks of white and brown scales (Fig. 2a,b) as already observed from the optical analysis. The SEM images of the white and brown scales show the typical oval shape with a width of 100 μm and length of 200 μm. Both of them consist of grating-like ridges with a typical distance of (2 ± 0.2) μm. The brown scales feature thin membranes between the ridges (Fig. 2d) while the white ones do not (Fig. 2c). Cross-ribs across the ridges create open areas with a size of (2 ± 0.2) μm × (1 ± 0.1) μm. Such windows created by cross-ribs are commonly termed alveoli and found in Papilionids 6 . The sides of the ridges are covered with microribs. The cross-sectional SEM image of the blue region shown in Fig. 2e reveals longitudinal ridges of cover and ground scales both consisting of very small microribs. The ridges are standing on a single thin film (lower lamina) with a thickness of around (190 ± 20) nm. Thin membranes can be observed in the windows of the brown scales between the ridges (Fig. 2d,e).
Optical spectroscopy of scales on the wing. Total diffusive reflection spectra were measured in different regions of the Hypolimnas salmacis wings with an integrating sphere. Figure 3 shows the obtained spectra of blue, white and brown regions marked in the inset. Blue areas feature a weak reflection of only 20% reflectance with a broad peak at ≈ 430 nm. In the reflection spectrum of the white area, there is no particular peak in the visible regime, resulting in the overall white appearance. Nonetheless, a considerably broad reflection peak can be noticed in the UV at ≈ 375 nm. A similar UV reflection was reported in the white patch of H. bolina 22 . Although the overall structure of the brown scales of H. salmacis is comparable to that of the white scales, the brown area has no reflection peak neither in UV nor in the visible spectral regime. This is presumably due to high melanin pigmentation in the membranes and ridges of the brown scales leading to high absorption.
Optical spectroscopy of single scales. In order to understand the observed scale stacking effects in more details, we performed also micro-spectrometry on individual scales and stacks of scales. All the reflection measurements are performed on black background to avoid stray reflection/scattering. The resulting spectra are plotted in Fig. 4. The white scale is almost transparent in the visible regime with a high transmittance of ≈ 90% (dashed line, Fig. 4a. Thus, the white scale can be considered as nearly pigment-free. However, an isolated white scale has a reflection peak at 420 nm, i.e., in the blue spectral area (solid line, Fig. 4a). We carried out similar experiments on a single isolated brown scale (Fig. 4b). The resultant spectra indicate very low reflecting and transmitting properties of the brown scale and therefore point out its strong absorbing behaviour.
To explain how the appearance of white and blue is created by scale stacking, we first measured the reflection spectrum of white scales lying on the wing membrane of Hypolimnas salmacis (Fig. 4c). In this arrangement, the white appearance indeed coincides with a broad spectrum (grey line) over all visible wavelengths. The spectrum of the wing membrane itself shows also a broadband reflection and whitish colouration (not shown). Defining the individual reflectance of a white scale, a brown scale and a wing membrane as R white , R brown and R membrane , respectively and the transmittance of white scale as T white , the reflectance of a white scale stacking on the wing membrane can be calculated from w,m w hite white m embrane w hite assuming non-coherent scattering during the light propagation in the stack. The resulting spectrum (black dashed line) is shown in Fig. 4c with a concise schematic noting the terms as inset. The calculated spectrum fits well with the measured stacked spectrum. However, some small mismatch can be noticed at long wavelengths, which might be due to a partial coherence effect during the light propagation which is not considered in the equation above 23 .
In a next step we placed a white scale on top of a brown scale and the measured reflection spectrum (solid blue line, Fig. 4d) shows a reflection peak at 420 nm in the same blue spectral area as observed in the reflection spectrum of the isolated white scale. These observations suggest that a white scale on an absorbing background exhibits blue colouration. To check the consistency of our measurements and assumptions we also calculated the non-coherent scattering of a white scale on a brown scale The calculated spectrum (black dashed line) of the stacked system in this case matches the measured spectrum completely as depicted in Fig. 4d. Hence, the overall result explains the scale stacking phenomena satisfactorily.
Translucency of white scales. The above presented micro-spectroscopy of a white scale resulted in a transmittance of about 90%. However, this experiment does not provide the scattering properties, i.e. whether white scales are transparent or translucent. In order to examine this property, we passed laser light with two different wavelengths (blue with 445 nm and red with 635 nm) through the white region of a butterfly wing and captured the resultant diffraction patterns on a screen (Fig. 5). A transparent medium does not allow diffuse light scattering and diffracts only the zeroth order. In our case, however, two diffracted orders including the zeroth order are observed on the screen indicating large diffuse light scattering at the micro-ridge patterns (2 ± 0.1 μm) of the white scales 24 . The diffraction pattern of a single white scale shown in Fig. 5c was obtained in transmission mode using a K-space imaging system with Bertrand lens 25 . This conoscopic imaging allows to record the directionality of the scattered beam, i.e. to record the Fourier plane. The zeroth order corresponds to a direct transmission of the white light source through the scale. For higher orders, colours with increasing wavelengths (from blue to red) are diffracted at increasing angles. However, the higher order diffraction signals are relatively broader in angle due to Simulation of the thin film interference of the white scales. The experimental results presented above already demonstrated that the effect of the blue colouration is caused by scale stacking which amplifies the broad blue peak of the white scales. As the upper lamina works as a diffuser, we speculate that the lower feature-less thin lamina is the origin of this blue peak. To prove that, we simulated the reflection of a thin film with where δ = (2πn c d cos θ)/λ is the phase delay introduced by the film thickness of d, and r is the reflection coefficient at the air-chitin boundary governed by Fresnel's equation for a given polarisation, i.e., r = (cos θ − n c cos(sin −1 (sin θ/n c )))/(cos θ + n c cos (sin −1 (sin θ/n c ))) for s-polarisation or r = (cos(sin −1 (sin θ/n c )) − n c cos θ)/ (cos(sin −1 (sin θ/n c )) + n c cos θ) for p-polarisation. Additionally, we have to consider that the thickness of the lamina is not perfectly constant. We observed in the electron microscopy images that it varies locally from 170 to 210 nm within the scale. In order to include these local variations of the thin film, we modeled the film thickness by a Gaussian distribution with mean thickness d and variance σ d . In this way, the local thickness variation of the lamina can be easily combined with Eq. (3) and the averaged reflectance of the wavy membrane for a given polarisation can be calculated from With this equation, first we calculated the thin film reflection for a mean thickness of 190 nm and varied the variance σ d at normal angle of incidence (Fig. 6a). For σ d = 0 nm, the calculated spectrum is simply the thin film reflectance for a 190 nm slab surrounded by air (dashed line). The effect of local variation on the reflection properties by the roughness factor σ d is indicated by dotted arrows. The corresponding reflection spectrum is shown in Fig. 6a and compared with the single white scale reflectance obtained from the micro-spectrometry. The simulated thin film reflectance with a variance σ d = 31 nm agrees well with the experimental data. Furthermore, we calculated the reflection spectra in unpolarised light condition by averaging out the s-and p-polarisation for various oblique angles of incidence and compared with the experimental ones in Fig. 6b. The roughness factor of σ d = 31 nm in the model also corresponds well with the experimental data at off-normal light condition. A blue-shift is noticed in the peak wavelength of reflection spectra towards UV spectral region at large angles of incidence. However, the inclusion of roughness factor of σ d = 31 nm in the thin film model red-shifts the peak wavelength of reflection of a flat thin film (σ d = 0 nm) by 10 nm (Fig. 6b). Overall, the theoretical thin film model verifies that the blue colouration results from the thin film nature of the lower lamina.

Discussion
Very little research on the butterfly Hypolimnas salmacis has been published so far although some other Hypolimnas species were studied extensively 19,22,28 . We observed structural UV reflection from the white patches owing to the multilayer microrib architecture. UV reflection from ridge-microrib structures has been already observed in nymphalids, pierids and other butterfly families 29,30 . However, to create blue reflection from a similar structure, longitudinal and transverse dimensions of the microribs need to be sufficiently large. This statement is supported by the Morpho butterfly structural pattern as a very good natural example of such architecture, as well as by several experimental biomimetic structures 12,31,32 . It was also reported that the origin of the intense and directional blue reflection of Hypolimnas bolina is similar kind of complex structures 22 .
However, we demonstrated that the colouration of the blue areas of the Hypolimnas salmacis wing is caused by scale stacking where the origin of the blue colour is the thin lower lamina of the white scales. Our experimental analysis as well as theoretical analytical modelling confirm that the phase delay introduced by the thin film lower lamina causes light interference in blue spectrum. Indeed, the lower lamina works as a one dimensional photonic crystal which is commonly found in butterfly wing scales 33,34 . However, due to the low index contrast between chitin (n = 1.56) and air (n = 1), the thin lamina can not form a complete photonic bandgap. It rather creates a pseudo-bandgap which causes the iridescence of the Hypolimnas salmacis 35 . The pseudo-photonic bandgap in the visible spectrum created by photonic crystals is responsible for iridescence in many other butterflies and insects 5,34 . Hence, the proposition of thin film interference explains the violet-purple appearance of the wing at oblique angles as shown in Fig. 1c. The theoretical modelling of a thin film including surface variation also confirms this hypothesis (Fig. 6b).
The local variation of the laminar thickness can be modelled by a Gaussian distribution and the variance (standard deviation) can act as a roughness factor. Such surface roughness smoothens the reflection spectrum of a single thin flat film (Fig. 6a). This explains the dull appearance of the blue colour. Although an isolated white scale has a peak blue reflectance of around 10%, blue areas on the wing reach up to 20% due to multiple scattering of overlapping scales 23 . Moreover, the peak wavelength of reflection encounters a red-shift of 10 nm because of the surface roughness (Fig. 6b). This can be explained by the incoherent effect caused by the local surface variation that disrupts partially the coherent scattering of a thin 1D photonic crystal i.e. the lamina. Hence, the local thickness variation of the lower lamella weakens the iridescent property of the scales towards UV spectral region.
Micro-ridges in butterfly scales usually act as good scatters for longer wavelengths 36 . Again, windows created by cross-ribs are often reported to be responsible for the diffusion of the incoming or outgoing light 6 . This can explain the translucency of the white scales when white light passes through the upper lamina. Due to the high translucency of the white scales, the weak blue reflection from the lower thin lamina is eliminated by the underlying incoherent diffuse scattering of the wing membrane. The broadband absorbing pigment melanin in black scales effectively reduces stray-light and back-scattering, at the same time uncovers the blue appearance of the white scales. The overall mechanism of structural colouration by scale stacking in Hypolimnas salmacis wing is sketched in Fig. 7. The pigment melanin is also found in other blue, e.g. Morpho butterflies and mainly prevents the decrease in saturation of the colour 37 . The thin lower lamina in Morpho butterflies works as a thin reflector too, but the intense blue reflection of the scales is dominated by the upper lamina multi-layered lamellar architecture 38 . In contrast, the upper lamina of Hypolimnas salmacis white scales works as a broadband light scatterer.
The typical colours of butterfly wing scales may be solely produced from either micro-and nanoarchitectures, or pigments, but most frequently from a combination of both 37,39 . This is in fact also true for other animals in nature [40][41][42] . Often the cover scales of butterfly wings consist of complex architectures with ridges, lamella, microribs, crossribs with or without pigments and contribute strongly to the wing colouration. Otherwise, the cover scales are reported to be transparent and scatter the underlying ground scale reflection 26 . Only a few examples of scale stacking were reported so far and mainly described as an enhancer of scattering properties in terms of perception and brightness 23,43 . Spectral alteration by scale stacking was only recently observed in small regions of European Nymphaline butterflies 44 . A similar colouration mechanism is also found in the feathers of the bird Steller's Jay where the colour difference of white and blue feathers arises primarily due to the inherent melanin pigment content difference below spongy nanostructures 45,46 .
In summary, we experimentally demonstrated that the blue wing regions of the Hypolimnas salmacis originate from scale stacking of white translucent scales on top of brown absorbing scales. Our single-scale micro-spectrometry and thin film simulation showed that the blue colouration is caused by the lower thin lamina of the white scales. Our detailed study also revealed the cause of the weak iridescence of Hypolimnas salmacis and might lead to better understanding of mating preferences and signal variation in other Hypolimnas butterflies 3,19 . Moreover, such a mechanism of colour interplay with different thin plates might be useful for technical applications in the field of nano-optics and photonics. For instance, by tuning the specular and/or diffuse reflection factor of a top optical layer, and the absorption properties of a bottom film, different optical signatures might be encoded.

Methods
Sample preparation and imaging. Dried samples of Hypolimnas salmacis were kindly supplied by the Stadtpark Mannheim GmbH, Germany and a dried sample was bought from Bug Under Glass©, USA. Scales were carefully removed from the wings with tweezers for subsequent imaging by optical microscopy. A stereo-microscope (SteREO Discovery.V8, Carl Zeiss Microscopy GmbH, Germany) was used in reflection mode to image the scales under epi-illumination condition (Fig. 1) For the imaging by scanning electron microscopy (SEM), the examined regions of the butterfly wings were coated with a 15 nm thin gold layer (K575X sputter coater, Quorum Technologies Ltd.). Surface patterns were subsequently imaged by SEM (SUPRA ® 60 VP, Carl Zeiss Microscopy GmbH, Germany) operated at 5 kV (see Fig. 2a-d).
For cross-section imaging, small pieces of wings were embedded in epoxide resin. First, the pieces were dipped in a combination of 70% acetone and 30% epoxide mix (42.4 g Glycidether, 29.6 g DDSA, 18.4 g MNA, all chemicals from SERVA Electrophoresis GmbH, Germany). Subsequently, they were exposed to vacuum for a few minutes to remove air bubbles. Afterwards, the mixture was shaken for an hour. This step was repeated first with a combination of 30% acetone and 70% epoxide mix, then twice with 100% epoxide mix to make sure the viscous resin penetrated into all the tiny cavities of the butterfly scales. The resin-infiltrated wing pieces were placed into a silicone mold and covered with epoxide mix plus accelerator (10 g epoxide mix and 0.265 g BDMA) and baked at 65 °C for 2 days. The polymerized block was removed from the mold and trimmed to expose the wing piece. Thin (≈ 70 nm) sections from the block-face were prepared using an ultramicrotome (Leica Microsystems, Germany) and transferred to small pieces of silicon wafer for SEM imaging. The cross section shown in Fig. 2e) was imaged at 1.5 kV in a SEM (Ultra, Carl Zeiss Microscopy GmbH, Germany).
Optical spectroscopy. The macroscopic reflection spectra of an intact Hypolimnas salmacis wing shown in Fig. 3 were recorded with a UV-Vis spectrometer (Lambda 1050, PerkinElmer Inc., USA). The total diffuse reflection was measured with an InGaAs 150 mm integrating sphere averaging over a 2 mm 2 area on different regions (blue, white, brown) of an intact wing.
A customized Zeiss Axio microscope was used for the micro-spectroscopic analysis of individual scales (Fig. 4) with a spot size of ≈ 25 μm in bright field (BF) reflection mode with a halogen lamp in Koehler illumination. Unpolarised light from the halogen lamp was illuminated via a 10X objective (EC Epiplan-APOCHROMAT, Zeiss) with a numerical aperture of NA = 0.3. The reflected light was collected with a spectrometer (AvaSpec-HS2048, Avantes, UK) through a 200 μm core optical fiber (Avantes, UK) mounted in confocal configuration.
The angular-resolved specular reflection was measured using home-built optical goniometric setup (Fig. 6b). A light source from a stabilised Tungsten light source (SLS201, Thorlabs, USA) is collimated with a pinhole and a long working distance objective lens to form a 50 μm wide parallel incident beam that illuminates a single scale at a fixed angle. The specularly reflected light is detected at different angles with an aperture of 2° and coupled into an optical fiber connected to the spectrometer (AvaSpec-ULS2048x64-USB2 Avantes, USA). All the spectra reported are referenced to a lambda/20 UV fused silica mirror (Thorlabs, USA).