The iridescent features of the butterfly species Apatura iris (Linnaeus, 1758) and A. ilia (Denis & Schiffermüller, 1775) were studied. We recognized the structural color of scales only on the dorsal side of both the fore and hind wings of males of both of the aforementioned butterfly species. The scale dimensions and microstructure were analyzed by a scanning electron microscope (SEM) and transmission electron microscope (TEM). The optical properties were measured and it was found that the peak reflectivity is around 380 nm, with a spectral width (full width at half maximum) of approximately 50 nm in both species. The angular selectivity is high and a purple iridescent color is observed within the angular range of only 18 degrees in both species.
©2011 Optical Society of America
The diurnal active members of the order Lepidoptera (butterflies) are considered to be the most attractive insects, together with representatives of the order Coleoptera (beetles). The delicate beauty of butterfly wings is a consequence of several phenomena: selective absorption by pigments, scattering, fluorescence and iridescence. The phenomenon of bright iridescence attracted much attention [1–7] and is observed in a great number of butterfly species, mostly tropical ones. Butterflies are known as the masters of mimicry (the type of camouflage which serves to avoid predators) and aposemy (warning coloration which is usually associated with an unpleasant taste to potential predators). In some species sexual dimorphism is observed, as in the two species analyzed in this paper.
Structural coloration investigations have been very popular in the last few decades and butterfly microstructure has been thoroughly investigated [8–10]. The following aspects have been studied as well: nanostructure and optical properties of wing scales [1, 11, 12], interference and diffraction in butterflies , ultraviolet reflection , structural blackness and whiteness in butterfly scales , fluorescence emission , and coherent scattering-induced structural color of scales . The optical properties of butterfly scales have been thoroughly measured and modeled  and their nanoscale structures could encourage further developments in artificial material manufacturing .
The butterfly species Apatura iris (Linnaeus, 1758) and A. ilia (Denis & Schiffermüller, 1775) (Fig. 1a and b ) are distributed from Europe to Eastern Asia (China). The most obvious difference between the two species is an extra eye spot on the fore wings of A. ilia. The males of Apatura spp. possess the iridescent color on the dorsal side of their wings [18, 19]. This is probably connected with intrasexual communication between males, rather than intersexual communication and attraction . The structural color of males is visible in flight when the movements of the wings are noticeable within a certain range of angles. This kind of iridescent coloration represents an excellent contrast to forest canopy – a natural habitat of Apatura spp. Apart from structural coloration, pigment coloration is present as well.
The flight behavior of male Apatura iris was recently studied and it consisted mainly of perching and patrolling flights. The daily aggregation of males at favored landmark sites from approximately midday was observed as well .
Here we present a detailed study of two Apatura species with respect to their optical properties and the relationship of these properties to the microscopic structure of the wing scales.
2. Materials and methods
2.1 Collecting data
Specimens of the species Apatura iris and A. ilia were used for optical investigations (Fig. 1). Both species were collected from the Balkan Peninsula: A. iris – Mt. Stara Planina, Southeastern Serbia (July 2009, leg. D. Stojanović), and Apatura ilia – Mt. Fruška Gora, Northern Serbia (July 2009, leg. D. Stojanović). The specimens were kept in the collection of the Institute of Zoology, Faculty of Biology, University of Belgrade, Serbia.
2.2 SEM procedure
The specimens of Apatura iris and A. ilia were rinsed with diethyl-ether to obtain a clear surface of the wings. The wings of the males were cut into rectangular shape (surface area of several mm2). This was followed by dehydration in order to obtain dry samples, fixed on a test-bed and subsequently covered with gold. Prepared samples were analyzed by a scanning electron microscope (SEM) (JSM-6460LV, JEOL, Tokyo, Japan).
2.3 TEM procedure
Wings were cut into small pieces, fixed in 3% glutaraldehyde in 0.1M phosphate buffer (pH 7.2) and postfixed in 1% osmium tetroxide in the same buffer. The specimens were dehydrated with serial ethanol solutions of increasing concentration and embedded in Araldite (Fluka, Germany). For electron microscopic examination, the tissue blocks were trimmed and cut with diamond knives (Diatome, Switzerland) on an UC6 ultramicrotome (Leica, Austria). The thin sections were mounted on copper grids, stained with uranyl acetate and lead citrate (Ultrastain, Leica, Austria) and examined on a Philips CM 12 transmission electron microscope (TEM) (Eindhoven, the Netherlands) equipped with a Megaview III digital camera (Soft Imaging System, Münster, Germany).
2.4 Spectrometric measurements
A HR2000CG-UV-NIR Fiber spectrometer was used (Ocean Optics Inc., Dunedin, USA) to collect the reflection spectra of the investigated butterflies. Wing samples were positioned on a computer-controlled rotation platform and illuminated with a tungsten halogen lamp. Thus, we were able to record the reflection spectrum of the wings as a function of the angle of incidence. A MIRA titanium-sapphire laser with frequency doubler (Coherent Inc., USA) was used to investigate the spectral dependence of the wing scattering pattern in the blue and UV part of the spectrum. A diode-pumped Nd-YAG laser at 532 nm and diode laser at 630 nm were used as well. The wings were irradiated with a laser beam and the scattered radiation was photographed on the cylindrical screen by Canon EOS 50D camera. Rigorous coupled-wave analysis was used to calculate the spectral reflectivity.
As in all butterfly species, the scales are positioned like roof tiles covering the entire dorsal and ventral sides of the wing (Fig. 2a ). We recognized two types of scales on the dorsal side of both the fore and hind wings of Apatura iris and A. ilia. The cover scales are on top, while ground scales are situated below. We found that the cover scales are responsible for the blue iridescence of these two butterfly species (Fig. 2b) due to their much denser microscopic structure in comparison to the ground scales. We have found that iridescent scales are pigmented (see brownish scales in lower half of Fig. 2c).
The white areas of the dorsal wing side are also interesting. Examination of the reflective property revealed that the scales uniformly scatter light (Fig. 2b), while in transmission they look completely transparent (Fig. 2c). Scale overlapping increases the scattering, which can be seen as dark areas in upper half of Fig. 2c. We have found that there is a cumulative effect of overlapping. A single scale transmits around 90% and we measured the transmission of the overlapping scales to be about 60%.
By magnifying the iridescent butterfly scale surface of Apatura iris we observed long parallel ridges, each having a number of lamellas positioned one over another (see Fig. 3 for SEM images in two different views). The rows are mutually connected with orthogonally positioned cross ribs. TEM images were used to obtain the exact morphological and dimensional characteristics of each ridge and its lamellae (Fig. 4 ). The lamellae in cross section exhibit a multilayer structure, conifer-like, with six pairs of lateral projections which are not widened distally and are triangularly pointed. The scale structure of Apatura ilia is similar, except that the ridge density is slightly higher.
Electron microscope images were used to construct a geometrical model of the structure which was further utilized for theoretical analysis of the optical properties. Characteristic dimensions can be seen in Fig. 5 . Ridges form a surface relief diffraction grating, with an 820 nm period and approximately the same depth (830 nm). On the other hand, the lamellae form a volume Bragg grating, with a roughly 75 nm period, with each lamella being 40 nm thick.
Butterfly wings were studied spectroscopically in relation to the illumination and observation angles. Several spectra were recorded for different angular orientations (Fig. 6a ). Maximum reflectivity is observed in the UV part of the spectrum (380 nm) and does not depend on the observation angle. Spectral width is small (50 nm FWHM) compared with more visually spectacular species, such as Morpho butterflies . There is a slight spectral shift (in the order of 10-20 nm) as a function of the angle of illumination. All of this apparently provides an evolutionary advantage when the butterfly reflectivity spectra (with UV maximum) are compared with the canopy spectra (with almost no reflectivity in UV region, a peak at 550 nm, and a plateau in the IR region) . UV reflectivity makes the butterfly very visible to its own species and considerably less visible to all other animals, especially potential predators. The intensity of reflected light at visible and IR wavelengths is much lower, making the butterfly appear dull brown from almost all directions – an excellent camouflage in forest, where Apatura species is living.
The iridescence of Apatura iris is observed in a rather narrow angular range (18 degrees); this is much narrower compared to other butterfly species . Iridescence at 380 nm as a function of the angle of incidence is shown in Fig. 6b. Nearly identical spectral properties were observed in Apatura ilia.
In order to observe the spatial distribution of iridescent light, we irradiated the butterfly wing with a laser beam from a tunable Ti-Sapphire laser coupled to frequency doubler. We were able to continuously tune the laser wavelength from 365 to 450 nm. A simple experimental setup is shown in Fig. 7a and 7b, and the typical spatial distribution of iridescence can be seen in Fig. 7c. It should be emphasized that the images were recorded by virtue of the natural fluorescence of the paper screen. In order to obtain an improved spatial distribution we applied pseudo-coloring of the recorded images.
Four images recorded at 365, 387, 405 and 450 nm are shown in Figs. 8a -8d. In comparison to other wavelengths, the spatial distribution at 387 nm is much narrower, indicating that at this particular wavelength the radiation is very directional. At 532 nm the directionality of scattered radiation is almost completely lost, as can be seen in Fig. 9 , which was recorded by a diode-pumped Nd-YAG laser at 532 nm.
The results presented in this study show that the iridescence of Apatura spp. butterflies is spectrally and directionally constrained. With respect to the butterfly body, the radiation is directed as shown in Fig. 10 in three orthogonal projections. This particular feature is a consequence of the mutual orientations of lamellae with respect to the scale, and the scale with respect to the wing membrane (Fig. 11 ). There is a critical angle of incidence (γ + 2α + 2β = π/2) when the radiation is Bragg-reflected along the wing surface. At a greater angle, the radiation cannot be further reflected (i.e., it is directed inside the material). In the case of Apatura butterflies, the angle α of the scale is large (~20°), as can be verified by the strong shadow cast by each scale in Figs. 2a and 2b.
The spectral selectivity of the Apatura spp. scale was analyzed using rigorous coupled-wave analysis using simplified geometry as shown in Fig. 12a . We were able to correctly reproduce the spectral reflectivity (as shown in Fig. 12b) using dimensions presented in Fig. 5 and the refractive index of 1.56 for chitin . Angular dependence of iridescence was calculated too and presented in Fig. 12c. Theoretical results slightly depart from experimental data, but this is due to idealized nature of calculation. In reality the butterfly grating is stochastically distorted, and the effect is averaged among many scales, inside illuminated wing area.
This phenomenon is in contrast with the reflection of Morpho butterfly wings, which direct radiation sideways and in a broad angular range. Also, the spectral maximum of iridescence shifts significantly with the angle of observation . The difference between Morpho spp. and Apatura spp. is due to the different orientations of butterfly scales with respect to wing membrane. In Morpho butterflies the scales are almost parallel to the membrane (for example, Morpho aega), while in the Apatura species the scales are strongly inclined. On the nanoscopic level, ridges on the Apatura scale are not as dense as in Morpho butterflies. Even though the number of lamellas is almost the same in Morpho helenor  and Apatura, their cross sectional profile (as seen in TEM) is quite different. All these factors lead to radically different optical properties - Apatura iridescence is spectrally very pure, and the angular pattern is narrow.
Apatura ilia and Apatura iris are visually quite similar. Apatura ilia males have an iridescent purple color on the wings that dorsally arise from a fully ordered 3D structure, and a yellowish-brown color produced by pigments on the wings ventrally. On the other hand the males of A. iris have the same purple iridescent color on the dorsal side of the wings and a brownish color on the ventral side.
The photonic-type nanostructures consisting of chitin, occurring in the butterfly wing scales of the male individuals of the species Apatura iris and A. ilia, were investigated by both scanning and transmission electron microscopy and reflectance spectroscopy. A tunable laser was used to analyze the variation of spatial distribution of iridescence.
As in all butterfly species, the architecture of the scales is complex. They possess numerous alternating air and cuticle layers responsible for iridescence. From an optical point of view, both analyzed species behave similarly. Maximum reflectivity is observed in the UV region of the spectrum for both species and depends to a certain extent on the observation angle. We have found that the scale iridescence is remarkably narrow, both spectrally and angularly, in the studied butterfly species. This is the consequence of the interplay between scale structure and inclination with respect to the wing membrane. Iridescence is observed in a rather narrow angular range (18 degrees for both analyzed Apatura species while it is much greater in other butterfly species previously studied). The spectral width of the iridescence is small (around 50 nm FWHM for both analyzed Apatura species and is much greater in tropical Morpho butterflies).
We are grateful to Mr. Miloš Bokorov (Faculty of Science, University of Novi Sad, Novi Sad, Serbia) for helping prepare the SEM photographs. Mrs. Anita Lazarević (Center for Electron Microscopy, Faculty of Biology, University of Belgrade, Belgrade, Serbia) assisted in the production of TEM micrographs. Finally, Dejan Stojanović, M.Sc. (Fruška Gora National Park, Sremska Kamenica, Serbia) provided some butterfly specimens for the investigations. The study was financially supported by the Serbian Ministry of Science and Technological Development (projects 141003, 143053, 143050, 45016, 171038, 173038, and 173055).
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