Wing Scale Microstructures and Nanostructures in Butterflies 109

Journal of Microscopy, Vol. 224, Pt 1 October 2006, pp. 108–110

WBlackwell Publishinging Ltd scale microstructures and nanostructures in butterﬂies − natural photonic crystals

Z. VÉRTESY*, Zs. BÁLINT†, K. KERTÉSZ*, J. P. VIGNERON‡, V. LOUSSE‡ & L. P. BIRÓ* *Research Institute for Technical Physics and Materials Science, H-1525 Budapest, POB 49, Hungary †Hungarian Natural History Museum, H-1088 Budapest, Baross str. 13, Hungary ‡Facultés Universitaires Notre-Dame de la Paix, Rue de Bruxelles 61, B-5000 Namur, Belgium

Key words. Butterﬂy wing, natural photonic crystals, scale nanostructure, structural colour.

have Urania-type wing scales that show open microcells formed Summary by longitudinal ridges, cross-ribs and basal membrane, or The aim of our study was to investigate the correlation between microcells filled with nanostructured layers, the so-called structural colour and scale morphology in butterflies. Detailed pepper-pot structure. correlations between blue colour and structure were investigated Since 1995 (Joannopoulos et al., 1995), researchers have in three lycaenid subfamilies, which represent a monophylum intensively studied photonic crystals − dielectric materials that in the butterfly family Lycaenidae (Lepidoptera): the Coppers change the propagation of light. Photonic crystals are composites (Lycaeninae), the Hairstreaks (Theclinae) and the Blues exhibiting a periodic distribution of refractive indexes. In (Polyommatinae). Complex investigations such as spectral photonic crystals, well-defined frequency ranges exist in which measurements and characterization by means of light micro- light cannot propagate through the structure. These frequency scopy, scanning electron microscopy and transmission electron ranges are also called photonic band gaps. Light of frequencies microscopy enabled us to demonstrate that: (i) a wide array of within the forbidden gap of an ideal photonic crystal is completely nanostructures generate blue colours; (ii) monophyletic groups reflected by the structure. Potential applications in optical use qualitatively similar structures; and (iii) the hue of the blue filtering and computing or in the design of very compact lasers colour is characteristic for the microstructure and nanostruc- provide the motivation for producing artificial photonic crystals. ture of the body of the scales. Natural photonic crystals also form a focus of attention (Parker, 2002). Photonic crystal structures occur in almost all animal phyla (Parker, 2003). Butterfly wings provide an excellent Introduction example of how living organisms can manipulate light. The Many authors have shown that two mechanisms are responsible colouration of certain species is produced by natural photonic for colour generation in butterfly wings (Mason, 1926; Vukusic, crystals; that is, colouration arises from the interaction of light 1998; Tilley & Eliot, 2002). Colour arises either from the pig- with three-dimensional microstructures and nanostructures mentation (chemical colour) or from the structure (physical of the scales that cover their wings (Biró et al., 2003). colour) of the wing scales. Physical colours depend on the struc- The colours that appear in butterflies are diverse and serve ture of the surface and the volume of the scales. Chemical colour different functions. Colour can be involved in thermal regula- is less intense than structural colour. Melanin and pterin, pig- tion as well as signalling. Bálint & Johnson (1997) formulated ments frequently found in butterflies, produce yellow, red, black a hypothesis that discoloration is connected with the thermal and brown colours. To the best of our knowledge, pigments only regulation of butterflies living at high altitudes or latitudes, cannot produce iridescent blue, violet, green and golden colours. where solar energy is restricted because of climatic conditions. The Lepidoptera family Lycaenidae, the Lycaenids, is well This hypothesis was tested experimentally (Biró et al., 2003) known for its vivid dorsal wing colouration, which represents by investigation of a cluster of sister species belonging to the practically the entire visible spectrum. Many species belonging genus Polyammatus (Polyommatinae) living at different altitudes. to this family have blue colouration of different hues. Lycaenids Combined information gathered from thermal and spectral measurements, investigations of the scale morphology and numerical Correspondence to: Zofia Vértesy. Tel: +36 1 392 2222; fax: +36 1 392 2226; simulation supported the important role of discoloration in e-mail: vertesyz@ mfa.kfki.hu thermal regulation and in enhancing chances of survival.

Colour signals are important factors in interactions between that scales are made of] walls and holes in the microcell. The individuals that often occur together in the same habitat at the microcell is defined as the volume framed by two consecutive same time. This is why butterflies have to have species-specific ridges, two consecutive cross-ribs, and the basal membrane of scales generating different colours. the scale. The Blues and Coppers are butterflies that live in open habitats, where the sunlight is strong and direct. There- fore, they have developed scales that manipulate the light using Materials and methods similar structures. The Hairstreaks have differently structured The characterization of butterfly scales was achieved using scales. The cross-ribs of the microcells and the layers with several complementary methods. We carried out spectral circular holes lie very deep (Fig. 1d,e). They are not parallel to measurements and performed investigations with light micro- the basal membrane, but generally form concave surfaces scopy, scanning electron microscopy and transmission electron inside the microcells. microscopy techniques. The hue of the reflected light is correlated with the nano- Reflection and/or transmission spectra of entire wings or structure of the body of the scales. This could be demonstrated individual scales were obtained using an Avaspec 2048/2 fibre with examples of nanostructures in Coppers. Heliophorus sena optic spectrometer (Avantes, Eerbeek, The Netherlands), work- (Fig. 1c) and L. alciphron have very similar nanostructures ing in the wavelength range from 200 to 1100 nm. Reflection and generate a violet colour, as shown in the reflectance graph spectra were measured in the perpendicular direction or using (Fig. 1g). These nanostructures are different from the nano- an integration sphere in order to record all the reflected light. structures of H. tamu, L. heterona, Polyommatus bellargus and The morphology of the scales was investigated by means of Polyommatus icarus, which generate blue colours. These scanning electron microscopy and transmission electron micro- species all have scale nanostructures similar to the nanostruc- scopy. Areas of interest were chosen by means of light micro- tures found in Polyommatus icarus scales (Fig. 1a). scopy. Small fragments of forewings were fixed to double-sided We also compared the structures of scales in Pseudolycaena carbon tape and Au coated for scanning electron microscopy marsyas and Morpho thamyris (Nymphalidae: Morphinae). investigations. Cross-sections were prepared for transmis- Morpho-type scales (Mason, 1926) are characterized by pos- sion electron microscopy observations by embedding pieces sessing only longitudinal ridges and not forming microcells. of scales in epoxy resin and cutting using an ultramicrotome. Both species generate similar vivid blue colours, but the micro- The investigated samples were scientific specimens held in structure and nanostructure of their scales are essentially the collections of the Hungarian Natural History Museum. different. Morphos and blue Hairstreaks primarily inhabit for- ested tropical habitats. Owing to the weaker illumination from the sun in these habitats, a more efficient light-manipulation Results and Discussion mechanism is needed as compared with open habitats, where Investigations of the correlations between colour and morpho- more solar energy is available. Because of special light conditions, logy were carried out on groups of lycaenid butterflies repre- distantly related species evolve similar behaviours, character- senting three subfamilies: the Coppers (Lycaeninae: Heliophorus ized by directional reflectance, giving a metallic aspect to the sena, H. tamu, Lycaena alciphron, L. heterona), the Hairstreaks butterfly wing colour, but use different types of structure to (Theclinae: Denivia hemon, Pseudolycaena marsyas, Theritas achieve light manipulation. paupera) and the Blues (Polyommatinae: Polyommatus bellargus, Polyommatus icarus, Polyommatus versicolor, Pseudolucia plumbea). Conclusions We gathered detailed information about the microstructure and nanostructure of the scales using scanning electron The results of our investigations can be briefly summarized as microscopy and transmission electron microscopy, and meas- follows: ured the corresponding reflection spectra. First, we noticed a 1A wide variety of nanostructures generate blue colours. correlation between reflectance intensity and direction. An 2 Monophyletic groups use qualitatively similar nanostructures. especially strong dependence on direction was observed for 3 The hue of the various blue colours is determined by L. alciphron and H. sena. differences in the nanostructure of the body of scales. We found that a variety of nanostructures generate blue 4 Similar hues of blue can be generated by qualitatively different colour in butterflies of the family Lycaenidae, and only mono- structures, as for example in Hairstreaks and Morphos, and phyletic groups use a qualitatively similar type of structure. this indicates phylogenetic distance. The Blues and Coppers (Fig. 1a–c) generally have scales that can be characterized by parallel layers of cuticles filling the Acknowledgements microcell of the individual scale. These layers have circular holes. The difference between the nanostructures of different This work was supported by the Hungarian Scientific Research species and subfamilies is mainly determined by the filling Fund (T-042972) and the EU FP6 NEST/PATHFINDER/ − factor, i.e. the ratio of chitin [(C8H13O5N)n the polysaccharide BIOPHOT- 012915 project.

Fig. 1. Nanostructure of scales and corresponding reﬂectance spectra measured by means of integration sphere: nanostructure characteristic for (a) Polyommatus icarus, (c) Heliophorus sena and (d) Pseudolycaena marsyas. (b) Transmission electron microscopy image of scale cross-section shown in (a). (e) Transmission electron microscopy image of scale cross-section shown in (d). (f–h) Reﬂectance spectra for families Polyammatinae (f), Lycaeninae (g) and Theclinae (h). All cross-sections are perpendicular to the longitudinal ridges. The tops of the images correspond to the up surface of the scales.

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