158 entomologische berichten 67(5) 2007 Colourful butterfly wings: scale stacks, iridescence and sexual dichromatism of Pieridae Doekele G. Stavenga Hein L. Leertouwer KEY WORDS Coliadinae, Pierinae, scattering, pterins Entomologische Berichten 67 (5): 158-164 The colour of butterflies is determined by the optical properties of their wing scales. The main scale structures, ridges and crossribs, scatter incident light. The scales of pierid butterflies have usually numerous pigmented beads, which absorb light at short wavelengths and enhance light scattering at long wavelengths. Males of many species of the pierid subfamily Coliadinae have ultraviolet-iridescent wings, because the scale ridges are structured into a multilayer reflector. The iridescence is combined with a yellow or orange-brown colouration, causing the common name of the subfamily, the yellows or sulfurs. In the subfamily Pierinae, iridescent wing tips are encountered in the males of most species of the Colotis-group and some species of the tribe Anthocharidini. The wing tips contain pigments absorbing short-wavelength light, resulting in yellow, orange or red colours. Iridescent wings are not found among the Pierini. The different wing colours can be understood from combinations of wavelength-dependent scattering, absorption and iridescence, which are characteristic for the species and sex. Introduction often complex and as yet poorly understood optical phenomena The colour of a butterfly wing depends on the interaction of encountered in lycaenids and papilionids. The Pieridae have light with the material of the wing and its spatial structure. But- two main subfamilies: Coliadinae and Pierinae. Within Pierinae, terfly wings consist of a wing substrate, upon which stacks of the tribes Pierini and Anthocharidini are distinguished, together light-scattering scales are arranged. The colour of the scales is with the Colotis-group. This paper describes and interprets the usually determined by pigments. The colour then is called a pig- various colours displayed by pierid butterflies, with special at- mentary or chemical colour. Additional colouration sometimes tention to colour differences between the sexes. occurs because of the interaction of light with periodic structu- res, for instance when the scales contain multilayers, resulting The large white and the small white (Pierini) in iridescence. The colour then is called a structural or physical colour. The colours of butterfly wings can be understood by ana- The large white, Pieris brassicae (Linnaeus), deserves its name, lyzing the different contributions of light scattering, pigment because its large wings are dorsally brilliant white (figure 1a). absorption and iridescence (technical terms are explained in The intense white light reflection is non-directional and there- the glossary in Box 1). fore the wings must have strongly scattering elements. The Butterflies of the family Pieridae are attractive for develo- fraction of back-scattered light, the reflectance, can be measu- ping our understanding of butterfly wing colouration, because red with a spectrophotometer. The reflectance spectrum is low their colouration methods are relatively simple compared to the in the ultraviolet, at wavelengths below 400 nm, and high in the 1. a Large white, Pieris brassicae. b Reflectance spectra of a white area of the wings of a male Pieris rapae measured with a micros- pectrophotometer, before and after the application of a drop of immersion fluid (xylene; modified from Stavenga et al. 2004). a Groot koolwitje, Pieris brassicae. b Reflectiespectra van een wit gebied van de vleugels van een manne- tje Pieris rapae gemeten met een microspectrofotometer, voor en na opbrengen van een druppel immer- sievloeistof (xyleen; gewijzigd van Stavenga et al. 2004). entomologische berichten 159 67(5) 2007 2. Electron microscopic photographs of scales of a male small white, Pieris rapae. a Cover sca- les partly overlap ground scales (scanning electron microscopy, SEM). The upper lamina of the scales features longitudinal ridges. b Part of a white scale (SEM). The ridges are connected by crossribs, which are adorned by numerous oval-shaped beads. c Transmission electron microscopic (TEM) section of a white scale, showing the beads at the upper lamina of the scale. The lower lamina of the scale is more or less flat. d A scale from one of the black spots (SEM), showing that in this case the crossribs do not have beads (a, bar: 25 μm; b-d, bar: 2 μm; modified from Stavenga et al. 2004). Electronenmicroscopische foto's van schubben van een mannetje klein koolwitje Pieris rapae. a Dekschubben overlappen deels de onderschubben (scanning-electronenmicroscopie, SEM). b Deel van een witte schub (SEM). De richels zijn verbonden door dwarsribben, waaraan talrijke ovale kralen hangen. c Transmissie-electronenmicroscopische (TEM) foto van een witte schub, waarin de kralen aan de bovenlaag van de schub te zien zijn. De onderlaag van de schub is min of meer vlak. d Een schub van een van de zwarte vlekken (SEM), waarbij te zien is dat de kralen aan de dwarsribben ontbreken (a, maatstreep: 25 μm; b-d, maatstreep: 2 μm; gewijzigd van Stavenga et al. 2004). and the lower lamina is virtually smooth (figure 2c). The upper lamina rests via pillars, so-called trabeculae, on the lower lami- na. The scales at the dorsal side of the wings of P.e rapae ar vir- tually all white, but some scales are black. The colour difference is related to a structural difference: the crossribs of the white scales carry oval-shaped beads, which contain pigment that ab- sorbs exclusively ultraviolet light (figure 2b, c), whereas the black scales do not have beads (figure 2d). The ridges and cross- ribs of the black scales contain melanin pigment, which absorbs ultraviolet as well as visible light (Stavenga et al. 2004). Structures with a refractive index different from their envi- ronment scatter incident light. The material of the ridges and crossribs of butterfly wing scales is chitin, which has a refracti- ve index of about 1.57, distinctly higher than the refractive in- dex value 1 of air. The beads probably have a still higher refracti- ve index, but that is as yet uncertain. It is clear that the scales act as light scatterers, because the scattering by the ridges, crossribs as well as the beads of the white scales is substantially reduced when an immersion fluid like xylene, which has a re- fractive index 1.49, replaces the air, so that the difference in re- fractive difference distinctly drops. In other words, the immersi- on fluid causes a reduced reflectance (figure 1b). The beads of the scales of P. rapae contain a pigment that ab- sorbs in the ultraviolet (UV) and this causes a low UV-reflectan- visible wavelength range, at wavelengths above 450 nm, as shown in the closely related small white P. rapae (Linnaeus) (fi- gure 1b). Insight into the physical basis of the reflectance can be ob- tained by applying immersion fluid. The reflectance spectrum (figure 1b, time t = -1 s) changes abruptly when a drop of immer- sion fluid, for instance xylene, is applied to the wing (at time t = 0 s). It causes an immediate decrease in the long-wavelength reflectance, but the reflectance gradually recovers when the im- mersion fluid evaporates (figure 1b, times t = 60, 120, 180 s). This result can be readily understood from the structure of the wings 3. Reflectance spectra of various parts of the wings of the male black jezebel, Delias nigrina (inset: up – dorsal side, and down – ven- (Ghiradella 1984, Nijhout 1991). The wings of pierids are, like tral side), measured with a fiber optic spectrometer (modified from those of all butterflies (and moths), studded with scales, partly Stavenga et al. 2006). overlapping as tiles on a roof (figure 2a). The scales are flat and Reflectiespectra van verschillende delen van de vleugels van een man- netje Delias nigrina (inzet: boven – dorsale zijde, onder – ventrale zijde), measure about 50 x 200 μm2. The upper lamina features charac- gemeten met een fiber-optische spectrometer (gewijzigd van Stavenga teristic longitudinal ridges, connected by crossribs (figure 2b, d), et al. 2006). 160 entomologische berichten 67(5) 2007 4. Reflectance spectra from the dorsal forewing of the male orange tip, Anthocharis cardamines. Inset: up - normal RGB photograph; down - UV photograph. Scale bar: 1 cm. Reflectiespectra van de dorsale voorvleugel van een mannetje oranje- tip, Anthocharis cardamines. Inzet: boven - normale kleurenfoto, onder – UV foto. Maatstreep: 1 cm. cations 1 and 3 at the dorsal side of the wing. These locations were opposed at the ventral side by red bands, whereas location 2 of the dorsal side of the wing had brown-black scales at the ventral side. Reflectance spectrum 2 (of location 2) is more or less flat at all wavelengths above 450 nm, but spectra 1 and 3 are only flat between 450 and 550 nm. The latter spectra rise above 550 nm, to flatten again at wavelengths above 700 nm. The re- flectance rise happens precisely in the wavelength range where the reflectance of the red band at the ventral wing side rapidly increases. The explanation of the rise in the reflectance spectra 1 and 3 above 550 nm is therefore that the dorsal scales are slightly transmittant. Long-wavelength incident light is obvi- ously strongly reflected, but a fraction is transmitted, and this ce (figure 1b). The human eye does not see into the ultraviolet light is back-scattered by the red scales at the ventral side of the wavelength range and therefore the high reflectance throug- wings and subsequently partly passes the dorsal scales. This hout the visible wavelength range of the wings of the whites transmitted light slightly but significantly contributes to the re- causes the observed white colour. Butterflies, as all insects, see flectance measured at the dorsal side (Stavenga et al. 2006). well into the UV and will therefore detect the low UV reflectan- ce of the Pieris wings.