Butterfly blues and greens caused by subtractive colour mixing of carotenoids and bile pigments.

Butterflies often have conspicuously patterned wings, due to pigmentary and/or structurally wing scales that cover the wing membrane. The wing membrane of several butterfly species is also pigmentary coloured, notably by the bile pigments pterobilin, pharcobilin and sarpedobilin. The absorption spectra of the bilins have bands in the ultraviolet and red wavelength range, resulting in blue-cyan colours. Here, a survey of papilionoid and nymphalid butterflies reveals that several species with wings containing bile pigments combine them with carotenoids and other short-wavelength absorbing pigments, e.g., papiliochrome II, ommochromes and flavonoids, which creates green-coloured patterns. Various uncharacterized, long-wavelength absorbing wing pigments were encountered, particularly in heliconiines. The wings thus exhibit quite variable reflectance spectra, extending the enormous pigmentary and structural colouration richness of butterflies.

Subtractive colour mixing with bile pigments creates the rich wing palette of Graphium weiskei butterflies.

The wings of the purple spotted swallowtail Graphium weiskei are marked by an unusual bright colour pattern. Spectrophotometry on G. weiskei wings demonstrated the presence of a pigment with an absorption spectrum (peak wavelength λmax=676 nm) similar to that of the bile pigment sarpedobilin in the wings of the congeneric Graphium sarpedon (λmax=672 nm). Sarpedobilin alone causes cyan-blue wing areas, but the green-coloured areas of G. sarpedon wings result from subtractive colour mixing with the carotenoid lutein. Reflectance spectra of the blue-coloured areas of G. weiskei wings indicate that sarpedobilin is mixed with the short-wavelength-absorbing papiliochrome II. An enigmatic pigment, tentatively called weiskeipigment (λmax=580 nm), enhances the saturation of the blue colour. Weiskeipigment causes a purple colour in areas where the sarpedobilin concentration is low. The wings of the related papilionid Papilio phorcas contain the bile pigment pharcobilin (λmax=604 nm), as well as another sarpedobilin (λmax=663 nm). The cyan to greenish wings of P. phorcas are due to phorcabilin and sarpedobilin mixed with papiliochrome II. A survey of known subspecies of G. weiskei as well as of congeneric Graphium species of the 'weiskei' group shows various degrees of subtractive colour mixing of bilins and short-wavelength absorbers (carotenoids and/or papiliochromes) in their wings. This study illuminates the underestimated role of bile pigments in butterfly wing colouration.

Pigmentary colouration of hairy carpenter bees, genus Xylocopa.

Carpenter bees can display distinct colouration patterns due to structural coloured wings and/or coloured hairs on their bodies. Females of the sexually dichromatic Xylocopa caerulea are marked by strongly blue-pigmented hairs on the head, thorax and abdomen. The thorax of female X. confusa is covered by yellow-pigmented hairs. The diffuse pigmentary colouration of the blue and yellow hairs is effectively enhanced by strongly scattering granules. The absorption spectrum of the blue pigment of X. caerulea has a maximum at 605 nm and is probably a bilin (a bile pigment). The absorption spectrum of the yellow pigment of X. confusa has a maximum at 445 nm and may be a pterin. The thoracic hairs of female X. confusa contain also a minor amount of the bilin. The reflectance spectra of the pigmented hairs suggest that the pigments are tuned to the spectral sensitivity of the bees' photoreceptors and provide spectral contrast with a green background.

Butterfly Wing Translucence Enables Enhanced Visual Signaling.

The light reflected by the dorsal side of butterfly wings often functions as a signal for, e.g., mate choice, thermoregulation, and/or predator deterrence, while the ventral wing reflections are generally used for crypsis and camouflage. Here, we propose that transmitted light can also have an important role in visual signaling because, in many butterfly species, the dorsal and ventral wing sides are similarly patterned and locally more or less translucent. Extreme examples are the Japanese yellow swallowtail (Papilio xuthus Linnaeus, 1758) and the Yellow glassy tiger (Parantica aspasia Fabricius, 1787). Their wings exhibit a similar color pattern in reflected and transmitted light, which allows enhanced visual signaling, especially in flight. Contrasting cases in which the coloration and patterning of dorsal and ventral wings strongly differ are the papilionid Papilio nireus Linnaeus, 1758, and the pierid Delias nigrina Fabricius, 1775. The wings observed in reflected or transmitted light then show very different color patterns. Wing translucence thus will strongly affect a butterfly's visual signal.

Circadian rhythm entrainment of the jewel wasp, Nasonia vitripennis, by antagonistic interactions of multiple spectral inputs.

Circadian light entrainment in some insects is regulated by blue-light-sensitive cryptochrome (CRY) protein that is expressed in the clock neurons, but this is not the case in hymenopterans. The hymenopteran clock does contain CRY, but it appears to be light-insensitive. Therefore, we investigated the role of retinal photoreceptors in the photic entrainment of the jewel wasp Nasonia vitripennis. Application of monochromatic light stimuli at different light intensities caused phase shifts in the wasp's circadian activity from which an action spectrum with three distinct peaks was derived. Electrophysiological recordings from the compound eyes and ocelli revealed the presence of three photoreceptor classes, with peak sensitivities at 340 nm (ultraviolet), 450 nm (blue) and 530 nm (green). An additional photoreceptor class in the ocelli with sensitivity maximum at 560-580 nm (red) was found. Whereas a simple sum of photoreceptor spectral sensitivities could not explain the action spectrum of the circadian phase shifts, modelling of the action spectrum indicates antagonistic interactions between pairs of spectral photoreceptors, residing in the compound eyes and the ocelli. Our findings imply that the photic entrainment mechanism in N. vitripennis encompasses the neural pathways for measuring the absolute luminance as well as the circuits mediating colour opponency.

Polarized iridescence of the tropical carpenter bee, Xylocopa latipes.

The tropical carpenter bee, Xylocopa latipes, has metallic-reflecting, iridescent wings. The wing reflectance spectra for TE- and TM-polarized light depend on the angle of light incidence in a way characteristic for dielectric multilayers. Anatomy indicates the presence of melanin multilayers in the wing's chitinous matrix. A simple optical model of melanin multilayers explains the angle dependence of the wing reflectance spectra. The wing reflections that occur upon oblique illumination exhibit colourful and strongly polarized light patterns, which may mediate intraspecific signaling and mutual recognition by conspecifics.

Automated Charting of the Visual Space of Housefly Compound Eyes.

This paper describes the automatic measurement of the spatial organization of the visual axes of insect compound eyes, which consist of several thousands of visual units called ommatidia. Each ommatidium samples the optical information from a small solid angle, with an approximate Gaussian-distributed sensitivity (half-width on the order of 1˚) centered around a visual axis. Together, the ommatidia gather the visual information from a nearly panoramic field of view. The spatial distribution of the visual axes thus determines the eye's spatial resolution. Knowledge of the optical organization of a compound eye and its visual acuity is crucial for quantitative studies of neural processing of the visual information. Here we present an automated procedure for mapping a compound eye's visual axes, using an intrinsic, in vivo optical phenomenon, the pseudopupil, and the pupil mechanism of the photoreceptor cells. We outline the optomechanical setup for scanning insect eyes and use experimental results obtained from a housefly, Musca domestica, to illustrate the steps in the measurement procedure.

The wing scales of the mother-of-pearl butterfly, Protogoniomorpha parhassus, are thin film reflectors causing strong iridescence and polarization.

The dorsal wings of the mother-of-pearl butterfly, Protogoniomorpha parhassus, display an angle-dependent pink, structural color. This effect is created by light interference in the lower lamina of the wing scales, which acts as an optical thin film. The scales feature extremely large windows that enhance the scale reflectance, because the upper lamina of ridges and cross-ribs is very sparse. Characteristic for thin film reflectors, the spectral shape of the reflected light strongly depends on the angle of light incidence, shifting from pink to yellow when changing the angles of illumination and observation from normal to skew, and also the degree of polarization strongly varies. The simultaneous spectral and polarization changes serve a possibly widespread, highly effective system among butterflies for intraspecific communication during flight.

Evolution of Insect Color Vision: From Spectral Sensitivity to Visual Ecology.

Color vision is widespread among insects but varies among species, depending on the spectral sensitivities and interplay of the participating photoreceptors. The spectral sensitivity of a photoreceptor is principally determined by the absorption spectrum of the expressed visual pigment, but it can be modified by various optical and electrophysiological factors. For example, screening and filtering pigments, rhabdom waveguide properties, retinal structure, and neural processing all influence the perceived color signal. We review the diversity in compound eye structure, visual pigments, photoreceptor physiology, and visual ecology of insects. Based on an overview of the current information about the spectral sensitivities of insect photoreceptors, covering 221 species in 13 insect orders, we discuss the evolution of color vision and highlight present knowledge gaps and promising future research directions in the field.

Coloration principles of the Great purple emperor butterfly (Sasakia charonda).

The dorsal wings of male Sasakia charonda butterflies display a striking blue iridescent coloration, which is accentuated by white, orange-yellow and red spots, as well as by brown margins. The ventral wings also have a variegated, but more subdued, pattern. We investigated the optical basis of the various colors of intact wings as well as isolated wing scales by applying light and electron microscopy, imaging scatterometry and (micro)spectrophotometry. The prominent blue iridescence is due to scales with tightly packed, multilayered ridges that contain melanin pigment. The scales in the brown wing margins also contain melanin. Pigments extracted from the orange-yellow and red spots indicate the presence of 3-OH-kynurenine and ommochrome pigment. The scales in the white spots also have multilayered ridges but lack pigment. The lower lamina of the scales plays a so-far undervalued but often crucial role. Its thin-film properties color the majority of the ventral wing scales, which are unpigmented and have large windows. The lower lamina acting as a thin-film reflector generally contributes to the reflectance of the various scale types.

Polymorphism of Colias croceus from the Azores caused by differential pterin expression in the wing scales.

The pierid butterfly Colias croceus (Geoffroy in Fourcroy, 1785), established in the Azores archipelago, is polymorphic with six forms, C. croceus f. croceus ♂ and ♀, C. c. f. cremonae ♂ and ♀, C. c. f. helice ♀, and C. c. f. cremonaehelice ♀. We investigated the optical mechanisms underlying the wing colouration of the butterflies by performing spectrophotometry and imaging scatterometry of the variously coloured wing areas and scales. The scale colouration is primarily due to wavelength-selective absorption of incident light by pterins expressed in granular beads in the wing scales, but thin film reflections of the scales' lower lamina and scale stacking also contribute. Three forms (croceus ♂ and ♀ and helice ♀) are consistent with the patterns of the well-known 'alba' polymorphism. We postulate the coexistence of a second polymorphism, 'cremonae', to understand the three other forms (cremonae ♂ and ♀, and cremonaehelice ♀), which are characterized by the absence of red pigment, presumably due to the differential blocking of erythropterin expression.

The lesser purple emperor butterfly, Apatura ilia: from mimesis to biomimetics.

Until now, hues as dynamic as those adorning the Apatura emperor butterflies have never been encountered in the painting world. Unlike and unmatched by the chemical pigments traditionally found on the painter's palette, the emperor's wings are studded with strongly reflecting iridescent scales that are structured like those of the iconic Morpho butterflies. The scale ridges act as diffractive multilayers, giving rise to narrow-band reflectance spectra. All scales together create a vividly purple iridescent wing colouration that is observed within a narrow angular range only. Recently, synthetic structures analogous to the multilayer reflectors found on butterfly wings have been developed, referred to as effect pigments. Artists can obtain vital clues for how to adapt and adopt these challenging new materials for painting, by tracing the origin of biomimetics back to the ancient concept of mimesis and building on the knowledge accumulated by optical studies. By selecting various effect pigments, and using the lesser purple emperor butterfly, Apatura ilia, as exemplar, we have accurately mimicked the butterfly's iridescence in art. The resulting artwork, like the butterfly, fluctuates in perceived colour depending on the direction of illumination and viewing. These nature-inspired-colouration and biomimetic-application methods extend the canon of art.

Highly different flavonol content explains geographic variations in the UV reflecting properties of flowers of the corn poppy, Papaver rhoeas (Papaveraceae).

Papaver rhoeas, the corn poppy, is a very common weed in cereal fields all over the world. Its flowers generally display a bright red coloration, but their reflectance in the ultraviolet (UV) wavelength range varies geographically. Whereas the UV reflectance of East Mediterranean flowers is minor, that of Central European ones is substantial. By comparing the pigmentation of the differently reflecting flowers, we found that only East Mediterranean flower petals contain high amounts of UV absorbing flavonol glycosides. The most abundant compounds were isolated by solid phase extraction and preparative HPLC, and their structures were elucidated by NMR and HRESI-MS, yielding seven kaempferol and quercetin glycosides, mostly unknown in P. rhoeas petals. Additionally, reflectance and transmittance measurements revealed that wavelength-selective scattering effects do not contribute to the flower color differences observed within this species. Possible abiotic and biotic factors influencing the UV reflecting properties of East Mediterranean and Central European poppies are discussed.

Biophotonics of diversely coloured peacock tail feathers.

Peacock feathers feature a rich gamut of colours, created by a most sophisticated structural colouration mechanism. The feather barbules contain biophotonic structures consisting of two-dimensionally-ordered lattices of cylindrical melanosomes and air channels embedded in keratin. Here, we study the reflectance characteristics of the various peacock tail feather colours by applying bifurcated-probe- and micro-spectrophotometry and imaging scatterometry. We compare the experimental results with published anatomical SEM and TEM data, using a transfer-matrix based effective-medium multilayer model that includes the number and diameter of the melanosome rodlets and air channels, the lattice spacing and the keratin cortex thickness, together with the recently determined wavelength-dependence of the refractive indices of keratin and melanin. Slight variations in the parameter values cause substantial changes in the spectral position and shape of the reflectance bands. We find that the number of layers crucially determines the number of peaks in the reflectance spectra. For a small number of melanosome layers, the reflectance band shape is particularly sensitive to the properties of the uppermost layer, which provides a simple mechanism for tuning the feather colours.

Enhanced UV-Reflection Facilitated a Shift in the Pollination System of the Red Poppy, Papaver rhoeas (Papaveraceae).

Evolutionary change is considered a major factor influencing the invasion of new habitats by plants. Yet, evidence on how such modifications promote range expansion remains rather limited. Here we investigated flower color modifications in the red poppy, Papaver rhoeas (Papaveraceae), as a result of its introduction into Central Europe and the impact of those modifications on its interactions with pollinators. We found that while flowers of Eastern Mediterranean poppies reflect exclusively in the red part of the spectrum, those of Central European poppies reflect both red and ultraviolet (UV) light. This change coincides with a shift from pollination by glaphyrid beetles (Glaphyridae) to bees. Glaphyrids have red-sensitive photoreceptors that are absent in bees, which therefore will not be attracted by colors of exclusively red-reflecting flowers. However, UV-reflecting flowers are easily detectable by bees, as revealed by visual modeling. In the North Mediterranean, flowers with low and high UV reflectance occur sympatrically. We hypothesize that Central European populations of P. rhoeas were initially polymorphic with respect to their flower color and that UV reflection drove a shift in the pollination system of P. rhoeas that facilitated its spread across Europe.

Conical epidermal cells cause velvety colouration and enhanced patterning in Mandevilla flowers.

The majority of angiosperms have flowers with conical epidermal cells, which are assumed to have various functions, such as enhancing the visual signal to pollinators, but detailed optical studies on how conical epidermal cells determine the flower's visual appearance are scarce. Here we report that conical epidermal cells of Mandevilla sanderi flowers effectively reduce surface gloss and create a velvety appearance. Owing to the reduction in surface gloss, the flower further makes more efficient use of floral pigments and light scattering structures inside the flower. The interior backscattering yields a cosine angular dependence of reflected light, meaning that the flowers approximate near-perfect (Lambertian) diffusers, creating a visual signal that is visible across a wide angular space. Together with the large flowers and the tilted corolla tips, this generates a distinct visual pattern, which may enhance the visibility to pollinators.

Bogong Moths Are Well Camouflaged by Effectively Decolourized Wing Scales.

Moth wings are densely covered by wing scales that are assumed to specifically function to camouflage nocturnally active species during day time. Generally, moth wing scales are built according to the basic lepidopteran Bauplan, where the upper lamina consists of an array of parallel ridges and the lower lamina is a thin plane. The lower lamina hence acts as a thin film reflector having distinct reflectance spectra that can make the owner colorful and thus conspicuous for predators. Most moth species therefore load the scales' upper lamina with variable amounts of melanin so that dull, brownish color patterns result. We investigated whether scale pigmentation in this manner indeed provides moths with camouflage by comparing the reflectance spectra of the wings and scales of the Australian Bogong moth (Agrotis infusa) with those of objects in their natural environment. The similarity of the spectra underscores the effective camouflaging strategies of this moth species.

Ultra-dense, curved, grating optics determines peacock spider coloration.

Controlling light through photonic nanostructures is important for everyday optical components, from spectrometers to data storage and readout. In nature, nanostructured materials produce wavelength-dependent colors that are key for visual communication across animals. Here, we investigate two Australian peacock spiders, which court females in complex dances with either iridescent color patterns (Maratus robinsoni) or an approximately angle-independent blue coloration (M. nigromaculatus). Using light microscopy, FIB-SEM imaging, imaging scatterometry, and optical modeling, we show that both color displays originate from nanogratings on structured 3D surfaces. The difference in angle-dependency of the coloration results from a combination of the local scale shape and the nanograting period. The iridescence of M. robinsoni arises from ordered gratings on locally flat substrates, while the more stable blue colors of M. nigromaculatus originate from ultra-dense, curved gratings with multiscale disorder. Our results shed light on the design principle of the peacock spiders' scales and could inspire novel dispersive components, e.g. used in spectroscopic applications.

Coloration of Flowers by Flavonoids and Consequences of pH Dependent Absorption.

Flavonoid pigments are key determinants of flower colors. As absorption spectra of flavonoids are known to be severely pH-dependent, cellular pH will play a crucial role in flower coloration. The flavonoids are concentrated in the vacuoles of the flowers' epidermal cells, and thus the pigments' absorption spectra are modulated by the vacuolar pH. Here we study the pH dependence of flavonoid absorption spectra in extracts from flowers of two poppy species Papaver dubium (red) and Meconopsis cambrica (orange), and a white and red Mandevilla sanderi variety. In the red poppy and Mandevilla flowers, absorption spectra of the cyanidin- and pelargonidin-based anthocyanins peak in the blue-green-wavelength range at low pH, but exhibit a distinct bathochromic shift at higher pH. This shift to longer wavelengths is not found for the blue-absorbing nudicaulin derivatives of M. cambrica, which have a similar absorption spectrum at low and high pH. The pH-dependent absorption changes of the white M. sanderi's flavonoid remained restricted to the UV. An analysis of the spectra with logistic functions suggests that the pH-dependent characteristics of the basic states of flavonols and anthocyanins are related. The implications of tuning of pH and pigment absorption spectra for studies on flower color evolution are discussed.

Vividly coloured poppy flowers due to dense pigmentation and strong scattering in thin petals.

The flowers of poppies (Papaveraceae) exhibit bright colours, despite their thin and floppy petals. We investigated the optical properties of flowers of Papaver rhoeas, P. dubium, Meconopsis cambrica and Argemone polyanthemos using a combined approach of anatomy, spectrophotometry and optical modelling. The petals of Papaver flowers are composed of only three cell layers, an upper and lower epidermal layer, which are densely filled with pigment, and an unpigmented mesophyll layer. Dense pigmentation together with strong scattering structures, composed of serpentine cell walls and air cavities, cause the striking poppy colours. We discuss how various aspects of the optical signal contribute to the flower's visibility to pollinators.