Elucidating nanostructural organization and photonic properties of butterfly wing scales using hyperspectral microscopy.

Biophotonic nanostructures in butterfly wing scales remain fascinating examples of biological functional materials, with intriguing open questions with regard to formation and evolutionary function. One particularly interesting butterfly species, Erora opisena (Lycaenidae: Theclinae), develops wing scales that contain three-dimensional photonic crystals that closely resemble a single gyroid geometry. Unlike most other gyroid-forming butterflies, E. opisena develops discrete gyroid crystallites with a pronounced size gradient hinting at a developmental sequence frozen in time. Here, we present a novel application of a hyperspectral (wavelength-resolved) microscopy technique to investigate the ultrastructural organization of these gyroid crystallites in dry, adult wing scales. We show that reflectance corresponds to crystallite size, where larger crystallites reflect green wavelengths more intensely; this relationship could be used to infer size from the optical signal. We further successfully resolve the red-shifted reflectance signal from wing scales immersed in refractive index liquids with varying refractive index, including values similar to water or cytosol. Such photonic crystals with lower refractive index contrast may be similar to the hypothesized nanostructural forms in the developing butterfly scales. The ability to resolve these fainter signals hints at the potential of this facile light microscopy method for in vivo analysis of nanostructure formation in developing butterflies.

Simple and complex, sexually dimorphic retinal mosaic of fritillary butterflies.

Butterflies have variable sets of spectral photoreceptors that underlie colour vision. The photoreceptor organization may be optimized for the detection of body coloration. Fritillaries (Argynnini) are nymphalid butterflies exhibiting varying degrees of sexual dimorphism in wing coloration. In two sister species, the females have orange (Argynnis paphia) and dark wings (Argynnis sagana), respectively, while the males of both species have orange wings with large patches of pheromone-producing androconia. In spite of the differences in female coloration, the eyes of both species exhibit an identical sexual dimorphism. The female eyeshine is uniform yellow, while the males have a complex retinal mosaic with yellow and red-reflecting ommatidia. We found the basic set of ultraviolet-, blue- and green-peaking photoreceptors in both sexes. Males additionally have three more photoreceptor classes, peaking in green, yellow and red, respectively. The latter is the basal R9, indirectly measured through hyperpolarizations in the green-peaking R1-2. In many nymphalid tribes, including the closely related Heliconiini, the retinal mosaic is complex in both sexes. We hypothesize that the simple mosaic of female Argynnini is a secondary reduction, possibly driven by the use of olfaction for intraspecific recognition, whereas vision remains the primary sense for the task in the males. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Opponent processing in the retinal mosaic of nymphalid butterflies.

The eyes of nymphalid butterflies, investigated with incident illumination, show colourful facet reflection patterns-the eye shine-which is uniform or heterogeneous, dependent on the species. Facet colours suggest that the ommatidia contain different sets of photoreceptors and screening pigments, but how the colours and the cell characteristics are associated has not been clearly established. Here, we analyse the retinae of two nymphalids, Apatura ilia, which has a uniform eyeshine, and Charaxes jasius, a species with a heterogeneous eye shine, using single-cell recordings, spectroscopy and optical pupillometry. Apatura has UV-, blue- and green-sensitive photoreceptors, allocated into three ommatidial types. The UV- and blue-sensitive cells are long visual fibres (LVFs), receiving opponent input from the green-sensitive short visual fibres (SVFs). Charaxes has an expanded set of photoreceptors, allocated into three additional, red-reflecting ommatidial types. All red ommatidia contain green-sensitive LVFs, receiving opponent input from red receptors. In both species, the SVFs do not receive any opponent input. The simple retina of Apatura with three ommatidial types and two colour-opponent channels can support trichromatic vision. Charaxes has six ommatidial types and three colour-opponent channels. Its expanded receptor set can support tetrachromatic vision. This article is part of the theme issue 'Understanding colour vision: molecular, physiological, neuronal and behavioural studies in arthropods'.

Red-green opponency in the long visual fibre photoreceptors of brushfoot butterflies (Nymphalidae).

In many butterflies, the ancestral trichromatic insect colour vision, based on UV-, blue- and green-sensitive photoreceptors, is extended with red-sensitive cells. Physiological evidence for red receptors has been missing in nymphalid butterflies, although some species can discriminate red hues well. In eight species from genera Archaeoprepona, Argynnis, Charaxes, Danaus, Melitaea, Morpho, Heliconius and Speyeria, we found a novel class of green-sensitive photoreceptors that have hyperpolarizing responses to stimulation with red light. These green-positive, red-negative (G+R-) cells are allocated to positions R1/2, normally occupied by UV and blue-sensitive cells. Spectral sensitivity, polarization sensitivity and temporal dynamics suggest that the red opponent units (R-) are the basal photoreceptors R9, interacting with R1/2 in the same ommatidia via direct inhibitory synapses. We found the G+R- cells exclusively in butterflies with red-shining ommatidia, which contain longitudinal screening pigments. The implementation of the red colour channel with R9 is different from pierid and papilionid butterflies, where cells R5-8 are the red receptors. The nymphalid red-green opponent channel and the potential for tetrachromacy seem to have been switched on several times during evolution, balancing between the cost of neural processing and the value of extended colour information.

Neural mechanism of spatio-chromatic opponency in the Drosophila amacrine neurons.

Visual animals detect spatial variations of light intensity and wavelength composition. Opponent coding is a common strategy for reducing information redundancy. Neurons equipped with both spatial and spectral opponency have been identified in vertebrates but not yet in insects. The Drosophila amacrine neuron Dm8 was recently reported to show color opponency. Here, we demonstrate Dm8 exhibits spatio-chromatic opponency. Antagonistic convergence of the direct input from the UV-sensing R7s and indirect input from the broadband receptors R1-R6 through Tm3 and Mi1 is sufficient to confer Dm8's UV/Vis (ultraviolet/visible light) opponency. Using high resolution monochromatic stimuli, we show the pale and yellow subtypes of Dm8s, inheriting retinal mosaic characteristics, have distinct spectral tuning properties. Using 2D white-noise stimulus and reverse correlation analysis, we found that the UV receptive field (RF) of Dm8 has a center-inhibition/surround-excitation structure. In the absence of UV-sensing R7 inputs, the polarity of the RF is inverted owing to the excitatory input from the broadband photoreceptors R1-R6. Using a new synGRASP method based on endogenous neurotransmitter receptors, we show that neighboring Dm8s form mutual inhibitory connections mediated by the glutamate-gated chloride channel GluClα, which is essential for both Dm8's spatial opponency and animals' phototactic behavior. Our study shows spatio-chromatic opponency could arise in the early visual stage, suggesting a common information processing strategy in both invertebrates and vertebrates.

The red admiral butterfly's living light sensors and signals.

We studied the wing colouration and the compound eyes of red admiral butterflies with optical methods. We measured reflectance spectra of the wing and scales of Vanessa atalanta and modelled the thin film reflectance of the wing membrane and blue scales. We utilized the eyeshine in the compound eye of Vanessa indica to determine the spectral and polarisation characteristics of its optical sensor units, the ommatidia. Pupil responses were measured with a large-aperture optophysiological setup as reduction in the eyeshine reflection caused by monochromatic stimuli. Processing of spectral and polarisation responses of individual ommatidia revealed a random array with three types of ommatidia: about 10% contain two blue-sensitive photoreceptors, 45% have two UV-sensitive photoreceptors, and 45% have a mixed UV-blue pair. All types contain six green receptors and a basal photoreceptor. Optical modelling of the rhabdom suggests that the basal photoreceptors have a red-shifted sensitivity, which might enhance the red admiral's ability to discriminate red colours on the wing. Under daylight conditions, the red shift of the basal photoreceptor is ∼30 nm, compared to the rhodopsin spectrum template peaking at 520 nm, while the shift of green photoreceptors is ∼15 nm.

Diffusive structural colour in Hoplia argentea.

Nature's nanostructures can bring about vivid and iridescent colours seen in many insects, notably in beetles and butterflies. While the intense structural colours can be advantageous for display purposes, they may also be appealing to predators and therefore constitute an evolutionary disadvantage. Animals often employ absorption and scattering in order to reduce the directionality of the reflected light and thereby enhance their camouflage. Here, we investigated the monkey beetle Hoplia argentea using microspectrophotometry, electron microscopy, fluorimetry and optical modelling. We show that the dull green dorsal colour comes from the nanostructured scales on the elytra. The nanostructure consists of a multi-layered photonic structure covered by a filamentous layer. The filamentous layer acts as a spatial diffuser of the specular reflection from the multilayer and suppresses the iridescence. This combination leads to a colour-stable and angle-independent green reflection that probably enhances the camouflage of the beetles in their natural habitat.

Horsefly object-directed polarotaxis is mediated by a stochastically distributed ommatidial subtype in the ventral retina.

The ventral compound eye of many insects contains polarization-sensitive photoreceptors, but little is known about how they are integrated into visual functions. In female horseflies, polarized reflections from animal fur are a key stimulus for host detection. To understand how polarization vision is mediated by the ventral compound eye, we investigated the band-eyed brown horsefly Tabanus bromius using anatomical, physiological, and behavioral approaches. Serial electron microscopic sectioning of the retina and single-cell recordings were used to determine the spectral and polarization sensitivity (PS) of photoreceptors. We found 2 stochastically distributed subtypes of ommatidia, analogous to pale and yellow of other flies. Importantly, the pale analog contains an orthogonal analyzer receptor pair with high PS, formed by an ultraviolet (UV)-sensitive R7 and a UV- and blue-sensitive R8, while the UV-sensitive R7 and green-sensitive R8 in the yellow analog always have low PS. We tested horsefly polarotaxis in the field, using lures with controlled spectral and polarization composition. Polarized reflections without UV and blue components rendered the lures unattractive, while reflections without the green component increased their attractiveness. This is consistent with polarotaxis being guided by a differential signal from polarization analyzers in the pale analogs, and with an inhibitory role of the yellow analogs. Our results reveal how stochastically distributed sensory units with modality-specific division of labor serve as separate and opposing input channels for visual guidance.

Compound eyes of the small white butterfly Pieris rapae have three distinct classes of red photoreceptors.

The two subspecies of the small white butterfly, the European Pieris rapae rapae and the Asian P. r. crucivora, differ in wing colouration. Under ultraviolet light, the wings of both male and female P. r. rapae appear dark, whereas the wings of male P. r. crucivora are dark and those of females are bright. It has been hypothesized that these sexually dimorphic wing reflections in P. r. crucivora may have induced the evolution of a fluorescing-screening pigment in the violet-opsin-expressing photoreceptors of males, thus facilitating greater wavelength discrimination near 400 nm. Comparing the compound eyes of the two subspecies using genetic, microscopical, spectrographic, and histological methods revealed no differences that would meaningfully affect photoreceptor sensitivity, suggesting that the fluorescing-screening pigment did not evolve in response to sexually dimorphic wing reflections. Our investigation further revealed that (i) the peri-rhabdomal reddish-screening pigments differ among the three ommatidial types; (ii) each of the ommatidial types exhibits a unique class of red photoreceptor with a distinct spectral peak; and (iii) the blue, green, and red photoreceptors of P. rapae exhibit a polarization sensitivity > 2, with red photoreceptors allowing for a two-channel opponency form of polarization sensitivity.

The giant butterfly-moth Paysandisia archon has spectrally rich apposition eyes with unique light-dependent photoreceptor dynamics.

The palm borer moth Paysandisia archon (Burmeister, 1880) (fam. Castniidae) is a large, diurnally active palm pest. Its compound eyes consist of ~ 20,000 ommatidia and have apposition optics with interommatidial angles below 1°. The ommatidia contain nine photoreceptor cells and appear structurally similar to those in nymphalid butterflies. Two morphological ommatidial types were identified. Using the butterfly numbering scheme, in type I ommatidia, the distal rhabdom consists exclusively of the rhabdomeres of photoreceptors R1-2; the medial rhabdom has contributions from R1-8. The rhabdom in type II ommatidia is distally split into two sub-rhabdoms, with contributions from photoreceptors R2, R3, R5, R6 and R1, R4, R7, R8, respectively; medially, only R3-8 and not R1-2 contribute to the fused rhabdom. In both types, the pigmented bilobed photoreceptors R9 contribute to the rhabdom basally. Their nuclei reside in one of the lobes. Upon light adaptation, in both ommatidial types, the rhabdoms secede from the crystalline cones and pigment granules invade the gap. Intracellular recordings identified four photoreceptor classes with peak sensitivities in the ultraviolet, blue, green and orange wavelength regions (at 360, 465, 550, 580 nm, respectively). We discuss the eye morphology and optics, the photoreceptor spectral sensitivities, and the adaptation to daytime activity from a phylogenetic perspective.

Classical lepidopteran wing scale colouration in the giant butterfly-moth Paysandisia archon.

The palm borer moth Paysandisia archon (Castniidae; giant butterfly-moths) has brown dorsal forewings and strikingly orange-coloured dorsal hindwings with white spots surrounded by black margins. Here, we have studied the structure and pigments of the wing scales in the various coloured wing areas, applying light and electron microscopy and (micro)spectrophotometry, and we analysed the spatial reflection properties with imaging scatterometry. The scales in the white spots are unpigmented, those in the black and brown wing areas contain various amounts of melanin, and the orange wing scales contain a blue-absorbing ommochrome pigment. In all scale types, the upper lamina acts as a diffuser and the lower lamina as a thin film interference reflector, with thickness of about 200 nm. Scale stacking plays an important role in creating the strong visual signals: the colour of the white eyespots is created by stacks of unpigmented blue scales, while the orange wing colour is strongly intensified by stacking the orange scales.

Red-shift of spectral sensitivity due to screening pigment migration in the eyes of a moth, Adoxophyes orana.

BACKGROUND: We have found that the spectral sensitivity of the compound eye in the summer fruit tortrix moth (Adoxophyes orana) differs in laboratory strains originating from different regions of Japan. We have investigated the mechanisms underlying this anomalous spectral sensitivity. METHODS: We applied electrophysiology, light and electron microscopy, opsin gene cloning, mathematical modeling, and behavioral analysis. RESULTS: The ERG-determined spectral sensitivity of dark-adapted individuals of all strains peaks around 520 nm. When light-adapted, the spectral sensitivity of the Nagano strain narrows and its peak shifts to 580 nm, while that in other strains remains unchanged. All tested strains appear to be identical in terms of the basic structure of the eye, the pigment migration in response to light- and dark-adaptation, and the molecular structure of long-wavelength absorbing visual pigments. However, the color of the perirhabdomal pigment clearly differs; it is orange in the Nagano strain and purple in the others. The action spectrum of phototaxis appears to be shifted towards longer wavelengths in the Nagano individuals. CONCLUSIONS: The spectral sensitivities of light-adapted eyes can be modeled under the assumption that this screening pigment plays a crucial role in determining the spectral sensitivity. The action spectrum of phototaxis indicates that the change in the eye spectral sensitivity is behaviorally relevant.

A fast multispectral light synthesiser based on LEDs and a diffraction grating.

Optical experiments often require fast-switching light sources with adjustable bandwidths and intensities. We constructed a wavelength combiner based on a reflective planar diffraction grating and light emitting diodes with emission peaks from 350 to 630 nm that were positioned at the angles corresponding to the first diffraction order of the reversed beam. The combined output beam was launched into a fibre. The spacing between 22 equally wide spectral bands was about 15 nm. The time resolution of the pulse-width modulation drivers was 1 ms. The source was validated with a fast intracellular measurement of the spectral sensitivity of blowfly photoreceptors. In hyperspectral imaging of Xenopus skin circulation, the wavelength resolution was adequate to resolve haemoglobin absorption spectra. The device contains no moving parts, has low stray light and is intrinsically capable of multi-band output. Possible applications include visual physiology, biomedical optics, microscopy and spectroscopy.

Four photoreceptor classes in the open rhabdom eye of the red palm weevil, Rynchophorus ferrugineus Olivier.

The red palm weevil (RPW) is a severe palm pest with high dispersal capability. Its visual sense allows it to navigate long distances and to discriminate among differently colored traps. We investigated the RPW compound eyes with anatomical and electrophysiological methods. The ommatidia are composed of eight photoreceptor cells in an open rhabdom arrangement with six peripheral and two central photoreceptors. The photoreceptor signals are relatively slow and noisy. The majority of recorded photoreceptors have broad spectral sensitivity with a peak in the green, at 536 nm. Three minor classes of photoreceptors have narrower spectral sensitivities with maxima in the UV (366 nm), green (520 nm) and yellow (564 nm). Sensitivity below 350 nm is very low due to filtering by the UV-absorbing cornea. The set of photoreceptors represents the retinal substrate for putative trichromatic color vision.

A cute and highly contrast-sensitive superposition eye - the diurnal owlfly Libelloides macaronius.

The owlfly Libelloides macaronius (Insecta: Neuroptera) has large bipartite eyes of the superposition type. The spatial resolution and sensitivity of the photoreceptor array in the dorsofrontal eye part was studied with optical and electrophysiological methods. Using structured illumination microscopy, the interommatidial angle in the central part of the dorsofrontal eye was determined to be Δϕ=1.1 deg. Eye shine measurements with an epi-illumination microscope yielded an effective superposition pupil size of about 300 facets. Intracellular recordings confirmed that all photoreceptors were UV-receptors (λmax=350 nm). The average photoreceptor acceptance angle was 1.8 deg, with a minimum of 1.4 deg. The receptor dynamic range was two log units, and the Hill coefficient of the intensity-response function was n=1.2. The signal-to-noise ratio of the receptor potential was remarkably high and constant across the whole dynamic range (root mean square r.m.s. noise=0.5% Vmax). Quantum bumps could not be observed at any light intensity, indicating low voltage gain. Presumably, the combination of large aperture superposition optics feeding an achromatic array of relatively insensitive receptors with a steep intensity-response function creates a low-noise, high spatial acuity instrument. The sensitivity shift to the UV range reduces the clutter created by clouds within the sky image. These properties of the visual system are optimal for detecting small insect prey as contrasting spots against both clear and cloudy skies.

Spatial reflection patterns of iridescent wings of male pierid butterflies: curved scales reflect at a wider angle than flat scales.

The males of many pierid butterflies have iridescent wings, which presumably function in intraspecific communication. The iridescence is due to nanostructured ridges of the cover scales. We have studied the iridescence in the males of a few members of Coliadinae, Gonepteryx aspasia, G. cleopatra, G. rhamni, and Colias croceus, and in two members of the Colotis group, Hebomoia glaucippe and Colotis regina. Imaging scatterometry demonstrated that the pigmentary colouration is diffuse whereas the structural colouration creates a directional, line-shaped far-field radiation pattern. Angle-dependent reflectance measurements demonstrated that the directional iridescence distinctly varies among closely related species. The species-dependent scale curvature determines the spatial properties of the wing iridescence. Narrow beam illumination of flat scales results in a narrow far-field iridescence pattern, but curved scales produce broadened patterns. The restricted spatial visibility of iridescence presumably plays a role in intraspecific signalling.

Spectral reflectance properties of iridescent pierid butterfly wings.

The wings of most pierid butterflies exhibit a main, pigmentary colouration: white, yellow or orange. The males of many species have in restricted areas of the wing upper sides a distinct structural colouration, which is created by stacks of lamellae in the ridges of the wing scales, resulting in iridescence. The amplitude of the reflectance is proportional to the number of lamellae in the ridge stacks. The angle-dependent peak wavelength of the observed iridescence is in agreement with classical multilayer theory. The iridescence is virtually always in the ultraviolet wavelength range, but some species have a blue-peaking iridescence. The spectral properties of the pigmentary and structural colourations are presumably tuned to the spectral sensitivities of the butterflies' photoreceptors.