The actin cytoskeleton plays multiple roles in structural colour formation in butterfly wing scales.

Vivid structural colours in butterflies are caused by photonic nanostructures scattering light. Structural colours evolved for numerous biological signalling functions and have important technological applications. Optically, such structures are well understood, however insight into their development in vivo remains scarce. We show that actin is intimately involved in structural colour formation in butterfly wing scales. Using comparisons between iridescent (structurally coloured) and non-iridescent scales in adult and developing H. sara, we show that iridescent scales have more densely packed actin bundles leading to an increased density of reflective ridges. Super-resolution microscopy across three distantly related butterfly species reveals that actin is repeatedly re-arranged during scale development and crucially when the optical nanostructures are forming. Furthermore, actin perturbation experiments at these later developmental stages resulted in near total loss of structural colour in H. sara. Overall, this shows that actin plays a vital and direct templating role during structural colour formation in butterfly scales, providing ridge patterning mechanisms that are likely universal across lepidoptera.

Wing coupling mechanism in the butterfly Pieris rapae (Lepidoptera, Pieridae) and its role in taking off.

The small white cabbage butterfly (Pieris rapae) flaps its fore- and hindwings in synchrony as the wings are coupled using a wing "coupling mechanism". The coupling mechanism of butterflies includes an enlarged humeral area located at the anterior of the hindwing base and a corresponding basal posterior part of the forewing, of which the former component dorsally contacts the ventral side of the latter one. The coupling mechanism allows for the fore- and hindwings sliding in contact along the span and chord. It is of interest that butterflies still take off successfully and fly, when their wing couplings are clipped, but they are unable to properly synchronize the fore- and hindwing motions. Compared with the regular takeoff trajectory of intact butterflies that always first fly backwards and then forwards, the coupling-clipped butterflies took off in a random trajectory. Due to the clipping of the coupling mechanism, the initiation of the hindwing flapping and the abdomen rotation from upward to downward during takeoff was postponed. The coupling-clipped butterflies changed their stroke plane in upstroke to a more vertical position and strengthened the abdominal undulation. We believe our work, which for the first time investigates the influence of coupling mechanism removal on insect flight, extends our understanding on the working principle of wing coupling in insects and its significance on the flapping flight.

Wolbachia affects mitochondrial population structure in two systems of closely related Palaearctic blue butterflies.

The bacterium Wolbachia infects many insect species and spreads by diverse vertical and horizontal means. As co-inherited organisms, these bacteria often cause problems in mitochondrial phylogeny inference. The phylogenetic relationships of many closely related Palaearctic blue butterflies (Lepidoptera: Lycaenidae: Polyommatinae) are ambiguous. We considered the patterns of Wolbachia infection and mitochondrial diversity in two systems: Aricia agestis/Aricia artaxerxes and the Pseudophilotes baton species complex. We sampled butterflies across their distribution ranges and sequenced one butterfly mitochondrial gene and two Wolbachia genes. Both butterfly systems had uninfected and infected populations, and harboured several Wolbachia strains. Wolbachia was highly prevalent in A. artaxerxes and the host's mitochondrial structure was shallow, in contrast to A. agestis. Similar bacterial alleles infected both Aricia species from nearby sites, pointing to a possible horizontal transfer. Mitochondrial history of the P. baton species complex mirrored its Wolbachia infection and not the taxonomical division. Pseudophilotes baton and P. vicrama formed a hybrid zone in Europe. Wolbachia could obscure mitochondrial history, but knowledge on the infection helps us to understand the observed patterns. Testing for Wolbachia should be routine in mitochondrial DNA studies.

The evolution of structural colour in butterflies.

Butterflies display some of the most striking examples of structural colour in nature. These colours originate from cuticular scales that cover the wing surface, which have evolved a diverse suite of optical nanostructures capable of manipulating light. In this review we explore recent advances in the evolution of structural colour in butterflies. We discuss new insights into the underlying genetics and development of the structural colours in various nanostructure types. Improvements in -omic and imaging technologies have been paramount to these new advances and have permitted an increased appreciation of their development and evolution.

Photonic effects in natural nanostructures on Morpho cypris and Greta oto butterfly wings.

Photonic crystals are some of the more spectacular realizations that periodic arrays can change the behavior of electromagnetic waves. In nature, so-called structural colors appear in insects and even plants. Some species create beautiful color patterns as part of biological behavior such as reproduction or defense mechanisms as a form of biomimetics. The interaction between light and matter occurs at the surface, producing diffraction, interference and reflectance, and light transmission is possible under suitable conditions. In particular, there are two Colombian butterflies, Morpho cypris and Greta oto, that exhibit iridescence phenomena on their wings, and in this work, we relate these phenomena to the photonic effect. The experimental and theoretical approaches of the optical response visible region were studied to understand the underlying mechanism behind the light-matter interaction on the wings of these Colombian butterflies. Our results can guide the design of novel devices that use iridescence as angular filters or even for cosmetic purposes.

Diverse nanostructures underlie thin ultra-black scales in butterflies.

Recently, it has been shown that animals such as jumping spiders, birds, and butterflies have evolved ultra-black coloration comparable to the blackest synthetic materials. Of these, certain papilionid butterflies have reflectances approaching 0.2%, resulting from a polydisperse honeycomb structure. It is unknown if other ultra-black butterflies use this mechanism. Here, we examine a phylogenetically diverse set of butterflies and demonstrate that other butterflies employ simpler nanostructures that achieve ultra-black coloration in scales thinner than synthetic alternatives. Using scanning electron microscopy, we find considerable interspecific variation in the geometry of the holes in the structures, and verify with finite-difference time-domain modeling that expanded trabeculae and ridges, found across ultra-black butterflies, reduce reflectance up to 16-fold. Our results demonstrate that butterflies produce ultra-black by creating a sparse material with high surface area to increase absorption and minimize surface reflection. We hypothesize that butterflies use ultra-black to increase the contrast of color signals.

Structures related to pheromone storage in alar androconia and the female abdominal scent gland of Heliconius erato phyllis, Heliconius ethilla narcaea, and Heliconius besckei (Lepidoptera: Nymphalidae: Heliconiinae).

We describe the morphology of alar androconia and the female abdominal scent gland of Heliconius erato phyllis, Heliconius ethilla narcaea, and Heliconius besckei. Androconial scales of Heliconius, which are arranged in overlapping wing bands, release pheromones during courtship, probably through vibratory movements of male wings over the female to induce her to mate. An antiaphrodisiac is produced by glands located in the valves of the male and is transferred during copulation to the yellow dorsal abdominal sac present in the virgin female, causing this sac to emit a scent that reduces the attractiveness of the female for courtship with other males. Stereomicroscopy, SEM, and TEM analyses were conducted to describe the morphology of the internal and external scales and the external abdominal scent sac. The findings revealed different sizes of external androconial scales and an internal group of porous structural vesicles that are probably related to the preservation of internal space, reception and storage of secretions, and elimination of volatiles when the male is actively involved in courtship. Translucent projections on the female abdominal scent sac create open reservoirs for the reception, storage, and emission of antiaphrodisiac volatiles along with stink clubs. Male valve denticles vary in form and probably attach securely to the female sac during mating, thus ensuring secretion transfer. These features are discussed in the context of a comparative analysis.

High accuracy of color-generating nanoarchitectures is kept in lowland and mountainous populations of Polyommatus dorylas (Lepidoptera: Lycaenidae: Polyommatinae).

It is known that the size of the scales covering the surface of the Lepidoptera wings is in correlation with body size: larger species possess larger scales. However, butterfly individuals representing the various generations of the same species but differing in body size were not investigated in this respect. Similarly, the question whether different scale size may influence structural color generation based on nanoarchitectures in the scale lumen was never addressed. Populations of lowland (environment of Budapest, Hungary) and upland (Carpathian Mountains, Romania) Polyommatus dorylas were compared in terms of voltinism, wing and scale size, and the structural origin of blue coloration. Data analysis showed that the univoltine upland population exhibits a larger wing and scale size. On the other hand, the nanomorphology of the blue color-generating scales was identical when compared between univoltine and bivoltine populations. Coloration was also identical when measured with a spectrophotometer under ultraviolet and visible light. This high accuracy present in the male structural coloration suggests that it is controlled genetically. Body size alteration for enhanced thermal fitness has no influence on the fine structure of the nanoarchitecture present in the scale lumen.

A SEM study of unexplored wax microtubes produced by caterpillar living on common banana leaves.

The primary concern of this research is to perform a microscopic analysis of the natural wax covering banana skipper caterpillar. The scanning electron microscopic (SEM) analysis shows that the wax exhibits an unprecedented microtubular form, consisting of carbon and oxygen as main elements as revealed by energy dispersive X-ray (EDX) analysis. The Fourier Transform Infra-Red (FTIR) spectroscopic analysis, which reveals the presence of peaks at 2914, 2890, 1469, and 722 cm-1 and the absence of peaks around 1700 cm-1, indicates that the analyzed natural wax is a polyethylene wax without the CO functional group.

Analysis of color shift on butterfly wings by Fourier transform of images from atomic force microscopy.

Butterfly wings have complex structure lending it several interesting properties. Coloration of the wing is one of the first things to encounter and the overall visual effect is in fact influenced by several factors. Chemical pigments set the base color of the wing, topographical structures on the wing scales cause color shift by interference and their arrangement into diffraction grating causes iridescence. The thin film interference can be attributed to microscopic ridges covering wing scales. Observation and calculation of the color shift on wings of Euploea mulciber species using Fourier transform of images obtained by atomic force microscopy is the focus of this article.

Plasticity and divergence in ultraviolet reflecting structures on Dogface butterfly wings.

The vast diversity of animal coloration is generated through a combination of pigment and structural colors. These colors can greatly influence the fitness and life history of an organism. Butterflies and their wing colors are an excellent model to study how these colors can impact the development and success of an organism. In this study, we explore species differences in structurally-based ultraviolet coloration in the Zerene butterfly. We show clear species differences in ultraviolet (UV) pattern and reflectance spectra. By varying larval diet, we show evidence for developmental plasticity in the structure and organization of UV reflecting scales in Zerene cesonia. We further show that feeding the larval host plant of Zerene eurydice to Z. cesonia does not result in greater similarity in scale structure or UV coloration to the sister species. These results not only demonstrate a connection between plasticity in a male ornamentation, UV wing pattern, and larval resource acquisition, but also identify candidate structural and organizational changes in wing scales responsible for the trait variation.

Ultrastructure of the proboscis sensilla of ten species of butterflies (Insecta: Lepidoptera).

The ultrastructure of the sensilla on the proboscis of ten species of butterflies, Iphiclides podalirius, Parara guttata, Colias fieldii, Celastrina oreas, Sasakia charonda, Tirumala limniace, Acraea issoria, Stichophthalma neumogeni, Callerebia suroia, and Libythea celtis, among five families were investigated using scanning electron microscopy. They were compared to reveal the morphological differences in the proboscis sensilla among these butterflies. Four distinct types of sensilla were found on the proboscis among these species. The types of proboscis sensilla of I. podalirius and T. limniace were sensilla chaetica, sensilla coeloconica, and sensilla basiconica. The types in the other eight species were sensilla chaetica, sensilla styloconica, and sensilla basiconica. The number of sensilla styloconica on the proboscis of non-flower-visiting species was greater than that of flower-visiting species.

Haze smoke impacts survival and development of butterflies.

The Southeast Asian transboundary haze contains a mixture of gases and particles from forest fires and negatively impacts people's health and local economies. However, the effect of the haze on organisms other than humans has not yet been sufficiently studied. Insects are important members of food webs and environmental disturbances that affect insects may impact whole ecosystems. Here we studied how haze directly and indirectly affects the survival, growth, and development of insects by rearing Bicyclus anynana butterflies under artificially generated smoke as well as reared in clean air but fed on plants previously exposed to smoke. Direct haze exposure significantly increased the mortality of caterpillars, increased larval development time, and decreased pupal weight, while indirect haze exposure, via ingestion of haze-exposed food plants, also affected development time and pupal weight. No smoke particles were found in the tracheae of subjects from the smoke treatment suggesting that the increase in development time and mortality of B. anynana under smoke conditions might be due to toxic smoke gases and toxic food, rather than particulate matter. These results document significant deleterious effect of haze smoke to the development, adult size, and survival of insects, key players in food-webs.

Comparative study on nanostructured order-disorder in the wing eyespots of the giant owl butterfly, Caligo memnon.

A characteristic feature of the giant owl butterfly, i.e. Caligo memnon, is its big wing eyespot. This feature could serve as deceiving functionality for the butterfly against predators. As evidenced by scanning electron microscope (SEM) image on black part of eyespot, the scales on wing eyespot contain nanostructured ridges and cross-ribs. Applying direct measurement, statistical method, and Fourier analysis, the authors evidence that these nanostructures display order-disorder in their shape and position. The autocorrelation of SEM image provides average values of characteristic periods of the order-disorder nanostructures together with an estimation of corresponding correlation lengths. Linecuts obtained from the Fourier transform of SEM image were also analysed with the Hosemann function to extract similar information. These analyses indicate that the nanostructured order-disorder may contribute to blackness on wing eyespot. The authors thus conclude that the blackness on wing eyespot of C. memnon could be attributed to contributions from both the nanostructured order-disorder and melanin pigment.

Multifractal Characterization of Butterfly Wings Scales.

A lot of insect families have physical structures created by evolution for coloration. These structures are a source of ideas for new bio-inspired materials. The aim of this study was to quantitatively characterize the micromorphology of butterfly wings scales using atomic force microscopy and multifractal analysis. Two types of butterflies, Euploea mulciber ("striped blue crow") and Morpho didius ("giant blue morpho"), were studied. The three-dimensional (3D) surface texture of the butterfly wings scales was investigated focusing on two areas: where the perceived colors strongly depend on and where they do not depend on the viewing angle. The results highlight a correlation between the surface coloration and 3D surface microtexture of butterfly wings scales.

Self-assembly of the butterfly proboscis: the role of capillary forces.

The proboscis of butterflies and moths consists of two C-shaped fibres, the galeae, which are united after the insect emerges from the pupa. We observed that proboscis self-assembly is facilitated by discharge of saliva. In contrast with vertebrate saliva, butterfly saliva is not slimy and is an almost inviscid, water-like fluid. Butterfly saliva, therefore, cannot offer any viscoelastic adhesiveness. We hypothesized that capillary forces are responsible for helping butterflies and moths pull and hold their galeae together while uniting them mechanically. Theoretical analysis supported by X-ray micro-computed tomography on columnar liquid bridges suggests that both concave and convex liquid bridges are able to pull the galeae together. Theoretical and experimental analyses of capillary forces acting on natural and artificial proboscises show that these forces are sufficiently high to hold the galeae together.

The Only Blue Mimeresia (Lepidoptera: Lycaenidae: Lipteninae) Uses a Color-Generating Mechanism Widely Applied by Butterflies.

The butterfly Mimeresia neavei (Joicey & Talbot, 1921) is the only species in the exclusively African subtribal clade Mimacraeina (Lipteninae: Lycaenidae: Lepidoptera) having sexual dimorphism expressed by structurally blue-colored male and pigmentary colored orange-red female phenotypes. We investigated the optical mechanism generating the male blue color by various microscopic and experimental methods. It was found that the blue color is produced by the lower lamina of the scale acting as a thin film. This kind of color production is not rare in day-flying Lepidoptera, or in other insect orders. The biological role of the blue color of M. neavei is not yet well understood, as all the other species in the clade lack structural coloration, and have less pronounced sexual dimorphism, and are involved in mimicry-rings.

Histological and electron microscopy observations on the testis and spermatogenesis of the butterfly Dione juno (Cramer, 1779) and Agraulis vanillae (Linnaeus, 1758) (Lepidoptera: Nymphalidae).

Lepidopteran species present an interesting case of sperm polymorphism and testicular fusion. The study of these features are of great importance in understanding the reproductive biology of these insects, especially in the case of those considered pests. Dione juno and Agraulis vanillae stand out as the most important pests of passion fruit (Passiflora sp.) crops in Brazil. Therefore, the objective of the present study was to characterize the testes and germ cells of Dione juno and Agraulis vanillae at different life stages, using light microscopy and scanning and transmission electron microscopy, to understand the maturation mechanisms of the male gametes in these species. The study showed that the larvae of both species have a pair of brown kidney-shaped testes, covered by epithelial cells which divide the organ into four follicles. The testes are full of spermatogonia which begin to differentiate in the third larval instar. In the fifth larval instar, spermatozoa can be observed. When they enter the prepupal stage the testes begin a fusion process that is completed in the adult insects, where they present as spherical organs divided into eight follicles, containing all the cells of the germ line. Spermatogenesis occurs centripetally, and in both species, sperm dimorphism is observed, where two different types of spermatozoa are formed, eupyrene (nucleated) and apyrene (anucleate), which differ in morphology and function. Apart from contributing to scientific basic research on the reproductive biology of these insects, the present study provides important data that can aid in research on the physiology, systematics, and control of these species.

Wing scale ultrastructure underlying convergent and divergent iridescent colours in mimetic Heliconius butterflies.

Iridescence is an optical phenomenon whereby colour changes with the illumination and viewing angle. It can be produced by thin film interference or diffraction. Iridescent optical structures are fairly common in nature, but relatively little is known about their production or evolution. Here we describe the structures responsible for producing blue-green iridescent colour in Heliconius butterflies. Overall the wing scale structures of iridescent and non-iridescent Heliconius species are very similar, both having longitudinal ridges joined by cross-ribs. However, iridescent scales have ridges composed of layered lamellae, which act as multilayer reflectors. Differences in brightness between species can be explained by the extent of overlap of the lamellae and their curvature as well as the density of ridges on the scale. Heliconius are well known for their Müllerian mimicry. We find that iridescent structural colour is not closely matched between co-mimetic species. Differences appear less pronounced in models of Heliconius vision than models of avian vision, suggesting that they are not driven by selection to avoid heterospecific courtship by co-mimics. Ridge profiles appear to evolve relatively slowly, being similar between closely related taxa, while ridge density evolves faster and is similar between distantly related co-mimics.

Morphological fine tuning of the feeding apparatus to proboscis length in Hesperiidae (Lepidoptera).

The form and function of the hesperiid feeding apparatus was studied in detail. The butterflies in the family Hesperiidae are of particular interest because the longest proboscis ever recorded in Papilionoidea was found in the Neotropical genus Damas. We focused on the functional morphology by comparing proboscis morphology as well as size and composition of both the stipes pump and the cibarial suction pump in skippers with short and extremely long proboscis. Results revealed that all studied Hesperiidae have the same proboscis micromorphology and sensilla endowment regardless of the proboscis length. However, the numbers of internal muscles of the proboscis, the morphology of the stipes pump as well as the pumping organs for nectar uptake are related to the proboscis length. We conclude that the low number of tip sensilla compared to proboscis length is responsible for remarkably longer manipulation times of long-proboscid species during flower visits. The organs for proboscis movements and nectar uptake organs are well tuned to the respective proboscis length and are accordingly bigger in species with a proboscis that measures twice the body length.