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.

Potential for identification of wild night-flying moths by remote infrared microscopy.

There are hundreds of thousands of moth species with crucial ecological roles that are often obscured by their nocturnal lifestyles. The pigmentation and appearance of moths are dominated by cryptic diffuse shades of brown. In this study, 82 specimens representing 26 moth species were analysed using infrared polarimetric hyperspectral imaging in the range of 0.95-2.5 µm. Contrary to previous studies, we demonstrate that since infrared light does not resolve the surface roughness, wings appear glossy and specular at longer wavelengths. Such properties provide unique reflectance spectra between species. The reflectance of the majority of our species could be explained by comprehensive models, and a complete parametrization of the spectral, polarimetric and angular optical properties was reduced to just 11 parameters with physical units. These parameters are complementary and, compared with the within-species variation, were significantly distinct between species. Counterintuitively to the aperture-limited resolution criterion, we could deduce microscopic features along the surface from their infrared properties. These features were confirmed by electron microscopy. Finally, we show how our findings could greatly enhance opportunities for remote identification of free-flying moth species, and we hypothesize that such flat specular wing targets could be expected to be sensed over considerable distances.

The forewing of a black cicada Cryptotympana atrata (Hemiptera, Homoptera: Cicadidae): Microscopic structures and mechanical properties.

Insects in nature flap their wings to generate lift force and driving torque to adjust their attitude and control stability. An insect wing is a biomaterial composed of flexible membranes and tough veins. In this paper, we study the microscopic structures and mechanical properties of the forewing of the black cicada, Cryptotympana atrata. The thickness of the wing membranes and the diameter of veins varied from the wing root to the tip. The thickness of the wing membranes ranged from 6.0 to 29.9 µm, and the diameter of the wing veins decreased in a gradient from the wing root to the tip, demonstrating that the forewing of the black cicada is a nonuniform biomaterial. The elastic modulus of the membrane near the wing root ranged from 4.45 to 5.03 GPa, which is comparable to that of some industrial membranes. The microstructure of the wing vein exhibited a hollow tubular structure with flocculent structure inside. The "fresh" sample stored more water than the "dry" sample, resulting in a significant difference in the elastic modulus between the fresh and dried veins. The different membrane thicknesses and elastic moduli of the wing veins near the root and tip resulted in varied degrees of deformation on both sides of the flexion line of the forewing during twisting. The measurements of the forewing of the cicada may serve as a guide for selecting airfoil materials for the bionic flapping-wing aircraft and promote the design and manufacture of more durable bionic wings in the future. RESEARCH HIGHLIGHTS: The distribution of the wing vein diameter and the wing membrane thickness indicated that the forewing of Cryptotympana atrata is composed of heterogeneous materials. The wing membrane and the outer wall of the wing vein are the layered structure with multilayer fibers, which has a great significance for improving the ability of the forewing to sustain aerodynamic loads. The elastic modulus of the wing membrane near the wing root is in the range of 4.45-5.03 GPa, which is comparable to that of membranes manufactured by industries. This is a suitable reference for selecting materials for making bionic aircraft wings. We proved that the elastic moduli of the "fresh" and "dry" wing veins differ greatly compared with those of the wing membrane. Because the wing vein microstructure exhibits an internal hollow tubular structure with flocculent structure inside, the "fresh" sample stores more water than the "dry" sample. The wing membrane near the wing root is thicker and reinforced by the main wing vein with a high elastic modulus. This renders the region near the wing root difficult to deform. The membrane far from the wing root is thinner and the elastic modulus of the nearby wing veins is smaller, making them more flexible.

Novel flight style and light wings boost flight performance of tiny beetles.

Flight speed is positively correlated with body size in animals1. However, miniature featherwing beetles can fly at speeds and accelerations of insects three times their size2. Here we show that this performance results from a reduced wing mass and a previously unknown type of wing-motion cycle. Our experiment combines three-dimensional reconstructions of morphology and kinematics in one of the smallest insects, the beetle Paratuposa placentis (body length 395 µm). The flapping bristled wings follow a pronounced figure-of-eight loop that consists of subperpendicular up and down strokes followed by claps at stroke reversals above and below the body. The elytra act as inertial brakes that prevent excessive body oscillation. Computational analyses suggest functional decomposition of the wingbeat cycle into two power half strokes, which produce a large upward force, and two down-dragging recovery half strokes. In contrast to heavier membranous wings, the motion of bristled wings of the same size requires little inertial power. Muscle mechanical power requirements thus remain positive throughout the wingbeat cycle, making elastic energy storage obsolete. These adaptations help to explain how extremely small insects have preserved good aerial performance during miniaturization, one of the factors of their evolutionary success.

In vivo visualization of butterfly scale cell morphogenesis in Vanessa cardui.

During metamorphosis, the wings of a butterfly sprout hundreds of thousands of scales with intricate microstructures and nano-structures that determine the wings' optical appearance, wetting characteristics, thermodynamic properties, and aerodynamic behavior. Although the functional characteristics of scales are well known and prove desirable in various applications, the dynamic processes and temporal coordination required to sculpt the scales' many structural features remain poorly understood. Current knowledge of scale growth is primarily gained from ex vivo studies of fixed scale cells at discrete time points; to fully understand scale formation, it is critical to characterize the time-dependent morphological changes throughout their development. Here, we report the continuous, in vivo, label-free imaging of growing scale cells of Vanessa cardui using speckle-correlation reflection phase microscopy. By capturing time-resolved volumetric tissue data together with nanoscale surface height information, we establish a morphological timeline of wing scale formation and gain quantitative insights into the underlying processes involved in scale cell patterning and growth. We identify early differences in the patterning of cover and ground scales on the young wing and quantify geometrical parameters of growing scale features, which suggest that surface growth is critical to structure formation. Our quantitative, time-resolved in vivo imaging of butterfly scale development provides the foundation for decoding the processes and biomechanical principles involved in the formation of functional structures in biological materials.

Programmed Scale Detachment in the Wing of the Pellucid Hawk Moth, Cephonodes hylas: Novel Scale Morphology, Scale Detachment Mechanism, and Wing Transparency.

No scales of most lepidopterans (butterflies and moths) detach from the wings through fluttering. However, in the pellucid hawk moth, Cephonodes hylas, numerous scales detach from a large region of the wing at initial take-off after eclosion; consequently, a large transparent region without scales appears in the wing. Even after this programmed detachment of scales (d-scales), small regions along the wing margin and vein still have scales attached (a-scales). To investigate the scale detachment mechanism, we analyzed the scale detachment process using video photography and examined the morphology of both d- and a-scales using optical and scanning electron microscopy. This study showed that d-scale detachment only occurs through fluttering and that d-scales are obviously morphologically different from a-scales. Although a-scales are morphologically common lepidopteran scales, d-scales have four distinctive features. First, d-scales are much larger than a-scales. Second, the d-scale pedicel, which is the slender base of the scale, is tapered; that of the a-scale is columnar. Third, the socket on the wing surface into which the pedicel is inserted is much smaller for d-scales than a-scales. Fourth, the d-scale socket density is much lower than the a-scale socket density. This novel scale morphology likely helps to facilitate scale detachment through fluttering and, furthermore, increases wing transparency.

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.

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.

Morphological and histological examination of short-wing formation in the winter moth Protalcis concinnata (Insecta: Lepidoptera, Geometridae).

Winter geometrid moths exhibit sexual dimorphism in wing length and female-specific flightlessness. Female-specific flightlessness in insects is an interesting phenomenon in terms of sexual dimorphism and reproductive biology. In the winter geometrid moth, Protalcis concinnata (Wileman), adult females have short wings and adult males have fully developed wings. Although the developmental process for wing reduction in Lepidoptera is well studied, little is known about the morphology and the developmental pattern of short-winged flightless morphs in Lepidoptera. To clarify the precise mechanisms and developmental processes that produce short-winged morphs, we performed morphological and histological investigations of adult and pupal wing development in the winter geometrid moth P. concinnata. Our findings showed that (a) wing development in both sexes is similar until larval-pupal metamorphosis, (b) the shape of the sexually dimorphic wings is determined by the position of the bordering lacuna (BL), (c) the BL is positioned farther inward in females than in males, and (d) after the short pupal diapause period, the female pupal wing epithelium degenerates to approximately two-thirds its original size due to cell death. We propose that this developmental pattern is a previously unrecognized process among flightless Lepidoptera.

Increased lateral tension is sufficient for epithelial folding in Drosophila.

The folding of epithelial sheets is important for tissues, organs and embryos to attain their proper shapes. Epithelial folding requires subcellular modulations of mechanical forces in cells. Fold formation has mainly been attributed to mechanical force generation at apical cell sides, but several studies indicate a role of mechanical tension at lateral cell sides in this process. However, whether lateral tension increase is sufficient to drive epithelial folding remains unclear. Here, we have used optogenetics to locally increase mechanical force generation at apical, lateral or basal sides of epithelial Drosophila wing disc cells, an important model for studying morphogenesis. We show that optogenetic recruitment of RhoGEF2 to apical, lateral or basal cell sides leads to local accumulation of F-actin and increase in mechanical tension. Increased lateral tension, but not increased apical or basal tension, results in sizeable fold formation. Our results stress the diversification of folding mechanisms between different tissues and highlight the importance of lateral tension increase for epithelial folding.

Linear and geometric morphometric analyses of variation of the plectrum in four species of bess beetles, tribe Proculini (Coleoptera: Passalidae).

Bess beetles are sapro-xillophagous insects that inhabit decaying logs. They display sub-social behavior and acoustic communication. Adults produce sounds with an abdomino-alary stridulatory system. The sixth abdominal tergum has lateral protuberances with spines (pars stridens), whereas the hind wings have spines in the radial cell (plectrum). The potential taxonomic utility of these spines has not been studied. We evaluated the variation of spines in the plectrum of adult females sampling four species of Proculini using traditional and geometric morphometric methods. We divided the plectrum into five zones to compare the density, size and shape of spines among zones and among species comparing each zone separately. Spine shape and size were quantified with 2D cartesian coordinates and four variables from SEM micrographs. Separate univariate and multivariate analyses of variance revealed that the size, density and shape of spines is different among zones in each of the four species. Also, spine shape and size in all zones is different among species, which would imply that plectrum spines might have taxonomic utility. Additionally, disparity analyses showed that the peripheral zones have a higher variability, and the disparity values in the middle and apex zones are higher in the two species with a wide distribution range.

How a raindrop gets shattered on biological surfaces.

Many biological surfaces of animals and plants (e.g., bird feathers, insect wings, plant leaves, etc.) are superhydrophobic with rough surfaces at different length scales. Previous studies have focused on a simple drop-bouncing behavior on biological surfaces with low-speed impacts. However, we observed that an impacting drop at high speeds exhibits more complicated dynamics with unexpected shock-like patterns: Hundreds of shock-like waves are formed on the spreading drop, and the drop is then abruptly fragmented along with multiple nucleating holes. Such drop dynamics result in the rapid retraction of the spreading drop and thereby a more than twofold decrease in contact time. Our results may shed light on potential biological advantages of hypothermia risk reduction for endothermic animals and spore spreading enhancement for fungi via wave-induced drop fragmentation.

Spectroscopy on the wing: Investigating possible differences in protein secondary structures in feather shafts of birds using Raman spectroscopy.

The central shaft of a bird's flight feather bears most of the aerodynamic load during flight and exhibits some remarkable mechanical properties. The shaft comprises two parts, the calamus and the rachis. The calamus is at the base of the shaft, while the rachis is the longer upper part which supports the vanes. The shaft is composed of a fibrous outer cortex, and an inner foam-like core. Recent nanoindentation experiments have indicated that reduced modulus values, Er, for the inner and outer regions of the cortex can vary, with the Er values of the inner region slightly greater than those of the outer region. In this work, Raman spectroscopy is used to investigate the protein secondary structures in the inner and outer regions of the feather cortex. Analysis of the Amide I region of Raman spectra taken from four birds (Swan, Gull, Mallard and Kestrel) shows that the ß-sheet structural component decreases between the inner and outer region, relative to the protein side-chain components. This finding is consistent with the proposal that Er values are greater in the inner region than the outer region. This work has shown that Raman spectroscopy can be used effectively to study the change in protein secondary structure between the inner and outer regions of a feather shaft.

Experimental method for 3D reconstruction of Odonata wings (methodology and dataset).

Insect wings are highly evolved structures with aerodynamic and structural properties that are not fully understood or systematically modeled. Most species in the insect order Odonata have permanently deployed high aspect ratio wings. Odonata have been documented to exhibit extraordinary flight performance and a wide range of interesting flight behaviors that rely on agility and efficiency. The characteristic three-dimensional corrugated structures of these wings have been observed and modeled for a small number of species, with studies showing that corrugations can provide significant aerodynamic and structural advantages. Comprehensive museum collections are the most practical source of Odonata wing, despite the risk of adverse effects caused by dehydration and preservation of specimens. Museum specimens are not to be handled or damaged and are best left undisturbed in their display enclosures. We have undertaken a systematic process of scanning, modeling, and post-processing the wings of over 80 Odonata species using a novel and accurate method and apparatus we developed for this purpose. The method allows the samples to stay inside their glass cases if necessary and is non-destructive. The measurements taken have been validated against micro-computed tomography scanning and against similar-sized objects with measured dimensions. The resulting publicly available dataset will allow aeronautical analysis of Odonata aerodynamics and structures, the study of the evolution of functional structures, and research into insect ecology. The technique is useable for other orders of insects and other fragile samples.

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.

Structure and tensile properties of the forewing costal vein of the honeybee Apis mellifera.

In this study, we investigated the morphological features and tensile properties of the forewing costal vein of the honeybee (Apis mellifera) under fresh, dry and in vitro-time varied conditions. The costal vein is composed of an outer sub-vein and an inner vein starting from the wing base to nearly 50% of the wing span and then they are fused into one vein extending to the wing tip. Confocal laser scanning microscopy revealed that the outer sub-vein with red autofluorescence is stiffer than the inner one with green autofluorescence, and the membrane in the gap between the sub-veins exhibited a long blue-autofluorescence resilin stripe. Considering the irregular cross-sectional shape of the costal vein, cross-sections of the tested specimens after tensile failure were analysed using scanning electron microscopy, to precisely calculate their cross-sectional areas by a customized MATLAB program. The Young's modulus and tensile strength of fresh specimens were ∼4.78 GPa and ∼119.84 MPa, which are lower than those of dry specimens (∼9.08 GPa and ∼154.45 MPa). However, the tensile strain had the opposite relationship (fresh: ∼0.031, dry: ∼0.018). Thus, specimen desiccation results in increasing stiffness and brittleness. The morphological features and material properties of the costal vein taken together represent a tradeoff between both deformability and stiffness. Our study provides guidance for material selection and bionic design of the technical wings of flapping micro aerial vehicles.

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.

Chondroitin sulfate proteoglycan Windpipe modulates Hedgehog signaling in Drosophila.

Proteoglycans, a class of carbohydrate-modified proteins, often modulate growth factor signaling on the cell surface. However, the molecular mechanism by which proteoglycans regulate signal transduction is largely unknown. In this study, using a recently developed glycoproteomic method, we found that Windpipe (Wdp) is a novel chondroitin sulfate proteoglycan (CSPG) in Drosophila. Wdp is a single-pass transmembrane protein with leucin-rich repeat (LRR) motifs and bears three CS sugar chain attachment sites in the extracellular domain. Here we show that Wdp modulates the Hedgehog (Hh) pathway. In the wing disc, overexpression of wdp inhibits Hh signaling, which is dependent on its CS chains and the LRR motifs. The wdp null mutant flies show a specific defect (supernumerary scutellar bristles) known to be caused by Hh overexpression. RNA interference knockdown and mutant clone analyses showed that loss of wdp leads to the up-regulation of Hh signaling. Altogether, our study demonstrates a novel role of CSPGs in regulating Hh signaling.

Ras2, the TC21/R-Ras2 Drosophila homologue, contributes to insulin signalling but is not required for organism viability.

Ras1 (Ras85D) and Ras2 (Ras64B) are the Drosophila orthologs of human H-Ras/N-Ras/K-Ras and R-Ras1-3 genes, respectively. The function of Ras1 has been thoroughly characterised during Drosophila embryonic and imaginal development, and it is associated with coupling activated trans-membrane receptors with tyrosine kinase activity to their downstream effectors. In this capacity, Ras1 binds and is required for the activation of Raf. Ras1 can also interact with PI3K, and it is needed to achieve maximal levels of PI3K signalling in specific cellular settings. In contrast, the function of the unique Drosophila R-Ras member (Ras2/Ras64B), which is more closely related to vertebrate R-Ras2/TC21, has been only studied through the use of constitutively activated forms of the protein. This pioneering work identified a variety of phenotypes that were related to those displayed by Ras1, suggesting that Ras1 and Ras2 might have overlapping activities. Here we find that Ras2 can interact with PI3K and Raf and activate their downstream effectors Akt and Erk. However, and in contrast to mutants in Ras1, which are lethal, null alleles of Ras2 are viable in homozygosis and only show a phenotype of reduced wing size and extended life span that might be related to reduced Insulin receptor signalling.

How flight feathers stick together to form a continuous morphing wing.

Variable feather overlap enables birds to morph their wings, unlike aircraft. They accomplish this feat by means of elastic compliance of connective tissue, which passively redistributes the overlapping flight feathers when the skeleton moves to morph the wing planform. Distinctive microstructures form "directional Velcro," such that when adjacent feathers slide apart during extension, thousands of lobate cilia on the underlapping feathers lock probabilistically with hooked rami of overlapping feathers to prevent gaps. These structures unlock automatically during flexion. Using a feathered biohybrid aerial robot, we demonstrate how both passive mechanisms make morphing wings robust to turbulence. We found that the hooked microstructures fasten feathers across bird species except silent fliers, whose feathers also lack the associated Velcro-like noise. These findings could inspire innovative directional fasteners and morphing aircraft.