Antennapedia regulates metallic silver wing scale development and cell shape in Bicyclus anynana butterflies

Butterfly wing scale cells can develop very intricate cuticular nanostructures that interact with light to produce structural colors such as silver, but the genetic basis of such nanostructures is mostly unexplored. Here, we address the genetic basis of metallic silver scale development by leveraging existing crispants in the butterfly Bicyclus anynana, where knockouts of five genes – apterous A, Ultrabithorax, doublesex, Antennapedia and optix – either led to ectopic gains or losses of silver scales. Most wildtype silver scales had low amounts of pigmentation and exhibited a common ultrastructural modification for metallic broadband reflectance, i.e., an undulatory air layer enclosed by an upper and lower lamina. Crispant brown scales differed from wildtype silver scales via the loss of the continuous upper lamina, increased lower lamina thickness, and increased pigmentation. The reverse was seen when brown scales became silver. On the forewings, we identified Antennapedia as a high-level selector gene, acting through doublesex to induce silver scale development in males and having a novel, post-embryonic role in the determination of ridge and crossrib orientation and overall scale cell shape in both sexes. We propose that apterous A and Ultrabithorax repress Antennapedia on the dorsal forewings and ventral hindwings, respectively, thereby repressing silver scale development, whereas apterous A activates the same GRN on the dorsal hindwings, promoting silver scales.

optix is involved in eyespot development via a possible positional information mechanism

optix, a gene essential and sufficient for eye development in Drosophila melanogaster, also plays important roles in the development of both the structure and pigmentation of butterfly wing scales. In particular, optix regulates wing scale lower lamina thickness and ommochrome pigment synthesis. Here we explore the role of optix in wing pattern development of Bicyclus anynana butterflies by examining its expression using immunostainings and testing its function via CRISPR-Cas9. We found Optix to be expressed in multiple domains, most prominently in the orange ring of the eyespots and in other scattered orange scales, and to regulate the pigmentation and the development of the upper lamina of the orange scales. We further explored the interaction of Optix with Spalt, a protein involved in the development of black scales in the eyespots, and expressed adjacent to the Optix domain. CRISPR knockouts of optix or spalt, followed by immunostainings, showed that Spalt represses optix expression in cells of the central black region of the eyespot. This regulatory interaction mimics that found in the anterior compartment of the wing disc where both genes respond to Decapentaplegic (Dpp) signaling and play a role in venation patterning. Using in situ hybridizations we show that dpp is expressed in the center of the eyespots and propose that this same circuit might have been recruited for eyespot development where Decapentaplegic acts as a central morphogen, activating optix and spalt at different concentration thresholds, and where spalt cross-regulates optix resulting in the formation of a sharp boundary between the two eyespot color rings.

The genetic basis of structural colour variation in mimetic Heliconius butterflies

Structural colours, produced by the reflection of light from ultrastructures, have evolved multiple times in butterflies. Unlike pigmentary colours and patterns, little is known about the genetic basis of these colours. Reflective structures on wing-scale ridges are responsible for iridescent structural colour in many butterflies, including the Müllerian mimics Heliconius erato and Heliconius melpomene. Here we quantify aspects of scale ultrastructure variation and colour in crosses between iridescent and non-iridescent subspecies of both of these species and perform quantitative trait locus (QTL) mapping. We show that iridescent structural colour has a complex genetic basis in both species, with offspring from crosses having wide variation in blue colour (both hue and brightness) and scale structure measurements. We detect two different genomic regions in each species that explain modest amounts of this variation, with a sex-linked QTL in H. erato but not H. melpomene. We also find differences between species in the relationships between structure and colour. Our results suggest that these species have followed different evolutionary trajectories in their convergent evolution of similar structural colour. This study provides a starting point for determining the genetic basis of structural colouration more broadly.

F-actin Re-organization Mediates Hierarchical Morphogenesis of Swallowtail Butterfly Wing Scale Nanostructures

The development of color patterning in lepidopteran wings is of fundamental interest in evolution and developmental biology. While significant advances have recently been made in unravelling the cell and molecular basis of lepidopteran pigmentary coloration, the morphogenesis of wing scales, often involved in structural color production, is not well understood. Contemporary research focuses almost exclusively on a few nymphalid model taxa (e.g., Bicyclus, Heliconius), despite an overwhelming diversity across lepidopteran families in the hierarchical nanostructural organization of the scale. Here, we present a time-resolved, comparative developmental study of hierarchical wing scale nanostructure in Parides eurimedes and other papilionid species. Our results uphold the putative conserved role of F-actin bundles in acting as spacers between developing ridges as previously documented in several nymphalid species. While ridges are developing, the plasma membrane manifests irregular crossribs, characteristic of Papilionidae, which delineate the accretion of cuticle into rows of planar disks in between ridges. Once ridges have grown, Arp2/3 appears to re-organize disintegrating F-actin bundles into a reticulate network that supports the extrusion of the membrane underlying the disks into honeycomb-like tubular lattices of air pores in cuticle. Our results uncover a previously undocumented role for F-actin in the morphogenesis of wing scale nanostructures prominently found in Papilionidae. They are also relevant to current challenges in engineering of mesophases, since understanding the diversity and biological basis of hierarchical morphogenesis may offer facile, biomimetic solutions.

Butterfly wing architectures inspire sensor and energy applications

Natural biological systems are constantly developing efficient mechanisms to counter adverse effects of increasing human population and depleting energy resources. Their intelligent mechanisms are characterized by the ability to detect changes in the environment, store and evaluate information, and respond to external stimuli. Bio-inspired replication into man-made functional materials guarantees enhancement of characteristics and performance. Specifically, butterfly architectures have inspired the fabrication of sensor and energy materials by replicating their unique micro/nanostructures, light-trapping mechanisms and selective responses to external stimuli. These bio-inspired sensor and energy materials have shown improved performance in harnessing renewable energy, environmental remediation and health monitoring. Therefore, this review highlights recent progress reported on the classification of butterfly wing scale architectures and explores several bio-inspired sensor and energy applications.

Distinguishing eastern North American forest moth pests by wing-scale ultrastructure: potential applications in paleoecology

The use of fossil moth wing scales has recently been introduced as a new method to reconstruct population histories of lepidopterans and provide a proxy for insect disturbance. We investigated the potential for using wing-scale ultrastructure to distinguish between the five most common outbreak species of moth pests in eastern North America: spruce budworm ( Choristoneura fumiferana Clemens), hemlock looper ( Lambdina fiscellaria Guenée), forest tent caterpillar ( Malacosoma disstria Hübner), blackheaded budworm ( Acleris variana Fernie), and jack pine budworm ( Choristoneura pinus Freeman). Using scanning electron images of scales, we made qualitative and quantitative comparisons of morphological traits at the ultrastructural level. We found that hemlock looper and eastern blackheaded budworm scales could be categorically separated from each other and from the three other species. We developed a quadratic discriminant function using measurements of ultrastructure traits that distinguishes scales of the three remaining species with an overall accuracy of 66%. We found that forest tent caterpillar could be well separated based on these traits, but we were less confident in distinguishing the closely related jack pine and spruce budworm. Our method offers potential advantages in scale identification for future studies in paleoecology, while providing the additional advantage of not requiring intact, unfolded, and undamaged scales.

Thin-film structural coloration from simple fused scales in moths

The metallic coloration of insects often originates from diverse nanostructures ranging from simple thin films to complex three-dimensional photonic crystals. In Lepidoptera, structural coloration is widely present and seems to be abundant in extant species. However, even some basal moths exhibit metallic coloration. Here, we have investigated the origin of the vivid metallic colours of the wing scales of the basal moth Micropterix aureatella by spectrophotometry and scanning electron microscopy. The metallic gold-, bronze- and purple-coloured scales share a similar anatomy formed of a fused lower and upper lamina resulting in a single thin film. The optical response of this thin-film scale can be attributed to thin-film interference of the incident light, resulting in the colour variations that correlate with film thickness. Subtle variations in the wing scale thickness result in large visible colour changes that give Micropterix moths their colourful wing patterns. This simple coloration mechanism could provide a hint to understand the evolution of structural coloration in Lepidoptera.