Developmental, cellular, and biochemical basis of transparency in the glasswing butterfly Greta oto

Numerous species of Lepidoptera have transparent wings, which often possess scales of altered morphology and reduced size, and the presence of membrane surface nanostructures that dramatically reduce reflection. Optical properties and anti-reflective nanostructures have been characterized for several ‘clearwing’ Lepidoptera, but the developmental basis of wing transparency is unknown. We apply confocal and electron microscopy to create a developmental time-series in the glasswing butterfly, Greta oto, comparing transparent and non-transparent wing regions. We find that scale precursor cell density is reduced in transparent regions, and cytoskeletal organization differs between flat scales in opaque regions, and thin, bristle-like scales in transparent regions. We also reveal that sub-wavelength nanopillars on the wing membrane are wax-based, derive from wing epithelial cells and their associated microvillar projections, and demonstrate their role in enhancing-anti-reflective properties. These findings provide insight into morphogenesis of naturally organized micro- and nanostructures and may provide bioinspiration for new anti-reflective materials

Evolution de la transparence des ailes chez les Lepidoptères mimétiques: propriétés optiques, modifications structurales et sélection pour de la convergence

Müllerian mimicry is a positive interaction involving co-occurring defended prey that share a common aposematic colour pattern advertising predators of their toxicity. In Lepidoptera, aposematic species typically harbour bright and contrasted colour patterns that are convergent between co-mimetic species. Surprisingly, some mimetic aposematic Lepidoptera have partially transparent wings, raising the question of the implication of transparent patches in the aposematic signal and of the structural features underlyings transparency. This work lays at the interface between physics and biology and focuses on the evolution of transparent wing in mimetic Neotropical Lepidoptera: ithomiine butterflies (Nymphalidae: Danainae) and their co-mimics. The tribe Ithomiini comprises 393 toxic aposematic species, 80% of which have partially transparent wings, combining transparent and brightly coloured patches. In the first chapter, we unravel an unsuspected diversity of micro- and nanostructures in transparent wings, with different efficacies in transmitting light. Using comparative analyses, we suggest that transmittance of transparent wing areas is under selection for convergence in co-mimetic species. In the second chapter, using mimicry data and ecological data on altitudinal repartition and flight height in ithomiines we show that the evolution of optical properties of transparent patches is more driven by mimicry than by other ecological factors. In the third chapter, using spectrophotometry and electronic microscopy to characterise wing nanostructures, we demonstrate the antireflective properties of the nanostructures of transparent areas and reveal that nipple, pillar and sponge nanostructure types are the most effective in limiting light reflection. In the fourth chapter, using a dense taxonomic sampling of the Ithomiini tribe, we observe the same diversity in nanostructural features as previously described, and confirm that light transmission increases when wing membrane coverage by microstructures (i.e. scales) decreases and coverage by nanostructures increases. Finally, we present in appendix a draft phylogeny based on ultra-conserved elements for the arctiine neotropical subtribe Pericopina, many species of which engage in mimicry with ithomiines, in the perspective of future studies on mimicry pattern evolution in this clade, and comparison with ithomiine butterflies. Overall, this work revealed a wide diversity of structures causing transparency, established a link between structural features and optical properties of transparent wings, and highlighted transparency as part of the aposematic signal.Le mimétisme mullérien est une interaction positive où des proies possédant des défenses et vivant au même endroit partagent un même patron de coloration, qui avertit les prédateurs de leur toxicité. Chez les Lépidoptères, les espèces aposématiques présentent généralement des patrons de coloration vifs et contrastés qui convergent entre espèces co-mimes. Etonnamment, certains lépidoptères aposématiques et mimétiques ont des ailes partiellement transparentes, ce qui soulève la question de l’implication des zones transparentes dans le signal aposématique et de la façon dont la transparence est réalisée. Cette thèse à l’interface entre la biologie évolutive et l’optique porte sur l’évolution de la transparence chez des lépidoptères mimétiques néotropicaux : les papillons ithomiines (Nymphalidae : Danainae) et leurs co-mimes. La tribu des Ithomiini comprend 393 espèces toxiques et aposématiques, dont 80% possèdent des ailes partiellement transparentes, dont le motif est composé d’une mosaïque de zones colorées et transparentes. Dans le chapitre 1, nous révélons une diversité insoupçonnée de micro- et nanostructures dans les zones transparents, qui sont plus ou moins efficaces pour transmettre la lumière. A l’aide d’analyses comparatives, nous suggérons que les propriétés de transmission des patchs transparents sont convergentes entre espèces co-mimes. Dans le chapitre 2, grâce à l’exploitation de données de niche altitudinale et de hauteur de vol chez les ithomiines, nous montrons que l’évolution des propriétés optiques des zones transparentes est plus influencée par le mimétisme que d’autres facteurs écologiques. Dans le chapitre 3, à l’aide de mesures spectrophotométriques et de la caractérisation des nanostructures alaires en microscopie électronique, nous démontrons les propriétés antireflets des nanostructures des zones transparentes et nous révélons que les nanostructures de type ‘nipples’, ‘pillars’ et ‘sponge’ sont plus efficaces pour limiter la réflexion de la lumière. Dans un quatrième chapitre, en utilisant un échantillonnage dense de la tribu Ithomiini, nous observons la même diversité de nanostructures que celle précédemment décrite et nous confirmons que la transmission de la lumière à travers l’aile augmente quand la couverture de la membrane par les microstructures (i.e. les écailles) diminue et quand la couverture de la membrane par les nanostructures augmente. Enfin, nous présentons en annexe une phylogénie préliminaire basée sur des éléments ultra conservés de la sous-tribu néotropicale des Pericopina (Erebidae), dont beaucoup d’espèces sont engagées dans des interactions mimétiques avec des ithomiines, dans la perspective d’études futures de l’évolution des patrons de coloration dans ce clade, en comparaison à celle des ithomiines. Globalement, ce travail aura révélé une importante diversité de structures à l’origine de la transparence, établi un lien entre ces structures et les propriétés optiques, et distingué la transparence en tant que constituant du signal aposématique

Developmental, cellular and biochemical basis of transparency in clearwing butterflies

International audienceThe wings of butterflies and moths (Lepidoptera) are typically covered with thousands of flat, overlapping scales that endow the wings with colorful patterns. Yet, numerous species of Lepidoptera have evolved highly transparent wings, which often possess scales of altered morphology and reduced size, and the presence of membrane surface nanostructures that dramatically reduce reflection. Optical properties and anti-reflective nanostructures have been characterized for several ‘clearwing’ Lepidoptera, but the developmental processes underlying wing transparency are unknown. Here, we applied confocal and electron microscopy to create a developmental time series in the glasswing butterfly, Greta oto, comparing transparent and non-transparent wing regions. We found that during early wing development, scale precursor cell density was reduced in transparent regions, and cytoskeletal organization during scale growth differed between thin, bristle-like scale morphologies within transparent regions and flat, round scale morphologies within opaque regions. We also show that nanostructures on the wing membrane surface are composed of two layers: a lower layer of regularly arranged nipple-like nanostructures, and an upper layer of irregularly arranged wax-based nanopillars composed predominantly of long-chain n-alkanes. By chemically removing wax-based nanopillars, along with optical spectroscopy and analytical simulations, we demonstrate their role in generating anti-reflective properties. These findings provide insight into morphogenesis and composition of naturally organized microstructures and nanostructures, and may provide bioinspiration for new anti-reflective materials

Hydrophobicity in clearwing butterflies and moths: impact of scale micro and nanostructure, and trade-off with optical transparency

Abstract In opaque butterflies and moths, scales ensure vital functions like camouflage, thermoregulation, and hydrophobicity. Wing transparency in some species – achieved via modified or absent scales – raises the question of whether hydrophobicity can be maintained and of it dependence on scale microstructural (scale presence, morphology, insertion angle, and coloration) and nanostructural (ridge spacing and width) features. To address these questions, we assessed hydrophobicity in 23 clearwing species differing in scale micro and nanofeatures by measuring static contact angle (CA) of water droplets in the opaque and transparent patches of the same individuals at different stages of evaporation. We related these measures to wing structures (macro, micro, and nano) and compared them to predictions from Cassie-Baxter and Wenzel models. We found that overall, transparency is costly for hydrophobicity and this cost depends on scale microstructural features: transparent patches are less hydrophobic and lose more hydrophobicity with water evaporation than opaque patches. This loss is attenuated for higher scale densities, coloured scales (for erect scales), and when combining two types of scales (piliform and lamellar). Nude membranes show lowest hydrophobicity. Best models are Cassie-Baxter models that include scale microstructures for erect scales, and scale micro and nanostructures for flat scales. All findings are consistent with the physics of hydrophobicity, especially on multiscale roughness. Finally, wing hydrophobicity negatively relates to optical transparency. Moreover, tropical species have more hydrophobic transparent patches but similarly hydrophobic opaque patches compared to temperate species. Overall, diverse microstructures are likely functional compromises between multiple requirements. Significance Statement Water repellency is vital for terrestrial organisms. Yet, how microstructural diversity may impact hydrophobicity is unknown. Bridging the gap between biology and physics, we exploit the microstructural diversity found in clearwing butterflies and moths to assess its impact on hydrophobicity, and its ecological relevance. Within a physical framework, we bring experimental and modelling evidence for a major role of microstructures (scale morphology, insertion angle, coloration) and multiscale roughness in determining wing hydrophobicity, with a role of nanostructures restricted to flat scales and nude membrane. For the first time, we evidence some costs for transparency, and a trade-off between optics and hydrophobicity. Beyond novel biological results, this study gives new sources of bioinspiration for applied research on transparent materials in physics

Convergence in light transmission properties of transparent wing areas in clearwing mimetic butterflies

Müllerian mimicry is a positive interspecific interaction, whereby co-occurring defended prey species share a common aposematic signal that advertises their defences to predators. In Lepidoptera, aposematic species typically harbour conspicuous opaque wing colour pattern, which have convergent optical properties, as perceived by predators. Surprisingly, some aposematic mimetic species have partially or totally transparent wings, which raises the question of whether optical properties of such transparent areas are also under selection for convergence. To answer this question and to investigate how transparency is achieved in the first place, we conducted a comparative study of optics and structures of transparent wings in neotropical mimetic clearwing Lepidoptera. We quantified transparency by spectrophotometry and characterised clearwing microstructures and nanostructures by microscopy imaging. We show that transparency is convergent among co-mimics in the eyes of predators, despite a large diversity of underlying micro- and nanostructures. Notably, we reveal that nanostructure density largely influences light transmission. While transparency is primarily produced by modification of microstructure features, nanostructures may provide a way to fine-tune the degree of transparency. This study calls for a change of paradigm in transparent mimetic lepidoptera: transparency not only enables camouflage but can also be part of aposematic signals.Müllerian mimicry is a positive interspecific interaction, whereby co-occurring defended prey species share a common aposematic signal that advertises their defences to predators. In Lepidoptera, aposematic species typically harbour conspicuous opaque wing colour pattern, which have convergent optical properties, as perceived by predators. Surprisingly, some aposematic mimetic species have partially or totally transparent wings, which raises the question of whether optical properties of such transparent areas are also under selection for convergence. To answer this question and to investigate how transparency is achieved in the first place, we conducted a comparative study of optics and structures of transparent wings in neotropical mimetic clearwing Lepidoptera. We quantified transparency by spectrophotometry and characterised clearwing microstructures and nanostructures by microscopy imaging. We show that transparency is convergent among co-mimics in the eyes of predators, despite a large diversity of underlying micro-and nanostructures. Notably, we reveal that nanostructure densit

Wing transparency in butterflies and moths: structural diversity, optical properties, and ecological relevance

International audienceIn water, transparency seems an ideal concealment strategy, as testified by the variety of transparent aquatic organisms. By contrast, transparency is nearly absent on land, with the exception of insect wings, and knowledge is scarce about its functions and evolution, with fragmentary studies and no comparative perspective. Lepidoptera (butterflies and moths) represent an outstanding group to investigate transparency on land, as species typically harbor opaque wings covered with colored scales, a key multifunctional innovation. Yet, many Lepidoptera species have evolved partially or fully transparent wings. At the interface between physics and biology, the present study investigates wing transparency in 123 Lepidoptera species (from 31 families) for its structural basis, optical properties, and biological relevance in relation to visual detection (concealment), thermoregulation, and protection against UV. Our results suggest that transparency has likely evolved multiple times independently. Efficiency at transmitting light is largely determined by clearwing microstructure (scale shape, insertion, coloration, dimensions, and density) and macrostructure (clearwing area, species size, or wing area). Microstructural traits, scale density and dimensions, are tightly linked in their evolution, with different constraints according to scale shape, insertion, and coloration. Transparency appears highly relevant for concealment, with size-dependent variations. Links between transparency and latitude are consistent with an ecological relevance of transparency in thermoregulation, and not so for protection against UV radiation. Altogether, our results shed new light on the physical and ecological processes driving the evolution of transparency on land and underline that transparency is a more complex coloration strategy than previously thought

Mimicry can drive convergence in structural and light transmission features of transparent wings in Lepidoptera

International audienceMüllerian mimicry is a positive interspecific interaction, whereby co-occurring defended prey species share a common aposematic signal. In Lepidoptera, aposematic species typically harbour conspicuous opaque wing colour patterns with convergent optical properties among co-mimetic species. Surprisingly, some aposematic mimetic species have partially transparent wings, raising the questions of whether optical properties of transparent patches are also convergent, and of how transparency is achieved. Here, we conducted a comparative study of wing optics, micro and nanostructures in neotropical mimetic clearwing Lepidoptera, using spectrophotometry and microscopy imaging. We show that transparency, as perceived by predators, is convergent among co-mimics in some mimicry rings. Underlying micro- and nanostructures are also sometimes convergent despite a large structural diversity. We reveal that while transparency is primarily produced by microstructure modifications, nanostructures largely influence light transmission, potentially enabling additional fine-tuning in transmission properties. This study shows that transparency might not only enable camouflage but can also be part of aposematic signals

Phylogeny of 33 Ithomiini species

Phylogeny of the 33 Ithomiini species used in the study and extracted from the 340 species Ithomiini phylogeny by Chazot et al. (accepted in Global Ecology and Biogeography

Data of transparency & detectability of 33 Ithomiini species, and PA concentration and measures of unpalatability for 10 of these species

1-Average transparency and detectability by avian predators (chromatic & achromatic contrast in just noticeable difference units [JNDs] for UVS- & VS-vision in large gap & forest shade light conditions) for the 33 Ithomiini species of the study. 2-Data for behavioural experiments with chicks to test unpalatability of 10 Ithomiini taxa (indicated in bold red in the first list) are also provided, including the number (& assigned colour ) of experimental pellets attacked at each trial (for a total of 12 trials per chick) for all 6 chicks tested for each butterfly species. 3-PA concentrations (μg/mg) measured for individual butterflies of the 10 selected taxa are also given