Interactions of Melanin with Electromagnetic Radiation: From Fundamentals to Applications.

Melanin, especially integumentary melanin, interacts in numerous ways with electromagnetic radiation, leading to a set of critical functions, including radiation protection, UV-protection, pigmentary and structural color productions, and thermoregulation. By harnessing these functions, melanin and melanin-like materials can be widely applied to diverse applications with extraordinary performance. Here we provide a unified overview of the melanin family (all melanin and melanin-like materials) and their interactions with the complete electromagnetic radiation spectrum (X-ray, Gamma-ray, UV, visible, near-infrared), which until now has been absent from the literature and is needed to establish a solid fundamental base to facilitate their future investigation and development. We begin by discussing the chemistries and morphologies of both natural and artificial melanin, then the fundamentals of melanin-radiation interactions, and finally the exciting new developments in high-performance melanin-based functional materials that exploit these interactions. This Review provides both a comprehensive overview and a discussion of future perspectives for each subfield of melanin that will help direct the future development of melanin from both fundamental and applied perspectives.

Hydric environment and chemical composition shape non-avian reptile eggshell absorption.

The amniotic egg fulfils a critical role in reproduction by serving as an interface between the external environment and the embryo. Because non-avian reptiles are rarely incubated, they must be heated by, and absorb water from, the oviposition site for the developing embryo. The mechanisms by which they absorb sufficient, but not excess, water and how these mechanisms vary with local habitat is largely unknown, despite its significance to their evolution. Here, we first performed histology, Fourier transform infra-red spectroscopy and dynamic vapor sorption experiments to elucidate the mechanisms of eggshell absorption for 56 reptile species. Then, we used phylogenetic comparative analyse to test the hypothesis that absorptive capacity of reptile eggshells increases with aridity of the environment. We found that water absorption increases in the presence of a superficial mucopolysaccharide layer and decreases with increased calcium content. We found that eggs from arid environments have highly absorbent eggshells, but only in species with weakly calcified shells. Our results suggest that reptile eggshells have over evolutionary time tuned absorptive capacity to environmental moisture level. Since these eggs often must sustain conflicting constraints, they may serve as inspirations for new biomimetic materials, such as water filtering membranes or humidity sensors.

Nanoscale millefeuilles produce iridescent bill ornaments in birds.

Colors are well studied in bird plumage but not in other integumentary structures. In particular, iridescent colors from structures other than plumage are undescribed in birds. Here, we show that a multilayer of keratin and lipids is sufficient to produce the iridescent bill of Spermophaga haematina. Furthermore, that the male bill is presented to the female under different angles during display provides support for the hypothesis that iridescence evolved in response to sexual selection. This is the first report of an iridescent bill, and only the second instance of iridescence in birds in which melanosomes are not involved. Furthermore, an investigation of museum specimens of an additional 98 species, showed that this evolved once, possibly twice. These results are promising, as they suggest that birds utilize a wider array of physical phenomena to produce coloration and should further stimulate research on nonplumage integumentary colors.

The evolution of multiple colour mechanisms is correlated with diversification in sunbirds (Nectariniidae).

How and why certain groups become speciose is a key question in evolutionary biology. Novel traits that enable diversification by opening new ecological niches are likely important mechanisms. However, ornamental traits can also promote diversification by opening up novel sensory niches and thereby creating novel inter-specific interactions. More specifically, ornamental colours may enable more precise and/or easier species recognition, and may act as key innovations by increasing the number of species-specific patterns and promoting diversification. While the influence of colouration on diversification is well-studied, the influence of the mechanisms that produce those colours (e.g. pigmentary, nanostructural) is less so, even though the ontogeny and evolution of these mechanisms differ. We estimated a new phylogenetic tree for 121 sunbird species and combined colour data of 106 species with a range of phylogenetic tools to test the hypothesis that the evolution of novel colour mechanisms increases diversification in sunbirds, one of the most colourful bird clades. Results suggest that (1) the evolution of novel colour mechanisms expands the visual sensory niche, increasing the number of achievable colours. (2) Structural colouration diverges more readily across the body than pigment-based colouration, enabling an increase in colour complexity. (3) Novel colour mechanisms might minimize trade-offs between natural and sexual selection such that colour can function both as camouflage and conspicuous signal. (4) Despite structural colours being more colourful and mobile, only melanin-based colouration is positively correlated with net diversification. Together, these findings explain why colour distances increase with increasing number of sympatric species, even though packing of colour space would predict otherwise.

Back in black: melanin-rich skin colour associated with increased net diversification rates in birds.

Evolutionary biologists have long been interested in understanding the factors that promote diversification in organisms, often focussing on distinct and/or conspicuous phenotypes with direct effects on natural or sexual selection such as body size and plumage coloration. However, multiple traits that potentially influence net diversification are not conspicuous and/or might be concealed. One such trait, the dark, melanin-rich skin concealed beneath the feathers, evolved more than 100 times during avian evolution, frequently in association with white feathers on the crown and UV-rich environments, suggesting that it is a UV-photoprotective adaptation. Furthermore, multiple species are polymorphic, having both light and dark skin potentially aiding occupation in different UV radiation environments. As such these polymorphisms are predicted to occur in species with large latitudinal variation in their distribution. Furthermore, by alleviating evolutionary constraints on feather colour, the evolution of dark skin may promote net diversification. Here, using an expanded dataset on bird skin coloration of 3033 species we found that more than 19% of species had dark skin. In contrast to our prediction, dark skinned birds have smaller distribution ranges. Furthermore, both dark skin and polymorphism in skin coloration promote net diversification. These results suggest that even concealed traits can influence large scale evolutionary events such as diversification in birds.

Influence of Core Type and Shell Thickness on Avian-Inspired Structural Colors Produced from Melanin Nanoparticle Assemblies.

Hollow melanosomes found in iridescent bird feathers, including violet-backed starlings and wild turkeys, enable the generation of diverse structural colors. It has been postulated that the high refractive index (RI) contrast between melanin (1.74) and air (1.0) results in brighter and more saturated colors. This has led to several studies that have synthesized hollow synthetic melanin nanoparticles and fabricated colloidal nanostructures to produce synthetic structural colors. However, these studies use hollow nanoparticles with thin shells (<20 nm), even though shell thicknesses as high as 100 nm have been observed in natural melanosomes. Here, we combine experimental and computational approaches to examine the influence of the varying polydopamine (PDA, synthetic melanin) shell thickness (0-100 nm) and core material on structural colors. Experimentally, a concomitant change in overall particle size and RI contrast makes it difficult to interpret the effect of a hollow or solid core on color. Thus, we utilize finite-difference time-domain (FDTD) simulations to uncover the effect of shell thickness and core on structural colors. Our FDTD results highlight that hollow particles with thin shells have substantially higher saturation than same-sized solid and core-shell particles. These results would benefit a wide range of applications including paints, coatings, and cosmetics.

Mechanism of structural colors in binary mixtures of nanoparticle-based supraballs.

Inspired by structural colors in avian species, various synthetic strategies have been developed to produce noniridescent, saturated colors using nanoparticle assemblies. Nanoparticle mixtures varying in particle chemistry and size have additional emergent properties that affect the color produced. For complex multicomponent systems, understanding the assembled structure and a robust optical modeling tool can empower scientists to identify structure-color relationships and fabricate designer materials with tailored color. Here, we demonstrate how we can reconstruct the assembled structure from small-angle scattering measurements using the computational reverse-engineering analysis for scattering experiments method and use the reconstructed structure in finite-difference time-domain calculations to predict color. We successfully, quantitatively predict experimentally observed color in mixtures containing strongly absorbing nanoparticles and demonstrate the influence of a single layer of segregated nanoparticles on color produced. The versatile computational approach that we present is useful for engineering synthetic materials with desired colors without laborious trial-and-error experiments.

Biomimetic pheomelanin to unravel the electronic, molecular and supramolecular structure of the natural product.

Herein, we investigate synthetic routes to a close mimic of natural pheomelanin. Three different oxidative polymerization routes were attempted to generate synthetic pheomelanin, each giving rise to structurally dissimilar materials. Among them, the route employing 5-cysteinyl-dihydroxyphenylalanine (5-CD) as a monomer was verified as a close analogue of extracted pheomelanin from humans and birds. The resulting biomimetic and natural pheomelanins were compared via various techniques, including solid-state Nuclear Magnetic Resonance (ssNMR) and Electron Paramagnetic Resonance (EPR). This synthetic pheomelanin closely mimics the structure of natural pheomelanin as determined by parallel characterization of pheomelanin extracted from multiple biological sources. With a good synthetic biomimetic material in hand, we describe cation-π interactions as an important driving force for pheomelanogenesis, further advancing our fundamental understanding of this important biological pigment.

Body size and substrate use affect ventral, but not dorsal, brightness evolution in lizards.

Substrate properties can affect the thermal balance of organisms, and the colored integument, alongside other factors, may influence heat transfer via differential absorption and reflection. Dark coloration may lead to higher heat absorption and could be advantageous when substrates are cool (and vice versa for bright coloration), but these effects are rarely investigated. Here, we examined the effect of substrate reflectance, specific heat capacity (cp), and body size on the dorso-ventral brightness using 276 samples from 12 species of cordylid lizards distributed across 26 sites in South Africa. We predicted, and found, that bright ventral colors occur more frequently in low cp (i.e., drier, with little energy needed for temperature change) substrates, especially in larger body-sized individuals, possibly to better modulate heat transfer with the surrounding environment. By contrast, dorsal brightness was not associated with body size nor any substrate thermal property, suggesting selection pressures other than thermoregulation. Ancestral estimation and evolutionary rate analyses suggest that ventral brightness rapidly differentiated within the Cordylinae starting 25 Mya, coinciding with an aridification period, further hinting at a thermoregulatory role for ventral colors. Our study indicates that substrate properties can have a direct role in shaping the evolution of ventral brightness in ectotherms.

How keratin cortex thickness affects iridescent feather colours.

The bright, saturated iridescent colours of feathers are commonly produced by single and multi-layers of nanostructured melanin granules (melanosomes), air and keratin matrices, surrounded by an outer keratin cortex of varying thicknesses. The role of the keratin cortex in colour production remains unclear, despite its potential to act as a thin film or absorbing layer. We use electron microscopy, optical simulations and oxygen plasma-mediated experimental cortex removal to show that differences in keratin cortex thickness play a significant role in producing colours. The results indicate that keratin cortex thickness determines the position of the major reflectance peak (hue) from nanostructured melanosomes of common pheasant (Phasianus colchicus) feathers. Specifically, the common pheasant has appropriate keratin cortex thickness to produce blue and green structural colours. This finding identifies a general principle of structural colour production and sheds light on the processes that shaped the evolution of brilliant iridescent colours in the common pheasant.

Hydrophobic Melanin via Post-Synthetic Modification for Controlled Self-Assembly.

Allomelanin is a class of nitrogen-free melanin mostly found in fungi and, like all naturally occurring melanins, is hydrophilic. Herein, we develop a facile method to modify synthetic hydrophilic allomelanin to yield hydrophobic derivatives through post-synthetic modifications. Amine-functionalized molecules of various kinds can be conjugated to allomelanin nanoparticles under mild conditions with high loading efficiencies. Hydrophobicity is conferred by introducing amine-terminated alkyl groups with different chain lengths. We demonstrate that the resulting hydrophobic allomelanin nanoparticles undergo air/water interfacial self-assembly in a controlled fashion, which enables the generation of large-scale and uniform structural colors. This work provides an efficient and tunable surface chemistry modification strategy to broaden the scope of synthetic melanin structure and function beyond the known diversity found in nature.

A combination of red structural and pigmentary coloration in the eyespot of a copepod.

While the specific mechanisms of colour production in biological systems are diverse, the mechanics of colour production are straightforward and universal. Colour is produced through the selective absorption of light by pigments, the scattering of light by nanostructures or a combination of both. When Tigriopus californicus copepods were fed a carotenoid-limited diet of yeast, their orange-red body coloration became faint, but their eyespots remained unexpectedly bright red. Raman spectroscopy indicated a clear signature of the red carotenoid pigment astaxanthin in eyespots; however, refractive index matching experiments showed that eyespot colour disappeared when placed in ethyl cinnamate, suggesting a structural origin for the red coloration. We used transmission electron microscopy to identify consecutive nanolayers of spherical air pockets that, when modelled as a single thin film layer, possess the correct periodicity to coherently scatter red light. We then performed microspectrophotometry to quantify eyespot coloration and confirmed a distinct colour difference between the eyespot and the body. The observed spectral reflectance from the eyespot matched the reflectance predicted from our models when considering the additional absorption by astaxanthin. Together, this evidence suggests the persistence of red eyespots in copepods is the result of a combination of structural and pigmentary coloration.

Pterosaur melanosomes support signalling functions for early feathers.

Remarkably well-preserved soft tissues in Mesozoic fossils have yielded substantial insights into the evolution of feathers1. New evidence of branched feathers in pterosaurs suggests that feathers originated in the avemetatarsalian ancestor of pterosaurs and dinosaurs in the Early Triassic2, but the homology of these pterosaur structures with feathers is controversial3,4. Reports of pterosaur feathers with homogeneous ovoid melanosome geometries2,5 suggest that they exhibited limited variation in colour, supporting hypotheses that early feathers functioned primarily in thermoregulation6. Here we report the presence of diverse melanosome geometries in the skin and simple and branched feathers of a tapejarid pterosaur from the Early Cretaceous found in Brazil. The melanosomes form distinct populations in different feather types and the skin, a feature previously known only in theropod dinosaurs, including birds. These tissue-specific melanosome geometries in pterosaurs indicate that manipulation of feather colour-and thus functions of feathers in visual communication-has deep evolutionary origins. These features show that genetic regulation of melanosome chemistry and shape7-9 was active early in feather evolution.

Enhanced photothermal absorption in iridescent feathers.

The diverse colours of bird feathers are produced by both pigments and nanostructures, and can have substantial thermal consequences. This is because reflectance, transmittance and absorption of differently coloured tissues affect the heat loads acquired from solar radiation. Using reflectance measurements and heating experiments on sunbird museum specimens, we tested the hypothesis that colour and their colour producing mechanisms affect feather surface heating and the heat transferred to skin level. As predicted, we found that surface temperatures were strongly correlated with plumage reflectivity when exposed to a radiative heat source and, likewise, temperatures reached at skin level decreased with increasing reflectivity. Indeed, nanostructured melanin-based iridescent feathers (green, purple, blue) reflected less light and heated more than unstructured melanin-based colours (grey, brown, black), as well as olives, carotenoid-based colours (yellow, orange, red) and non-pigmented whites. We used optical and heat modelling to test if differences in nanostructuring of melanin, or the bulk melanin content itself, better explains the differences between melanin-based feathers. These models showed that the greater melanin content and, to a lesser extent, the shape of the melanosomes explain the greater photothermal absorption in iridescent feathers. Our results suggest that iridescence can increase heat loads, and potentially alter birds' thermal balance.

The evolution of darker wings in seabirds in relation to temperature-dependent flight efficiency.

Seabirds have evolved numerous adaptations that allow them to thrive under hostile conditions. Many seabirds share similar colour patterns, often with dark wings, suggesting that their coloration might be adaptive. Interestingly, these darker wings become hotter when birds fly under high solar irradiance, and previous studies on aerofoils have provided evidence that aerofoil surface heating can affect the ratio between lift and drag, i.e. flight efficiency. However, whether this effect benefits birds remains unknown. Here, we first used phylogenetic analyses to show that strictly oceanic seabirds with a higher glide performance (optimized by reduced sink rates, i.e. the altitude lost over time) have evolved darker wings, potentially as an additional adaptation to improve flight. Using wind tunnel experiments, we then showed that radiative heating of bird wings indeed improves their flight efficiency. These results illustrate that seabirds may have evolved wing pigmentation in part through selection for flight performance under extreme ocean conditions. We suggest that other bird clades, particularly long-distance migrants, might also benefit from this effect and therefore might show similar evolutionary trajectories. These findings may also serve as a guide for bioinspired innovations in aerospace and aviation, especially in low-speed regimes.

Morphogenesis of Iridescent Feathers in Anna's Hummingbird Calypte anna.

Color is a phenotypic trait of utmost importance, particularly in birds, which are known for their diverse color signals and color-producing mechanisms including pigment-based colors, light scattering from nanostructured feather tissues and combinations thereof. Bright iridescent plumage colors of hummingbirds are caused by light scattering by an organized array of flattened, pigment organelles, containing air-filled vesicles, called melanosomes. These hollow platelets are organized in multilayer arrays that contain numerous sharp air/melanin refractive index interfaces, producing brilliant iridescent colors. Despite their ecological significance and potential for inspiration of new optical materials, how platelets form and spatially arrange in nanostructures in growing feathers remains unknown. Here, we tested the hypothesis that melanosome formation and organization occurs mostly through passive self-assembly processes by assembling a developmental time series of growing hummingbird feathers using optical and electron microscopy. We show that hummingbird platelets contain air bubbles or vesicles upon their formation in pigment-producing cells, melanocytes. When melanosomes are transferred to neighboring keratinocytes (the cells shaping barbule structure) they drastically expand in size; and variation in this enlargement appears to be driven by physical constraints caused by the placement of the melanosomes within the barbule plate and their proximity to other melanosomes. As the barbule elongates and narrows, polymerizing feather corneous beta-protein orients melanosomes unilaterally, forcing them into a stacked configuration. These results reveal potentially novel forces driving the self-assembly of the nanostructures producing some of the brightest colors in nature.

Feather Gene Expression Elucidates the Developmental Basis of Plumage Iridescence in African Starlings.

Iridescence is widespread in the living world, occurring in organisms as diverse as bacteria, plants, and animals. Yet, compared to pigment-based forms of coloration, we know surprisingly little about the developmental and molecular bases of the structural colors that give rise to iridescence. Birds display a rich diversity of iridescent structural colors that are produced in feathers by the arrangement of melanin-containing organelles called melanosomes into nanoscale configurations, but how these often unusually shaped melanosomes form, or how they are arranged into highly organized nanostructures, remains largely unknown. Here, we use functional genomics to explore the developmental basis of iridescent plumage using superb starlings (Lamprotornis superbus), which produce both iridescent blue and non-iridescent red feathers. Through morphological and chemical analyses, we confirm that hollow, flattened melanosomes in iridescent feathers are eumelanin-based, whereas melanosomes in non-iridescent feathers are solid and amorphous, suggesting that high pheomelanin content underlies red coloration. Intriguingly, the nanoscale arrangement of melanosomes within the barbules was surprisingly similar between feather types. After creating a new genome assembly, we use transcriptomics to show that non-iridescent feather development is associated with genes related to pigmentation, metabolism, and mitochondrial function, suggesting non-iridescent feathers are more energetically expensive to produce than iridescent feathers. However, iridescent feather development is associated with genes related to structural and cellular organization, suggesting that, while nanostructures themselves may passively assemble, barbules and melanosomes may require active organization to give them their shape. Together, our analyses suggest that iridescent feathers form through a combination of passive self-assembly and active processes.

Anisotropic Synthetic Allomelanin Materials via Solid-State Polymerization of Self-Assembled 1,8-Dihydroxynaphthalene Dimers.

Melanosomes in nature have diverse morphologies, including spheres, rods, and platelets. By contrast, shapes of synthetic melanins have been almost entirely limited to spherical nanoparticles with few exceptions produced by complex templated synthetic methods. Here, we report a non-templated method to access synthetic melanins with a variety of architectures including spheres, sheets, and platelets. Three 1,8-dihydroxynaphthalene dimers (4-4', 2-4' and 2-2') were used as self-assembling synthons. These dimers pack to form well-defined structures of varying morphologies depending on the isomer. Specifically, distinctive ellipsoidal platelets can be obtained using 4-4' dimers. Solid-state polymerization of the preorganized dimers generates polymeric synthetic melanins while maintaining the initial particle morphologies. This work provides a new route to anisotropic synthetic melanins, where the building blocks are preorganized into specific shapes, followed by solid-state polymerization.

Evolution of eggshell structure in relation to nesting ecology in non-avian reptiles.

Amniotic eggs are multifunctional structures that enabled early tetrapods to colonize the land millions of years ago, and are now the reproductive mode of over 70% of all terrestrial amniotes. Eggshell morphology is at the core of animal survival, mediating the interactions between embryos and their environment, and has evolved into a massive diversity of forms and functions in modern reptiles. These functions are critical to embryonic survival and may serve as models for new antimicrobial and/or breathable membranes. However, we still lack critical data on the basic structural and functional properties of eggs, particularly of reptiles. Here, we first characterized egg shape, shell thickness, porosity, and mineralization of eggs from 91 reptile species using optical images, scanning electron microscopy, and micro computed tomography, and collected data on nesting ecology from the literature. We then used comparative analyses to test hypotheses on the selective pressures driving their evolution. We hypothesized that eggshell morphology has evolved to protect shells from physical damage and desiccation, and, in support, found a positive relationship between thickness and precipitation, and a negative relationship between porosity and temperature. Although mineralization varied extensively, it was not correlated with nesting ecology variables. Ancestral state reconstructions show thinning and increased porosity over evolutionary time in squamates, but the opposite in turtles and crocodilians. Egg shape, size, porosity and calcification were correlated, suggesting potential structural or developmental tradeoffs. This study provides new data and insights into the morphology and evolution of reptile eggs, and raises numerous questions for additional research.

Untangling the structural and molecular mechanisms underlying colour and rapid colour change in a lizard, Agama atra.

With functions as diverse as communication, protection and thermoregulation, coloration is one of the most important traits in lizards. The ability to change colour as a function of varying social and environmental conditions is thus an important innovation. While colour change is present in animals ranging from squids, to fish and reptiles, not much is known about the mechanisms behind it. Traditionally, colour change was attributed to migration of pigments, in particular melanin. More recent work has shown that the changes in nanostructural configuration inside iridophores are able to produce a wide palette of colours. However, the genetic mechanisms underlying colour, and colour change in particular, remain unstudied. Here we use a combination of transcriptomic and microscopic data to show that melanin, iridophores and pteridines are the main colour-producing mechanisms in Agama atra, and provide molecular and structural data suggesting that rapid colour change is achieved via melanin dispersal in combination with iridophore organization. This work demonstrates the power of combining genotypic (gene expression) and phenotypic (microscopy) information for addressing physiological questions, providing a basis for future studies of colour change.