Structural color and its interaction with other color-producing elements

Structural color and its interaction with other color-producing elements: perspectives from spiders

Bor-Kai Hsiung*, Todd A Blackledge, and Matthew D Shawkey Department of Biology and Integrated Bioscience Program, The University of Akron, Akron, Ohio 44325, USA ABSTRACT Structural color is produced when nanostructures alter light in contrast with pigment-based colors that are produced by selective absorption of certain wavelengths of light. Research on biogenic photonic nanostructures has primarily focused on bird feathers, butterfly wings and beetle elytra, and not diverse groups such as spiders. We argue that spiders are a good model system to study the functions and evolution of colors in nature for the following reasons. First, these colors clearly function in some spiders outside of sexual selection, which is likely the dominant driver of the evolution of structural colors in birds and butterflies. Second, within more than 44,000 currently known spider species, a hugely diverse set of colors is produced using the same materials. Using spiders, we can study how colors evolve to serve different functions under a variety of selective pressures, and how those colors are produced within a relatively simple system. Here, we first review the different color-producing materials and mechanisms (i.e., light absorbing, reflecting and emitting) in birds, butterflies and beetles, the interactions between these different elements, and the functions of colors in different organisms. We then summarize the current state of knowledge of spider colors and compare it with that of birds and insects. We then raise questions including: 1. Could spiders use fluorescence as a mechanism to protect themselves from UV radiation, if they do not have the biosynthetic pathways to produce melanins? 2. What functions could color serve for nearly blind tarantulas? 3. Why are only multilayer nanostructures (thus far) found in spiders, while birds and butterflies use many diverse nanostructures? And, does this limit the diversity of structural colors found in spiders? Addressing any of these questions in the future will bring spiders to the forefront of the study of structural colors in nature. Keyword list: structural color; pigment; bird; insect; spider; interference; scattering; diffraction; interaction; biomimicry

1. INTRODUCTION Structural colors are produced by coherent or incoherent interaction between light and materials with different refractive indices that are structured at nano-scales. This contrasts with most colors that are produced by selective absorption of light by pigments. Biological structural colors were first described, independently, by Hooke and Newton when they investigated the tail feathers of peacocks1,2. Since then, many more examples of structural colors in animals have been described3, particularly in birds (Class: Aves), butterflies and beetles (Class: Insecta)4. These are familiar, colorful organisms and hence it is not surprising that they are well studied. In contrast, our knowledge of structural colors in spiders (Class: Arachnida) is limited5. While the vast majority of spiders are relatively dull and brown, some have bright and conspicuous iridescent colorations. In particular, tarantulas (Family: Theraphosidae) and jumping spiders (Family: Salticidae) display the most color varieties6. Here, we review our current understanding of color-producing materials and mechanisms in birds, butterflies, beetles and spiders. Mainly, we focus on birds and anthropods, because in those taxa, structural colors are produced outside of living cells by hard biomaterials, such as keratins or chitins respectively, and we have a detailed understanding about them due to previous research5,7. On the other hand, structural colors in other taxa, such as cephalopods and plants, often occur in soft living tissues and/or have not been extensively researched5.

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The Nature of Light: Light in Nature V, edited by Rongguang Liang, Joseph A. Shaw, Proc. of SPIE Vol. 9187, 91870B · © 2014 SPIE · CCC code: 0277-786X/14/$18 · doi: 10.1117/12.2060831

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By comparing and contrasting the materials and mechanisms that produce colors in spiders with those of butterflies and beetles, and those of more distantly related birds, we raise some interesting and important questions that can only be answered by investigating spiders in more detail, such as: 1. Why do spiders not produce melanins? 2. Do schemochromes, other than multilayers, exist in spiders? 3. How and why do diverse colors evolve when they do not seem to serve any obvious function to the host organisms (e.g., nearly blind tarantulas)? Furthermore, we provide thoughts on how answering those questions could increase our understanding of color evolution and color-producing mechanisms in nature. Finally, we discuss future directions for investigating structural colors in spiders and how this could inspire future biomimetic applications. There are many reviews providing general backgrounds about colors, color theory, different ways to produce colors in nature and also the optical principles involved in structural color production3,5,8-11. Here, we focus on the three different color-producing strategies found in spiders: pigment-based colors, structural colors and fluorescent colors. Basic definitions for terminology in this article are in Box 1. Box 1. Glossary 1. Interference: Waves of light superposed to form a resultant wave is defined as interference. Interference usually occurs between waves of light that are from the same source or have the same frequency. Interference can alter the composition of light, enhancing or decreasing the intensity of certain wavelengths of light, and hence produce colors. 2. Diffraction: Diffraction is the change in the directions and intensities of a group of waves after passing by an obstacle or through an aperture whose size is approximately the same as the wavelength of the waves. Diffraction per se does not alter the wavelength of the diffracted waves. 3. Scattering: While interference and diffraction consider light as a form of wave, scattering emphasizes its particulate nature. Scattering is the changing of directions of propagation of photons after colliding with scatterers. After light is scattered, its wavelength may change due to inelastic scattering, or elastic scattering with moving scatterers (e.g., Doppler effect). 4. Coherence: When the phase differences among interacting waves of light are constant, it is called coherence. The phase relationships do not need to be exactly in-phase or out-of-phase to be coherent. When the distances between scatterers are smaller than the wavelengths of light, waves scattered from nearby scatterers are nonrandom in phase, making the waves coherent. 5. Incoherence: When the phase relationships among interacting waves of light are random, it is called incoherence. When the distances between scatterers are larger than the wavelengths of light, those scatterers are spatially independent. A spatially independent scatterer array is an incoherent array. 6. Pigments: Molecules, often insoluble, that produce colors as results of selectively absorbing specific wavelengths of light due to their chemical structures. 7. Biochromes: Biological pigments. 8. Chromatophores: Cells or tissue that contain biochrome granules inside. 9. Iridophores: Cells or tissue containing granules which producing iridescent appearances. 10. Structural colors: Colors produced by light interacting with nanostructures through optical principles. 11. Schemochromes: Biogenic nanostructures that producing colors when interacting with light. 12. Fluorophores: Things that absorb light of certain wavelengths and then re-emit light at longer wavelengths later.

In the following sections, we will first review the materials that act as building blocks for color-producing mechanisms in nature independently.

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2. COLOR-PRODUCING MATERIALS 2.1 Pigments 2.1.1 Biochromes that are common but missing in spiders Biochromes can be grouped into different classes based on their core chemical structures that result from varying biosynthetic pathways. Common biochromes are summarized in Table 1. Table 1. Summary for common biochromes and their colors Type Subtype Chemistry Cross-linked 5,6-dihydroxyindole (DHI) and Eumelanin 5,6-dihydroxyindole-2-carboxylic acid Melanin (DHICA) copolymers. Pheomelanin Benzothiazine and benzothiazole oligomer. Depending on the position Xanthophyll C40HxOy and length of their conjugated Carotenoid double bound system, they absorb light ranging from Carotene C40Hx 400-550 nm. Low molecular weight, Ommatin soluble in alkalis. Tryptophan Ommochrome metabolites High molecular weight, Ommin insoluble in alkalis. Bilin Tetrapyrroles Highly conjugated double bond system, strongly absorb visible light. Porphyrin Usually bond with metals (porphyrins that bond with irons are hemes). Pterin

-

Derivatives of 2-keto, 4-amino pteridine.

Color black, brown

Organism Common, but not found in arachnids.

red, brown yellow orange, red yellow, orange, red red, brown, black green, blue

Common. Only found in some mites among arachnids. Invertebrates (first found as visual pigments) Common

red

Common, but not found in arachnids.

yellow, orange, red

Common (used as visual pigments in birds)

The two most common classes of biochromes used in animal colors are melanins and carotenoids. Melanin absorbs light non-selectively across the visible wavelengths and can produce colors ranging from blacks and browns to reds12. Melanin’s absorption efficiency is greatest towards shorter (UV) wavelengths so that it is used for protection against UV radiation by many animals13. Carotenoids produce a series of colors ranging from light yellows and oranges to dark reds14. Unlike most other endogenous biochromes, animals are not able to synthesize carotenoids and must acquire them from food (either by eating plants directly or by consuming herbivores). Therefore, colors produced by carotenoids are often used as indicators for health or foraging ability of the animal15. In addition to melanins and carotenoids, birds use many other common biochromes, such as porphyrins and minor biochromes like bile pigments, psittacofulvins, turacins to produce colorful feathers16 and eggs17 (see Table 1). None of these biochromes has been found so far in spiders, and indeed only two classes of biochromes are currently known in them: ommochromes and bilins18. It is unusual to have such a limited range of biochromes relative to other animals. Carotenoid-based colors are relatively sparsely distributed across animals, so it is not surprising that they are absent (or at least undetected as yet) in spiders. However, the absence of melanins in spiders is unexpected. Melanins are nearly universally distributed across fungi and bacteria to vertebrates, including in all classes of arthropods except arachnids. Moreover, eumelanin is important for pathogen encapsulation in the innate immune system of insects and crustaceans19. The innate immune systems of spiders was also thought to use melanin20, but recent genomic research suggests that spiders are not capable of synthesizing melanins21 . The absence of melanin in spiders raises numerous questions: 1. What alternative mechanisms do spiders use in their immune systems? 2. How do spiders protect themselves from UV wavelengths? 3. Why did arachinids lose the ability to synthesize melanins? Devising research to answer these questions may inspire new anti-bacterial drugs, sunscreens, and could shed some light on the driving forces for spider color evolution. Surprisingly, previous genomic research also suggests that some spiders should be able to synthesize pterins, a class of biochromes that has not yet been biochemically identified in spiders21. Determining if pterin biosynthesis pathways are widespread in all spiders or isolated only in certain groups of spiders would be a reasonable next step. Determining presence of pterins by analytical techniques, such as LC-MS would be important too.

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2.1.2 The main biochromes in spiders: ommochromes Ommochromes produce melanin- and carotenoid-like colors, ranging from yellows, oranges, reds, browns to blacks, in spiders22(Fig. 1A-C). For example, ommochromes produce all the color variations of the crab spiders (family Thomisidae)23 that range from yellow to brown (Fig. 1A), and the blacks and reds on ladybug-mimicking spiders (Paraplectana sp.) (Fig. 1B) and black widow spiders (Latrodectus variolus)22 (Fig. 1C). Crab spiders are one of the few groups of spiders24 that can dramatically change their colors, with some species changing from white to bright yellow on different floral backgrounds25. Crab spiders change color by controlling the metabolism of ommochromes and also the arrangement and distribution of different types of ommochrome granules in the hyperdermis23,26. 2.1.3 Bilins and other biochromes The only other class of biochromes described in spiders so far are the green bilin pigments27(Fig. 1D, E). Bilins can be highly concentrated in exoskeletons (Fig. 1D), or diffusely present in the hemolymph, so that the green color is observed through the translucent chitin exoskeleton of spiders (Fig. 1E). Green colors are probably related to the colors of the plant substrates in their natural habitats, and aid with crypsis. Almost no biochromes produce blue color in nature (Table 1). For biochromes to appear blue, they need to absorb longer wavelengths of light (500~750 nm), but reflect or scatter shorter wavelength (400~500 nm). This requires biochromes with unusually large conjugated double bond systems; either a very long chain of alternating carbon-carbon double bonds (-[-C=C-C-]-)n or a much larger and complicated aromatic ring systems than for ommochromes, making blue biochromes difficult and energetically costly to synthesize. UV/blue visual pigments, on the other hand, actively absorb at shorter wavelengths, e.g., anthracene (C14H10) absorbs efficiently ~380 nm, and could be used as a UV/blue visual pigment if it occurs in biological systems. When used as a colorant, anthracene appears to be transparent (liquid) or white (powder). Therefore, UV/blue visual pigments are relatively simple in structure and easier to synthesize. The perception of blue light is thus almost ubiquitous across animal taxa28. Hence, blue color can be important in signaling and other mechanisms are often used to produce blue coloration in animals to compensate for the lack of blue biochromes29,30 (see 2.4 Schemochromes). 2.2 Fluorophores Fluorophores are pigments that absorb light and release most of the energy absorbed by emitting light at longer wavelengths than those absorbed. After conventional pigments absorb light, they dissipate the absorbed energy in the form of heat and reflect any remaining wavelengths of light. The colors produced by fluorophores largely depend on the wavelengths of light they emit. Although fluorescence is not common among animals, many spiders31, all scorpions32, some insects33,34, and even some birds35,36 fluoresce under UV light (Fig. 1F, I). We will discuss possible interactions and functions of spider fluorescence in the following sections (see Sec 3.2 and Sec 4). The chemistry of the fluorophores in spiders largely remains unknown and awaits further investigation. 2.3 Guanine crystals Guanine is a purine that is typically excreted or metabolized immediately after its production. However, in spiders, some fishes and lizards, guanine is deposited and stored in crystalline form in vesicles of specialized cells called guanocytes37,38, where the guanine crystals are used as colorants39. Some consider guanine crystals to be a kind of “pigment”, like titanium dioxide (TiO2) particles in white paints40, but both guanine crystals and TiO2 particles produce color by reflecting and scattering light randomly, rather than through absorption. Hence, technically they are not pigments. Random ordering of guanine crystals is the most common mechanism for producing of matte white (Fig. 1G) to silver mirror-like (Fig. 1H) colors in spiders depending on the shape and orientation of the crystals in guanocytes37. In lizard iridophores, guanine crystals can be both orderly and disorderly arranged. Blue/green hues can be produced by the coherent interaction between light and orderly arranged guanine crystals in those iridophores38. 2.4 Schemochromes The speed of light is reduced when travelling in transparent/translucent materials relative to a vacuum (c). The refractive index (n) of a material is defined as the amount light slows in that material (n = c/vlight in material). The propagation of light is altered at the interface of materials with different refractive indices. When interfaces form structures at a scale similar to or smaller than the wavelength of light, colors can be produced. Such colors are called structural colors and the nanostructures producing them are called schemochromes. Schemochromes are most frequently reported in bird feathers

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and arthropod exocuticles. The materials from which these schemochromes are formed differ for birds and arthropods7. In birds, beta-keratin, a fibrous structural protein that is also the major component of feathers, and melanin, are arranged in various morphologies to produce structural colors7. Schemochromes made by keratin (n ~ 1.56) and air voids (n = 1.0) alone are enough to produce structural colors41,42. However, melanin (n ~ 2.0), deposited in specialized organelles called melanosomes that range in shape from spherical to rod-like, also plays a critical role in color production43. Though melanin in bulk strongly absorbs light, when deposited into organized structures at nanometer scales, it instead reflects and scattered light. Melanosomes can also be completely filled with melanin (solid) or with air voids at the core of the organelle (hollow)44,45. The incorporation of melanin increases the contrast of refractive indices at the interfaces between materials (especially for hollow melanosomes; nmelanin:nair ~ 2:1), thereby enriching the brightness and range of colors the schemochromes can produce45. In other words, feathers with hollow melanosomes in the keratin matrices can produce more diverse and vivid colors than feathers with solid melanosomes and feathers without melanosomes45. Chitin, a long-chain polymer of N-acetylglucosamine, is often referred to as the building block of arthropod exocuticles46. However, this is oversimplified. Unlike feather keratins, which are made of almost pure keratin proteins, the chitin in arthropod exocuticles is layered with significant and varied amounts of proteins and minerals7. Therefore, the exocuticles of arthropods are chitin/protein composites. The exact composition varies in different body parts within the same organism, between different organisms within the same species, and among different species. As a consequence, the refractive indices of exocuticles can vary immensely from one body part of an organism to different body parts of the same or different organisms. Still, the refractive indices of exocuticles are most often cited as ~ 1.56, on average7. However, even with just a handful of material building blocks and a limited number of optic principles, the color palette is nearly unlimited for schemochromes. New types of nanostructures are still being found and described in birds and insects7. Table 2 summarizes the most common and important structural designs found in birds and insects. Table 2. List of schemochromes and their optical mechanisms found in birds and insects. Type of Optic principles Color Organisms Engineering examples nanostructures Interference, Artificially recreated Christmas tree Butterflies47,48 Diffraction butterfly scale models48 Birds49, Multilayer Interference Optical mirrors51/filters52 Insects50 Photonic crystals Iridescent colors (2D, 3D, gyroid, Interference, Birds53, Photonic crystal fibers (PCFs)56, close packed 54,55 Scattering Insects solar cells57 [fcc/hcp], diamond lattice) Diffraction, CDs, Diffraction grating Beetles58 Interference diffractive beam splitter59 Quasi-ordered Scattering, Birds41,42,60, Non-iridescent photonic Non-iridescent colors photonic crystal Interference Insects61,62 “pigment”63 16 Birds , TiO2 in paints40, Disorder structure Incoherent scattering Blue and white Insects64,65 colloidal suspension66

Diffraction gratings are seldom used as a major color-producing strategy in nature11, although they are often found in combination with interference mechanisms to produce complex optical effects67,68. Based on optics and engineered materials, we know that diffraction gratings cause iridescent effects across the entire visible spectrum as viewing angle changes, producing rainbow-like iridescence58, such as the colors on the surface of a CD or the beam splitter inside a spectrometer. The rarity of rainbow-like iridescence in living organisms supports the notion that diffraction gratings are rarely used by themselves to produce structural colors, even though the fossil record suggests that they are one of the most ancient structural color-producing mechanisms4,69. Diffraction grating derived rainbow-like structural colors occur in the sea shrimp (Azygocypridina lowryi)70, nacres of mollusks71, and some beetles58. Rainbow-like iridescence is also present in the feathers of some birds, such as bronzewing pigeons (Phaps chalcoptera); however, this rainbow-like iridescence is produced by multilayer interference (Fig. 2A), rather than by diffraction gratings, due to a small but well-coordinated spacing gradient between layers of melanosomes72. Each barbule in one of those feathers only exhibits iridescent colors within a narrow spectrum (i.e. different greens) when changing viewing angle. The rainbow-like effect is caused by an orderly combination of barbules of different iridescent colors across a barb. Rainbow-like iridescence

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also occurs in some spiders, especially in rainbow jumping spiders (Maratus robinsoni)(Fig. 3). Unlike bronzewings, where the rainbow-like effect is spread across many barbules attached to a barb, a single iridescent scale from the opisthosoma (abdomen) of M. robinsoni can show colors varied across the whole spectrum with changing viewing angle (Fig. 3A, B). Therefore, it is likely that diffraction gratings cause the rainbow-like iridescence in M. robinsoni. However, this requires further examination. Multilayer interference is the most common origin for structural colors, especially in arthropods3. Structural colors caused by interference are usually iridescent within a narrow range of wavelengths. They are also strongly directional, and typically very bright. Finally, multilayer interference can relatively easily produce short wavelength colors like blues and purples68. The blue color of Morpho butterflies’ wings is a classic example of multilayer interference48. This kind of color is most suitable for sending signals to specific recipients to convey specific messages because of the highly directional iridescence. However, many other types of schemochromes exist (Table 2) that use a variety of principles to produce colors in birds and insects, probably for many different functions. Compared to birds and butterflies, our understanding of structural colors in spiders is limited5. So far, most schemochromes observed in spiders are multilayer structures6,73-75 (Fig. 2B, C), some with grooves or ridges that presumably could act as diffraction gratings6,67 (Hsiung, unpublished data). However, the optical mechanisms for most of the known schemochromes in spiders have not been investigated in detail. As we continue to investigate structural colors in spiders, we may discover new types of schemochromes and maybe biochromes. Otherwise, we will need to determine why only multilayer schemochromes can be found in spiders.

3. INTERACTION BETWEEN COLOR-PRODUCING ELEMENTS In this section, we summarize four different types of interaction related to structural colors: 1. chromatophore-schemochrome interactions, 2. fluorophore-schemochrome interactions between organisms, 3. schemochrome interactions across different spatial scales, and 4. the interactions between structural regularity and irregularity. 3.1 Chromatophore – schemochrome interaction Structural colors and pigment-based colors were historically treated as independent mechanisms for color production. Researchers tended to distinguish the origin of coloration from living organisms as either structurally-based or pigment-based coloration and focused their investigations solely on one or the other7. As research has advanced, more and more evidence supports that pigment-based colors and structural colors interact. The interaction between chromatophores and schemochromes can be as simple as color mixing. For example, the green body coloration in some lizards and frogs results from mixing of yellow chromatophores and blue schemochromes29. Other than color mixing, biochromes can function to absorb nonspecific scattered light caused by schemochromes, which enhances the colors produced and makes them appear more saturated and conspicuous76,77. This effect can be achieved in many different ways. First, biochromes can be deposited inside vesicles or granules in chromatophores aggregated together beneath schemochromes, analogous to how birds use basal melanosome layers to assist non-iridescent structural color production77,78. Alternately, biochromes can be diffusely distributed and form a homogeneous layer beneath schemochromes as in many butterfly wing scales47 and highly iridescent hairs from the chelicerae of the tarantula Ephebopus cyanognathus6. The pigment layers can be observed in Transmission Electron Microscopy (TEM) as areas of a more electron-dense (darker) material in the micrographs. Moreover, biochromes can be mixed homogeneously with the building materials (such as chitin) of those schemochromes, as shown in examples from some butterfly wing scales79. In this case, biochromes are diffused homogeneously throughout entire schemochromes, so TEM micrographs are not useful for determining the presence of these pigments. Rather, chemical analyses, involving biochrome extractions and further analysis using techniques like LC-MS are needed to determine biochrome presence in schemochromes. For example, hairs from the tarantulas Poecilotheria metallica and Lampropelma violaceopes have similar amorphous sponge-like structures, except that hairs from P. metallica are yellow, and hairs from L. violaceopes are black. TEM micrographs for cross sections of hairs from both tarantulas show homogenous structures without any darkened area6 (Hsiung, unpublished data). However, it is difficult to believe that the black appearance can result solely from nanostructures and that the chitin-based hairs lack pigments. Many spiders’ exoskeletons are translucent (Fig. 1E, F), because chitin has low absorption to visible light. However, black can only be produced through absorption. Therefore,

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the most reasonable explanation for the color difference in the hairs of P. metallica and L. violaceopes is that ommochromes are diffusely present in those hairs and different subclasses of ommochromes are responsible for either yellow or black coloration of the hairs. Therefore, follow up chemical analyses are needed to determine if ommochromes exist in these hairs. Generally, when chromatophores interact with schemochromes, chromatophores are located in the inner layers beneath the schemochromes, which are proximal to the skin surface or cortex. This kind of spatial relationship works when the function of schemochromes is to selectively reflect light, producing the color that we see, while the chromatophores absorb non-specifically scattered light and minimize the background noise. However, there are exceptions. Some of the lighter biochromes, such as those that produce yellows and reds, do not have enough tint strength to be perceived on their own against dark backgrounds. Another type of colorant needs to work in concert with those biochromes to make them more conspicuous. This kind of colorant interaction is nearly ubiquitous in paints. TiO2 is used as a basal colorant in most paints while additional pigments are added to produce other colors80. TiO2 scatters light across the entire visible spectrum, producing a white appearance. However, when TiO2 acts in concert with other color pigments in paints, it increases brightness of the pigments and the ability to mask the surface beneath it, so the color of the paints can be perceived faithfully on any kind of surface. Guanine crystals are natural colorants that scatter light like TiO2 with similar functional implications. Guanine crystals that are deposited in guanocytes of spiders37,39 and in iridophores of some lizards38 interact with chromatophore layers above them, thereby increasing brightness and making the colors of the chromatophores more conspicuous (Fig. 1C inlet, the red hourglass). As previously discussed, based on the mechanism of how color is produced, guanine crystals should be categorized as schemochromes rather than chromatophores. Therefore, in the case of guanine crystals, schemochromes are buried underneath chromatophores. Guanine is a metabolic intermediate from nutrient digestion. Storing guanine crystals inside the bodies may cost more energy for animals compared to excreting it39. Therefore, lizards that have brighter body coloration as a result of guanine crystals may have higher reproductive success81. For web weaving spiders, conspicuous patterns produced by guanine crystals may increase foraging success by attracting more prey to the web82. 3.2 Fluorophore – schemochrome interaction: colors produced from interactions between organisms Color-producing mechanisms almost always arise from a single organism, although ambient light in the environment clearly plays a key role83. However, in some instances color signals arise due to interactions between organisms. The jumping spider, Cosmophasis umbratica, shows this kind of interaction between male and female during courtship84. Jumping spiders have photoreceptors with peak sensitivity in the UV-green range27. Male C. umbratica reflect UV due to schemochromes on their exoskeletons. In response, female C. umbratica‘s palps become excited by that UV and fluoresce back in green light. Males judge the quality of their mates by how strongly the females fluoresce while the females depend on the intensity of the UV light that their mates reflect from the ambient environment. This interaction is probably a conspecific recognition mechanism during courtship. 3.3 Interaction across spatial scales The third type of interaction involves structures interact across different spatial scales. How do nano-scale schemochromes interact with larger micro-scale structures to produce overall macro-scale body colorations? Little research has touched upon these interactions62,85. A well known example is the green wing scales of the male Papilio palinurus butterfly, where the green color is a mixture of yellow and blue caused by a nano-scale multilayer structure and a micro-scale concavity from the shape of a single butterfly wing scale86. Since natural structures are often hierarchical across several orders of scales87, these kinds of interaction could be widespread and require further investigation. Indeed, blue structurally-colored hairs on some tarantulas, such as P. metallica and L. violaceopes, show conspicuous hierarchical cylindrical groove-like structures along their length (Fig. 4), in addition to the multilayer nanostructure (similar to Fig. 2B, C) that likely produce the blue6. We hypothesize that this hierarchical microstructure reduces angle dependency from the iridescent multilayer nanostructures resulting in less iridescent blue coloration. 3.4 Regularity – irregularity interaction The amount of regularity vs. irregularity in nanostructures also affects structural color production11,88. Schemochromes likely form by self-assembly processes under only loose control by cellular machinery41,89,90. It is difficult for self-assembly processes to create perfect periodic structures over large areas without defects. Therefore, a large single crystal is rare in nature. For example, bigger gemstones are disproportionately more likely to have defects in their crystal structures than small crystals. Quasi-ordered photonic crystals41,42,60,61 and many schemochromes in insects55,58,88,91,92 are

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examples of this principle. For example, the nearly angle-independent green iridescence from scales of the weevil Lamprocyphus augustus results from many irregularly oriented, micrometer-sized single-crystalline diamond lattice domains in the scale93. Even simple multilayer schemochromes in spiders (Fig. 2 B, C) are not perfectly “multilayered”. The multilayer schemochromes are filled with disruptive gaps and channels, and the thickness of the layers also varies. All natural photonic structures probably incorporate some degree of irregularity. The critical question is: how does this irregularity affect color production and is irregularity itself a key design feature? New artificial photonic crystal design and manufacturing processes may be inspired by truly understanding the interaction between regularity and irregularity as it resides in natural photonic crystals. This could potentially lead to reducing the energy and precision required for manufacture, or developing more bottom-up fabrication approaches.

4. FUNCTIONS OF STRUCTURAL COLORS 4.1 General biological functions of color From fossil evidence and research on extant organisms, we know that appearance coevolved with the development of vision69,94. To understand color’s function, it is important to have a basic understanding about the potential recipients and their visual systems95,96. For example, most birds can see UV light, hence UV-reflecting plumages are widespread in birds. However, humans cannot see UV light, so many plumages that are sexually dichromatic in the UV region were mistakenly categorized as sexually monochromatic97. Birds, butterflies, and beetles all have good color vision98,99, hence color signals are an important part of their communication. In all of these taxa, colors are used for intraspecific communication, such as conspecific recognition, sex recognition and mate choice. Spiders, on the other hand, are generally considered to have poor eyesight and lack color vision, especially for web weavers, with a few notable exceptions such as jumping spiders. Colors usually serve in non-signaling functions, such as thermoregulation, or in interspecific signaling functions, such as prey attraction or crypsis18. For example, the white and silver on the dorsal sides of many web weavers allow thermoregulation, while the conspicuous yellow/red patches on many species may attract prey or make them cryptic when dropped in the grass82,100. Interspecific color signals are usually more generalized, nonspecific, broadcasting signals, and hence the colors usually do not vary much between different species of web weavers. Therefore, generally speaking, spiders are not considered to be colorful organisms. Many spiders fluoresce under UV radiation but the function of this fluorescence is unknown31. Since environmental UV radiation comes from sunlight, the intensity for UV peaks at daytime. Naturally, spider fluorescence peaks during daytime as well. The intensity of fluorescence cannot be compared with sunlight, hence any fluorescent signal is most likely masked during daytime. If the major function for spider fluorescence is not for signaling, could it be for UV protection by absorbing the more harmful UV radiation and converting that energy to less harmful visible light101? Do spiders (and scorpions) use this strategy for UV protection because of their lack of melanin? These questions should be pursued in future research. Certain species of crab spiders can change their body colors to match their backgrounds (Fig. 1A). In other members of the same family, body coloration and patterns usually match their natural habitats well, but cannot change (Fig. 1F). Research suggests that crab spiders have color vision102,103, but there is no behavioral evidence that they use color for intraspecific signaling. For the few species of crab spiders that can change their body colorations, the function of color change was thought to be crypsis104. Recent research, however, shows that even though they match color in the visible wavelengths, they still have high UV contrast against the background105. Hence, for their major predators and prey items (i.e., birds and insects), they are still conspicuous. These colors could thus be used for crypsis to organisms that cannot see UV, but as prey attraction using UV signals106. In the following, we will discuss the two groups of spiders that commonly have colorful iridescent hairs: tarantulas and jumping spiders. 4.2 Tarantulas vs. jumping spiders While iridescence occurs in other groups of spiders (e.g., lynx spiders, Family: Oxyopidae), they are most common in two distantly related taxa – tarantulas and jumping spiders – that are also quite different in their ecologies. Tarantulas are large, with leg span of some species reaching 20 cm or more, whereas jumping spiders are small and usually less then 1 cm (Fig. 3C). Tarantulas have poor eyesight and lack color vision107, while jumping spiders have high visual acuity, depth perception and color vision27,108,109. Tarantulas are usually nocturnal, sit-and-wait opportunistic predators, while jumping spiders are mostly diurnal, stalk-and-strike predators. Because of these differences, colors likely evolved and function differently between these two groups. For example, schemochromes so far have only been observed in the

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specialized hairs of tarantulas, but not in their cuticles; whereas they have been described both in specialized hairs/scales and in the cuticles of jumping spiders6. 4.3 Potential functions of color in tarantulas While many tarantulas are drab, species within several genera show a variety of structural and pigment-based colors110. All kinds of color can be found in a single genus, including: whites, yellows (golds), oranges, reds (rare), greens, blues, purples, browns and blacks (Fig. 5). Coloration also varies between different stages of life and different organisms. In some genera, colors are more vibrant and vivid in juveniles, and then fade away gradually as the spiders mature. In others, they remain dull until they are fully matured. The colors in some species are sexually dimorphic and some are not. Tarantulas do not possess color vision and are therefore unlikely to use colors for intraspecific signaling like sex recognition. Invertebrates with color vision can usually perceive colors better than vertebrates under low light conditions. Some nocturnal moths can see colors even under dim starlight111. Could tarantula colors attract potential prey that can see colors under dim light, such as nocturnal moths? Even this hypothesis seems unlikely as most species are ambush predators remaining at the entrance to their retreats while hunting. More behavioral and ecological studies for those tarantulas are needed to answer the questions and identify the potential recipients of their colors. Could all colors in tarantulas instead be incidental byproducts of selection on some other aspect of cuticle performance? It is possible, but what would the main function of those nanostructures be if not color production? They could enhance mechanical integrity of the exoskeleton. For example, melanin enhances the mechanical properties in bird feathers, especially in flight feathers112 , so that at least some aspects of bird coloration did not evolve for communication per se. Multilayers and a smooth outer surface in hairs of blind golden moles produce colors, but likely evolved to enhance wear-resistance and movement through soil113. Thus, tarantulas offer an opportunity to investigate mechanisms for structural color evolution in a system where communication is unlikely to be the selective force. 4.4 Functions of color in jumping spiders Despite the small body sizes of jumping spiders, they possess an unusually large repertoire of intraspecific interactions and behaviors, including male-male competition and courtship behaviors. Jumping spiders have good color vision27, color memory108 and are highly visually active during courtship. Evidence also supports that the diversification of jumping spiders is driven by sexual selection114. Jumping spiders often display sexual dimorphism with males being much more colorful than females. Hence, functions of color in jumping spiders are more evident. Colors are used for conspecific signaling in jumping spiders84,115 ,particularly with the shorter wavelength colors, as their vision is most sensitive to these wavelengths. Therefore, many jumping spiders can reflect UV light or have green/blue iridescent body coloration, especially on their chelicerae74. Males in one particular group of jumping spiders – the peacock spiders (Maratus spp.) are especially colorful (Fig. 6)116. The courtship behavior of peacock spiders closely resembles the courtship display of peacocks and birds of paradise in that they intentionally flash their iridescent ornaments directly in the female’s face117. Surprisingly, patterns in red are also observed in many species of peacock spiders (Fig. 5 A-D), although it is thought that jumping spiders are insensitive to these colors. Given the small body size of peacock spiders, it is also interesting to consider how sizes affect color production. For example, the white coloration in longhorn beetle (Anoplophora graafi) is composed by scales of many different colors; because the scales are too small for our eyes to discriminate, we perceive the overall color as white62. On the other hand, scales of many colors on the opisthosoma of peacock spiders are conspicuous enough for us to distinguish by the naked eye (Fig. 5). Signaling during courtship by jumping spiders is multimodal in that they also use chemical cues and vibrational signals simultaneously with visual signals118. Color alone is unlikely to determine successful courtship. This gives us an opportunity to study how colors interact and evolve with other types of signaling in jumping spiders and to study the theory of color evolution in general.

5. FUTURE PERSPECTIVES AND APPLICATIONS We are only beginning to understand the mechanistic basis of structural color, its function and evolution in spiders. All currently observed spider schemochromes are multilayer-based nanostructures6. If multilayer interference is the only optical mechanism that spiders utilize to produce structural colors, does it limit the capability for spiders to produce a variety of colors? How does the color space comprised with all the colors observed in spiders compare to that of birds, butterflies or even beetles? If the color space of spider colors is not significantly less than that of birds or insects, why do birds and insects utilize so many different types of nanostructures? Could new biogenic photonic structures be found in spiders? In addition to their intriguing biological implications, answering these questions could also help technological innovations. Biomimicry, or biomimetics, is a field that seeks to understand the underlying principles in targeted

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biological systems, abstract those principles, then modify and apply them into engineered systems to suit human purposes and to improve from the original engineering designs. Photonic crystals have a wide array of technology applications, ranging from optical instruments, sensing and even computing (See Table 2). Hence, this field is a natural candidate to incorporate biomimetic approaches. New knowledge gained by studying biogenic photonic structures may someday be translated to new bio-inspired designs or fabrication processes for artificial photonic crystals. For example, we know that engineered diamond lattice structures are best for creating photonic crystals with complete photonic band gaps119. However, current top-down fabrication approaches can only produce structures with photonic band gaps that are in the range of infrared (IR) and it is difficult to create diamond lattices with a scale small enough to produce photonic band gaps within the range of visible wavelengths. Solutions for this may come from studying how the diamond lattice schemochromes are made in beetle scales93. The same applies to studying structural colors in spiders. The study of spider color is just beginning and will be fertile ground for research in and across many disciplines.

ACKNOWLEDGEMENT We would like to thank Jürgen Otto (Australia), Nicky Bay (Singapore), Rong-Haur Tsai (Taiwan), Thomas Bresson (USA) and Matthias Foellmer (Adelphi University, USA) for giving permissions to use their valuable photos in this manuscript. We thank AFOSR (grant FA9550-13-1-0222) and HFSP (grant RGY0083) for funding. This work was supported by the National Science Foundation.

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Fig 1. Different spider colorations. A) Crab spiders (Misumena vitia), large yellow female at the center, with orange stripes on both sides of its opisthosoma (abdomen); small dark red male at the center right, on the female’s opisthosoma. B) Ladybug-mimic spider (Paralectana sp.). C) Adult female black widow spider (Latrodectus mactans), guanocyte granules can be seen under the red hourglass pattern (inlet). A-C) Typical colors by ommochromes. D) A feeding Araneus seminiger. E) Tetragnatha squamata, its legs and prosoma (cephalothorax) are translucent. D,E) Bilins originated geen colors. F) A crab spider (Tmarus sp.) sitting on a twig, its body coloration is similar to background for camouflage. G) A white crab spider on a flower. H) A dewdrop spider (Argyrodes sp.). G,H) White and silver are caused by guanine crystals. I) The same spider as in (F) fluoresce under UV light at night. Copyright: via Wikimedia Commons: 1. Under CC BY-SA 3.0: (C) by Shenrich91, (E) by Masaki Ikeda; 2. Under CC BY 3.0: (G) by Thomas Bresson. (A) Courtesy of Dr. Matthias Foellmer; (D) Courtesy of Rong-Haur Tsai; (H) By Ryan Kaldari (public domain); (B), (F), (I) ©2014 Nicky Bay http://sgmacro.blogspot.com, taken and adapted with permission.

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Fig. 3. Sections of the blue setae, viewed with the transmiss The peripheral multilayer as seen (a) through a section a and (b) through a section nearly parallel to the axis. The period on these sections confirm the values obtained from images. The slight opacity of the lighter layers is interpre embedding medium between the darker cylindrical chitin

of these planar structure, which has its counterpart – slightl the case of a curved, cylindrical, surface.

in phase

out of phase

4.2. Bristle’s cross-section, as observed in Transmission E

The SEM observations and data are confirmed by transm images. However, as we will now see, some details call for Fig. 3. Sections of the blue setae, viewed with the transmission electron microscope (TEM). Samples were by embedding Fig 2. Thin film interference and observed structuresThe in spiders. A multilayer treated as a stack of prepared thin weaxis individual bristles peripheralA) multilayer as seencan (a)be through a section at right anglefilms. from Here the seta infiltrate the structure at a constant temperature of 35◦C fo illustrate thin film interference for simplisity (grey: higher n; white: lower n). Different thickness of a thin film selectively reflect light and (b) through a section nearly parallel to the axis. The measurement of the multilayer for 72 more hours. Then, slices (90 at different wavelengths (top: blue, bottom: red) becauseperiod of constructive interference. lights are syncing in phase those nm thick) were cut pe on these sections confirm Reflected the values obtained from scanning electronatmicroscopy examined with a FEI Tecnai Transmission Electron Micro images. The slight opacity of the lighter layers is interpreted as the presence of wavelengths due to the optical path difference is in integer multiples of the wavelengths. Other wavelengths of light either get infiltrated partial shown in Fig. 3. antinous Figure 3(a) shows the structure of the m embedding structure medium between the darker chitin sheets. or complete destruction due to phase differences. B, C) multilayer observed in the cylindrical blueare setae (hairs) of P. under 75 gives a view of the same multila cross-section. 3(b) TEM with different sectioning orientations: B) perpendicular to the axis, C) parallel to the axis (Simonis, P., etFigure al. 2013 ). to the bristle’s axis. The examination of these views confirm of these planar structure, which has its counterpart slightly hollow more complicated, see below, – in The– internal cylindrical volume is homogeneous; (3 Taken and adapted with permission. the case of a curved, cylindrical, surface. more bilayers) lies below the external surface; (4) The out multilayer. 4.2. Bristle’s cross-section, as observed in Transmission Electronpart Microscopy The interesting is the multilayer, which shows alter doubt that theelectron dark layers are made(TEM) of the same chitin The SEM observations and data are confirmedlittle by transmission microscope cortex internal homogeneous images. However, as we will now see, some details calland forthe caution andhollow a careful discussion. cylinder: the multilayer, opacity ofresin, the “clear” separating the d Samples were prepared by embedding individual bristlesthe in an epoxy lettinglayers this resin ◦ C for ◦ C bristle. This same as the of the resin outside infiltrate the structure at a constant temperaturethe of 35 48 opacity hours, and polymerize at 60the different from the material imbedded for 72 more hours. Then, slices (90 nm thick) were cut perpendicular to the bristle’sbetween axis andthe dark she the interstitial material in the structure Since SEM sh examined with a FEI Tecnai Transmission Electron Microscope (TEM). Two typicalrises. images between sheetsinare non-embedded are shown in Fig. 3. Figure 3(a) shows the structure of thechitin multilayer theempty planein ofthe thedry, bristleopacities by assuming in the transport process into t cross-section. Figure 3(b) gives a view of the same multilayer, along athat, section nearly parallel ing the long polymerizing time, the infiltrated to the bristle’s axis. The examination of these views confirms that: (1) The bristle is hollow; (2)resin has gat slightly changed its cylindrical contrast with respect to(3the The internal hollow cylindrical volume is homogeneous; (3) A thin multilayer or pure, outerto be difficult to avoidthan when treating a complex str more bilayers) lies below the external surface; is (4)likely The outer layer is thicker those in the multilayer. The interesting part is the multilayer, which shows alternate clear and dark layers. There is little doubt that the dark layers are made of the same chitinous material as the outer protective cortex and the internal hollow homogeneous cylinder: the electron opacity is the same. In the multilayer, the opacity of the “clear” layers separating the dark chitinous lamellae is not exactly 2013This OSAmay suggest that the outer resin 25 March the same as the opacity of the resin outside the (C) bristle. is 2013 / Vol different from the material imbedded between the dark sheets and the question of the nature of the interstitial material in the structure rises. Since SEM shows unambiguously that the spaces between chitin sheets are empty in the dry, non-embedded structure, we interpret the different opacities by assuming that, in the transport process into the multilayer’s void spaces or during the long polymerizing time, the infiltrated resin has gathered some absorbing material and slightly changed its contrast with respect to the pure, outer-lying resin. This unwanted staining is likely to be difficult to avoid when treating a complex structure that has been preserved dry.

constructive

destructive

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Fig 3. Rainbow jumping spider (Maratus robinsoni) A) Rainbow-like iridescent scales are on the opisthosoma of male M. robinsoni. B) Zoom in view of the M. robinsoni opisthosoma, noted the positions of the colors changed after changing the viewing angles. C) A M. robinsoni on a human fingernail to give a sense of relative scale. All photos taken and adapted with permission ©Jürgen Otto.

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Micro-grooves (arrow) Micro-appearance: more iridescent

Adult♀ L. violaceopes

Nano-grooves (arrow)

Macro-appearance: less iridescent

Fig 4. Optical and scanning electron microscopy (SEM) images of blue structural colored hairs from adult female L. violaceopes. The blue color is less iridescent when observing the tarantula with naked eyes then observing the hairs under optical microscope. SEM micrographs show hierarchical grooves (arrow) along the length of a single structural colored hair.

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Fig 5. Diversity of body coloration in tarantulas A) Typhochlena curumim sp. n., female. B) Typhochlaena costae sp. n., female. C) Typhochlaena seladonia, female. D) Pterinochilus murinus. E) Poecilotheria metallica. F) Chromatopelma cyaneopubescens. Photos taken and adapted via Wikimedia Commons under: 1. CC BY 3.0: (A), (B), (C) by Bertani, R. 2012110; 2. Public domain: (D) by Morkelsker;

3. CC BY-SA 2.0: (E) by MLursus; 4. CC BY 2.0: (F) by snakecollecor.

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Fig 6. Colorful male peacock spiders t . -: A) M. splendens. B) M. volans. C) M. mungaich. D) M. speciosus. E) M. spicatus. F) M. chrysomelas. All photos taken and adapted with permission ©Jürgen Otto.

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