Structural Diversity of Arthropod Biophotonic Nanostructures SpansAmphiphilic Phase-SpaceVinodkumar Saranathan,*,†,‡,⊥,¶ Ainsley E. Seago,§ Alec Sandy,∥ Suresh Narayanan,∥

Simon G. J. Mochrie,#,¶ Eric R. Dufresne,#,∇,○,¶ Hui Cao,#,∇,¶ Chinedum O. Osuji,◆,¶

and Richard O. Prum*,⊥,¶

†Division of Physics and Applied Physics, School of Physical and Mathematical Sciences, Nanyang Technological University, 21Nanyang Link, 637371, Singapore‡Edward Grey Institute of Field Ornithology, Department of Zoology, University of Oxford, South Parks Road, Oxford, OX1 3PS,United Kingdom§CSIRO Ecosystem Sciences, GPO Box 1700, Canberra, Australian Capital Territory 2601, Australia∥Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 60439, United States⊥Department of Ecology and Evolutionary Biology, and Peabody Museum of Natural History, #Department of Physics, ∇Departmentof Applied Physics, ○Department of Mechanical Engineering and Materials Science, ◆Department of Chemical and EnvironmentalEngineering, and ¶Center for Research on Interface Structures and Phenomenon (CRISP), Yale University, New Haven, Connecticut06520, United States

*S Supporting Information

ABSTRACT: Many organisms, especially arthropods, produce vividinterference colors using diverse mesoscopic (100−350 nm) integumentarybiophotonic nanostructures that are increasingly being investigated fortechnological applications. Despite a century of interest, precise structuralknowledge of many biophotonic nanostructures and the mechanismscontrolling their development remain tentative, when such knowledge canopen novel biomimetic routes to facilely self-assemble tunable, multifunc-tional materials. Here, we use synchrotron small-angle X-ray scattering andelectron microscopy to characterize the photonic nanostructure of 140integumentary scales and setae from ∼127 species of terrestrial arthropodsin 85 genera from 5 orders. We report a rich nanostructural diversity,including triply periodic bicontinuous networks, close-packed spheres,inverse columnar, perforated lamellar, and disordered spongelikemorphologies, commonly observed as stable phases of amphiphilic surfactants, block copolymer, and lyotropic lipid−watersystems. Diverse arthropod lineages appear to have independently evolved to utilize the self-assembly of infolding lipid-bilayermembranes to develop biophotonic nanostructures that span the phase-space of amphiphilic morphologies, but at optical lengthscales.

KEYWORDS: Biophotonic nanostructures, structural colors, iridescence, self-assembly, membrane-folding, biomimetics

Organismal colors are commonly produced by pigmentsthat selectively absorb specific wavelengths of light and

re-emit others.1 However, many organisms also routinelyproduce vivid structural colors by constructive interference oflight scattered by mesoscale (100−350 nm) biophotonicnanostructures that are diverse in form and optical function.1−4

Arthropods, insects in particular, are the most abundant,diverse, and colorful animals on Earth. Arthropod structuralcolors function in a variety of contexts including social andsexual signaling, camouflage, and warning or aposematiccommunication.5−8 A burgeoning number of studies demon-strate that arthropods are an excellent source of biologicalinspiration for emerging technologies, such as in sensing, andphotonics2−4,7,9−13 (also see Supporting Information Table S1for list of references).

In terrestrial arthropods, structural colors are often producedby the well-characterized class of one-dimensional biophotonicnanostructures comprising thin-film, lamellar (multilayer)reflectors, or diffraction gratings in the cuticle of theintegument.2−4,7 However, various butterflies (Lepidoptera:Lycaenidae, Papilionidae),14,15 weevils (Coleoptera: Curculio-nidae),7 longhorn beetles (Coleoptera: Cerambycidae),7 bees(Hymenoptera: Apidae),16 jumping spiders (Araneomorphae:Salticidae),17 and tarantulas (Mygalomorphae: Theraphosi-dae)18 (see Supporting Information Table S1 for a full list of

Received: January 18, 2015Revised: April 30, 2015Published: May 4, 2015

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references) have structurally colored integumentary scales orsetae on their wings, elytra, abdomen, and legs. Thesetrichogen-based scales and setae possess a variety of complex,three-dimensional biophotonic nanostructures composed of thepolysaccharide chitin, cuticular proteins, and air (reviewed inrefs 2−4, 7, and 15). Despite the growing body of research onarthropod structural colors (Supporting Information Table S1),we currently lack accurate structural characterizations of morethan a few species. Although knowledge of the development ofbiophotonic crystals in arthropods is growing,14,19−21 we stilllack a comparative developmental framework to understand therich biodiversity of arthropod cuticular biophotonic nanostruc-tures (discussed in refs 14 and 15) or how these biologicalsignals function and evolve in these organisms. These complexnanostructures are too large to be developing via classicalmorphogenesis as seen in molecular/cell biology, and much toosmall to be understood using a multicellular developmentalbiology framework.14 They are also of broader biomimeticinterest, because synthetic fabrication of three-dimensionalphotonic nanostructures at these rather large optical lengthscales remains challenging.22−25

Recently, we applied synchrotron small-angle X-ray scatter-ing (SAXS) to identify single gyroid (I4132) photonic crystalsin wing scales of five well-studied butterflies from two families14

and single diamond (Fd3m) photonic crystals in the coverscales of a fossil weevil Hypera diversipunctata.26 Here, we applySAXS to structurally characterize the biophotonic nanostruc-tures present within scales and setae from 140 distinctly coloredintegumentary patches of diverse land arthropod taxa withprominent structural coloration. We supplemented our SAXSassays with electron microscopy (EM) for 44 out of the 140patches. Our sample includes 129 different color patches from∼98 species in 75 genera belonging to 9 families of beetles(Coleoptera), single patches from 3 species in 2 genera of bees(Hymenoptera: Apidae: Apinae), single patches from 6 speciesin 6 genera belonging to 2 families of spiders (Araneae), in

addition to one lycaenid butterfly (Lepidoptera: Lycaenidae)and a bee fly (Diptera: Asiloidea: Bombyliidae) (SupportingInformation Table S1).

■ RESULTSWe find no evidence of a coherently scattering nanostructurepresent in 19 out of the 140 distinctly colored integumentarypatches (Supporting Information Figure S1 and Table S1). TheSAXS patterns from the rest of the arthropod scales and setaeexamined ranged from a large number of discrete Bragg spots inthe azimuthal directions (Figure 1m−o,q and SupportingInformation Figure S1), characteristic of a polycrystallinenanostructure, to a series of concentric powder-like (orDebye−Scherrer) diffraction rings (Figure 1p,r and SupportingInformation Figure S1), characteristic of an amorphous orquasi-ordered nanostructure with only short-range isotropicorder.14,27,28 The presence of a large number of sharp, higher-order diffraction spots up to 8 or 9 orders from some weeviland longhorn beetle scales (Figures 1m−o, 2a−c, andSupporting Information Figure S1) underscores a high degreeof spatial order for a biological system. However, like SAXSexperiments on synthetic soft materials,27 40 of the 121 casesexhibited SAXS spectra with variable or ambiguous features thatwere intermediate between the idealized predictions ofcrystalline structures. This could be due to both variation inlength scale of peak structural correlations due to finite long-range ordering, which may broaden and flatten diagnosticspectral peaks, or owing to variation in nanostructure betweendifferent scales of the same species or even different domainswithin a single species. For these cases, we present ananostructural diagnosis based on the predominant SAXSspectra distribution, but we recognize these tentative diagnoseswith an * in Table S1 (also see Supporting Information).In the following, we present our SAXS nanostructural

diagnoses based on assigning the symmetry that is mostconsistent with the indexed Bragg peaks in the azimuthal

Figure 1. Representative morphology of structural color producing arthropod cuticular nanostructures. (a−f) Light micrographs, (g−l) electronmicrographs, and (m−r) representative 2D SAXS patterns from the photonic scales or setae of the following: (a,g,m) Platyaspistes venustus(Curculionidae), single gyroid (I4132) (see Supporting Information Figure S1.105); (b,h,n) Eupholus quintaenia (Curculionidae), single diamond(Fd3 m) + face-centered orthorhombic (Fddd) (see Supporting Information Figures S1.41 and S1.42); (c,i,o) Sternotomis pulchra (Cerambycidae),simple cubic or single primitive (Pm3 m) (see Supporting Information Figure S1.130); (d,j,p) Anoplophora graaf i (Cerambycidae), quasi-ordered +fcc opal (see Figure 2d and Supporting Information Figures S1.123 and S1.124); (e,k,q) Amegilla cingulata (Apidae), inverse 2D hexagonal columnar(see Figure 2i and Supporting Information Figure S1.137); and (f,l,r) Thyreus nitidulus (Apidae), inverse 2D twisted columnar (see Figure 2h, andSupporting Information S1.139). False color SAXS patterns (unmasked) depict the logarithm of scattering intensity as a function of the scatteringwave vector, q. The radii of the concentric circles are given by the peak scattering wave vector (qpk) times the moduli of the assigned hkl indices ofpermitted Bragg reflections for the assigned space groups. Crystallographic motifs seen in the SEM images are identified in panels g−i. Panel k andits inset respectively show longitudinal SEM, and cross-sectional TEM views of the setae interior. Scale Bars: a, 25 μm; b−f, 50 μm; g,h, 500 nm; i,250 nm (inset, 100 nm); j, 100 nm; k, 250 nm (inset, 1 μm), l−600 nm (inset, 500 nm); m−r, 0.05 nm−1. Abbreviations: c, chitin; a, air void.

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profiles according to IUCr conventions,29 complemented withreal-space information from electron microscopy for a third ofthe assays. The diversity of arthropod cuticular nanostructuresis best summarized taxonomically because different lineageshave evolved distinct classes of biophotonic nanostructures(Figures 1−3 and Supporting Information Figure S1 and TableS1). Triply periodic, bicontinuous networks were relativelyrestricted in their distribution. Single gyroid (I4132) networksoccurred only in photonic scales of snout weevils (Curculio-nidae; e.g., Platyaspistes venustus, Figure 1a,g,m) and in wingscales of the Lycaenid butterfly Lycaena kasyapa (Lycaenidae;Supporting Information Figure S1.1); However, single gyroid

photonic crystals are so far known in the wing scales of onlyfour other genera within lycaenid and papilionid butter-flies.14,15,30,31 Single diamond (Fd3 m) and sheared, face-centered orthorhombic (Fddd32,33) networks were also foundonly in snout weevils (e.g., Eupholus quintaenia; Figure 1b,h,nand Supporting Information Figure S1.42). In Lamprocyphusweevils (Supporting Information Figures S1.72−80 and TableS1) for instance, both single diamond and single gyroidphotonic crystals were identified on the same individual andsometimes within different domains in the same scale.Spongelike or quasi-ordered versions of single diamondnetworks with varying degrees of disorder were also present

Figure 2. Structural diagnoses of representative SAXS profiles of arthropod cuticular photonic nanostructures. The normalized, azimuthally averagedSAXS profiles were calculated from the respective 2D SAXS patterns and shown on a log−log scale (top to bottom). Curculionidae: (a)Pachyrrhynchus reticulatus, single diamond (Fd3m) (see Supporting Information Figure S1.84), (b) Chloropholus nigropunctatus, single gyroid (I4132)(see Supporting Information Figure S1.113). Cerambycidae: (c) Sternotomis mirabilis, simple cubic or single primitive (Pm3m) (see SupportingInformation Figure S1.132), (d) Anoplophora graaf i, quasi-ordered + fcc opal, (see Figure 1d,j,p and Supporting Information Figures S1.123 andS1.124), (e) A. birmanica, bcc spheres (see Supporting Information Figure S1.119), (f) A. versteegi, quasi-ordered spheres (see SupportingInformation Figure S1.122). Apidae: (g) Thyreus pictus, sponge (see Supporting Information Figure S1.138), (h) T. nitidulus, inverse 2D twistedcolumnar (see Figure 1f,l,r, and Supporting Information Figure S1.139), (i) Amegilla cingulata, inverse 2D hexagonal columnar (see Figure 1e,k,q andSupporting Information Figure S1.137), (j) Porod (q−4) background. The colored vertical lines correspond to the expected Bragg peak positionalratios for various alternative crystallographic space groups, presented together for direct comparison. The numbers above the vertical lines aresquares of the moduli of the Miller indices (hkl) for the allowed reflections of specific space-groups. The normalized positional ratios of the scatteringpeaks are indexed to the predictions of specific crystallographic space groups or symmetries according to IUCr conventions.29

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in weevils, sometimes also within the same individual (e.g.,Eupholus bennetti; Supporting Information Figures S1.39 andS1.40 and Table S1).By contrast, the photonic scales of longhorn beetles

(Coleoptera: Cerambycidae) possessed nanostructures essen-tially based on ball-and-stick arrangements of chitin spheres.One class of nanostructure found in the photonic scales oflonghorn beetles consisted of ordered (face-centered cubic, fcc;body-centered cubic, bcc) and quasi-ordered close-packing ofdiscrete chitin spheres sometimes connected by thin necks(Coleoptera: Cerambycidae; e.g., Anoplophora graaf i; Figures1d,j,p and 2d and Supporting Information Figures S1.123−124). Single primitive or simple cubic (Pm3 m) triply periodicbicontinuous networks as well as quasi-ordered or sponge-likeversions of the same were present only in the Sternotominitribe of longhorn beetles (Coleoptera: Cerambycidae; e.g.,Sternotomis pulchra bifasciata, Figure 1c,i,o and SupportingInformation Figures S1.130−131). The scale photonic

nanostructure of the longhorn beetle Prosopocera lactator wasdiagnosed as a bcc (Im3 m) network of connected spheres (cf ref34). Interestingly, these appear to be structurally intermediatebetween the close-packed sphere nanostructures of otherlonghorn beetles and bicontinuous network nanostructures ofSternotomine longhorns.Quasi-ordered arrays of chitin spheres were also identified

within scales of two basal taxa of beetles, Toxonotus sp.(Coleoptera: Curculionoidea: Anthribidae) and Isacantha sp.(Coleoptera: Curculionoidea: Belidae).A two-dimensional, inverse hexagonal columnar morphology

(i.e., air pores in chitin) was diagnosed in the iridescent setae ofa digger bee, Amegilla cingulata (Hymenoptera: Apidae:Anthophorini, Figures 1e,k,q and 2i)16 and in a jumping spider(Araneae: Salticidae, Supporting Information Figure S1.141). A2D, Bouligand-like,35 twisted inverse columnar morphology wasidentified in setae of a cuckoo bee, Thyreus nitidulus(Hymenoptera: Apidae: Melectini, Figures 1e,k,q and 2i, and

Figure 3. A biophysical framework for classifying the diversity of arthropod cuticular photonic nanostructures. (a) A schematic of the lyotropic phasebehavior depicting the well-known equilibrium inverse lipid phases in order of increasingly positive Gaussian curvature (see ref 51), starting with theflat lamellar phase (Lα), whose interfacial mean and Gaussian curvature is trivially zero. These include the inverse bicontinuous cubic phases (QII:double gyroid Ia3d, double diamond Pn3m, and double primitive Im3m), perforated lamellae (L3), inverse hexagonal columnar cylinders (HII) as wellas inverse micelles or vesicles that can be jammed or close-packed into cubic (III) or amorphous (L2) arrays. (b) Topological decomposition oflyotropic phases in (a) into constant mean curvature surfaces: plane, saddle (Schoen’s gyroid G, Schwarz’s diamond D, and Schwarz’s primitive P),lamellar-helicoid (Riemann’s minimal surface shown; image credit: Matthias Weber), cylinder, and sphere, along with the corresponding interfacialmean (H) and Gaussian or saddle-splay curvature (KG). Note the first three are minimal surfaces. (c) The diversity and taxonomic distribution ofarthropod cuticular photonic nanostructures.

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Supporting Information Figure S1.139), and in scales of Hopliascarab beetles (Coleoptera: Scarabaeidae, Supporting Informa-tion Figures S1.3−4).36Amorphous or quasi-ordered, spongelike networks were

diagnosed in the photonic setae of another species of the samegenus of cuckoo bee, T. pictus (Supporting Information FigureS1.138). Lastly, the photonic nanostructures in purple-bluesetae of tarantulas (Araneae: Theraphosidae, SupportingInformation Figures S1.142−145) were characterized asperforated lamellae, as in many butterflies.15,18,20,37,38

The SAXS structural data also confirm that the arthropodcuticular nanostructures are sufficiently ordered at theappropriate length scales to produce the observed colors viaconstructive interference (Supporting Information Figure S2).We have thus assayed the cuticular scale and setae

nanostructure of ∼127 species of land arthropods in 85 generabelonging to 4 orders of insects and two suborders of spidersusing SAXS and EM, including ∼66 genera that have not to ourknowledge been previously investigated. The analysis hasresolved uncertainties in the diagnoses of the biophotonicnanostructure in previous studies (Supporting InformationTable S1). Compared to birds,28,39,40 lizards and frogs,41

cephalopods and other aquatic animals,13,42 terrestrial arthro-pods, especially insects, exhibit a breath-taking array ofbiophotonic nanostructures within scales and setae.Intriguingly, the diversity of arthropod photonic nanostruc-

tures documented here exhibits the very same, rich poly-morphism found in amphiphilic macromolecules, such as blockcopolymers,43−46 surfactants,47 and lipids.48,49 Biological lipidbilayer membranes are known to self-assemble in aqueousmedia into a variety of inverse or type-II lyotropic liquidcrystalline phases or morphologies50,51 (Figure 3a). In additionto their crystallographic space group symmetries, these self-assembled membrane phases can be classified based on thevariation in their interfacial curvature.48,50−52 The fundamentalgeometry of these lyotropic phases can then be topologicallyclassified into sphere, cylinder, or the associate (Bonnet)families of lamellar-helicoid (Riemann’s) and saddle (Schwarz’sD, P, and Schoen’s G) surfaces. All of these surfaces arecharacterized by a constant mean curvature (H = (c1 + c2)/2)but they can be differentiated on the basis of their Gaussian orsaddle-splay curvature (KG = c1c2, where c1 and c2 are theprincipal curvatures along orthogonal planes perpendicular tothe surface)53,54 (Figure 3b). The Gaussian curvature of asphere is positive, zero for cylinders and planes, and negativefor a saddle. Furthermore, lamellar, lamellar-helicoid, andsaddle morphologies are examples of minimal surfaces, that is,they have zero mean curvature. Even in a quasi-ordered spongeor a perforated lamellar morphology, the pores may be thoughtof as a Riemann’s minimal surface with helicoid-like bridgesconnecting adjacent, asymptotic, and parallel (lamellar)planes55−57 (Figure 3b).The similarity of arthropod cuticular nanostructures to

amphiphilic block copolymer, surfactant, and lyotropic lipidphase states is not merely a structural or geometrical analogy.Biological lyotropic lipid membrane morphologies, includingthe triply periodic, bicontinuous cubic phases (double gyroid,double diamond, and double primitive morphologies) are well-known in membrane-bound organelles of living cells.57−66

However, these arthropod biophotonic nanostructures aremuch larger in size (lattice parameters of 50−500 nm)50,61,65

than those produced by typical lipid−water systems (≤20nm).51

The self-assembly of biological lipid bilayer membranes intolyotropic morphologies is hypothesized to be regulated by theenergetics of membrane curvature.50,63,67 Living cells controlthe curvature and shape of their membranes with membrane-binding proteins that vary in shape, molecular weight, andelectrostatic properties.52,63−65,67,68 We hypothesize that multi-ple lineages of arthropods have independently evolved to utilizethe intrinsic capacity of membrane-bound organelles to self-assemble into lyotropic phases to template the precursors ofcolor producing nanostructures. Specifically, we posit that thecontrolled expression of membrane-binding proteins withdifferent bilayer bending (κ) and Gaussian or saddle-splay (κ)moduli51,52 in arthropod scales and setae cells could similarlyfacilitate the development of various lyotropic precursortemplates of biophotonic nanostructures.50,61,65,66,68 It is alsolikely that the curvature of these biological nanostructures arestabilized by intermembrane binding proteins.68

Interestingly, each arthropod lineage studied has evolved tooccupy different portions of the total lyotropic phase space(Figure 3c). For instance, many different arthropod familiesexhibit quasi-ordered sponge-like networks or perforatedlamellae. But single gyroid and single diamond nanostructuresare found only in curculionid weevils, and lycaenid andpapilionid butterflies. Likewise, quasi-ordered and orderedclose-packings of chitin spheres are found only in longhornbeetles and in two basal families of weevils. Thus, it appearslikely that within each lineage the molecular structure of themembrane-binding proteins has evolved to express only alimited range of the total possible physical effects on membranecurvature and stability.50−52 The plausibility of this model issupported by the existence of the highly conserved superfamilyof BAR-domain proteins, which have been shown to affect andstabilize membrane curvature in endocytic spherical or tubularinvaginations,67,69 for example, in three-dimensional, cubicmembrane (t-tubule) networks in striated skeletal musclecells.58,70 This model suggests a likely biophysical frameworkfor understanding the developmental basis and biodiversity ofarthropod photonic nanostructures (Figure 3). The distinctlynonrandom distribution of the cuticular biophotonic nano-structures in arthropods also implies apomorphic (lineage-specific) differences in membrane-folding mechanisms (seebelow).In butterflies, the single gyroid photonic crystals in wing scale

cells have been hypothesized to develop from a core−shelldouble gyroid (Ia3d) template made by tandem infolding of thesmooth endoplasmic reticulum (SER) and plasma mem-branes,14,19−21 as in core−shell morphologies seen in triblockcopolymer systems.71,72 After development of the core−shelldouble gyroid template, chitin is deposited into the extracellularspace, which is continuous with one of the two single gyroidcores. Upon maturation, the rest of the scale cell dries up anddies, leaving behind a single gyroid (I4132) network comprisingchitin and air of the appropriate size to constructively reflect aspecific visible color.14 Double gyroids can be directly self-assembled, unlike single gyroids (which have superior opticalproperties73). Therefore, butterflies have evolved to self-assemble a double gyroid template from the interactions ofplasma and SER membranes, but use only one of the two singlegyroid compartments to bioengineer the final opticalnanostructure.14

The development of biophotonic nanostructure within scalecells has been investigated only in a few butterflies.19−21,74

Nevertheless, given the homology of the trichogen (shaft-

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forming cells) across all arthropods,75 the development ofphotonic nanostructures in scales and setae of other insects andspiders likely proceeds via similar membrane-folding mecha-nisms. However, the diversity of optical nanostructuresobserved in arthropod integument requires recognition oftwo broad classes of biophysical developmental mechanisms.The first class can lead to self-assembly starting from a singlelipid bilayer interface dividing two aqueous volumes.44,45,51

These nanostructures include spongelike quasi-ordered net-works, perforated lamellae, inverse (hexagonal and twisted)columnar phases, and quasi-ordered and ordered (fcc, bcc)sphere packings. These morphologies likely develop withinscale cells from the infolding of the plasma membrane into alyotropic template, followed by extracellular chitin depositionand the maturation of the cell. By contrast, the second classleads to the self-assembly of single gyroid and single diamondnanostructures of snout weevils from double gyroid or doublediamond precursor templates. This process requires thecollaborative infolding of both the plasma membrane andSER, as observed in lycaenid butterflies.14,21

These developmental hypotheses are further supported bythe observation that single gyroid and single diamond networksin butterfly14 and weevil scales have relatively low chitin fillingfractions (mean 0.2; Supporting Information Table S1). Thelow volume of chitin results from the fact that the final cuticularnanostructure consists of only one of a pair of chiralinterpenetrating networks. The similarity of weevil scaledevelopment to that of butterflies is further supported by thepresence of one or more submicron vesicles in weevil scales(e.g., see Supporting Information Figure S1.44) that appear tobe vestiges of SER fragments hypothesized to play a role inorganizing the parallel membranes.21 On the other hand, thebiophotonic nanostructures in longhorn beetles and bees havesignificantly higher (unpaired t = 7.6, df = 41, P < 0.0001)chitin filling fractions (mean 0.48; Supporting InformationTable S1). Therefore, unlike butterflies or weevils, these largerchitin volume fractions eliminate the physical space for theparallel infolding of a second lipid bilayer within the developingscale cells. Interestingly, like other longhorn beetles, the singleprimitive or simple cubic triply periodic bicontinuous networksof Sternotomis longhorn beetles also have high chitin fillingfractions (0.4, on average; Supporting Information Table S1),which would prevent the infolding of a second parallel bilayermembrane during their development. So, they appear to share acommon developmental mechanism with other longhornbeetles. This underscores the phylogenetic constraints on thedevelopment and evolution of biophotonic nanostructures.We illustrate these possible developmental scenarios by

analogy to the microphase separation of block copolymers(Figure 4). As in butterflies,14 we posit that single gyroid andsingle diamond nanostructures in snout weevil scales developvia the tandem infolding of parallel plasma and SER bilayermembranes, akin to a linear ABC triblock copolymer that iscompositionally asymmetrical about the midplane, that is,ABCB′A′ (Figure 4a).76 A core−shell double gyroid (Ia3d) or adouble diamond (Pn3m) precursor thus self-assembled istransformed into a single gyroid or single diamond bybackfilling only one of the core volumes (which is continuouswith the extra-cellular space) with chitin. By contrast, wepropose that scale or setae nanostructures in longhorn beetles,bees, and spiders develop only by the invagination of theplasma membrane, similar to an AB diblock copolymer that iscompositionally asymmetrical about the midplane, that is, ABA′

or ABC (Figure 4b) to produce nanostructures closer to 50%volume fractions. In both cases, chitin, which is an extracellularpolymer, is then deposited into the extracellular space (A), andthe subsequent desiccation and degeneration of the rest of thedying cell results in the final photonic nanostructure. Futureobservations and experiments on the development of scale andsetae nanostructures in weevils, longhorn beetles, and otherarthropods are necessary to test these hypotheses.Cellular control of the physical mechanisms of lipid bilayer

self-assembly has likely enabled many arthropod lineages toevolutionarily explore most of the phase space of lyotropic oramphiphilic materials for a photonic function at optical lengthscales not easily achieved in synthetic soft matter systems.45,51

The repeated evolutionary co-option of the biologicalmembrane self-assembly provides a novel, generalized explan-ation for the explosive diversity of photonic nanostructures inarthropod scale and setae cells. Future experiments shouldfocus on identifying putative proteins that control membranecurvature and invagination within scale or setae cells duringdevelopment.These diverse arthropod biophotonic scales may offer

convenient biotemplates to serve in functional applications

Figure 4. Self-assembly of arthropod cuticular photonic nanostruc-tures, by analogy to microphase separation in block copolymers. (a) Asin butterflies,14 single gyroid (I4132) (illustrated) and single diamond(Fd3m) scale nanostructures in snout weevils likely develop viatandem infolding of parallel plasma and SER bilayer membranes into aprecursor, core−shell double gyroid (Ia3d) (illustrated) or doublediamond (Pn3m) template within the perimeter of the scale cell. Thisis akin to a linear ABC triblock copolymer, which is compositionallyasymmetrical about the midplane (ABCB′A′). (b) Scale and setaenanostructures in longhorn beetles, bees, and spiders likely develop bythe invagination of the plasma membrane only, similar to an ABdiblock copolymer that is compositionally asymmetrical about themidplane (ABA′/ABC) to produce nanostructures with the observed∼50% volume fractions (as illustrated for the Pm3m nanostructure). Inboth panels a and b, the extracellularly synthesized biopolymer chitin isdeposited into only one of the interpenetrating volumes (A), which iscontinuous with extracellular space, and the subsequent desiccationand degeneration of the rest of the dying cell results in the finalphotonic nanostructure (middle and right columns). The biologicalcomponents of the developing scale or setae cell are shown color-coded to the different monomer blocks of a hypothetical linearnoncentrosymmetric ABCB′A′ or ABA′/ABC block copolymer(leftmost column).

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such as sensing10 and photonics77 (after appropriate dielectricinfiltration). At the same time, protein-mediated membraneself-assembly of synthetic bilayer membranes,68 and the phasebehavior of a linear, noncentrosymmetric pentablock copoly-mer obtained by the mixing of a ternary triblock and a binarydiblock copolymer with suitably optimized molecular weightsto maximize the lattice parameters76 are two perhaps promisingbioinspired (if not biomimetic) approaches to synthesizingtunable mesophases with large lattice parameters, includingmorphologies like single gyroid and single diamond currentlynot accessible via direct synthetic self-assembly.24

■ ASSOCIATED CONTENT*S Supporting InformationDetailed SAXS structural diagnoses, SAXS optical reflectancepredictions, and coherence length estimates of arthropodcuticular photonic nanostructures, and materials and methods.The Supporting Information is available free of charge on theACS Publications website at DOI: 10.1021/acs.nano-lett.5b00201.

■ AUTHOR INFORMATIONCorresponding Authors*E-mail: Vinodkumar.Saranathan@aya.yale.edu.*E-mail: Richard.Prum@yale.edu.Present Addresses(V.S.) Life Sciences, Yale-NUS College, 6 College Avenue East,138614, Singapore(A.E.S.) Agricultural Scientific Collections Unit, NSW Depart-ment of Primary Industries, Orange Agricultural Institute, 1447Forest Road, Orange NSW 2800, AustraliaAuthor ContributionsV.S., A.E.S., A.S., S.N., and R.O.P. designed the research; V.S.and A.E.S. performed research; all authors discussed and/oranalyzed the data; V.S. and R.O.P wrote the manuscript withinputs from S.G.J.M, E.R.D., and C.O.O.FundingSAXS data collection at 8-ID, Advanced Photon Source,Argonne National Laboratory, was supported by the U.S.Department of Energy, Office of Science, Office of Basic EnergySciences, under Contract DE-AC02−06CH11357. This workwas supported with seed funding from the US National ScienceFoundation (NSF) Materials Research Science and EngineeringCenter (DMR 1119826) and NSF grants to S.G.J.M. (DMR-0906697), H. C. (PHY-0957680), a Royal Society NewtonFellowship and Linacre College EPA Junior Research Fellow-ship to V.S. as well as Yale University W. R. Coe Funds toR.O.P. R.O.P. acknowledges the support of the IkerbasqueScience Fellowship and the Donostia International PhysicsCenter.NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe thank Bill Krinsky, Steve Lingafelter, Rick Leschen, andRolf Oberprieler for assistance with arthropod identification,Pietro De Camilli for stimulating discussions on protein−membrane interactions, Antonia Monteiro, and two anony-mous reviewers for helpful comments on this manuscript.Arthropod specimens were kindly provided by the YalePeabody Museum of Natural History Entomology Collections(Larry Gall), University of Oxford Natural History Museum

Hope Entomological Collections (James Hogan, Ray Gabriel,and Darren Mann), Smithsonian U.S. National MuseumEntomology Collections (Steve Lingafelter), and CSIROAustralian National Arthropod Collection (ANIC). We thankNick Terrill and Tobias Richter for help with SAXS datacollection at beamline I22 of the Diamond Light Source thatcontributed to some of the results presented here, as well asStephen Mudie, Sarah Weisman, and Tara Sutherland forcomments and support regarding specimen preparation forSAXS at the Australian Synchrotron.

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