Letter

pubs.acs.org/NanoLett

Structural Diversity of Biophotonic Nanostructures Spans Amphiphilic Phase-Space † ‡ ⊥ ¶ § ∥ ∥ Vinodkumar 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, 21 Nanyang 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, ∥ 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, Department ○ ◆ of Applied Physics, Department of Mechanical Engineering and Materials Science, Department of Chemical and Environmental ¶ Engineering, and Center for Research on Interface Structures and Phenomenon (CRISP), Yale University, New Haven, Connecticut 06520, United States

*S Supporting Information

ABSTRACT: Many organisms, especially , produce vivid interference colors using diverse mesoscopic (100−350 nm) integumentary biophotonic nanostructures that are increasingly being investigated for technological applications. Despite a century of interest, precise structural knowledge of many biophotonic nanostructures and the mechanisms controlling their development remain tentative, when such knowledge can open novel biomimetic routes to facilely self-assemble tunable, multifunc- tional materials. Here, we use synchrotron small-angle X-ray scattering and electron microscopy to characterize the photonic nanostructure of 140 integumentary scales and setae from ∼127 species of terrestrial arthropods in 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 spongelike morphologies, commonly observed as stable phases of amphiphilic surfactants, block copolymer, and lyotropic lipid−water systems. Diverse arthropod lineages appear to have independently evolved to utilize the self-assembly of infolding lipid-bilayer membranes to develop biophotonic nanostructures that span the phase-space of amphiphilic morphologies, but at optical length scales. KEYWORDS: Biophotonic nanostructures, structural colors, iridescence, self-assembly, membrane-folding, biomimetics

rganismal colors are commonly produced by pigments In terrestrial arthropods, structural colors are often produced O that selectively absorb specific wavelengths of light and by the well-characterized class of one-dimensional biophotonic 1 re-emit others. However, many organisms also routinely nanostructures comprising thin-film, lamellar (multilayer) produce vivid structural colors by constructive interference of reflectors, or diffraction gratings in the cuticle of the light scattered by mesoscale (100−350 nm) biophotonic 2−4,7 fl 1−4 integument. However, various butter ies (Lepidoptera: nanostructures that are diverse in form and optical function. Lycaenidae, Papilionidae),14,15 weevils (Coleoptera: Curculio- Arthropods, in particular, are the most abundant, 7 7 nidae), longhorn (Coleoptera: Cerambycidae), diverse, and colorful on Earth. Arthropod structural (: ),16 jumping spiders (Araneomorphae: colors function in a variety of contexts including social and 17 sexual signaling, camouflage, and warning or aposematic Salticidae), and tarantulas (Mygalomorphae: Theraphosi- − 18 communication.5 8 A burgeoning number of studies demon- dae) (see Supporting Information Table S1 for a full list of strate that arthropods are an excellent source of biological inspiration for emerging technologies, such as in sensing, and Received: January 18, 2015 − − photonics2 4,7,9 13 (also see Supporting Information Table S1 Revised: April 30, 2015 for list of references). Published: May 4, 2015

© 2015 American Chemical Society 3735 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742 Nano Letters Letter

Figure 1. Representative morphology of structural color producing arthropod cuticular nanostructures. (a−f) Light micrographs, (g−l) electron micrographs, and (m−r) representative 2D SAXS patterns from the photonic scales or setae of the following: (a,g,m) Platyaspistes venustus (), single gyroid (I4132) (see Supporting Information Figure S1.105); (b,h,n) Eupholus quintaenia (Curculionidae), single diamond (Fd3m̅) + face-centered orthorhombic (Fddd) (see Supporting Information Figures S1.41 and S1.42); (c,i,o) Sternotomis pulchra (Cerambycidae), simple cubic or single primitive (Pm3m̅) (see Supporting Information Figure S1.130); (d,j,p) Anoplophora graafi (Cerambycidae), quasi-ordered + fcc opal (see Figure 2d and Supporting Information Figures S1.123 and S1.124); (e,k,q) 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, and Supporting Information S1.139). False color SAXS patterns (unmasked) depict the logarithm of scattering intensity as a function of the scattering wave 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 of permitted Bragg reflections for the assigned space groups. Crystallographic motifs seen in the SEM images are identified in panels g−i. Panel k and its 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. references) have structurally colored integumentary scales or addition to one lycaenid butterfly (Lepidoptera: Lycaenidae) setae on their wings, elytra, abdomen, and legs. These and a fly (Diptera: Asiloidea: Bombyliidae) (Supporting trichogen-based scales and setae possess a variety of complex, Information Table S1). three-dimensional biophotonic nanostructures composed of the polysaccharide chitin, cuticular proteins, and air (reviewed in ■ RESULTS − refs 2 4, 7, and 15). Despite the growing body of research on We find no evidence of a coherently scattering nanostructure arthropod structural colors (Supporting Information Table S1), present in 19 out of the 140 distinctly colored integumentary we currently lack accurate structural characterizations of more patches (Supporting Information Figure S1 and Table S1). The than a few species. Although knowledge of the development of 14,19−21 SAXS patterns from the rest of the arthropod scales and setae biophotonic crystals in arthropods is growing, we still examined ranged from a large number of discrete Bragg spots in lack a comparative developmental framework to understand the the azimuthal directions (Figure 1m−o,q and Supporting rich biodiversity of arthropod cuticular biophotonic nanostruc- Information Figure S1), characteristic of a polycrystalline tures (discussed in refs 14 and 15) or how these biological nanostructure, to a series of concentric powder-like (or signals function and evolve in these organisms. These complex Debye−Scherrer) diffraction rings (Figure 1p,r and Supporting nanostructures are too large to be developing via classical Information Figure S1), characteristic of an amorphous or morphogenesis as seen in molecular/cell biology, and much too quasi-ordered nanostructure with only short-range isotropic small to be understood using a multicellular developmental order.14,27,28 The presence of a large number of sharp, higher- 14 biology framework. They are also of broader biomimetic order diffraction spots up to 8 or 9 orders from some weevil interest, because synthetic fabrication of three-dimensional and longhorn scales (Figures 1m−o, 2a−c, and photonic nanostructures at these rather large optical length Supporting Information Figure S1) underscores a high degree 22−25 scales remains challenging. of spatial order for a biological system. However, like SAXS Recently, we applied synchrotron small-angle X-ray scatter- experiments on synthetic soft materials,27 40 of the 121 cases ing (SAXS) to identify single gyroid (I4132) photonic crystals exhibited SAXS spectra with variable or ambiguous features that 14 in wing scales of five well-studied butterflies from two families were intermediate between the idealized predictions of and single diamond (Fd3m̅) photonic crystals in the cover crystalline structures. This could be due to both variation in scales of a fossil weevil Hypera diversipunctata.26 Here, we apply length scale of peak structural correlations due to finite long- SAXS to structurally characterize the biophotonic nanostruc- range ordering, which may broaden and flatten diagnostic tures present within scales and setae from 140 distinctly colored spectral peaks, or owing to variation in nanostructure between integumentary patches of diverse land arthropod taxa with different scales of the same species or even different domains prominent structural coloration. We supplemented our SAXS withinasinglespecies.Forthesecases,wepresenta assays with electron microscopy (EM) for 44 out of the 140 nanostructural diagnosis based on the predominant SAXS patches. Our sample includes 129 different color patches from spectra distribution, but we recognize these tentative diagnoses ∼98 species in 75 genera belonging to 9 families of beetles with an * in Table S1 (also see Supporting Information). (Coleoptera), single patches from 3 species in 2 genera of bees In the following, we present our SAXS nanostructural (Hymenoptera: Apidae: ), single patches from 6 species diagnoses based on assigning the symmetry that is most in 6 genera belonging to 2 families of spiders (Araneae), in consistent with the indexed Bragg peaks in the azimuthal

3736 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742 Nano Letters Letter

Figure 2. Structural diagnoses of representative SAXS profiles of arthropod cuticular photonic nanostructures. The normalized, azimuthally averaged SAXS 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 (Fd3m̅) (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 (Pm3m̅) (see Supporting Information Figure S1.132), (d) Anoplophora graafi, quasi-ordered + fcc opal, (see Figure 1d,j,p and Supporting Information Figures S1.123 and S1.124), (e) A. birmanica, bcc spheres (see Supporting Information Figure S1.119), (f) A. versteegi, quasi-ordered spheres (see Supporting Information Figure S1.122). Apidae: (g) Thyreus pictus, sponge (see Supporting Information Figure S1.138), (h) T. nitidulus, inverse 2D twisted columnar (see Figure 1f,l,r, and Supporting Information Figure S1.139), (i) Amegilla cingulata, inverse 2D hexagonal columnar (see Figure 1e,k,q and Supporting Information Figure S1.137), (j) Porod (q−4) background. The colored vertical lines correspond to the expected Bragg peak positional ratios for various alternative crystallographic space groups, presented together for direct comparison. The numbers above the vertical lines are squares of the moduli of the Miller indices (hkl) for the allowed reflections of specific space-groups. The normalized positional ratios of the scattering peaks are indexed to the predictions of specific crystallographic space groups or symmetries according to IUCr conventions.29 profiles according to IUCr conventions,29 complemented with photonic crystals are so far known in the wing scales of only real-space information from electron microscopy for a third of four other genera within lycaenid and papilionid butter- the assays. The diversity of arthropod cuticular nanostructures flies.14,15,30,31 Single diamond (Fd3̅m) and sheared, face- is best summarized taxonomically because different lineages centered orthorhombic (Fddd32,33) networks were also found have evolved distinct classes of biophotonic nanostructures only in snout weevils (e.g., Eupholus quintaenia; Figure 1b,h,n (Figures 1−3 and Supporting Information Figure S1 and Table and Supporting Information Figure S1.42). In Lamprocyphus S1). Triply periodic, bicontinuous networks were relatively weevils (Supporting Information Figures S1.72−80 and Table restricted in their distribution. Single gyroid (I4132) networks S1) for instance, both single diamond and single gyroid occurred only in photonic scales of snout weevils (Curculio- photonic crystals were identified on the same individual and nidae; e.g., Platyaspistes venustus, Figure 1a,g,m) and in wing sometimes within different domains in the same scale. scales of the Lycaenid butterfly Lycaena kasyapa (Lycaenidae; Spongelike or quasi-ordered versions of single diamond Supporting Information Figure S1.1); However, single gyroid networks with varying degrees of disorder were also present

3737 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742 Nano Letters Letter

Figure 3. A biophysical framework for classifying the diversity of arthropod cuticular photonic nanostructures. (a) A schematic of the lyotropic phase behavior depicting the well-known equilibrium inverse lipid phases in order of increasingly positive Gaussian curvature (see ref 51), starting with the fl at lamellar phase (Lα), whose interfacial mean and Gaussian curvature is trivially zero. These include the inverse bicontinuous cubic phases (QII: double gyroid Ia3d̅, double diamond Pn3m̅, and double primitive Im3m̅), perforated lamellae (L3), inverse hexagonal columnar cylinders (HII) as well as inverse micelles or vesicles that can be jammed or close-packed into cubic (III) or amorphous (L2) arrays. (b) Topological decomposition of lyotropic 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 interfacial fi mean (H) and Gaussian or saddle-splay curvature (KG). Note the rst three are minimal surfaces. (c) The diversity and taxonomic distribution of arthropod cuticular photonic nanostructures. in weevils, sometimes also within the same individual (e.g., nanostructure of the Prosopocera lactator was Eupholus bennetti; Supporting Information Figures S1.39 and diagnosed as a bcc (Im3m̅) network of connected spheres (cf ref S1.40 and Table S1). 34). Interestingly, these appear to be structurally intermediate By contrast, the photonic scales of longhorn beetles between the close-packed sphere nanostructures of other (Coleoptera: Cerambycidae) possessed nanostructures essen- longhorn beetles and bicontinuous network nanostructures of tially based on ball-and-stick arrangements of chitin spheres. Sternotomine longhorns. One class of nanostructure found in the photonic scales of Quasi-ordered arrays of chitin spheres were also identified longhorn beetles consisted of ordered (face-centered cubic, fcc; within scales of two basal taxa of beetles, Toxonotus sp. body-centered cubic, bcc) and quasi-ordered close-packing of (Coleoptera: Curculionoidea: ) and Isacantha sp. discrete chitin spheres sometimes connected by thin necks (Coleoptera: Curculionoidea: ). (Coleoptera: Cerambycidae; e.g., Anoplophora graafi; Figures A two-dimensional, inverse hexagonal columnar morphology 1d,j,p and 2d and Supporting Information Figures S1.123− (i.e., air pores in chitin) was diagnosed in the iridescent setae of 124). Single primitive or simple cubic (Pm3m̅) triply periodic a digger bee, Amegilla cingulata (Hymenoptera: Apidae: bicontinuous networks as well as quasi-ordered or sponge-like Anthophorini, Figures 1e,k,q and 2i)16 and in a jumping spider versions of the same were present only in the Sternotomini (Araneae: Salticidae, Supporting Information Figure S1.141). A tribe of longhorn beetles (Coleoptera: Cerambycidae; e.g., 2D, Bouligand-like,35 twisted inverse columnar morphology was Sternotomis pulchra bifasciata, Figure 1c,i,o and Supporting identified in setae of a cuckoo bee, Thyreus nitidulus Information Figures S1.130−131). The scale photonic (Hymenoptera: Apidae: , Figures 1e,k,q and 2i, and

3738 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742 Nano Letters Letter

Supporting Information Figure S1.139), and in scales of Hoplia The self-assembly of biological lipid bilayer membranes into scarab beetles (Coleoptera: Scarabaeidae, Supporting Informa- lyotropic morphologies is hypothesized to be regulated by the tion Figures S1.3−4).36 energetics of membrane curvature.50,63,67 Living cells control Amorphous or quasi-ordered, spongelike networks were the curvature and shape of their membranes with membrane- diagnosed in the photonic setae of another species of the same binding proteins that vary in shape, molecular weight, and − of cuckoo bee, T. pictus (Supporting Information Figure electrostatic properties.52,63 65,67,68 We hypothesize that multi- S1.138). Lastly, the photonic nanostructures in purple-blue ple lineages of arthropods have independently evolved to utilize setae of tarantulas (Araneae: Theraphosidae, Supporting the intrinsic capacity of membrane-bound organelles to self- Information Figures S1.142−145) were characterized as assemble into lyotropic phases to template the precursors of perforated lamellae, as in many butterflies.15,18,20,37,38 color producing nanostructures. Specifically, we posit that the The SAXS structural data also confirm that the arthropod controlled expression of membrane-binding proteins with cuticular nanostructures are sufficiently ordered at the different bilayer bending (κ) and Gaussian or saddle-splay (κ)̅ appropriate length scales to produce the observed colors via moduli51,52 in arthropod scales and setae cells could similarly constructive interference (Supporting Information Figure S2). facilitate the development of various lyotropic precursor We have thus assayed the cuticular scale and setae templates of biophotonic nanostructures.50,61,65,66,68 It is also nanostructure of ∼127 species of land arthropods in 85 genera likely that the curvature of these biological nanostructures are belonging to 4 orders of insects and two suborders of spiders stabilized by intermembrane binding proteins.68 using SAXS and EM, including ∼66 genera that have not to our Interestingly, each arthropod lineage studied has evolved to knowledge been previously investigated. The analysis has occupy different portions of the total lyotropic phase space resolved uncertainties in the diagnoses of the biophotonic (Figure 3c). For instance, many different arthropod families nanostructure in previous studies (Supporting Information exhibit quasi-ordered sponge-like networks or perforated Table S1). Compared to birds,28,39,40 lizards and frogs,41 lamellae. But single gyroid and single diamond nanostructures cephalopods and other aquatic animals,13,42 terrestrial arthro- are found only in curculionid weevils, and lycaenid and pods, especially insects, exhibit a breath-taking array of papilionid butterflies. Likewise, quasi-ordered and ordered biophotonic nanostructures within scales and setae. close-packings of chitin spheres are found only in longhorn Intriguingly, the diversity of arthropod photonic nanostruc- beetles and in two basal families of weevils. Thus, it appears tures documented here exhibits the very same, rich poly- likely that within each lineage the molecular structure of the morphism found in amphiphilic macromolecules, such as block membrane-binding proteins has evolved to express only a − copolymers,43 46 surfactants,47 and lipids.48,49 Biological lipid limited range of the total possible physical effects on membrane − bilayer membranes are known to self-assemble in aqueous curvature and stability.50 52 The plausibility of this model is media into a variety of inverse or type-II lyotropic liquid supported by the existence of the highly conserved superfamily crystalline phases or morphologies50,51 (Figure 3a). In addition of BAR-domain proteins, which have been shown to affect and to their crystallographic space group symmetries, these self- stabilize membrane curvature in endocytic spherical or tubular assembled membrane phases can be classified based on the invaginations,67,69 for example, in three-dimensional, cubic − variation in their interfacial curvature.48,50 52 The fundamental membrane (t-tubule) networks in striated skeletal muscle geometry of these lyotropic phases can then be topologically cells.58,70 This model suggests a likely biophysical framework classified into sphere, cylinder, or the associate (Bonnet) for understanding the developmental basis and biodiversity of families of lamellar-helicoid (Riemann’s) and saddle (Schwarz’s arthropod photonic nanostructures (Figure 3). The distinctly D, P, and Schoen’s G) surfaces. All of these surfaces are nonrandom distribution of the cuticular biophotonic nano- characterized by a constant mean curvature (H =(c1 +c2)/2) structures in arthropods also implies apomorphic (lineage- but they can be differentiated on the basis of their Gaussian or specific) differences in membrane-folding mechanisms (see saddle-splay curvature (KG = c1c2, where c1 and c2 are the below). principal curvatures along orthogonal planes perpendicular to In butterflies, the single gyroid photonic crystals in wing scale the surface)53,54 (Figure 3b). The Gaussian curvature of a cells have been hypothesized to develop from a core−shell sphere is positive, zero for cylinders and planes, and negative double gyroid (Ia3d̅) template made by tandem infolding of the for a saddle. Furthermore, lamellar, lamellar-helicoid, and smooth endoplasmic reticulum (SER) and plasma mem- − saddle morphologies are examples of minimal surfaces, that is, branes,14,19 21 as in core−shell morphologies seen in triblock they have zero mean curvature. Even in a quasi-ordered sponge copolymer systems.71,72 After development of the core−shell or a perforated lamellar morphology, the pores may be thought double gyroid template, chitin is deposited into the extracellular of as a Riemann’s minimal surface with helicoid-like bridges space, which is continuous with one of the two single gyroid connecting adjacent, asymptotic, and parallel (lamellar) cores. Upon maturation, the rest of the scale cell dries up and 55−57 planes (Figure 3b). dies, leaving behind a single gyroid (I4132) network comprising The similarity of arthropod cuticular nanostructures to chitin and air of the appropriate size to constructively reflect a amphiphilic block copolymer, surfactant, and lyotropic lipid specific visible color.14 Double gyroids can be directly self- phase states is not merely a structural or geometrical analogy. assembled, unlike single gyroids (which have superior optical Biological lyotropic lipid membrane morphologies, including properties73). Therefore, butterflies have evolved to self- the triply periodic, bicontinuous cubic phases (double gyroid, assemble a double gyroid template from the interactions of double diamond, and double primitive morphologies) are well- plasma and SER membranes, but use only one of the two single − known in membrane-bound organelles of living cells.57 66 gyroid compartments to bioengineer the final optical However, these arthropod biophotonic nanostructures are nanostructure.14 much larger in size (lattice parameters of 50−500 nm)50,61,65 The development of biophotonic nanostructure within scale − than those produced by typical lipid−water systems (≤20 cells has been investigated only in a few butterflies.19 21,74 nm).51 Nevertheless, given the homology of the trichogen (shaft-

3739 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742 Nano Letters Letter forming cells) across all arthropods,75 the development of photonic nanostructures in scales and setae of other insects and spiders likely proceeds via similar membrane-folding mecha- nisms. However, the diversity of optical nanostructures observed in arthropod integument requires recognition of two broad classes of biophysical developmental mechanisms. The first class can lead to self-assembly starting from a single lipid 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 within scale cells from the infolding of the plasma membrane into a lyotropic template, followed by extracellular chitin deposition and the maturation of the cell. By contrast, the second class leads to the self-assembly of single gyroid and single diamond nanostructures of snout weevils from double gyroid or double diamond precursor templates. This process requires the collaborative infolding of both the plasma membrane and SER, as observed in lycaenid butterflies.14,21 Figure 4. Self-assembly of arthropod cuticular photonic nanostruc- tures, by analogy to microphase separation in block copolymers. (a) As These developmental hypotheses are further supported by fl 14 in butter ies, single gyroid (I4132) (illustrated) and single diamond the observation that single gyroid and single diamond networks ̅ fl 14 fi (Fd3m) scale nanostructures in snout weevils likely develop via in butter y and weevil scales have relatively low chitin lling tandem infolding of parallel plasma and SER bilayer membranes into a fractions (mean 0.2; Supporting Information Table S1). The precursor, core−shell double gyroid (Ia3d̅) (illustrated) or double low volume of chitin results from the fact that the final cuticular diamond (Pn3m̅) template within the perimeter of the scale cell. This nanostructure consists of only one of a pair of chiral is akin to a linear ABC triblock copolymer, which is compositionally interpenetrating networks. The similarity of weevil scale asymmetrical about the midplane (ABCB′A′). (b) Scale and setae development to that of butterflies is further supported by the nanostructures in longhorn beetles, bees, and spiders likely develop by presence of one or more submicron vesicles in weevil scales the invagination of the plasma membrane only, similar to an AB diblock copolymer that is compositionally asymmetrical about the (e.g., see Supporting Information Figure S1.44) that appear to ′ be vestiges of SER fragments hypothesized to play a role in midplane (ABA /ABC) to produce nanostructures with the observed 21 ∼50% volume fractions (as illustrated for the Pm3m̅ nanostructure). In organizing the parallel membranes. On the other hand, the both panels a and b, the extracellularly synthesized biopolymer chitin is biophotonic nanostructures in longhorn beetles and bees have fi deposited into only one of the interpenetrating volumes (A), which is signi cantly higher (unpaired t = 7.6, df = 41, P < 0.0001) continuous with extracellular space, and the subsequent desiccation chitin filling fractions (mean 0.48; Supporting Information and degeneration of the rest of the dying cell results in the final Table S1). Therefore, unlike butterflies or weevils, these larger photonic nanostructure (middle and right columns). The biological chitin volume fractions eliminate the physical space for the components of the developing scale or setae cell are shown color- parallel infolding of a second lipid bilayer within the developing coded to the different monomer blocks of a hypothetical linear ′ ′ ′ scale cells. Interestingly, like other longhorn beetles, the single noncentrosymmetric ABCB A or ABA /ABC block copolymer primitive or simple cubic triply periodic bicontinuous networks (leftmost column). of Sternotomis longhorn beetles also have high chitin filling fractions (0.4, on average; Supporting Information Table S1), or ABC (Figure 4b) to produce nanostructures closer to 50% which would prevent the infolding of a second parallel bilayer volume fractions. In both cases, chitin, which is an extracellular membrane during their development. So, they appear to share a polymer, is then deposited into the extracellular space (A), and common developmental mechanism with other longhorn the subsequent desiccation and degeneration of the rest of the beetles. This underscores the phylogenetic constraints on the dying cell results in the final photonic nanostructure. Future development and evolution of biophotonic nanostructures. observations and experiments on the development of scale and We illustrate these possible developmental scenarios by setae nanostructures in weevils, longhorn beetles, and other analogy to the microphase separation of block copolymers arthropods are necessary to test these hypotheses. (Figure 4). As in butterflies,14 we posit that single gyroid and Cellular control of the physical mechanisms of lipid bilayer single diamond nanostructures in snout weevil scales develop self-assembly has likely enabled many arthropod lineages to via the tandem infolding of parallel plasma and SER bilayer evolutionarily explore most of the phase space of lyotropic or membranes, akin to a linear ABC triblock copolymer that is amphiphilic materials for a photonic function at optical length compositionally asymmetrical about the midplane, that is, scales not easily achieved in synthetic soft matter systems.45,51 ABCB′A′ (Figure 4a).76 A core−shell double gyroid (Ia3d̅)ora The repeated evolutionary co-option of the biological double diamond (Pn3m̅) precursor thus self-assembled is membrane self-assembly provides a novel, generalized explan- transformed into a single gyroid or single diamond by ation for the explosive diversity of photonic nanostructures in backfilling only one of the core volumes (which is continuous arthropod scale and setae cells. Future experiments should with the extra-cellular space) with chitin. By contrast, we focus on identifying putative proteins that control membrane propose that scale or setae nanostructures in longhorn beetles, curvature and invagination within scale or setae cells during bees, and spiders develop only by the invagination of the development. plasma membrane, similar to an AB diblock copolymer that is These diverse arthropod biophotonic scales may offer compositionally asymmetrical about the midplane, that is, ABA′ convenient biotemplates to serve in functional applications

3740 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742 Nano Letters Letter such as sensing10 and photonics77 (after appropriate dielectric Hope Entomological Collections (James Hogan, Ray Gabriel, infiltration). At the same time, protein-mediated membrane and Darren Mann), Smithsonian U.S. National Museum self-assembly of synthetic bilayer membranes,68 and the phase Entomology Collections (Steve Lingafelter), and CSIRO behavior of a linear, noncentrosymmetric pentablock copoly- Australian National Arthropod Collection (ANIC). We thank mer obtained by the mixing of a ternary triblock and a binary Nick Terrill and Tobias Richter for help with SAXS data diblock copolymer with suitably optimized molecular weights collection at beamline I22 of the Diamond Light Source that to maximize the lattice parameters76 are two perhaps promising contributed to some of the results presented here, as well as bioinspired (if not biomimetic) approaches to synthesizing Stephen Mudie, Sarah Weisman, and Tara Sutherland for tunable mesophases with large lattice parameters, including comments and support regarding specimen preparation for morphologies like single gyroid and single diamond currently SAXS at the Australian Synchrotron. not accessible via direct synthetic self-assembly.24 ■ ASSOCIATED CONTENT ■ REFERENCES *S Supporting Information (1) Fox, D. L. Biochromes and Structural Colors; University of Detailed SAXS structural diagnoses, SAXS optical reflectance California Press: Berkeley, CA, 1976. 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3742 DOI: 10.1021/acs.nanolett.5b00201 Nano Lett. 2015, 15, 3735−3742