Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane

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Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane Incorporating a Low-Adhesion, Nonwetting, Superhydrophobic, Bactericidal, and Biocompatible Surface Gregory S. Watson,*,†,△ David W. Green,*,‡ Bronwen W. Cribb,§ Christopher L. Brown,∥ Christopher R. Meritt,∥ Mark J. Tobin,⊥ Jitraporn Vongsvivut,⊥ Mingxia Sun,# Ai-Ping Liang,# and Jolanta A. Watson† †

Faculty of Science, Health, Education and Engineering, University of the Sunshine Coast, Maroochydore DC, Queensland 4558, Australia ‡ Department of Oral Biosciences, Faculty of Dentistry, University of Hong Kong, The Prince Philip Dental Hospital, 34 Hospital Road, Sai Ying Pun, Hong Kong SAR, China § Centre for Microscopy & Microanalysis and School of Integrative Biology, The University of Queensland, Saint Lucia, Queensland 4072, Australia ∥ Queensland Micro & Nanotechnology Center, Griffith University, Brisbane, Queensland 4111, Australia ⊥ Infrared Microspectroscopy beamline, Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia # Key Laboratory of Zoological Systematics and Evolution, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China △ Department of Oral Biology, Yonsei University College of Dentistry, 250 Seongsanno, Seodaemun-gu, Seoul 120-752, Korea S Supporting Information *

ABSTRACT: Nature has produced many intriguing and spectacular surfaces at the micro- and nanoscales. These small surface decorations act for a singular or, in most cases, a range of functions. The minute landscape found on the lotus leaf is one such example, displaying antiwetting behavior and low adhesion with foreign particulate matter. Indeed the lotus leaf has often been considered the “benchmark” for such properties. One could expect that there are animal counterparts of this self-drying and self-cleaning surface system. In this study, we show that the planthopper insect wing (Desudaba danae) exhibits a remarkable architectural similarity to the lotus leaf surface. Not only does the wing demonstrate a topographical likeness, but some surface properties are also expressed, such as nonwetting behavior and low adhering forces with contaminants. In addition, the insect-wing cuticle exhibits an antibacterial property in which Gram-negative bacteria (Porphyromonas gingivalis) are killed over many consecutive waves of attacks over 7 days. In contrast, eukaryote cell associations, upon contact with the insect membrane, lead to a formation of integrated cell sheets (e.g., among human stem cells (SHED-MSC) and human dermal fibroblasts (HDF)). The multifunctional features of the insect membrane provide a potential natural template for manmade applications in which specific control of liquid, solid, and biological contacts is desired and required. Moreover, the planthopper wing cuticle provides a “new” natural surface with which numerous interfacial properties can be explored for a range of comparative studies with both natural and man-made materials. KEYWORDS: insect, lotus leaf, antiwetting, self-cleaning, superhydrophobic, bactericidal, biocompatible

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INTRODUCTION

numerous studies in relation to a range of properties (for example, antiwetting), the functions and functional efficiencies on countless other species and taxonomic groups have not been investigated. The multifunctional character of these natural surfaces, along with limited knowledge of the habits and behaviors of many organisms, adds to the complexity in

The countless living solutions at surfaces and interfaces programmed by natural selection offer scientists with a diverse library of micro- and nanostructures to study. These can be utilized as templates with which to enhance existing technologies and the performance of materials.1−3 Such “free” architectural adaptations are of specific interest for the design of surfaces because the delicate control of interfacial properties is a key aspect in many current and emerging industries. While the epidermal topography of many organisms has been examined in © 2017 American Chemical Society

Received: June 12, 2017 Accepted: June 22, 2017 Published: June 22, 2017 24381

DOI: 10.1021/acsami.7b08368 ACS Appl. Mater. Interfaces 2017, 9, 24381−24392

Research Article

ACS Applied Materials & Interfaces

Figure 1. Digital photographs of the Australian native lotus plant, Nelumbo nucifera, and the planthopper, Desudaba danae (a) Lotus leaf in its natural environment along the banks of the Ross River showing the typical orientation of leaves; (b) higher-resolution optical image showing the edge of a lotus leaf resting on the water; and (c) higher-resolution image of water droplets resting on the lotus leaf, demonstrating the superhydrophobic interaction with water with a contact angle exceeding 150°. This interaction is a consequence of the hydrophobic chemistry and the fine micro- and nanostructure of the leaf surface shown in Figure 2. (d, e) Digital photographs of the Australian and Northern Queensland native planthopper, D. danae, resting on the branch of the plant Planchonia careya. (f) Planthopper hindwing (left) and forewing (dorsal side; middle image, ventral side image on right). (g, h) Higher-magnification images of regions on the dorsal section of the forewing showing the pigmentation and vein structure at these scales.

determining all of the functions of these intriguing and potentially informative natural templates and materials. The natural lotus leaf surface has been intensively studied (Nelumbo sp.).4−11 This interface demonstrates a range of properties that afford high functional efficiency in cleanliness. This evolutionarily refined architecture presumably enhances the plant’s survival maintaining an upper surface largely free from solid and liquid contaminants, which can potentially interfere with plant functioning such as photosynthesis. The quintessential qualities of the lotus leaf, together with an indepth understanding of these features, makes it a common reference point for comparisons between natural and artificial self-cleaning surfaces. Indeed, the lotus leaf in many situations can be viewed as the “gold standard” for numerous surface features, particularly antiwetting and self-cleaning, e.g., refs 1, 3, and 9. Because the lotus leaf micro- and nanostructures represent a highly optimized natural surface adapted to its physical environment, from an evolutionary point of view, we could expect convergent adaptations in animals inhabiting similar environments. Insects in particular embody a likely pool of candidates because they represent such a diverse and vast number of specimens (over 950 000). The micro- and nanostructuring found thus far on such insects has proven to be astonishingly diverse and, for the majority of species, represents still-uncharted territory. However, based on existing studies, this complexity can be simplified into seven basic

categories composed of (1) simple microstructures, dome-like or pillars; (2) simple nanostructures, dome-like or pillars; (3) complex geometric microstructures (varied shape and form); (4) complex geometric nanostructures, varied shape and form; (5) scales; (6) hairs and setae; and (7) hierarchical structuring of categories 1−6.12 An intriguing group of small flying insects are planthoppers (Fulgoromorpha), which live in a diverse and, at times, hostile environment. Like many mobile insects, which live close to the ground, contact with liquid and solid surfaces and particles is unavoidable; for example, water droplets from rain, condensation, contaminants such as plant material, soil particles, micro-organisms (e.g., bacteria), and excrement from insects and other organisms. Continuous exposure of these environmental contaminants can potentially inhibit or degrade the functioning of the cuticle as a protective mechanical barrier. For example, as the growth of many micro-organisms is enhanced by increased water availability, attachment and proliferation may result from wetting property changes, which may necessitate some form of self-cleaning. In this study, we have examined the surface topographies of a small insect (a species of planthopper, Desudaba danae; Gerstaecker, 1895)13 and investigated their properties.13 This species was collected within 50 m of where Australian native lotus plants, Nelumbo nucifera, were growing. Thus, both species experienced similar environmental conditions, such as rainfall and types and frequency of exposure to airborne contaminants. Interestingly, D. danae possesses a topography at 24382


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ACS Applied Materials & Interfaces various regions of the wing cuticle membrane, which closely resembles the lotus leaf surface structures. This was the impetus for a comparative examination of topography and properties. Because the lotus leaf has previously demonstrated low adhesion with water and solid contaminants, we carried out an analogous evaluation of the planthopper cuticle, specifically on the insect wing cuticle membrane. Surface topography has also been associated with control of living cells based on its relation with extracellular matrix surface topographies or surface features. In this regard, we investigated interactions between the insect cuticle and bacteria and eukaryotic cells.

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RESULTS AND DISCUSSION Characterization of the Planthopper Wing Membrane (Topography and Chemistry). At first glance, at the macro level, the planthopper cuticle and lotus leaf bear little resemblance to each other (Figure 1). At the level of several millimeters in scale, the insect forewing and hindwing display a membrane with distinct regions characterized by a series of interconnecting veins and also separate colors and shades (Figure 1d−h). Color variation on insect wings has been speculated to serve a number of possible functions from communication to camouflage.14 The most brightly colored orange and red regions of the wings are located on the hindwing (similar in color to the insect abdomen; Figure 1e). The location of these brightly colored areas on the wings (basal region on the hindwing) may suggest that the insect has some control over whether to display this color to the outside world and thus may be associated with a specific time or activity in the insect life cycle. The spacing of veins on the forewing in particular can be quite small, in the several hundred micron range (Figure 1f,g). In contrast, the lotus leaf presents a surface with a typical green color from chlorophyll with a surface roughness certainly visible at the millimeter scale (Figure 1b,c). However, when we image at medium and high resolutions at the micro- and nanostructures on the lotus and insect surfaces, we immediately see stark similarities (Figure 2). The lotus leaf is covered with small papillae (microasperities), which, although they display a general random arrangement, are typically separated by a distance of several microns (greater than the width of the structures) and are slightly tapered, as shown in Figure 2a,b. Table 1 shows the average spacing, height, and width of the papillae. Similar-type microstructures have also been described for other plant species.4,7 The surfaces of the microprojections are also decorated at the nanoscale with tubule-like structures (Figure 2b−e). These smaller projections present a homogeneous dense carpet on the surface and appear to be randomly oriented, with some lying almost flat on the surface plane, while others are projecting orthogonal to the underlying surface. The tubules also appear to be fairly flat and angular at the apex rather than curved. Similar-type nanostructures have also been described for other plant species.4,7 The corresponding images of the planthopper hindwing regions of the membrane (Figure 2e−h) show a remarkably similar topographical configuration to the lotus architecture with a dual-scale landscape consisting of microprojections, which are furnished with smaller nano-protuberances. Table 1 shows that the two architectures on the plant and insect have comparative dimensions and spacing. The planthopper microstructuring is spaced slightly further apart and thus not as densely packed and are typically smaller than the lotus in height and width (Table 1). The nanostructuring on the insect is

Figure 2. Comparative scanning electron microscopy images of the lotus leaf (a−d) and planthopper hindwing (e−h) at various magnifications, illustrating the similar hierarchical organization, topographical features, and dimensions. (i) The topographical structuring on the forewing of the planthopper, again displaying a hierarchical architecture (dual layer) but without the well-defined microscale tapered projections. The highlighted regions show the upper-tier topography exhibiting isolated clumps protruding from the surface. See also Figure S1, which shows a larger field of view of panel i and a more-detailed wider field of view to highlight the heterogeneous and homogeneous aspects of the surface architecture.

around half the width and diameter, of similar length (Table 1), and typically more directional in orientation (i.e., a higher percentage project outward from the surface plane; see Figure 2f,h). The larger topographical features (microasperities) on the hindwing may potentially protect the inner nanotopography of that membrane from abrasion. The planthopper forewing interestingly did not display the same topography as the hindwing, as can be seen in Figure 2i (see also Figure S1). The wing does, however, still display two levels of roughness as seen by the grouped structures (isolated clumps) rising higher from the surface and also the smaller nanoscale roughness of various dimensions. The well-formed microstructuring in the form of distinct tapered microasperities, as seen on the lotus leaf and hindwing is prominently absent. Why the structuring differs on the two wing types is unclear at present; however, some studies have shown that function specific structuring can vary between wings and indeed on occasions on the same wing where topographical features of different scales is observed.15 It may be that the forewing structuring is more resilient to wear or economical in fabrication. While the surface properties (e.g., adhesion and wetting) of the lotus leaf and planthopper wing will be significantly influenced by the topographical landscape and scaling (Table 1), the chemical nature of the surfaces will also make a substantial contribution to such properties. To gain insights into surface chemistry of the insect wings, attenuated total reflection Fourier transform infrared (ATR-FTIR) measurements using a laboratory-based FTIR spectrometer and a 24383


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Table 1. Comparison of Dimensions and Spacing of the Micro- and Nanostructuring on the Lotus Leaf and the Planthopper Hindwing Membrane (e.g., Spacing, Height and Length, Width of Micron, and Nanometer Features)a planthopper lotus

height (μm)

basal width (μm)

spacing (μm)

density (no./10000 μm2)

length (nm)b

diameter/width (nm)b

6.5 ± 1.6 10.4 ± 0.8

3.8 ± 0.88 8.0 ± 2.4

14.4 ± 6.8 19.5 ± 12.5

57 35

520 ± 310 530 ± 150

47 ± 11 100 ± 30

a

Measurements of micron-scaled features have associated length scales of micrometers in the table. The smaller nanoscale topography has the units of nanometers assigned to them. bRefers to the nanometer size structuring (wax tubules of the lotus and nanoprotuberances of the planthopper wing).

particulates has been shown to contribute to various diseases including lung cancer.18 Pollen grains are typically one of the most common component among the floating particles in air (aeroplankton) surrounding most terrestrial animals and plants.19 Other major atmospheric particulates can originate from soils, such as naturally occurring silica particles, which can comprise as much as 95% of the sand and silt fraction of soil.20 Silica dust has not only been linked to lung disease but also as a contributor to silicosis, cancer, pulmonary tuberculosis, emphysema, and immunologic reactions.21,22 Thus, the interaction of these particles (silica particles and pollen grains and fragments) with various surfaces is of great interest in terms of distribution, transport, and capture. The planthopper wing architecture, with its dual lotus-like hierarchy, provides an interesting natural template with which to examine such contacts with different microsized contaminants. We have measured the adhesion of contaminating particles (silica particles of two different sizes and a species of pollen) on the planthopper hindwing (Figure 3). For comparison, we have also made similar interfacial contacts with one other insect wing membrane, a termite wing (Schedorhinotermes sp.), as well as a

synchrotron-based FTIR microscope were obtained (see Figure S2). As shown, the ATR-FTIR spectral features in the highwavenumber region revealed a broad O−H stretching band at ∼3300 cm−1, and the characteristic C−H stretching modes in forms of triplet bands were observed within 3000−2840 cm−1 range. Specifically, the bands centered at 2965 and 2875 and at 2925 and 2852 cm−1 are assigned to the asymmetric and symmetric ν(C−H) stretching vibrations of methyl (−CH3) and methylene (−CH2) functional groups of lipids and proteins, respectively. The presence of these C−H stretching features together with the relative prevalence of the methylene bands compared with the methyl bands is indicative of longchain hydrocarbons, which are attributed to the epicuticular lipids presented on the surface of the wing membrane. Such epicuticular lipids, which are found mainly in forms of waxes, are in fact known for decreasing the wettability of the insect wings giving them an excellent hydrophobic property.16 However, the major peaks found in the low-wavenumber region included amide I at ∼1660 cm−1 (i.e., CO stretching coupled to N−H bending modes) and amide II at ∼1550 cm−1 (i.e., C−N stretching coupled to N−H bending vibration), both of which were attributed to chitin and proteins.16 In addition the ν(CO) stretching band of carbonyl esters is clearly identifiable at ∼1740 cm−1 for the regions examined. In comparison, the lotus leaf also has a wax-type outer chemistry with an absorption band at ∼2930 cm−1 due to CH stretching vibrations of CH, CH2, and CH3 groups.7 Recent analysis shows that the wax of the upper side of the lotus leaf contains ca. 65% of various nonacosanediols and around 22% of nonacosan-10-ol.9 It is noteworthy that while the waxes of lotus leaves exist as tubules, on other leaves, waxes can be found to exist in the form of platelets or other morphologies.17 It is not surprising that the insect and plant surfaces have adapted their outer layers topographically because both of their chemistries are at the upper end of what nature can fabricate to achieve a low-energy surface. To investigate this optimization of surface features, we have caused the planthopper surface to interact with liquid and solid contacts to evaluate fouling in a variety of contexts. Adhesion. The hierarchical (dual tier) and open intricate architecture of the planthopper, as shown in Figure 2, suggests that the surface structuring may facilitate minimal adhesion with particles of various length scales. In addition, small adhesion forces between contaminating particles and the topographical plant equivalent to the planthopper (i.e., lotus leaf) have been measured in comparison with a number of other plant leaf surfaces.5,6 The atmospheric environment surrounding insects and plants contains a complex mixture of natural particulate matter including dust, algae, fungal spores and hyphae, bacteria, and pollens, which can potentially contaminate the surfaces. Exposure to individual, or a combination of various, air-borne

Figure 3. (a) Adhesive values between various particles (silica beads of two different size scales and the Australian wattle pollen, Acacia f imbriata) interacting with the planthopper, termite (Schedorhinotermes sp.), and a silica surface. (b) Diagram illustrating the changes in the interfacial contact area with particles of different-length scales. 24384


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Figure 4. Images of water droplets interacting with the planthopper dorsal side of the hind wing. (a) Small droplets of water (∼10 μL) on the dorsal region of the hindwing. (a) Apparent static droplet showing contact angle with water. The droplet footprint covers all three color domains, as shown in Figure 1f (far left wing in image). (b) Advancing droplet. (c) Receding droplet. (c−f) Impacting droplet of 2.2 mm in diameter on the planthopper hindwing (dorsal region) (v = 0.60 m/s and We = 5.4). (e, f) Images of water droplets (advancing and receding droplet) interacting with the planthopper dorsal side of the forewing. (g) Time evolution of an impacting droplet of 2.2 mm in diameter on the planthopper forewing (dorsal region) (v = 0.40 m/s and We = 2.4 (see eq 2)).

flat hydrophilic silica surface (Figure 3). The two insect surfaces investigated represent a contrast in topography with the termite wing membrane exhibiting a comparatively flat surface and low contact angle (>150° versus 70−82° for the termite). We have previously investigated the micro structure and wetting and adhesion properties of this termite, which provides a good comparison with other insect species.23 The planthopper recorded the lowest adhesional values for all types of particles in comparison with the other two surfaces. The highest adhesional values for all particles were measured on the flattest and most hydrophilic of the surfaces (silica, contact angle of 35°). The adhesion between the silica microspheres and the insect cuticles represents a high-surface energy contaminant particle coming into contact with low-energy hydrophobic (planthopper) and a higher-energy hydrophilic surface (termite). This is highlighted in Figure 3, showing the differences in adhesion between the two insect species. The higher adhesion values measured between the hydrophilic contacting surfaces (hydrophilic termite insect membrane and silicon surface) with the hydrophilic contaminants (silica particles) may reflect the menisci formation from water present on the surfaces. In addition, the relatively flattened and broadened structuring of the hydrophilic insect (termite) does not minimize the contact area to the degree of the hydrophobic planthopper species. The high adhesion values measured on the termite suggests a higher risk of wing contamination, although this may not be a critical problem because the insect will only utilize the wings for relatively short time periods before complete shedding.23 The adhesion of the larger-diameter silica particles (∼35 μm) is higher than the smaller microbeads (∼7 μm) on all surfaces as one would predict simply based on the predicted increase in contact area; however, the relative difference on the planthopper membrane is significantly lower. This may be related to the dual topography of the planthopper, in which the microasperities restrict access to contact with the underlying layer on the wing membrane for larger-sized particles as well as reduce the contact area (see Figure 3b). The adhesion on the

planthopper wing is significantly lower than literature values for lotus leaves (>150 nN) with similar particle contacts; however, the contact conditions (applied force loadings prior to adhesion measurements) may be different for those studies.5,6 The wattle pollen grain (Acacia f imbriata) is of a similar size scale to the 35 μm silica bead and thus is useful for comparative purposes and as gauge for contact with other pollens. Due to the rougher morphology and more-hydrophobic nature of the long chain polymers that composes the pollen sporopollenin, e.g., ref 24, adhesion between the pollen and the insect cuticles is significantly lower (see Figure 3). Even though the adhesion measured on the planthopper with particles is extremely low, for the total removal from the surface to take place some mechanism facilitating contaminant motion must occur. There are a number of self-cleaning processes, which can potentially clean the wing membrane, which include mechanical motion of the wings, gravity, and wind and water in various forms (e.g., rain, fog, and dew). All of these mechanisms will be facilitated if the adhesion of contaminants with the membrane is low. Thus, the very low measured adhesion of particulates we have measured suggests that removal mechanisms will operate at a high efficiency. Wetting Properties of the Planthopper Wing Membrane. The wetting behavior of a surface is an important consideration because it will dictate many of the mechanisms which an organism can use to interact with its environment. For example, an antiwetting surface may inhibit microbial growth by the absence of forming a thin water layer, as well as facilitate rolling droplets, which can transport contaminants from the surface. One of the principal parameters, which characterizes the wetting interaction of a surface is the apparent contact angle defined as the observable angle that a liquid makes with a solid. Insects demonstrate a large range of contact angles reflecting hydrophilic and hydrophobic interactions.15,23,25 The similarity in surface structure (Figure 2) and hydrophobic chemistry (waxes) of the planthopper with the lotus leaf suggests that they may have similar interactions with water. Indeed this is the case with apparent water contact angles on the planthopper 24385


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Figure 5. (a) Images of condensation drops autonomously propelling off the wing membrane (forewing and hindwing (far-right image)). (b) Sequence showing the evolution of a droplet self-propulsion from the forewing of the leafhopper and then returning to the surface under the influence of gravity and bouncing off at approximately 37.5 ms.

wings in the range of 153° ± 6°, which compares favorably with values reported in the literature for the lotus leaf (compare Figure 1b,c with Figure 4a).9,26 Hysteresis values (apparent) as shown in Figure 4b,c and e,f (hind and forewing, respectively) with advancing and receding contact angles of 158° ± 5° and 148° ± 7°, respectively, are also comparable to those of the lotus leaf.26 This also suggests that replication of the wing topography utilizing a chemistry of lower surface energy27 may optimize these values. The superhydrophobic planthopper membrane is also resistant to impacting droplets (emulating rain) as shown in Figure 4d, where the droplet rebounds from the surface as opposed to being pinned after impact. The wetting behavior of the forewing as shown in Figure 4e−g demonstrates that this alternative topography is just as efficient in acting as an antiwetting surface, where both apparent static, advancing, and receding angles and droplet impact behavior were closely matched. There are a number of theories to describe the superhydrophobic interaction, all of which have certain assumptions and limitations.28−30 Cassie and Baxter29 express the superhydrophobic state in terms of a number of interfaces; a liquid− air interface with the ambient environment surrounding the droplet and a surface under the droplet involving solid−air, solid−liquid, and liquid−air interfaces. Eq 1 expresses the contact angle (θC) formed with a rough surface: cos θC = f1 cos θ1 − f2

origin for the observed high contact angle and low adhesion of water (low hysteresis). The Cassie−Baxter wetting regime has been observed on other wings including pigeon feathers, in which the two-scaled structure of the feathers facilitates a waterproof surface.31 Simply based on the dimensionally smaller size of the planthopper microasperities in comparison to the lotus leaf (Figure 2); all other parameters being equal, one would expect less actual solid−liquid contact when interacting with droplets; however, the density of planthopper microasperities is higher (Table 1 and Figure 2) and thus acting to increase the overall interfacial contact. While apparent contact-angle measurements and related hysteresis measurements are useful for characterizing the surface, under normal environmental conditions, the planthopper cuticle will encounter rolling and impacting droplets from rain. It should be noted that such dynamic conditions entailing processes such as droplet pressure, bouncing, and vibrating droplets on many surfaces can lead to a wetting transition from the heterogeneous Cassie−Baxter state to the homogeneous Wenzel wetting state.32 A review of the mechanisms involved can be found in the literature.32 When a droplet impacts a surface, the kinetic energy can be stored in deformation and converted into surface energy and utilized for the rebound of the droplet from the surface, especially if it displays superhydrophobicity. The Weber number We (ratio of relative importance of kinetic energy to surface energy) is regularly used to characterize the droplet impact event as it entails the competition of these forces. This dimensionless value incorporates several parameters, which can be easily varied to investigate responses to altered conditions:

(1)

where f1 is the total area of solid under the drop per unit projected area under the drop, f 2 is defined in an analogous way viewing the liquid−air component, and θ1 is the contact angle on a smooth surface of the same material. The minimized contact area due to the multiscale topography of the planthopper contributing to the f1 and f 2 terms is the basic

We = 24386

ρw R dv 2 σ

(2) DOI: 10.1021/acsami.7b08368 ACS Appl. Mater. Interfaces 2017, 9, 24381−24392

Research Article

ACS Applied Materials & Interfaces where ρw, Rd, and v are the density, radius, and impact velocity of the droplet, respectively, and σ is the surface tension of the liquid. (Note that the characteristic length term in the Weber number equation is typically the diameter or radius of the droplet. We have used the radius in our investigation, which should be noted when comparing this with other studies.) When small water droplets (2.2 mm in diameter) were impacted with the wing dorsal and hindwing at a velocity of 0.4−0.6 m s−1 (We: 2.4−5.4) they rebounded from the surface instead of being pinned to the membrane upon impact (Figure 4d,g). At these velocities, the droplets maintained the form of a single droplet after the first and subsequent rebound events from the surface and transitioned to a number of forms such as a pyramidal (e.g., Figure 4d, second frame) and a toroidal shape (e.g., Figure 4g, 10 ms frame), which increase the contact area during the spreading event. Thus, deformation and the footprint of the droplet is significant at this point. For comparison, droplets have also been observed to rebound off the surface of a lotus leaf at similar velocities.33 The authors of that study suggested that the rebounding droplet, which underwent intensive vibrations when rebounding, was not pinned to the surface due to the irregular distribution of the micro structuring (papillae). Because the leafhopper also exhibits this variable microstructuring, this may aid the surface in repelling droplets of this origin. Droplets were observed to bounce up to five times on the surface before transitioning into mobile rolling droplets or bouncing off the wing (the small wing size restricted the number of rebounding events as droplets made contact with the wing edge on repetitive bounces). When the velocity of the droplet was increased to over 2 m s−1 (We > 60), the surface energy of the impacting droplet was no longer able to maintain a single droplet and formed several secondary droplets, all of which bounced or rolled off of the surface. In addition to impacting and rolling droplets from rain, the planthopper will also encounter water in the form of fog and condensation from atmospheric conditions. By the cooling of the planthopper wing below the dew point, surface condensation was induced, which formed microdroplets. As they grew, some merged and then self-propelled from the surface (Figure 5a,b). This process has also been observed on some natural (including the lotus leaf)10 and some artificial surfaces34 and takes place from the changes in surface energies (i.e., energy released as a result of two or more droplets possessing more surface area than a single droplet of comparable size). The droplet propulsion process can be expressed in a simplified manner by considering changes in the droplet surface energies. For example, if we consider the case in which two small water droplets merge on a superhydrophobic surface to form a larger droplet, the maximum height, Hmax, that can be attained by a droplet can be determined by integrating the velocity of a droplet over its time-of-flight, resulting in eq 3: Hmax =

Eq 3 predicts a maximum height over 10 mm for small-sized droplets; however, a recent study shows that various factors (such as viscous dissipation) significantly reduces this value, such that ∼25% of the energy released by the droplet merging is converted to the effective kinetic energy propelling the droplet.35 Regardless, these distances (of the millimeter range; see Figure 5a,b) have the potential for droplets to be removed totally from the cuticle and transported outside the membrane−air boundary layer. Importantly, the self-propulsion releases the droplet from the surface−air interface and associated adhesion and exposes it to external forces that could potentially transport the drop significant distances away (e.g., gravity and wind). Because the droplets are extremely small, their mass will be sufficiently low, such that very slight external wind forces are able to fully remove the droplet from the membrane once they have self-propelled above the surface. For water condensate droplets of 75 μm in diameter, a wind force of only several nano-Newtons is required to oppose the gravitational force. The self-propelled droplets will also be likely to aid in removal of some contaminants from the wing cuticle.34 The high contact angle, low hysteresis, and antiwetting behavior of droplets formed under various environmental conditions as shown in Figures 4 and 5 demonstrate that the wing-membrane surface can utilize the quintessential mechanisms, which are necessary for the self-cleaning of the surface.1−3,9,34 These features have been related to various high contact angles and low adhesion. Interaction with Bacteria and Eukaryotic Cells. Recently, it has been shown that the outer layer of some organisms can be potentially harmful to surface-associating bacteria. For example, the cuticles of some insect species have demonstrated a nanostructured surface, which can kill certain species of bacteria.36 These bacterial studies commenced from our field collecting and observation of minimal decay of some insect wings and investigations of these natural surfaces with contaminating solids (e.g., biological material such as pollens) and suggested exploration of such contacts in aqueous environments, e.g., refs 15 and 37. The specific mechanisms for such antibacterial effects are still not completely understood; however, the process presumably takes place via gravity and nonspecific forces.38 A recent theoretical study suggests that when nanopillars become sharper or spaced further apart (assuming that contact is maintained between some of the nanospikes and cell surface), the antibacterial properties (stretching) should be enhanced and, hence, maximize bacterial death.38 Another study suggests that more closely spaced structuring should be more effective in bacterial cell rupture.39 We suspect that both conditions may impair bacterial function, but the mechanistic process and contact conditions may differ for the two scenarios. The later study using a quantitative thermodynamic model considers the total free-energy change of bacterial cells. The critical factor contributing to the bacterial damage is the significant increase in contact adhesion area with the surface structuring. The analysis is composed of a number of terms, including the stretching free energy (by knowing the change of the cell membrane area), the bending energy of the membrane at the edge of the cell, and the bending energy of the initial cell membrane. The study shows that the surface energy is an important factor and that high adhesion energy leads to a large stretching degree of the bacterial membrane. In addition, the study shows that higher density of pillars and height lead to greater stretching of the membrane, which is caused by the increase of

⎡ ⎫⎤ 9ρ C Dϕ ⎧ (1 + f )2 ⎬⎥ ln⎢1 + a2 2 w ⎨ − 1 3ρa C D ⎢⎣ R mρw g ⎩ (1 + f 3 )2/3 ⎭⎥⎦ ρw R m

⎪

⎪

⎪

⎪

(3)

where ρa and ρw are the densities of air and water, respectively; Rm is the radius of the merged single droplet; CD is the drag coefficient of a droplet in air; ϕw is the water surface tension; g is the gravitational acceleration (9.8 m s−2); and f is the ratio of drop diameters. 24387


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observation (up to 7 days). The subtle differences in topography between the two surfaces (planthopper and lotus) provide an interesting opportunity for comparison with bacterial interaction in future studies. For example, the orientation and apex dimensions of the nanostructuring exhibit differences. From Figure 2, it is evident that a significant population of the wax crystals on the lotus are not orientated in a near vertical position. This is in stark contrast to the planthopper nanostructuring, in which a near-vertical orientation is assumed. In addition, the planthopper apex dimension and shape is slightly different from the lotus leaf; the lotus leaf has a nonrounded wax apex (Table 1 and Figure 2). A previous study using replicated lotus-type structuring has demonstrated an ability of the surface to retain coccoid bacteria such as Staphylococcus aureus and Planococcus maritimus, primarily from attachment at the crevices between the microstructuring.42 The authors suggested that air entrapped by the topographical features may have inhibited contact between the cells and the titanium substratum. The wide range of shape, spacing, height, and size of insect cuticle structures provides researchers with a comprehensive number of permutations of structured surfaces to explore a range of interactions of eukaryotic cells.12 These type of surfaces can be used to investigate specific cellular responses and may entail directional cell guidance (as insect structuring can be anisotropic, e.g., butterfly scales, some beetle elytra). The antibacterial nature of the planthopper surface suggests that this type of structuring may have applications for biomedical materials. With this in mind, coupled with the unique micro- and nanoarchitecture of the insect cuticle, we also examined the contact response of eukaryotic cells with the wing structuring. A pair of different cell lines were investigated (SHED-MSCs and human dermal fibroblasts (HDF)). SHEDMSCs are stem cells from the erupting tooth that harbor strong capabilities toward dental pulp and periodontal root tissue formation. Templates for stem cell division, expansion, and specialization are in high demand in biotechnology. Human dermal fibroblasts represent an important connective tissue cell type in demand for skin wound healing injuries. Platform technologies for their expansion and formation into realistic dermal cell layers could be significant inputs for burn treatments. In contrast to bacterial contacts, when we exposed the wing membrane to the eukaryotic cells, the planthopper surface demonstrated compatibility for natural attachment, division, and growth (Figure 6b−d). Physical templates with cell-growth promotion are a critical boost to cell and tissue therapy, addressing the problem of the slow expansion of cell populations in captivity. In the specific case of HDF cells, they spread into well-constructed cell sheets reminiscent of the organization in the natural dermal layer, although not in a 3D aspect (see Figure 6b,c). Additionally, stem cells proliferate into substantial 3D clusters on top of planthopper wing surface topography, more typical of a stem-cell niche architecture (Figure 6d). In a previous study to our bacterial observations, we have shown that another insect membrane, a cicada membrane nanostructure, can be biocompatible with eukaryotic cells.43 The planthopper represents a different surface topography in which, in addition to microprojections, we have a more-random nanostructure and a larger variation in height. Recent studies on fabricated structuring with a similar nanostructure spacing to the planthopper have demonstrated that cell lines can exhibit different responses, where particular

the contact adhesion area. Thus, the high density of planthopper nanostructuring combined with sufficient height may be significant contributors in bacterial death. The planthopper offers a new and interesting alternative topography to explore with cell contacts as not only is it hierarchical but also random in its nanostructuring (e.g., height and spacing). Contact of bacteria (Porphyromonas gingivalis) with the planthopper membrane was observed using confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM; Figure 6a). Although this particular bacteria

Figure 6. (a) Confocal laser scanning microscopy images of P. gingivalis on the planthopper wing membrane. Live cells and dead cells are stained; live is indicated in green, while dead cells are depicted in red. The inset in panel a shows, for comparison, the density and state of P. gingivalis on a silica nonstructured surface. (b) Confocal laser scanning microscopy images of live spindle-like HDF, a natural shape typical of in situ cells, adhered to the planthopper membrane and clustered into a mosaic. In many regions, they formed into cell sheets reminiscent of the natural 2D arrangement in human skin. (c) Display of the HDF monolayer growing on a relatively smooth TCP surface. (d) SHED-MSCs proliferate into substantial 3D clusters on top of Planthopper wing surface topography, more typical of a stem-cell niche architecture. Red: TO-PRO-3 iodide (642/661) nuclear stain; green: cell-tracker green CMFDA viability fluorescent probe.

is unlikely to come into contact with planthopper cuticle, it serves as a good illustrative example, reflecting the properties (especially cell-wall properties) of Gram-negative bacteria.40,41 In addition, these bacteria have significant implications for humans, and thus, control of these cells is of great interest. They are commonly found in the oral cavity, where they have been implicated in certain forms of periodontal disease as well as other areas of the human body.40,41 Exposure of these bacterial cells to the membrane resulted in death of the bacteria, as shown in Figure 6a, using CLSM. The green-stained cells represent living cells, while the red-colored cells represent dead cells. Dead cells were primarily composed of single bacterium as well as small cell aggregates or chains (end-to-end assembly) several microns in length. The metalcoated wing cuticle showed the same response (predominantly dead cells populating the surface). This suggests that the bactericidal mechanism is primarily related to the topographical structuring of the surface as the coated samples (different chemistry and near-identical topography) produced the same effect. For comparison, the inset shows a flat silica surface with predominantly living viable cells. Death of bacteria on the planthopper membrane was observed for the full period of 24388


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Figure 7. Properties and putative functions of the planthopper cuticle wing structuring. Superhydrophobicity and antiwetting attributes, dropletremoving on impact, droplet-removing rolling, antifogging and antidew qualities, self-cleaning of large rolling and impacting droplets and selfpropelling droplets, low adhesion of contaminants, antibacterial quality, and eukaryotic cell adhesion.

dimensions can provide favorable conditions for some cells (e.g., endothelial cells) while inhibiting others (e.g., fibroblasts).44,45 The density and surface area of contact seem to be key factors for cells to form mature focal adhesions.44−46 However, these fabricated architectures are typically nonhierarchical in design and cells interacting with the planthopper must contend with both micro- and nanocontact points. Further studies with other cell lines on the planthopper membrane may also demonstrate cell selection and specific growth orientation. Exposure to a range of bacterial and eukaryotic cells in future studies will help elucidate specific parameters and may provide insights into design for numerous biomedical surfaces in which it would be advantageous to enhance the growth of a desired cell type while inhibiting that of another (cell selectivity).

for which selective cell activity is required. In addition, the subtle differences between the lotus and planthopper structuring (e.g., orientation of nanostructures, shape of nanostructure apex, and spacing of microstructures) provides a framework and foundation for future comparative studies. It is also noteworthy that the multifunctional structuring on the planthopper may also enhance the insect in ways not demonstrated in this particular study. For example, a previous study has shown that inhomogeneous surface structuring may aid in antireflection (e.g., as found on the glasswing butterfly), for which reduced reflection by wings occurs even when viewed at high angles. This was shown to be a consequence of the nonregular arrangement of nanopillar structures featuring random heights and width distributions similar in dimensions and variations in height and spacing to the planthopper.47 Thus, the planthopper architecture may also exhibit this advantageous optical property, enhancing the camouflage ability of the insect by reducing reflection of light and thus predation. The dull appearance of wing membrane as shown in Figure 1d,e and observations from field collection of the samples (no observable wing reflections) lends supports to this premise. The development of low-adhesion, nonreflective, nonwetting, antibacterial, and biocompatible coatings is of immense interest from practical, commercial, and scientific perspectives worldwide. The planthopper membrane provides an interesting topographical template for multifunctional man-made designs, which may potentially aid in controlling various interfacial interactions in terrestrial, semiaquatic, and aquatic environments. The utility of this natural template may provide insights into improving a variety of applications and interfaces such as hospital surfaces; artificial liquid channels (e.g., syringes, catheters, inlet ports, central line ports, and next-generation animal capillaries); dental implants; contact lenses; woundhealing architectures; feeder-layer templates for stem cell cultivation; and model systems for healing, toxicology, and basic cell biology and membranes used in industrial applications (e.g., water-filtration systems). We also note that the planthopper wing and insect wings in general composed of a deformable thin membrane, which can potentially be modified to adopt numerous geometric configurations. This significantly increases the range of applications for such nanostructuring on surfaces. For example, one could speculate such applications as

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CONCLUSIONS The micro- and nanostructuring on the planthopper wing has evolutionary and multifunctional constrained factors that dictate the architecture, scaling, and form. In many ways, these insects’ micro- and nanostructures is an example of an animal analogue to the well-known and studied lotus-leaf topography. The common configuration of the planthopper and lotus leaf micro- and nanostructuring accounts for their many similar interfacial properties. The planthopper will encounter a range of environmental conditions, which are also similar to the lotus plant, including light and heavy rain, fog and dew conditions, and interaction with a range of microsolids. Thus, mechanistic processes congruent with these conditions are necessary for maintaining the integrity and functionality of the wing. Our study has investigated some of the associated properties and putative functions of this thin membrane with a focus on solid and liquid contamination, which are summarized in Figure 7. The antiwetting properties of the structuring will help maintain the surface free from a water film and thus inhibit the growth of many micro-organisms. The superhydrophobicity and architecture which limits contact with solids, also facilitates the removal of interactions with a range of water droplet sizes and mechanisms. The antibacterial action and human-cell biocompatibility are interesting aspects of the thin membrane and may represent a new line of inquiry into such interactions 24389


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Ge crystal until key bands were observed on a live spectrum. Other macro ATR-FTIR acquisition parameters used in OPUS 7.2 software suite (Bruker) included 4 cm−1 spectral resolution, Blackman-Harris 3Term apodization, Mertz phase correction, and zero-filling factor of 2 to obtain spectral data in the range of 3800−700 cm−1. In addition, the macro ATR-FTIR measurement using the synchrotron IR light source was performed using 12.5 μm diameter pinhole, and thereby, each macro ATR-FTIR spectrum represented the molecular information from a 3.1 μm diameter sampled area on the wing surface, taking into account the refractive index of Ge (nGe = 4.0). Adhesion Experiments. The adhesion measurements were carried with a ThermoMicroscope TMX-2000 Explorer. The instrument is based on detection of tip-to-surface forces through the monitoring of the optical deflection of a laser beam incident on a force-sensing and imposing lever. The analyses were carried out under air-ambient conditions (temperature of 22−26 °C and 50−60% RH). Beam-shaped probes (NT-MDT Ultrasharp) were used throughout the work. The normal force constant was determined by using resonance methods and the scanners were calibrated using atomically flat surfaces. Force versus distance (f−d) analysis was used to obtain adhesion data. Pollen grains and silica beads were utilized to measure particle and cuticle interactions and forces. The particle was held stationary at an x−y (sample plane) location and ramped along the zaxis, first in the direction of approach and contact with the surface and then in the reverse direction. The f−d curves were acquired at rates of translation in the z-direction in the range of 5−10 μm s−1. Each f−d curve consisted of 600 data points. A total of 20 measurements per particle were acquired for each location. A total of 2 particles were attached to cantilevers for each particle type (i.e., 4 silica beads and 2 pollens were used for adhesion measurements, each yielding 20 measurements for each sample). Adhesion was measured under the conditions of the two surfaces coming into contact with minimal applied loading force. That is, adhesion represented the primary force of attraction that the particle surface would experience, such that the deformation of structures is minimized and in which the main contributing force involved is primarily that of the adhesion of the particle to the surface. This was achieved by selecting a specific distance (as opposed to a predetermined set point) for the probe to travel toward the surface. This distance was fine-tuned and adjusted until an f−d curve was obtained in which no discernible loading force could be measured (i.e., the f−d data composed of an initial snap on force (e.g., short-range forces such as meniscus comprising the loading and applied force) and adhesion force in the retract curve). Water-Interaction Experiments. The contact angle measurements were determined using an optical microscope system with digital capturing. A total of 20 single measurements (Milli-Q water) were taken on the forewing and hindwing of the insect captured at ambient conditions of ∼25 °C and RH of 50−60%. Left and right angles between the sample surface and the tangent line to the droplet (5−10 μL) were considered as one measurement. Hysteresis measurements were determined by application of a droplet via syringe onto the surface for advancing and receding angles. The mobile droplet experiments consisted of droplet release using Milli-Q water from a microsyringe with a fine needle (SGA). The balance between droplet surface tension, needle adhesion, and gravitational force sets the droplet size, which is 1.1 mm in radius (R) within a 6% deviation. The droplet impact velocity was determined by video capture immediately before impact and also confirmed from the height of the released droplet, V = 2g (H − 2R ) , where g is the force of gravity and H is the distance between the needle tip and the cuticle surface. For condensation experiments, the wing membrane samples were mounted on a cooled copper element maintained at a temperature below the dew point (14−16 °C). The surface temperature of the membrane was monitored with a dual temperature meter HT-L13 with thermocouples (type K). Typical conditions of dew formation were achieved in the laboratory with humidity of 80−90% and ambient temperatures of 24−26 °C. Data for impacting droplets and

artificial red blood cell shaped membranes or microribbons attacking bacteria in fluidic systems (e.g., blood capillaries) or self-propelled membrane-shaped structures from intermittent contact with cold surfaces (e.g., a rotating wheel with contact fins moving on a flat surface propelled by self-propelled droplets).

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EXPERIMENTAL METHODS

Insect Collection Preparation. A total of 50 insect specimens (D. danae) were captured from local flora near the Ross river foreshore in Northern Queensland, Townsville, 19°22′58.475″ S, 146°44′9.321″ E, and 19°21′52.034″ S, 146°43′55.483″ E. The insects were predominantly found on only one tree species (Planchonia careya). Wing samples (forewings and hindwings) were excised from the deceased insect bodies using a sterile scalpel. Wing membrane surfaces were cleaned after being excised and prior to experimentation with a steady stream of Milli-Q water at a flow no greater than droplet impacts from natural occurrences and sources (i.e., rain droplets). Termite samples were collected from the private residences of Drs. G.S.W. and J.A.W. (authors) in Brisbane, Queensland, Australia, and Townsville, Queensland, Australia. Scanning Electron Microscopy. Both the hindwing and forewing of the planthopper were examined on four samples. A small section of insect membrane (approximately 2 mm × 3 mm) was excised and mounted on an aluminum pin-type stub with carbon-impregnated double-sided adhesive and then sputter coated with 7−10 nm of platinum before being imaged using a JEOL 6300 field-emission SEM at 8 kV. Lotus leaves (N. nucifera) were also examined using SEM (e.g., Figure 2). A small section of the upper leaf surface (3 samples from different plants, approximately 10 mm × 10 mm sections) was sectioned and mounted on a pin-type stub with carbon-impregnated double-sided adhesive and then sputter-coated with 7−10 nm of platinum before being imaged using a JEOL 6300 field-emission SEM at 8 kV. Both Au and Pt sputter-coated insect cuticles were also prepared for examination of the interaction with bacteria. Attenuated Total Reflection Fourier Transform Infrared. The ATR-FTIR spectra of the wing membrane samples (dorsal and ventral regions of the fore and hindwings including brown, yellow, black, and clear regions) were measured using two independent instruments. The first instrument was a FTIR spectrometer (Spectrum 100, PerkinElmer) equipped with an internal Globar infrared (IR) source, a deuterated triglycine sulfate (DTGS) detector, and a single-reflection diamond ATR accessory (UATR, PerkinElmer) with a 45° angle of incidence and a 2 mm × 2 mm active-sensing surface. A background spectrum was individually acquired on a clean surface of the diamond crystal prior to each sample measurement. Sample measurements were conducted by pressing the wing membrane against the sensing surface of the diamond ATR crystal using a metal clamp, and a few highquality spectra were collected from each wing sample using 16 coadded scans. Reproducible contact was achieved by the use of an electronic sensor plate available with the ATR unit. The other instrument was a Hyperion 2000 FTIR microscope (Bruker Optic GmbH, Ettlingen, Germany) at Australian Synchrotron’s infrared microspectroscopy (IRM) beamline (Clayton, Victoria, Australia), which used synchrotron IR radiation as an IR light source and was coupled to a Bruker Vertex V80v spectrometer and a liquid nitrogen-cooled narrow-band mercury cadmium telluride (MCT) detector. In this case, the ATR-FTIR spectra were acquired using an in-house developed macro ATR-FTIR device that was equipped with a germanium (Ge) ATR crystal having a 250 μm diameter active sensing surface. The region of interest on the wing membrane was selected visually based on the physical appearance observed through microscopic images, which were acquired using a Leica optical microscope (IC80 HD, Leica Microsystems Pty Ltd., North Ryde, Australia). For the macro ATR-FTIR measurement, a background spectrum was collected once through air prior to raising up the wing membrane sample to make contact with the Ge crystal using 128 co-added scans. Sample measurements were subsequently conducted using eight coadded scans by raising the sample up against the sensing surface of the 24390


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ACS Applied Materials & Interfaces condensation experiments was collected using high-speed video at 400 and 1200 frames per second (fps) at a 640 × 240 and a 320 × 120 pixel resolution, respectively, with frame rates of 2.50 and 0.833 ms per frame and up to 62.5 μs shutter speeds (exposure times) and video playback at 30 fps. Planthopper Wing Exposure to Bacterial and Eukaryotic Cells. P. gingivalis was subcultured onto blood agar plates prior to incubation in an anaerobic environment at 37 °C (85% N2, 10% CO2, and 5% H2) according to standard protocol. At 4 days, a specific working concentration of bacteria was calculated. The bacteria were harvested directly from the agar plate, picked up with a cotton bud, and then dipped into 10 mL of nutrient broth to produce a new suspension. The nutrient broth was made from 30 g of TSB, 5 g of yeast extract, 5 mg of hemin, and 1 mg of menadione dissolved in 1 L of distilled water. The bacterial suspension was reconfigured to fit the MacFarland standard by measuring the sample wavelength at 660 nm. The quantity of bacteria formulated and used was 1 × 107 bacteria cells. The planthopper wing samples (uncoated and coated) and smooth silica wafer controls were sliced into small sections measuring 5 mm × 5 mm. All specimens were placed into individual tissue 24 well culture plates. All experiments were cultured in triplicate. The bacterial suspension, measuring 2 mL, was added onto the samples inside of the wells. All sample cultures were placed within a controlled anaerobic environment at 37 °C for between 24 and 168 h. The broth covering each sample was replenished every 24 h to maintain purity and optimal growing environments. At the end of culturing, the samples were fixed and stained with a LIVE/DEAD BacLight bacterial viability kit (ThermoFisher Scientific). The fluorescently labeled surface-associating bacteria were viewed under a laser confocal microscope at 20× and 40× magnifications pooled across 5 randomly chosen areas of the wing surface. The final 3D images were made from 50 stacked laser slices that started 100 μm underneath the wing surface. The possibility of free-floating unattached bacteria in view was undone by washing the surface prior to imaging. Human Cell Culture on Wings. We applied two clinically relevant human cells (a stem cell and an epithelial cell) on the planthopper wings, both being known for their strong, positive induction by nanotopographies.48,49 Culture flasks were filled with primary, low-passage-number (6) SHED-MSCs previously extracted from fresh deciduous baby teeth (7−8 year olds) according to the method of Miura et al. 2003.50 The population of SHED-MSCs were previously tested for intrinsic “multipotency” using anti-Stro-1 monoclonal antibody recognition (a highly specific marker for the MSC populations).51,52 The two steps toward culture expansion consisting of two passages in Dulbecco’s modified Eagle medium (DMEM) led to the selection of homogeneous SHED-MSC populations. Primary human dermal fibroblasts from the normal dermis of human adult skin were cultured in fibroblast growth medium in identical incubator conditions as SHED-MSCs. Planthopper wing samples measuring 5 mm × 5 mm were cut using a size 10 scalpel blade. Single sections were placed into a 2.0 cm2 tissue culture well. SHED-MSC cells were trypsinised from the culture flasks, removed, and added into a 10 mL plastic tube and centrifuged at 1500 rpm for 10 min prior to resuspension in plain DMEM. A volume of SHED-MSC cell suspension containing 100 000 cells was directly seeded onto each of 12 prepared samples per individual SHED-MSC population. Samples were incubated for 4 h, at which time attachment to the TCP surface occurred. Absence of FBS proteins in the media prevented the formation of cellular attachment complexes on the insect membrane, which encourage cell adhesion. Thus, we could be confident that cell attachment was mediated by the physical topography and not adsorbed chemical components. At this stage, 1 mL of 10% FBS-supplemented DMEM was added to just cover the surface of every sample to maximize nutrient and gaseous exchanges. Cells were left to grow and divide for 7 days in an incubator at 37 °C in a 5% CO2 atmosphere. Media was exchanged with fresh DMEM every 48 h to remove excess cellular waste products. The fluorescent-labeled surface associating SHED-MSCs were viewed under a laser confocal microscope at 20× and 40× magnifications pooled across 5 randomly chosen areas of the wing surface. Finalized

images of surface associating human cells were composed of between 35 stacked laser slices that began 100 μm underneath the outer epidermal surface.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.7b08368. Figures showing topographical structuring on the forewing of the planthopper, an ATR spectrum, and synchrotron-sourced micro-ATR of the planthopper wing. (PDF)

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gregory S. Watson: 0000-0001-9843-9211 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Joy Taylor for her kind donation of the photographic equipment used in this study. REFERENCES

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Insect Analogue to the Lotus Leaf: A Planthopper Wing Membrane

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