A dynamic nanoindentation technique to investigate the nanomechanical properties of a colored beetle

Abstract

Special internal structures of the cuticle of a beetle have multifunctionality including self-protection and attraction of mates using structural coloration, and being lightweight with high strength, which can protect the body and membranous hindwings. Special optical properties were found in the black spots region (BSR) of the cuticle of the multicolored Asian lady beetle (Harmonia axyridis). Both the BSR and the orange region (OR) have alternating layers of chitin and melanoprotein, as detected using field emission scanning electron microscopy (FESEM). However, a parallel wavy line structure was found in the BSR via laser scanning confocal microscopy (LSCM). This special structural color may arise via a diffraction grating mechanism. To explore the relationship between the material and its optical properties, the dynamic nanoindentation approach (nano-DMA) was used. The viscoelastic properties of the cuticle were assessed including the storage modulus (E′), loss modulus (E′′) and loss tangent (tan δ), in the differently colored zones. The extent of protein cross-linking affects the cuticle's mechanical properties. We then demonstrated the applicability of a power-law frequency dependence analysis of E′ and tan δ. Furthermore, the frequency exponent of n and tan δ were discussed in relation to the BSR and OR. A lower wavelength of maximum reflectance was found in the BSR that had little frequency dependence on n and a larger tan δ value related to its extent of protein cross-linking. This is contrary to the results obtained in the OR. The results will help in designing lightweight, high strength, and color-changing micro air vehicles (MAVs).

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1. Introduction

Structural colors found in insects, birds, and plants do not fade and have iridescent, polarization, and UV effects that provide several functions. These functions include conspicuousness (including efficiency of reflection, aposematism, and attraction of conspecifics), camouflage, thermoregulation, and mirror/antireflection in photophores.^1,2 Structural colors recently have attracted substantial attention due to their potential applications in textiles, cosmetics, vehicle shells, anti-counterfeiting technologies (on banknotes, credit and debit cards, and branded goods), optical filters, and optical security devices.³ Inspired by the humidity-dependent color change observed in the Hercules beetle cuticle, a biomimetic thin-film-type humidity sensor with a nanoporous structure (3-D photonic crystals) was designed.⁴ The visible color of the fabricated humidity sensor changed from blue-green to red as environmental humidity increased. Most bioinspired fabrications or applications only focus on structural biomimetics.

The structural colors found in beetles include white, opal, orange scales, yellow or green iridescence, rows of brilliant spots, yellow and blue bands, greenish-white, mixed blue and violet colors, and bright white.^5–8 Recent research has focused on the effects of structures on color display. The most common mechanisms for structural colors are film interference, diffraction grating, scattering, and photonic crystals. However, the mechanical characteristics of various color zones and interfaces remain unknown primarily due to their small dimensions.

The Coccinellidae are a family of small beetles with markings (including black spots, curved lines, and shapes) on their bodies^9,10 that serve as an aposematic color scheme involving red or yellow coloring with black markings.¹¹ Coccinellidae has been studied extensively in the fields of genetics, evolution, population, and biological control.¹² The bright colors on members of Coccinellidae protect them from predators (birds, frogs, dragonflies, wasps, and spiders) by causing associating the colors with an unpleasant taste.¹³ The color polymorphism of Harmonia axyridis (Pallas) is likely associated with multiple alleles, phenotypic variability, larval diet, and temperature.¹² Carotenoid pigments largely control variations in the red elytra coloration of H. axyridis, and females with lighter black spots have greater amounts of harmonine than those with darker spots.¹⁴ The tanning process of Coccinellidae leads to the hardening (sclerotization) and pigmentation or coloration of the cuticle by changing interactions between cuticular proteins and oxidized catechols, which would lead to a change in mechanical properties and color.¹⁵ This change is the result of cross-linked proteins that stiffen the matrix; the extent of protein cross-linking affects the cuticle's mechanical properties.¹⁶ The structural, mechanical, and optical properties of their cuticles may provide useful information for scientists and engineers for more precisely modeling these differently colored zones and the relationships between their microstructural and mechanical properties.

Quasi-static nanoindentation testing is useful for investigating the mechanical properties of biomaterials in small, local, and specific regions.¹⁷ Quasi-static nanoindentation was primarily developed to test elastic materials, but substantial challenges imposed by the viscoelastic behavior of polymers and biomaterials can be overcome using a dynamic nanoindentation method.¹⁸ The development of dynamic nanoindentation has been applied successfully to determine accurately the viscoelastic properties of several materials such as polymers, bone, nacre, and soft tissue.^19–25 This technique involves sinusoidal loading, which is superimposed on the quasi-static loading during the nanoindentation test to investigate the dynamic properties of viscoelastic materials including the storage modulus (E′), loss modulus (E′′), and loss tangent (tan δ).^26,27

In this paper, the source of the structural coloration in the multicolored Asian lady beetle Harmonia axyridis was investigated by examining the optical properties and microstructures of the differently colored zones of its elytra. The relationships with the optical properties were then investigated and discussed. Their viscoelastic properties were measured using dynamic nanoindentation techniques to investigate potential bioinspired designs related to stealth engineering or pest control, and to explore the relationship between their optical and mechanical aspects.

2. Specimens and experimental methods

2.1 Materials

Multicolored Asian lady beetles, Harmonia axyridis Pallas, were collected in Changchun City, Jilin Province, China. A total of 10 beetles were acquired as samples, and each elytron was measured in the same location (the highest point of the curvature) via quasi-static and dynamic nanoindentation testing. The specimens were fixed on the slide glass using modified acrylate adhesive.

A laser scanning confocal microscope (LSCM, OLYMPUS OLS3000, Japan) was used to investigate the surface microstructures of the beetle elytra in the black spots region (BSR) and orange region (OR). Field emission scanning electron microscopy (FESEM, JEOL JSM-6700F, Japan) was used to investigate the microstructures of the BSR and OR cross-sections.

2.2 Optical method

Sample spectra were measured using an UV-Vis-NIR spectrophotometer (CARY5000, Agilent Technologies, USA) with a laser. The illumination source for spectrophotometry was direct sunlight to mimic the natural illumination conditions under which the optical mechanisms function. The laser was mounted parallel to the direction of the incoming sunlight, and the estimated diameter of the spot size from which reflected light was collected was 5 mm.

2.3 Indentation method

Dynamic mechanical analysis (DMA) refers to the application of an oscillatory deformation to a sample to measure its response as a function of time, temperature, or frequency.²⁸ DMA results consist of three parameters: (a) the dynamic storage modulus E′, (b) the dynamic loss modulus E′′, and (c) a mechanical damping factor or tan δ, which represents the ratio of the dynamic loss modulus to the dynamic storage modulus and is useful for determining the occurrence of a molecular mobility transition.²⁹ For viscoelastic materials, it is desirable to have the capability to characterize both E′ and E′′.³⁰ A frequency sweep is a powerful test for the characterization of viscoelastic materials.^8,31 The sinusoidal variation in time is typically described as a rate that is specified by the frequency (f in units of Hz or angular frequency, ω = 2πf rad s⁻¹). In dynamic nanoindentation tests, the indenter tip is oscillated with a small loading, and the resulting displacement is recorded. The recorded information is then used to determine the E′ and E′′ of the material. The viscoelastic materials have some phase angle, δ, between the applied stress and the responding strain that varies sinusoidally with time. Their molecular motion and relaxation lead to phase lag.³² These two moduli can be collectively described as E* (eqn (1)), where the phase angle δ is the angle between the real axis and the vector that represents E* in a phasor diagram; E′ is the real part of E*, and E′′ is the imaginary part. E′ describes the stiffness or phase response of the material to the external applied force and is related to the Young's modulus, E. This modulus describes the elastic recovery of the specimen and the amount of energy recovered from the specimen subsequent to a loading cycle. E′′ is related to the damping behavior of the material and can be determined from the time lag between the maximum force and the maximum displacement. This damping is the amount of energy applied to the sample during indentation that is dissipated by various processes that facilitate energy losses, primarily heat generation. The dynamic loss modulus is often associated with “internal friction”, and is sensitive to various types of molecular motion, relaxation processes, transitions, morphology, and other structural heterogeneities. Therefore, the dynamic properties of the material provide information beneficial to understanding the mechanical behavior of the polymer at the molecular level.^8,33 tan δ, which is the ratio between E′′ and E′, is the value of internal friction. The equations that represent these quantities are given below.^8,31

E* = E′ + iE′′ (1)

E′ = |E*|cos δ (4)

E′′ = |E*|sin δ = E′ tan δ (5)

where k_S and C_S are the stiffness and damping coefficients of the contact, respectively; ω is the angular frequency; and A_C is the contact area, which is a polynomial function of the contact depth h_C. In general, E′′ ≪ E′, and E′ approaches E*.

The dynamic nanoindentation tests were performed using a dynamic nanoindentation II™ adjunct of a TriboIndenter (Hysitron Incorporated) with a Berkovich diamond tip with nominal curvature radius of 100 nm. The loading process consisted of maintaining a constant mean contact force while applying a frequency sweep from 10 to 200 Hz with 100 cycles at each frequency. The static contact force for the cycles was 500 μN, and the dynamic load was 25 μN. Nine indentations were applied in a 3 × 3 square matrix under each load to obtain the average values with a separation distance of 5 μm from one indentation point to the next.

3. Results and discussion

The elytra of the studied beetles were lustrous orange with symmetrical black spots. All beetles were adults measuring 5–7 mm in length and 4–6 mm in width (Fig. 1a). Fig. 1b–e show the surface morphology and transverse microstructures of the beetle elytra in the BSR and OR. Fig. 1b shows a parallel wavy structure that is densely covered with dimples; setae are present in the larger dimples. Fig. 1d shows irregular marks and small dimples with setae that are larger than those in the BSR. There are clear alternating light- and dark-contrast layers, which are chitin and melanoprotein³⁴ in the BSR (Fig. 1c) and OR (Fig. 1e), respectively. In Fig. 1e, there are several highlighted spots scattered in the various layers.

Fig. 1 (A) Spotted lustrous orange morph of the multicolored Asian lady beetle, Harmonia axyridis Pallas. Laser scanning confocal microscope (LSCM) images acquired in BSR (B) and OR (D). Field emission scanning electron microscopy (FESEM) was used to investigate the transverse microstructure of the BSR (C) and OR (E).

The reflectivity of OR and BSR as a function of wavelength is shown in Fig. 2. The peak of the OR is centered at 600 nm, corresponding to an orange color (Fig. 2). There are subtle fluctuations in the spectrum of BSR; the melanin should have absorbed light to prevent reflection. This effect is similar to that seen in the black Lomaptera sp.: their spectrum has a more intense and less uniform broadband reflectance, which is due to a combination of specular reflectance and additional reflectance arising from diffraction grating.³⁵ Therefore, the bright patterned color of the cuticle of Asian lady beetles is apparently caused by both pigments and physical phenomena.

Fig. 2 Reflectivity spectra of OR and BSR of the Asian lady beetle at normal incidence.

There are three classes of mechanisms producing physical colors in beetles: multilayer reflectors, three dimensional photonic crystals, and diffraction gratings.⁵ A multilayer reflector is made of a series of layers that is usually alternately composed of two different materials of lower and higher refractive indexes. Interaction with light occurs when the spacing between layers approaches one quarter the wavelength of visible light. Hence, a stack of layers all having the same optical thickness will produce a constructive interference for the same wavelength, and their combined action will produce a more intense and brighter colors.³⁶ The wavelength of maximum reflectance at a normal incidence of a stack of thin layers of alternating types is³⁷

λ = 2(l₁ + l₂)m (6)

where λ is the wavelength of maximum reflectance at normal incidence, l₁ and l₂ are the thicknesses of the dark contrasted layer and the light contrasted layer, respectively, and m₁ and m₂ are refractive indexes of the dark contrasted layer and the light contrasted layer, respectively. m is the average refractive index at normal incidence.

The multilayer reflectors are assumed to be made up of dark-contrast chitin layers with a refractive index of 1.55 and light-contrast chitin layers with a refractive index of 1.68.^35,38 In Fig. 1c and e, l₁ is 53 ± 6 nm and 39 ± 8 nm and l₂ is 24 ± 6 nm and 26 ± 7 nm for BSR and OR, respectively. Using these lower estimates results in a predicted peak wavelength far lower than the value we obtained. We therefore utilized a higher value of 2.0 as the refractive index of our electron-dense layer (dark-contrast layer) and (consequently) 1.56 for our electron-lucent layer (light-contrast layer).¹¹ Therefore, the calculated peak wavelengths for BSR and OR are 245 nm and 208 nm, respectively. The peak wavelength calculated for the OR is 208 nm, which is not consistent with its observed orange color at normal angle of incidence and indicates that the electron-dense layer observed above the stack of layers is composed of the pigment melanin. Thus, the colors in the BSR and OR are due to pigmentation. A pigment analysis revealed that variations in orange coloration are due to the amount of erythropterin pigment, which is stored in intracellular granules.³⁸ Erythropterin was also found in black Lomaptera species, which contained a stack of thin layers; however, this stack had no optical properties consistent with a multilayer reflector.³⁵ We soaked the elytra in a concentration of 95% hydrogen peroxide for 12 hours; the OR color faded to light yellow, whereas the color of the BSR remained clear (Fig. 3). The experimental observation found that the reflection from BSR is due to an interaction between the pigment and diffraction grating mechanisms, of which the diffraction grating has greater efficiency in scattering light of lower wavelengths.

Fig. 3 Comparison of hydrogen peroxide-treated elytron (left) and untreated elytron (right).

To investigate the relationship between the structure and mechanical properties of BSR and OR, dynamic nanoindentation tests were conducted on both the BSR and OR. The root mean square roughness (R_q) and average roughness (R_a) of the BSR were 5.72 nm and 4.72 nm, respectively, greater than those of the OR (R_q = 1.57 nm, R_a = 1.20 nm). In dynamic nanoindentation tests, the contact depths of the BSR and OR were 260–300 nm and 130–240 nm, respectively, substantially larger than the respective roughness of the different surfaces (R_a and R_q). The testing contact area was 2.59 × 10⁵ ± 0.24 × 10⁵ nm² and 4.11 × 10⁵ ± 0.16 × 10⁵ nm² for the BSR and OR, respectively, which would not cause interactions between indents. The thickness of BSR and OR was 15.19 ± 2.20 μm and 14.39 ± 1.40 μm, respectively. Hence, the contact depths of BSR and OR were less than 10% of the thickness. So, the substrate and surface have no obvious effect on the dynamic nanoindentation results.

In the BSR and OR (Fig. 4a and b), the storage moduli exhibited a dependence on frequency that increased with increasing frequency (10 Hz < f < 252 Hz). For the BSR, E′′ was nearly independent of frequency; however, this quantity was observed to decrease with an increasing frequency for the OR. The average value of E′ and E′′ of the OR (7.195 ± 0.051 GPa and 0.0230 ± 0.015 GPa, respectively) is substantially larger than that for the BSR (1.335 ± 0.010 GPa and 0.083 ± 0.005 GPa, respectively). Those values are less than what acquired in our previous paper, which data were acquired by Modulus Mapping (MM) technique (the E′ and E′′ for BSR and OR are 2.94 ± 0.85, 0.23 ± 0.09 GPa and 4.99 ± 0.91, 1.17 ± 0.31 GPa, respectively).³⁹ In the MM technique, the dynamic load applied on the sample must be 1 μN so as to acquire mapping of a 5 μm × 5 μm region of elytra surface. The applied force is small enough to avoid tip penetration greater than 2–5 nm into the sample surface, and the elastic type of contact between the tip and the sample is realized with no plastic deformation involved. However, in this paper, the static and dynamic load are 500 μN and 25 μN, respectively, which are applied to make an indentation on the surface of the sample with the contact depths of the BSR and OR of 260–300 nm and 130–240 nm respectively. By eqn (2), E′ and E′′ are calculated by the contact area of the indentation, which are polynomial functions of contact depth. So, the larger contact depth in this paper lead to lower E′ and E′′ of BSR and OR.

Fig. 4 Variations in E′ and E′′ with oscillation frequency for (A) BSR and (B) OR.

In our previous tests,³⁹ by quasi-static indentation, the reduced modulus (E_r) and hardness (H) of BSR and OR were 1.66 ± 0.29 GPa and 0.28 ± 0.06 GPa, and 3.94 ± 0.19 GPa and 0.47 ± 0.03 GPa, respectively. The reduced modulus E_r of a test specimen is defined as follows: 1/E_r = (1 − υ_s²)/E_s + (1 − υ_i²)/E_i. In this equation, E_s and υ_s are the elastic modulus and Poisson's ratio, respectively, and E_i and υ_i are the elastic modulus and Poisson's ratio for a diamond tip and are equal to 1114 GPa and 0.07, respectively. In this paper, E′ is 7.195 ± 0.051 GPa for BSR, which is greater than the reduced modulus acquired by quasi-static tests. However, for OR, E′ is lower (1.335 ± 0.010 GPa). The loading process of dynamic indentation consisted of maintaining a constant mean contact force while applying frequency sweep. Cross-linked and uncross-linked materials respond differently to such frequency sweep. Hence, the elastic modulus and hardness of BSR are greater than those of OR, but BSR exhibits a substantially greater frequency dependence, and is not a cross-linked material.

As shown in Fig. 5, E_r and H of OR and BSR decrease with increasing load. Contact depths of the indenter for BSR and OR increase from 114.15 nm to 930.36 nm and 82.12 nm to 761.60 nm, respectively, with load increasing from 100 μN to 3000 μN. E_r of the outer layer is the highest (2.96 ± 0.35 GPa for BSR and 5.38 ± 0.66 GPa for OR) because it is the protective layer. H of the outer layer is 0.25 ± 0.04 GPa for BSR and 0.43 ± 0.1 GPa for OR. For BSR and OR, the internal multilayers are formed by alternating light and dark contrasting layers. With indentation depth increasing, the layers that directly contribute to the indentation results are more. The hard outer layer protects the membranous and delicate hindwings from mechanical stress, and the soft inner layer can reduce external impact force. The multilayer structure gives the cuticle toughness and flexibility, and the multilayer structure can absorb and store energy, which improves its the impact-resistance capabilities.⁴⁰

Fig. 5 Variations in E_r and H with load for (A) BSR and (B) OR.

Using the dynamic nanoindentation method, two important parameters, frequency exponent of n and tan δ, are obtained.^15,41 For polymeric materials, the relationship between E′ and frequency depends on the relaxation modes available to the constitutive polymer chains.⁴² E′ varies according to the strain wave oscillation frequency ω; both were fit to a power-law model, E′ ∼ ωⁿ, in which n is indicative of the cross-linking density of a polymeric sample.⁴³ A similar technique can be used to distinguish the extent of protein cross-linking.¹⁵ By fitting E′ and ω with a power-law relationship, the values of n for the BSR and OR were 0.0391 and 0.0282, respectively, over the investigated frequency range (10 Hz < f < 252 Hz) (Fig. 4). The cross-linking of a polymer leads to an increase in its elastic response relative to its viscous response to applied stress. Thus, E′ of more highly cross-linked materials are more independent from oscillation frequency.¹⁵ The higher the extent of cross-linking, the lower the value of n.⁴⁴ For a lightly cross-linked biopolymer, E′ is typically observed to have a weak power-law frequency dependence, E′(ω) ∼ ω^0.1–0.3.^15,45 Hence, the values of n observed for OR are consistent with those of cross-linked materials. In contrast, BSR exhibits a substantially greater frequency dependence, as indicated by 38.65% larger n value than that for OR, which is not a cross-linked material.

tan δ is particularly well suited to the detection of dissipative processes such as friction.⁴² A progressive decrease in tan δ with increasing frequency can be attributed to a decrease in internal chain friction at higher frequencies.³⁶ The value of tan δ is high or low on behalf of high vibrational damping or high elastic behavior.⁴⁶ The BSR exhibited significantly larger values of tan δ throughout the investigated frequency range than the OR (Fig. 6). The greater viscous damping of the BSR relative to the OR, as indicated by the larger tan δ, is consistent with its greater power-law frequency exponent n. An increased tan δ suggests that metabolic differences in the black mutant strain result in elytra of Tribolium castaneum that are less cross-linked and more pigmented than the other types.¹⁵ We speculate that this result can be explained as follows: as the frequency increases, the dry sliding friction between the chitin fibers cannot keep pace with the change in the vibration frequency. In other words, there is an insufficient slip phenomenon due to the reduced energy consumption of the dry material. In the case of the tan δ value of the BSR, there is little energy dissipation. The value of tan δ is inversely dependent on the vibration frequency and thus decreases with increasing vibration frequency. This frequency relationship results in lower load viscoelastic effects and greater rigidity at high frequencies, causing the elytron cuticle to exhibit lower tan δ values at high frequencies. There was a small variation in tan δ (a decrease of approximately 15%) in the BSR from 10 Hz to 205 Hz; conversely, over the investigated frequency range, the tan δ value of the OR proportionally decreased (by approximately 88%) as the frequency increased. The ratio of E′′/E′ is a function of molecular interconnectivity. Since cross-linking can reduce both the magnitude of tan δ and its inverse dependence on frequency,³³ the tan δ of BSR is substantially larger than that of OR. This is due to the different extents of cross-linking in the OR and BSR. OR has a larger wavelength of maximum reflectance and frequency dependence of tan δ; however, there is a lower power law frequency exponent value n and tan δ in the OR compared with the BSR. Consuming cuticular protein cross-linking for the natural production of melanin pigments is a possible reason for the observed mechanical behaviors.⁴⁶

Fig. 6 Variations in tan δ with oscillation frequency for BSR and OR.

The coloration and nanomechanical properties are both related to micro-structures of cuticle of BSR and OR, which are produced by different extents of protein cross-linking.

The elytra of the beetle is light-mass and hard, and has an area equivalent to about half of the beetle's whole body. It has multiple functions such as preventing water evaporation, regulating body temperature, protecting the body and membranous hind wings, reducing friction, and waterproofing. The surface of elytra shows biological coloration and characteristics that help the beetle protect itself and attract mates so as to adapt to the environment. The elytra surface also provides an inspiration for designing bionic composite color-changing materials. Special structures of the beetle cuticle provide both structural coloration and lightweight-high strength, which give inspiration for designs of new materials with those advantages. In this paper, we found that the internal microstructures resulting from the extent of protein cross-linking in the elytra not only provide structural coloration for the beetle, but also different nanomechanical properties. The results will help in the design of lightweight, high strength, and color-changing composite materials for MAVs.

4. Conclusion

New materials that incorporate natural polymers and structural hierarchical complexity have the potential to match or even surpass the properties of natural tissues and provide commercially viable alternatives to the materials used in a variety of aerospace, biomedical, and consumer product applications. In some beetle cuticles, they have both structural coloration and lightweight-high strength produced by their microstructures, which give inspiration for the design of new materials with those advantages. There are optical properties found in the BSR of the elytra of the multicolored Asian lady beetle, Harmonia axyridis Pallas. However, melanin absorbs light and prevents reflection from the stack of chitin layers underneath, a result of additional reflectance arising from the diffraction grating. The peak wavelength for the OR is 600, which is higher than that for the BSR. The viscoelastic properties of elytra in the differently colored zones were investigated using a dynamic nanoindentation technique. Furthermore, we demonstrated the applicability of a power-law frequency dependence analysis of E′. The values of n for the BSR and OR were 0.0391 and 0.0282, respectively. Average tan δ values were 0.062 ± 0.004 and 0.032 ± 0.003 for the BSR and OR, respectively. For the OR, tan δ is at a higher frequency dependence, which decreased as frequency increased. Conversely, BSR is less frequency-dependent. This mechanical behavior is related to the extent of protein cross-linking. The BSR and OR are not cross-linked and lightly cross-linked, respectively. The natural production of melanin pigments reduced the extent of cuticular protein cross-linking, which led to lower stiffness (4-fold and 2-fold lower E′ and E′′ than in the OR). These results suggest that elytra color patterns, the extent of protein cross-linking, and the mechanical properties have the potential to help scientists and engineers design biomimetic composed materials with specific color and mechanical properties.

Acknowledgements

This work was supported by the National Science & Technology Pillar Program of China in the Twelfth Five-year Plan Period (2014BAD06B03), by the National Natural Science Foundation of China (31172144), and by “Project 985” of Jilin University.

References

1. M. G. Meadows, M. W. Butler, N. I. Morehouse, L. A. Taylor, M. B. Toomey, K. J. McGraw and R. L. Rutowski, Iridescence: a functional perspective, J. R. Soc., Interface, 2009, 6, S107–S113.
2. B. Bhushan, Biomimetics: Bioinspired Hierarchical-Structured Surfaces for Green Science and Technology, Springer International, 2nd edn, 2016.
3. J. Y. Sun, B. Bhushan and J. Tong, Structural coloration in nature, RSC Adv., 2013, 3, 14862–14889.
4. J. H. Kim, J. H. Moon, S. Y. Lee and J. Park, Biologically inspired humidity sensor based on three-dimensional photonic crystals, Appl. Phys. Lett., 2010, 97, 103701.
5. A. E. Seago, P. Brady, J. P. Vigneron and T. D. Schultz, Gold bugs and beyond: a review of iridescence and structural colour mechanisms in beetles (Coleoptera), J. R. Soc., Interface, 2009, 6, S165–S184.
6. J. P. Vigneron and P. Simonis, Natural photonic crystals, Phys. B, 2012, 20, 4032–4036.
7. Y. Hiroshi, S. Yuta and S. Masatsugu, Unique light reflectors that mimic the structural colors of tiger beetles, Polym. J., 2014, 46, 212–215.
8. O. Franke, M. Göken, M. A. Meyers, K. Durst and A. M. Hodge, Dynamic nanoindentation of articular porcine cartilage, Mater. Sci. Eng., C, 2011, 31, 789–795.
9. R. A. Crowson. The Biology of the Coleoptera. Academic Press, London, 1981.
10. J. B. Chapin and V. A. Brou, Harmonia axyridis (Pallas), the third species of the genus to be found in the United States (Coleoptera: Coccinellidae), Proc. Entomol. Soc. Wash., 1991, 93, 630–635.
11. S. A. Fabricant, D. J. Kemp, J. Krajíček, Z. Bosáková and M. E. Herberstein, Mechanisms of color production in a highly variable shield-back stinkbug, Tectocoris diopthalmus (Heteroptera: Scutelleridae), and why it matters, PLoS One, 2013, 8, e64082.
12. R. L. Koch, The multicolored Asian lady beetle, Harmonia axyridis: A review of its biology, uses in biological control, and non-target impacts, J. Insect Sci., 2003, 3, 32.
13. H. R. Vincent and T. C. Ring, Encyclopedia of Insects, Academic Press, 2009.
14. A. L. Bezzerides, K. J. McGraw, R. S. Parker and J. Husseini, Elytra color as a signal of chemical defense in the Asian ladybird beetle Harmonia axyridis, Behavioral Ecology and Sociobiology, 2007, 61, 1401–1408.
15. J. Lomakin, Y. Arakane, K. J. Kramer, R. W. Beeman, M. R. Kanost and S. H. Gehrke, Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains, J. Insect Physiol., 2010, 56, 1901–1906.
16. J. F. V. Vincent and U. G. K. Wegst, Design and mechanical properties of insect cuticle, Arthropod Struct. Dev., 2004, 33, 187–199.
17. Y. F. Zhang, S. L. Bai, X. K. Li and Z. Zhang, Viscoelastic properties of nanosilica-filled epoxy composites investigated by dynamic nanoindentation, J. Polym. Sci., Polym. Phys. Ed., 2009, 47, 1030–1038.
18. J. L. Loubet, W. C. Oliver and B. N. Lucas, Measurement of the loss tangent of low-density polyethylene with nanoindentation technique, J. Mater. Res., 2000, 15, 1195–1198.
19. K. Hu, P. Radhakrishnan, R. V. Patel and J. J. Mao, Regional structural and viscoelastic properties of fibrocartilage upon dynamic nanoindentation of the articular condyle, J. Struct. Biol., 2001, 136, 46–52.
20. G. M. Odegard, T. Bandorawalla, H. M. Herring and T. S. Gates, Characterisation of viscoelastic properties of polymeric materials through nanoindentation, Exp. Mech., 2005, 45, 130–136.
21. N. Bouaita, S. J. Bull, J. F. Palacio and J. R. White, Dynamic nanoindentation of some polyolefins, Polym. Eng. Sci., 2006, 46, 1160–1172.
22. B. Mohanty, K. S. Katti, D. R. Katti and D. Verma, Dynamic nanomechanical response of nacre, J. Mater. Res., 2006, 21, 2045–2051.
23. A. Faingold, S. R. Cohen and H. D. Wagner, Nanoindentation of osteonal bone lamellae, J. Mech. Behav. Biomed. Mater., 2012, 9, 198–206.
24. Y. R. Jeng, C. P. Mao and K. T. Wu, Instrumented indentation investigation on the viscoelastic properties of porcine cartilage, J. Bionic Eng., 2013, 10, 522–531.
25. J. B. Pethica and W. C. Oliver, Tip surface interactions in STM and AFM, Phys. Scr., T, 1987, 19, 61–66.
26. S. A. S. Asif and J. B. Pethica, in Thin Films—Stresses and Mechanical Properties VII, Material Research Society Symposium Proceedings, ed. R. C. Cammarata, M. Nastasi, E. P. Busso and W. C. Oliver, 1998, vol. 505, p. 103.
27. S. A. Hayes, A. A. Goruppa and F. R. Jones, Dynamic nanoindentation as a tool for the examination of polymeric materials, J. Mater. Res., 2004, 19, 3298–3306.
28. B. Bhushan, Nanotribology and Nanomechanics II: Nanotribology, Biomimetics, and Industrial Applications, Springer, 2011.
29. R. C. Chang, F. Y. Chen and P. H. Yang, Dynamic mechanical properties of photo resist thin films, J. Mech. Sci. Tech., 2007, 21, 1739–1744.
30. S. A. S. Asif, K. J. Wahl and R. J. Colton, Nanoindentation and contact stiffness measurement using force modulation with a capacitive load–displacement transducer, Rev. Sci. Instrum., 1999, 70, 2408–2413.
31. S. A. S. Asif, K. J. Wahl, R. J. Colton and O. L. Warren, Quantitative imaging of nanoscale mechanical properties using hybrid nanoindentation and force modulation, J. Appl. Phys., 2001, 90, 1192–1200.
32. K. S. Kwan, The role of penetrant structure on the transport and mechanical properties of a thermoset adhesive, PhD thesis, Virginia Polytechnic Institute and State University, 1998.
33. Y. Arakane, J. Lomakin, R. W. Beeman, S. Muthukrishnan, S. H. Gehrke, M. R. Kanost and K. J. Kramer, Molecular and functional analyses of amino acid decarboxylases involved in cutile tanning in Tribolium castaneum, J. Biol. Chem., 2009, 284, 16584–16594.
34. F. Liu, H. Yin and B. Dong, et al., Inconspicuous structural coloration in the elytra of beetles Chlorophila obscuripennis (Coleoptera), Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys., 2008, 77, 981–984.
35. M. Xu, A. E. Seago, T. D. Sutherland and S. Weisman, Dual structural color mechanisms in a scarab beetle, J. Morphol., 2010, 271, 1300–1305.
36. M. Uliana, Chromatic patterns in Coleoptera and their evolution the Chrysolina clade (Coleoptera, Chrysomelidae), PhD thesis, University of Padova, 2009.
37. J. P. Vigneron, M. Rassart, C. Vandenbern, V. Lousse, O. Deparis, L. P. Biro, D. Dedouaire, A. Cornet and P. Defrance, Spectral filtering of visible light by the cuticle of metallic woodboring beetles and microfabrication of a matching bioinspired material, Phys. Rev. E: Stat., Nonlinear, Soft Matter Phys., 2006, 73, Thesis.
38. J. A. Noyes, P. Vukusic and I. R. Hooper, Experimental method for reliably establishing the refractive index of buprestid beetle exocuticle, J. Morphol., 2007, 15, 4351–4358.
39. J. Y. Sun, W. Wu, W. L. Xue, L. Ren, R. Akhtar and J. Tong, Quantitative nanomechanical properties of the cuticle of the multicolored Asian lady beetle using the modulus mapping technique, Curr. Nanosci., 2015, 11, 245–252.
40. J. Y. Sun, J. Tong and J. Zhou, Application of nano-indenter for investigation of the properties of the elytra cuticle of the dung beetle (Copris ochus Motschulsky), IEE Proc.: Nanobiotechnol., 2006, 153, 129–133.
41. J. D. Ferry, Visoelastic properties of Polymers, Wiley, NY, 1980.
42. H. H. Winter and F. Chambon, Analysis of linear viscoelasticity of a crosslinking polymer at gel point, J. Rheol., 1986, 30, 367–382.
43. M. L. Gardel, J. H. Shin, F. C. MacKintosh, L. Mahadevan, P. A. Matsudaira and D. A. Weitz, Scaling of F-actin network rheology to probe single filament elasticity and dynamics, Phys. Rev. Lett., 2004, 93, 188102.
44. M. F. Ashby, Overview No. 80: On the engineering properties of materials, Acta Metall., 1989, 37, 1273–1293.
45. H. J. Kong, E. Wong and D. J. Mooney, Independent control of rigidity and toughness of polymeric hydrogels, Macromolecules, 2003, 36, 4582–4588.
46. J. Lomakin, P. A. Huber, C. Eichler, Y. Arakane, K. J. Kramer, R. W. Beeman, M. R. Kanost and S. H. Gehrke, Mechanical properties of the beetle elytron, a biological composite material, Biomacromolecules, 2011, 12, 321–335.

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