Journal of Insect Physiology 56 (2010) 1901–1906
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Mechanical properties of elytra from Tribolium castaneum wild-type and body color mutant strains Joseph Lomakin a, Yasuyuki Arakane b,c, Karl J. Kramer b,d, Richard W. Beeman d, Michael R. Kanost b, Stevin H. Gehrke a,* a
Department of Chemical & Petroleum Engineering, The University of Kansas, 1530 W. 15th St., Lawrence, KS 66045, USA Department of Biochemistry, Kansas State University, Manhattan, KS 66506, USA Division of Plant Biotechnology, College of Agriculture and Life Science, Chonnam National University, Gwangju 500-757, Republic of Korea d Center for Grain and Animal Health Research, Agricultural Research Service, US Department of Agriculture, Manhattan, KS 66502, USA b c
A R T I C L E I N F O
A B S T R A C T
Article history: Received 5 October 2009 Received in revised form 11 August 2010 Accepted 11 August 2010
Cuticle tanning in insects involves simultaneous cuticular pigmentation and hardening or sclerotization. The dynamic mechanical properties of the highly modiﬁed and cuticle-rich forewings (elytra) from Tribolium castaneum (red ﬂour beetle) wild-type and body color mutant strains were investigated to relate body coloration and elytral mechanical properties. There was no statistically signiﬁcant variation in the storage modulus E0 among the elytra from jet, cola, sooty and black mutants or between the mutants and the wild-type GA-1 strain: E0 averaged 5.1 0.6 GPa regardless of body color. E0 is a power law function of oscillation frequency for all types. The power law exponent, n, averaged 0.032 0.001 for elytra from all genotypes except black; this small value indicated that the elytra are cross-linked. Black elytra, however, displayed a signiﬁcantly larger n of 0.047 0.004 and an increased loss tangent (tan d), suggesting that metabolic differences in the black mutant strain result in elytra that are less cross-linked and more pigmented than the other types. These results are consistent with the hypothesis that black elytra have a balanine-deﬁcient and dopamine-abundant metabolism, leading to greater melanin (black pigment) production, probably at the expense of cross-linking of cuticular proteins mediated by N-b-alanyldopamine quinone. ß 2010 Elsevier Ltd. All rights reserved.
Keywords: Tribolium castaneum Elytra Insect cuticle Cross-linking Tanning Sclerotization Pigmentation Dynamic mechanical analysis Melanin Catecholamine Red ﬂour beetle
1. Introduction Tanning is a complex process that involves hardening (sclerotization) and pigmentation or coloration of insect cuticle (Andersen et al., 1995, 1996; Sherald, 1980; Sugumaran, 1998). Changes in mechanical properties and coloration of the exoskeleton are governed primarily by interactions between cuticular proteins and oxidized catechols (Andersen, 2008; Hopkins and Kramer, 1992). Notably, the resulting cuticle may be quite variable in color and/or stiffness depending on the species, phenotype, body region and chemical composition. There are different measures of material ‘stiffness’, but the Young’s modulus (proportionality between stress and strain when Hooke’s Law holds) is the most commonly used. Young’s moduli of different cuticles can vary by more than two orders of magnitude at nearly constant density, a wider range than for any other common class of materials, whether biological or synthetic (Ashby and Johnson, 2002; Wegst and
* Corresponding author. Tel.: +1 785 864 4956; fax: +1 785 864 4967. E-mail address: [email protected]
(S.H. Gehrke). 0022-1910/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2010.08.012
Ashby, 2004). Cuticle can be soft and elastic, as in the case of a beetle’s dorsal abdominal cuticle, which can have a Young’s modulus of only 62 MPa but a fracture strain of 20% (Reynolds, 1975). However, a cuticular structure can also be very rigid, as in the case for a load-bearing grasshopper mandible, which has an elastic modulus measured by nanoindentation as high as 15,000 MPa (Cribb et al., 2008; Schoberl and Jager, 2006). Cuticle pigmentation may range from almost transparent and colorless to opaque and black (Hopkins and Kramer, 1991; Wittkopp and Beldada, 2009). In the red ﬂour beetle, Tribolium castaneum, a range of brown to black naturally occurring phenotypes has been reported (Wappner et al., 1995). The beetle’s color is believed to be derived from the particular combination of brown and black pigments. The black pigments are melanins formed from polymerization reactions of oxidized catecholamines. The reactions are initiated by oxidation of dopamine to its quinone (Czapla et al., 1988; Hopkins et al., 1984). Signiﬁcantly, the melanin pathway may be blocked by N-acylation of dopamine with acetate or b-alanine (Roseland et al., 1987; Kramer et al., 1989). In this case, the N-acyl derivatives N-acetyldopamine (NADA) and N-b-alanyldopamine (NBAD) become precursors of quinone tanning agents (Hopkins and
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Kramer, 1991; Hopkins and Kramer, 1992; Kramer et al., 2001). The N-acylated precursors can lead to the production of brown pigments. Hence, it is theorized that N-acylation and oxidative enzyme activities in vivo control the relative production of brown and melanic pigments, and that this balance determines the body color phenotypes of insects such as T. castaneum (Wittkopp and Beldada, 2009). The body color of insects has been implicated in sexual selection (Czapla et al., 1990; Lewis and Austad, 1994; Sokoloff et al., 1960), but coloration is also linked to survival. For example, increased melanization has been shown to increase the resistance of Drosophila strains to dehydration (Parkash et al., 2008). As a model tissue for use in the study of a relationship between color and mechanical properties, we have chosen the elytra or modiﬁed forewings of the red ﬂour beetle, T. castaneum. These composite dorsal appendages are cuticle-rich, serve as body covers to protect beetles against mechanical stress and dehydration, and are inherently important to insect viability, helping beetles to adapt to a variety of environments and stresses (Chen et al., 2007a,b). However, with the exception of basic puncture tests (Roseland et al., 1987), the mechanical properties of elytra from Tribolium wildtype strains and color body mutants remain unexplored. Since pigmentation and protein modiﬁcation reactions share common enzymes and organic precursors (Arakane et al., 2009; Kramer et al., 2001), we hypothesized that coloration changes may also indicate differences in cuticle mechanical properties. Thus the dynamic mechanical properties of elytra from several Tribolium body color phenotypes were determined for comparison with those of elytra from the GA1 wild-type strain. Dynamic mechanical analysis (DMA) can distinguish between elastic and viscous contributions to deformation resistance and thus can be utilized to evaluate the extent of cross-linking among the color phenotypes (Lomakin, 2009; Lomakin et al., submitted for publication). Hence, we hypothesize that DMA can help to establish correlations between a sample’s mechanical properties and the extent of protein cross-linking and pigment production.
removed with tweezers, allowed to equilibrate with the lab atmosphere for 10 h and tested. The specimens were then mounted with epoxy to strips of hard plastic that were clamped in the instrument grips. Devcon 1.5-Ton quick-setting epoxy cement was allowed to dry for 1 h prior to testing; ﬁxing such samples without damage was conﬁrmed optically and by reproducibility of results. The epoxy and plastic strips were conﬁrmed to be stiff enough not to compromise results by comparison with results obtained from mounting and testing in a comparable fashion both plastic and aluminum strips of known properties. The elytra were tested whole, without being cut into a particular test shape beforehand to avoid introducing sample defects. Geometric uniformity was maintained by clamping such that a rectangular portion of the elytron remained between the grips. Once the samples were mounted, the mechanical properties were determined via both dynamic strain and frequency sweeps (Lomakin, 2009). The strain-independent linear viscoelastic region determined by strain sweeps was maintained past a strain of 0.1% for all sample types tested; thus, a strain of 0.1% was used for all frequency sweep experiments. Frequency sweeps were performed from 0.1 to 600 rad/s to measure the storage (or elastic) modulus E0 , the loss (or viscous) modulus E00 and the ratio E00 /E0 , also known as the loss tangent, tan d. The variation of the storage modulus E0 with strain wave oscillation frequency v was ﬁt to the power law model E0 vn (r2 > 0.95) between 10 and 100 rad/s (Arakane et al., 2009; Kong et al., 2003; Winter and Chambon, 1986). The modulus calculations used an engineering or nominal stress based on the initial cross-sectional area of the sample, approximated as the product of the elytra’s centerline width measured by calipers (found to vary only by 20%) and its centerline thickness that was measured under an optical microscope with a digital ﬁlar micrometer. These dimensions were conﬁrmed in comparison to SEM measurements in the case of the wild-type strain (Lomakin, 2009). It was not feasible to measure the exact dimension of each sample tested, so the average cross-sectional area of a population was used in the stress calculations.
2. Materials and methods 3. Results 2.1. Materials Elytra from T. castaneum wild-type (GA1 strain) and naturally occurring body color mutant strains jet, cola, sooty and black (Sokoloff et al., 1960) were used in the experiments. Insects were reared at the USDA-ARS Center for Grain and Animal Health Research in Manhattan, KS, under standard conditions on a diet of organic wheat ﬂour fortiﬁed with 5% brewer’s yeast. Pupae were shipped overnight in capped vials to the University of Kansas, where they were allowed to ecdyse and mature into adults. Adults 7–10 days post-eclosion were considered to be fully tanned and were subsequently sacriﬁced and tested as noted below. 2.2. Methods A TA Instruments RSAIII dynamic mechanical analyzer was used to perform all of the mechanical measurements. The instrument utilizes a direct-drive linear motor to apply a strain and a force transducer to measure the resulting force (Macosko, 1994). This combination accounts for the high force resolution of the instrument, down to 104 N, and a strain resolution down to 1 nm, capabilities which enabled acquisition of reproducible results on these small specimens. A frequency range of 0.1– 600 rad/s was used for dynamic mechanical analysis. Prior to mechanical testing, the beetles were immobilized by chilling at 20 8C for 30 min (no signiﬁcant differences were seen in comparison to removal of elytra from live insects). Elytra were
The Tribolium body color phenotypes examined in this study are shown in Fig. 1, where the color mutants are displayed from lightest to darkest with the wild-type appearing lightest and most reddish-brown. An elytron from the wild-type strain is not a homogeneous structure. It consists of both thick dorsal and thinner ventral cuticular layers that are connected by small beam-like structures called trabeculae, with the tracheae positioned between the two layers (Chen et al., 2007a,b; Arakane et al., unpublished data). Furthermore, there are several ribs that run in parallel along the length of the dorsal side of the elytron, and also shallow circular cavities containing setae on the dorsal surface (Chapman, 1998; Hepburn, 1976). Elytra from the body color mutants, when examined individually via light microscopy, exhibit the same morphology, differing only in the degree of pigmentation as shown in Fig. 2. Dynamic mechanical experiments were done to determine the storage modulus E0 and the loss modulus E00 as a function of oscillation frequency and strain. E0 is a measure of the elastically recoverable deformation energy and hence is also known as the (dynamic) elastic modulus. In contrast, E00 is a measure of viscous energy dissipation (dampening) and hence is also known as the viscous modulus. The ratio E00 /E0 is known as the ‘‘loss tangent’’ or simply tan d, where d is the phase angle between sinusoidally applied stress and strain. For materials such as the elytra where E0 E00 , E0 is approximately equal to the Young’s modulus obtained from the slope of simple stress–strain measurements at the same
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Fig. 1. Macroscopic view of Tribolium body color mutant strains. Cola, sooty, jet: recessive, cause darkening of cuticle. Black: incompletely recessive, causes darkening of cuticle. Strains are pictured in order of lightest to darkest. All beetles pictured are homozygous. Ventral (left) and dorsal (right) views of each mutant are shown.
Fig. 2. Light micrographs of the ventral side of elytra from Tribolium body color mutants. Elytra from the body color mutants exhibit a similar morphology, varying only in degree of pigmentation. Black, white and green arrows indicate ribs, tracheae and hollow pits containing setae, respectively. These structures are located on or near the dorsal surface, but are easily visible ventrally through the translucent cuticle. Bar = 0.1 mm.
strain rate (Fried, 2003; Sperling, 2006). Hence, E0 is a measure of the stiffness of the elytra. The storage moduli at a reference frequency of 1 Hz (6.28 rad/s) for the variously colored elytra are listed in Table 1. The values vary from 4.4 to 5.8 GPa, which are values comparable to those of synthetic plastics like polystyrene (Fried, 2003; Sperling, 2006). Within the 95% conﬁdence intervals, there is no signiﬁcant variation in E0 among the differently colored elytra. The storage modulus for an individual elytron varied between 3 and 8 GPa with the 95% conﬁdence intervals about 30% of the mean values. Most of this measured variability is attributed to small differences in hydration levels and sample geometry as opposed to inherent differences in actual material properties among individual specimens. The variation of the storage modulus of the elytra with oscillation frequency at a strain of 0.1% was also determined. Representative frequency sweeps obtained by dynamic mechanical testing of elytra from different color mutant strains are shown in Fig. 3. The storage modulus E0 shows marked dependence on oscillation frequency at frequencies below 1 and above 200 rad/s. However, over the bulk of the frequency range tested, E0 displays only a weak power law dependence on oscillation frequency. The exponent of this power law dependence is an
indicator of the extent of cross-linking within a sample (Kong et al., 2003; Lomakin, 2009; Winter and Chambon, 1986). Although there are slight differences in the magnitude of E0 for reasons noted above, all of the elytra from the color mutant strains clearly have a similar frequency dependence as elytra from the wild-type strain with the exception of the black specimens, whose sweep crosses over those of sooty and cola. The characteristic
Table 1 Mechanical properties from frequency sweeps at 0.1% strain of Tribolium elytra.* Sample
E0 at 1Hz (MPa)
E0 frequency exponent (n)
tan d at 1 Hz
Wild-type Cola Sooty Jet Black
5.8 2.1 4.4 1.1 5.2 0.9 5.5 2.5 4.6 1.5
0.031 0.002 0.033 0.001 0.030 0.003 0.032 0.002 0.047 0.004
0.078 0.005 0.081 0.006 0.075 0.008 0.081 0.005 0.151 0.006
The values reﬂect the 95% conﬁdence intervals.
Fig. 3. Representative frequency sweeps at 0.1% strain of Tribolium elytra from wildtype and body color mutants, demonstrating the dependence of storage modulus (E0 ) on oscillation frequency.
J. Lomakin et al. / Journal of Insect Physiology 56 (2010) 1901–1906
Fig. 4. Representative frequency sweeps at 0.1% strain of Tribolium elytra from wildtype and body color mutants, demonstrating the dependence of E00 /E0 ratio (tan d) on oscillation frequency.
frequency exponents, n, for the Tribolium wild-type and color mutant elytra are summarized in Table 1. Wild-type and color mutant elytra all display statistically indistinguishable frequency exponents of about 0.030, with the exception of black. Black elytra exhibit a much more pronounced frequency dependence, indicated by an n value of 0.047 0.004. During the frequency sweeps, the loss moduli and tan d were also measured. Representative tan d curves as a function of frequency are shown in Fig. 4 and the mean values of tan d at 1 Hz are given in Table 1. Values of tan d are less variable than either E0 or E00 . This result indicates that the major contributor to uncertainty in E0 and E00 is the measurement of the cross-sectional area (and the fact that the crosssectional area is not perfectly rectangular). This conclusion is drawn because tan d is a ratio in which the cross-sectional areas cancel out, and hence it is independent of sample dimension measurement unlike the moduli. The data also suggest that variability in hydration was not a signiﬁcant problem, as this would also have affected the magnitude of tan d. The black elytron has a signiﬁcantly larger tan d across the recorded frequency range than that of any of the other color variants. At the representative frequency of 1 Hz, the black elytron displays a distinct tan d of 0.151 0.006, nearly twice that of the elytra from sooty, jet or cola. The latter three color mutant elytra exhibit a tan d statistically indistinguishable from each other and from the wild-type, ranging from 0.073 to 0.086. The greater viscous dampening of the black elytron relative to the others, as demonstrated by a greater tan d, is consistent with its greater power law frequency exponent n. 4. Discussion The cuticular tanning process involves simultaneous pigmentation, cuticular protein cross-linking and dehydration. Pigmentation polymers (Moses et al., 2006) and cross-links have demonstrated potential to modulate the physical properties of biological materials (Hopkins and Kramer, 1992). DMA provides a direct measure of fundamental material mechanical properties known and understood for many types of materials, which is an advantage over tests such as puncture resistance that may not yield parameters easily correlated with other types of mechanical tests. Since DMA tests impart only small strains and stresses, irreproducibility due to variability in
sample preparation, mounting and testing is minimized. Furthermore, two important parameters, tan d and the E0 frequency exponent n, obtained from DMA are independent of dimensional measurement and thus can be determined with a high level of accuracy. The value of n is a highly reproducible parameter, much more so than the E0 modulus itself, which depends on an accurate measurement of a sample’s dimensions. The variability observed in n is under 10%, whereas that in E0 is close to 50%. tan d is also highly reproducible. Although n varies with frequency, a single value of n is found to ﬁt one decade or more of frequencies with an r2 value of >0.98. The ﬁrst signiﬁcant result reported here was that the stiffness of the Tribolium elytron as quantiﬁed by the storage modulus E0 is independent of pigmentation. It may be that Tribolium adults need to have a value of E0 of several gigapascals to survive and that mutations that reduce the stiffness notably produce adults that are less viable. This is an observation consistent with our work on the manipulation of enzymatic pathways in Tribolium by RNA interference. When the cuticle does not notably tan, the insects do not survive to maturity (Arakane et al., 2009). The frequency sweep experiments on fully tanned wild-type and color mutant Tribolium elytra yield both the storage (E0 ) and the loss (E00 ) moduli as a function of the imposed oscillation stress frequency (v). The dependence of these moduli, as well as their ratio (tan d), in polymeric materials is in general a function of the relaxation modes available to the constitutive polymeric chains (Ferry, 1980). The storage modulus (E0 ) for an entangled but uncross-linked polymeric material is expected to increase notably with frequency, while E0 is expected to become frequencyindependent for a well cross-linked material at frequencies below 100 rad/s (Gardel et al., 2004; Koenderink et al., 2006; Kong et al., 2003). Experimentally, the storage modulus of lightly cross-linked biopolymers is observed to have a weak power law frequency dependence with a frequency exponent n about 0.1–0.3. The frequency exponent n decreases with increased cross-linking, which inhibits polymer relaxation in this frequency range (Hoffman et al., 2005; Kong et al., 2003). For alginate gels, n decreases from 0.94 to 0.01 as the cross-linking is increased. Hence, the small values of n observed for all elytral samples are consistent with those of cross-linked materials. The E0 frequency dependence of the color mutant elytra, with the notable exception of that of black, was found to be indistinguishable from that of the wild-type elytra. In contrast, the black elytral frequency exponent is over 50% greater than that of the wild-type. This result is consistent with the interpretation that black elytra have an overabundance of melanic pigments that may not be cross-linked but that have relatively high molecular weights, and that these melanic pigments are produced at the expense of cuticular protein cross-linking mediated through NBAD quinone. This conclusion is further supported by the observed E00 /E0 ratio for the elytra. This ratio, tan d, decreases with frequency, indicating that viscous effects become increasingly signiﬁcant at the lower end of the frequency range tested. In this range, entangled but uncross-linked polymeric chains are able to ﬂow past each other at sufﬁciently long time scales, which is observed as softening, whereas cross-links limit such motion. Furthermore, uncross-linked materials exhibit a relatively high viscous dissipation and hence a higher tan d throughout the frequency range. Cross-linking reduces the magnitude of tan d and hence tan d can be used as an indicator of the molecular interconnectivity in polymeric materials (Ferry, 1980). The tan d of the black elytron was determined to be over 80% greater than that of the wild-type, cola, sooty or jet elytron. This result is indicative of the higher dampening ability of the black elytron and leads to the conclusion that cuticle in the black elytron is less cross-linked than that in the wild-type even though both elytra exhibit the same degree of
J. Lomakin et al. / Journal of Insect Physiology 56 (2010) 1901–1906
stiffness (E0 ). However, the relative importance of pigmentation and cross-linking to the magnitude of E0 is not speciﬁcally established. This analysis also does not directly distinguish between physical cross-linking and covalent cross-linking. In other work, however, we argue that these dynamic mechanical results are best interpreted in terms of the catechol oxidation processes (Arakane et al., 2009; Lomakin, 2009; Lomakin et al., submitted for publication). Thus, dynamic mechanical analysis demonstrates that the black elytron is distinctly different from those of the other color mutants. Assuming that cuticle pigmentation and protein cross-linking are a direct result of catechol oxidation reactions, these results support the hypothesis that the black strain synthesizes catecholic metabolites that produce uncross-linked, high molecular weight melanic pigments at the expense of protein cross-links. It is possible that dopaminequinone, indole quinones and other short indole quinone polymers produced by the oxidation of dopamine during cuticle tanning may also cross-link cuticular proteins, although much less efﬁciently than the reactive quinones produced from N-acetyldopamine or N-b-alanyldopamine. The other color mutations, on the other hand, have no signiﬁcant effect on the mechanical or structural properties of the elytra. This indicates that the body color variations of cola, jet and sooty could be a matter of degree rather than of kind. Such distinctions could not be drawn based on observation of the coloration alone and demonstrates the value of DMA toward understanding differences in the metabolic pathways of these species variations. The pigmentation chemistry of the black mutant, however, is more extensive relative to that in the other color mutants and diminishes the cross-linking chemistry in the former. In summary, while the jet, sooty and cola Tribolium body color mutant elytra were found to be mechanically identical to the wildtype, the black elytron was found to be unique. Dynamic mechanical analysis showed that the black elytron, while being comparable in stiffness, has a greater degree of viscous energy dissipation. The increased frequency dependence of the storage modulus and tan d both suggest that the black body color mutation leads to a cuticle that is less cross-linked. This mechanical behavior suggests that the natural production of melanin pigments within the elytron comes at the expense of cuticular protein cross-linking. Like black, the sooty mutation can be rescued by injection of balanine (Roseland et al., 1987). However, the exact genetic and biochemical basis of the jet, sooty and cola body color variants is unknown. The fact that their tan d values resemble wild-type rather than black might indicate that there are alternative independent pathways that can lead to a black body pigmentation. Alternatively, it may be that incomplete suppression of b-alanine production is sufﬁcient to promote a noticeable darkening of the cuticle, whereas compete suppression of this pathway might be required before an effect on the dynamic mechanical properties becomes measurable. The black mutation used in this work is known to be deﬁcient of aspartate-1-decarboxlyase transcripts (Arakane et al., 2009), but the other dark body mutants might represent only partial knockouts of the respective genes, resulting in different degrees of body pigmentation. Cuticular pigmentation undoubtedly serves important functions beyond those involving the physical properties of the exoskeleton and the range of what is normal for cuticular coloration is almost limitless. Camouﬂage is probably the most important of these other functions, which also include mate recognition, regulation of heat absorption and water content and possibly others. Hence, DMA has shown that the elytra of the black strain are structurally different from those of the other color mutants or the wild-type strain. Further work on the black elytron may be valuable in determining the genes necessary for cuticle pigmentation and/or sclerotization as well as in deconvoluting the reactions responsible
for cuticular color changes from those responsible for cuticle hardening. Previously, NMR spectroscopic data were presented that was consistent with melanin and b-alanine being more abundant in the black and wild-type strains, respectively (Kramer et al., 1989). We have more recently reported that the black phenotype results from a deﬁciency in b-alanine, due to a low expression of aspartate1-decarboxylase and an increased amount of dopamine that is oxidized to produce melanin (Arakane et al., 2009). More generally, this work demonstrates the utility of dynamic mechanical analysis for probing the structure of biological composites such as the insect elytron. Certain parameters of such analysis such as the frequency exponent n and tan d show great reproducibility and sensitivity to material structural changes as they do not depend upon the accuracy of sample dimension measurements. Further application of DMA to natural and engineered tissues such as the cuticle phenotypes examined in this study will be useful in establishing structure–function relationships within other important biopolymer composites. Acknowledgements We thank Patricia A. Huber for assistance in preparation of this manuscript. This material is based upon work supported by the National Science Foundation under Grant No. IOS 0726412. Any opinions, ﬁndings, and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reﬂect the views of the National Science Foundation. This is contribution 10-068-J from the Kansas Agricultural Experiment Station. Mention of trade names or commercial products in this publication is solely for the purpose of providing speciﬁc information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis, without regard to race, color, national origin, religion, sex, age, marital status or handicap. References Andersen, S.O., 2008. Quantitative determination of catecholic degradation products from insect sclerotized cuticles. Insect Biochemistry and Molecular Biology 38, 877–882. Andersen, S.O., Hojrup, P., Roepstorff, P., 1995. Insect cuticular proteins. Insect Biochemistry and Molecular Biology 25, 153–176. Andersen, S.O., Peter, M.G., Roepstorff, P., 1996. Cuticular sclerotization in insects. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology 113, 689–705. Arakane, Y., Lomakin, J., Beeman, R.W., Muthukrishnan, S., Gehrke, S.H., Kanost, M.R., Kramer, K.J., 2009. Molecular and functional analyses of amino acid decarboxylases involved in cuticle tanning in Tribolium castaneum. Journal of Biological Chemistry 284, 16584–16594. Ashby, M.F., Johnson, K., 2002. Materials and Design: The Art and Science of Material Selection in Product Design. Butterworth-Heinemann, Boston, MA. Chapman, R.F., 1998. Insects: Structure and Function, 4th ed. Cambridge University Press, New York, NY. Chen, J., Dai, G., Xu, Y., Iwamoto, M., 2007. Optimal composite structures in the forewings of beetles. Composite Structures 81, 432–437. Chen, J., Ni, Q.Q., Xu, Y., Iwamoto, M., 2007. Lightweight composite structures in the forewings of beetles. Composite Structures 79, 331–337. Cribb, B.W., Stewart, A., Huang, H., Truss, R., Noller, B., Rasch, R., Zalucki, M.P., 2008. Insect mandibles-comparative mechanical properties and links with metal incorporation. Naturwissenschaften 95, 17–23. Czapla, T.H., Hopkins, T.L., Kramer, K.J., 1990. Cuticular strength and pigmentation of ﬁve strains of adult Blattella germanica (L.) during sclerotization: correlations with catecholamines, ß-alanine and food deprivation. Journal of Insect Physiology 36, 647–654. Czapla, T.H., Hopkins, T.L., Kramer, K.J., Morgan, T.D., 1988. Diphenols in hemolymph and cuticle during development and cuticle tanning of Periplaneta americana (L.) and other cockroach species. Archives of Insect Biochemistry and Physiology 7, 13–28. Ferry, J.D., 1980. Viscoelastic Properties of Polymers, 3rd ed. Wiley, New York, NY. Fried, J.R., 2003. Polymer Science and Technology, 2nd ed. PrenticeHall, Upper Saddle River, NJ. Gardel, M.L., Shin, J.H., MacKintosh, F.C., Mahadevan, L., Matsudaira, P.A., Weitz, D.A., 2004. Scaling of F-actin network rheology to probe single ﬁlament elasticity and dynamics. Physical Review Letters 93, 188102–188105.
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