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First published online January 31, 2006
Journal of Experimental Biology 209, 722-730 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02065
Local mechanical properties of the head articulation cuticle in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae)
Evolutionary Biomaterials Group, Department Arzt, Max-Planck-Institute for Metals Research, Heisenbergstr. 3, 70569, Stuttgart, Germany
* Author for correspondence (e-mail: enders{at}mf.mpg.de)
Accepted 27 December 2005
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15-20%
of the cuticle mass) led to an increase in hardness (from 0.1 to 0.49 GPa) and
elastic modulus (from 1.5 to 7.5 GPa). The lipid extraction caused a slight
further hardening (to 0.52 GPa) as well as stiffening (to 7.7 GPa) of the
material. The results are discussed in relation to the mechanical function of
the gula plate.
Key words: desiccation, gula plate, insect cuticle, mechanical property, nanoindentation
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;
Gorb, 2001). It serves
primarily as an exoskeleton that gives the body its shape and stability. The
cuticle is also a barrier against evaporation of water from the body and hence
protects the insect against desiccation
(Locke, 1964;
De Renobales et al., 1991;
Noble-Nesbitt, 1991).
Like most biological materials, cuticle is a fiber composite
(Hepburn and Chandler, 1980).
The fibers mainly consist of chitin, and the matrix is formed by proteins.
Chitin is a natural polymer composed of 300 nm-long and 3 nm-thick nanofibrils
(Vincent, 1980;
Vincent, 2002). Each
nanofibril contains 19 molecular chains
(Vincent, 1980;
Vincent, 2002) running
anti-parallel to one another (i.e. alpha chitin)
(Neville, 1975;
Vincent, 1980). The protein
matrix stabilizes the chitin fibers. It normally contains some amount of water
(in some cuticles it is 90% of the protein matrix mass). The function of
water in the cuticle is largely unknown, but presumably it is separation of
the two main components of the cuticle from each other
(Vincent, 1980).
Arthropod cuticle has a multilayer structure
(Neville, 1975;
Andersen, 1979)
(Fig. 1). It typically consists
of three main layers: epicuticle, exocuticle and endocuticle. The latter two
layers form the procuticle. In some insects, there is a layer of mesocuticle
located between the endocuticle and the exocuticle
(Neville, 1975;
Andersen, 1979;
Noble-Nesbitt, 1991). The
non-chitinous, tanned lipoproteinous epicuticle is the outer layer, which is
very thin and has a relatively high tensile strength
(Bennet-Clark, 1963). The
surface of the epicuticle is coated with wax and lipids. The exocuticle has a
dense chitin-protein structure and may become hard and stiff due to
sclerotization. The endocuticle is usually the thickest region of the cuticle
(Locke, 1964;
Neville, 1975).
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).
In many larvae, the cuticle is soft and colorless. During sclerotization, a
hard and colored integument is formed in most adults
(Fraenkel and Rudall, 1940).
The variation in mechanical properties depends on fractions of the main
constituents (Hepburn and Chandler,
1976).
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;
Chakhvorostov, 2004).
All mechanical investigations were carried out on the head part of the head
articulation called gula, which is normally in contact with the counter
surface of the thorax during the head movement. The gula is a median ventral
plate of the head, in some prognathous insects formed by a sclerotization of
the neck region proximal to the posterior tentorial pits, continuous with the
postmentum or submentum (Snodgrass,
1935). In the present study, the detailed information about the
surface and internal structure of the gula cuticle was obtained, and
mechanical properties (hardness and elasticity modulus) of its superficial
cuticle layers were measured using nanoindentation. The surface morphology and
ultrastructure of the cuticle were studied by means of scanning electron
microscopy (SEM). The gula samples were mechanically tested in the fresh, dry
and chemically treated conditions, in order to identify the influence of
desiccation (dry versus fresh conditions) and removal of an outer wax
layer (dry versus chemically treated conditions). Desiccation rate
measurements were also carried out, in order to obtain information about the
desiccation dynamics of the gula cuticle.
The aim of our study was to understand which structural features of the gula cuticle are responsible for its friction-reducing and wear-resistant properties. The following questions were asked. (1) Do the structure and local mechanical properties of the gula show any features of specialization for friction reduction and wear resistance? (2) How does the liquid content of the gula cuticle influence its mechanics? (3) How do cuticular surface waxes influence hardness and elastic modulus of the gula?
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Scanning electron microscopy
The samples were fixed in 2.5% glutaraldehyde in a phosphate buffer (pH
7.3). The specimens were dehydrated in an ascending series of ethanol and then
critical-point dried. Pieces of the dried material were fractured using a
razor blade. The prepared samples were mounted on holders, sputter-coated with
gold-palladium (10 nm thickness) and examined in a Hitachi S-800 scanning
electron microscope (SEM) at 20 kV.
Transmission electron microscopy
For transmission electron microscopy (TEM), fresh gula was fixed for 12 h
at 4°C in 2.5% glutaraldehyde (in 0.01 mol l-1 phosphate buffer
at pH 7.3) and postfixed for 1 h in 1% osmium tetroxide in phosphate buffer at
2°C (Gorb, 1998). After
washing in distilled water, the preparations were stained for 1 h at 4°C
in 0.1% aqueous uranyl-acetate solution, washed, dehydrated and embedded in a
low-viscosity resin (Spurr,
1969). Ultra-thin sections were picked up on copper grid slots
coated with formvar film (Plano GmbH, Wetzlar, Germany), contrasted with
uranyl acetate and lead citrate, and observed in TEM Philips CM10.
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Nanoindentation
Nanoindentation is a fast and reliable technique for evaluation of local
mechanical properties, such as hardness and elastic modulus, in very small
volumes of material (Oliver and Pharr,
1992; Bhushan and Li,
2003) (Fig. 3).
During the past decade, this method has become an important tool in the
characterization of inorganic materials. During nanoindentation, a
geometrically well-defined diamond pyramid is brought into contact with the
sample surface. The applied load and the displacement (indentation) into the
specimen are recorded simultaneously. The load-displacement curves are used to
determine the hardness and the elastic modulus of the material under
consideration.
Three key parameters of the load-displacement data are necessary for
determination of the hardness and elastic modulus: (1) the peak load
(Fmax), (2) the indentation depth at the peak load
(hmax) and (3) contact stiffness (S)
(Fig. 4)
(Oliver and Pharr, 1992).
The hardness (H) is defined as the ratio between the maximum load
and the contact area, A, generated during the indentation:
 | (1) |
A is given by:
 | (2) |
where k is a geometric constant of the tip (k=24.5 for the Berkovich tip),
and hc is the contact depth
(Fig. 5), which can be defined
as:
 | (3) |
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 | (4) |
where 
is a geometric constant of the indenter
(Oliver and Pharr, 1992;
Hay and Pharr, 2000).
The reduced elastic modulus, Er, for the
specimen/indenter system can be calculated using the equation:
 | (5) |
where Es and Ei are Young's moduli, and vs and vi are Poisson's ratios, for the specimen and for the indenter tip, respectively.
The relationship between the load-displacement data and the experimentally
measured contact stiffness (S) and the contact area (A) is:
 | (6) |
where ß is a constant depending on the tip geometry
(Oliver and Pharr, 1992;
Hay and Pharr, 2000)
(ß=1.034 for the Berkovich tip).
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), the
contact stiffness can be measured continuously during indentation, as a
function of depth. In this method, the indenter is periodically loaded and
unloaded at a frequency of 75 Hz, and hardness and elastic modulus values are
extracted at discrete points of the loading curve. This technique may also be
applied for soft materials, such as biological materials and polymers. Hardness and elastic modulus of the head cuticle in the beetle were measured using a Nano Indenter® SA2 system (MTS Nano Instruments, Oak Ridge, TN, USA) equipped with a Berkovich tip. The sides of the pyramid form an angle of 65.3° with the normal to the base (Fig. 5). Due to the high damping coefficient and high resonant frequency of the indenter, it was possible to perform measurements on materials with low contact stiffness and low damping coefficients.
The beetle head was mounted, using super glue, on a holder with the ventral surface of the gula facing up. We tested 10 heads with 15 indents each (N=150). The overall testing time for one head was 1.0-1.5 h. All indents were made on the top of the hemispherical surface of the gula and were separated from each other by 30-50 µm. The nanoindentation experiments were load-controlled. The maximum displacement was 3 µm. Since the biological samples contain liquid organic substances on the surface, the indenter tip was presumably contaminated during the test. Thus, the tip was cleaned and then recalibrated by indenting in the reference materials, such as Al and SiO2 (fused silica) samples, between the measurements.
Atomic force microscopy
The surface profiles of the samples were investigated by atomic force
microscopy (AFM) (DME, DualScopeTM C-21 with scanner DS 45-40 BIO; Danish
Micro Engineering A/S, Herlev, Denmark) after the indentation test, in order
to estimate the type of deformation behaviour (elastic, plastic,
visco-elastic).
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80 µm. A very thin (1-3
µm) and dense epicuticle can be easily distinguished (Figs
6B,
7A,B). Procuticle is composed
of two layers: 22 µm-thick, dense exocuticle and a thicker endocuticle
(50 µm) that is less dense than the exocuticle. Fibres are oriented
nearly perpendicular to the surface in the superficial part of the exocuticle
(Figs 6B,
7B,C) and parallel to the
surface in the deeper layers of the exocuticle
(Fig. 7B,D) and in the
endocuticle (Fig. 6B).
Desiccation
Measurements of the desiccation rate were carried out to monitor the water
loss in the samples during the indentation time (1.0-1.5 h). In the entire
head, the loss of water in the first hour was 20%
(Fig. 8). After 10 h, the
samples had lost 60% of their initial mass. Desiccation rate of the
dissected gula part was much higher than for the entire head. After
approximately 40 min, the samples had dried out completely, with a mass loss
of 18%.
Mechanical properties
The nanoindentation measurements revealed a strong dependence of the
mechanical behaviour on the preparation conditions (Figs
9,
10). Hardness and elastic
modulus values of the fresh, dry and chemically treated samples were compared
with each other at different indentation depths (250 nm, 500 nm, 1 µm and
1.5 µm). The results of statistical tests show that material properties of
the gula cuticle differ significantly for the fresh, dry and chemically
treated conditions (Table 1).
Only elastic modulus, measured in the dry and chemically treated samples at
indentation depths of 1 and 1.5 µm, did not differ significantly.
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The maximum displacement for the fresh samples was 3 µm, but for the dry and chemically treated samples it was only 2 µm, because the maximum load of the instrument had been reached. High values of hardness and elastic modulus in the first 50 nm of indentation are normally erroneous data caused by the contact formation behaviour between the indenter tip and sample due to the roughness and contamination. The data obtained at this depth were not included for further processing. For all samples, hardness and elastic modulus decreased slowly with indentation depth. However, after 1.7 µm, both parameters dropped rapidly for the dry and chemically treated specimens.
Hardness (H=0.10±0.07 GPa) of the fresh samples was significantly lower than in the dry (H=0.49±0.14 GPa) and the chemically treated (H=0.52±0.15 GPa) states (Fig. 10A) (ANOVA, P<0.0005; Tukey post test, P<0.0005). The difference between hardness values obtained on the dry (H=0.49±0.14 GPa) and chemically treated samples (H=0.52±0.15 GPa) was lower, but also significant (P<0.0005, ANOVA), especially in the range of depths of 200-1000 nm.
The same tendency was observed for the elastic modulus (Fig. 10B). Dry (E=7.50±1.80 GPa) and chemically treated (E=7.70±1.90 GPa) samples are significantly stiffer than fresh ones (E=1.50±0.80 GPa) (ANOVA, P<0.0005). In this case, the extraction of lipids caused a slight increase of the elastic modulus below 1 µm indentation depth. At depths higher than 1 µm, the elastic modulus values of the dry and chemically treated samples were not significantly different.
Atomic force microscopy
The surface profile of the gula cuticle was scanned using AFM after
indentation tests (Fig. 11).
Residual deformation of the cuticle surface after indentation is similar to an
elastic-plastic contact, which is typical for most engineering materials
(Bhushan and Li, 2003). The
deviation of the residual imprint from the perfect pyramidal shape results
from a visco-elastic relaxation, as is expected for soft biological materials
(Wainright et al., 1976; Vincent,
1990; Kohane et al.,
2003). This relaxation behaviour is especially evident on the
lateral surfaces of the imprint, where the material almost goes back to its
original condition (`cushion formation').
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). The
epicuticle is only 1-3 µm thick and consists of numerous very thin layers
running parallel to the surface. The exocuticle of the gula differs from the
layered pattern of regular cuticle, where the orientation of fibres is
parallel to the surface (Neville,
1975; Wainwright et al.,
1976; Vincent,
1990). The fibres in the external layers of the exocuticle are
oriented roughly perpendicular to the surface. A similar fibre orientation has
previously been found in the contact system of attachment pads of grasshoppers
(Gorb and Scherge, 2000);
however, the gula and adhesive pads have very different functions.
Desiccation
As expected, the gula cuticle had a higher desiccation rate than the entire
head. As the samples were ready for testing in 3-5 min, and the test duration
did not exceed 1.5 h, it can be concluded that the indentations were carried
out in an almost native condition of the material. The desiccation
measurements showed that the water content of the dissected gula cuticle was
about 18% mass. This value is comparable with literature data of approximately
12% for hard and tanned types of cuticle
(Vincent and Wegst, 2004).
Mechanical behaviour of the cuticle in different conditions
In general, soft and compliant cuticle contains more water than hard and
stiff cuticle (Vincent and Wegst,
2004). The gula belongs to this type of cuticle and bears a low
proportion of water. In spite of this, the results show that desiccation has a
great influence on the mechanical behaviour of the gula cuticle. After drying,
it becomes about five times harder and stiffer (H=0.49±0.14
GPa; E=7.5±1.8 GPa) than in the fresh state
(H=0.1±0.07 GPa; E=1.5±0.8 GPa). Water content
seems to be a crucial factor for the mechanical properties, as previously
reported by other authors (Andersen et al.,
1996; Arzt et al.,
2002; Enders et al.,
2004; Vincent and Wegst,
2004). While structural changes have been previously studied
during sclerotization (Andersen et al.,
1996), the mechanical effects of drying are unknown in detail. It
is obvious that water makes cuticle soft and compliant. Indentation tests on
different parts of insect cuticle display also a considerable difference in
the mechanical behaviour between fresh (hydrated) and dehydrated materials
(Hillerton et al., 1982;
Arzt et al., 2002;
Enders et al., 2004).
After drying, removal of cuticle lipids caused only small additional changes in the indentation results (a slight increase in hardness and stiffness), especially at a depth below 1 µm. This confirms the hypothesis that the mechanical behaviour of the dry material is determined mainly by proteins and chitin, which could not be removed by the lipid extraction. In order to understand the exact mechanical roles of the two main components (proteins and chitin), further studies are necessary.
Hardness and elastic modulus of the gula
Our previous nanoindentation tests on the excised cuticle of the dung
beetle Geotrupes stercorarius were performed using a Nano Indenter II
(MTS Nano Instruments) without continuous measurement of hardness and elastic
modulus, and therefore contained no information about gradients of the
material properties in the joint cuticle
(Arzt et al., 2002). In the
present study, continuous stiffness mode allowed testing cuticle mechanical
properties gradually at various indentation depths. SEM and TEM images were
used to obtain information about the material structure in the indented
region. The maximum indentation depth in our experiments was set to 3 µm,
and, therefore, mechanical properties were mostly estimated for the
epicuticle, whose thickness was in the range of 1-3 µm. According to models
used for hardness determination in thin metal films
(Jönsson and Hogmark,
1984; Burnett and Rickerby,
1987; McGurck et
al., 1994; Rother and Jehn,
1996; Korsunsky et al.,
1998), the results obtained for gula cuticle could also be
influenced by the underlying layers of the exocuticle. Hardness measured at
certain indentation depth is influenced by underlying layers of the material
located at up to 10 times greater depth
(Bueckle, 1965;
Bhushan and Li, 2003). In the
case of the gula, a 100-300 nm indentation depth can be assumed to
characterize entirely the epicuticle, which is 1-3 µm. At larger indenter
displacements, the data demonstrate combined properties of the epicuticle and
external layers of the exocuticle. This suggestion is true only for the
estimation of hardness. The elastic modulus is influenced by the entire
thickness of multilayered composite material.
According to our results, the gula cuticle can be considered as a softer
material coated by a harder film. At indentation depths larger than 1 µm,
both the e-modulus and hardness tend to decrease. This is an indication of an
increasing influence of the mechanical properties of the exocuticle. With
further penetration, the exocuticle properties become more and more dominant,
and, at displacements of h>2.5 µm, the data reach a certain
plateau (Fig. 10). This shows
clearly that the deeper cuticle layers are softer and more compliant than the
outer layers. The influence of the underlying layers on the mechanical
properties is even more pronounced for the dry and chemically treated samples.
During the drying process, the material shrinks, and thus the indenter tip can
penetrate layers that could not be reached in the fresh state because they
were located considerably deeper within the material. The sharp drop in
hardness values for the dry and chemically treated samples at the depth of 1.7
µm indicates the presence of even softer and more compliant layers in the
exocuticle. This result might also be due to the difference in desiccation
behaviour between the surface layers and the deeper ones due to a lower
density of the material (Locke,
1964; Neville,
1975).
In attempting to compare the present results with the literature data, it
is important to note that most experiments so far were performed on dry or
rehydrated samples, and only a few indentation measurements of the fresh
cuticle were reported. Previous microindentation experiments had relatively
low spatial resolution and/or did not apply continuous stiffness mode to allow
measurement of hardness and elastic modulus as a function of depth. The mean
hardness (0.1-0.52 GPa) and elastic modulus (1.5-7.7 GPa) values obtained in
our study are nevertheless similar to those of other sclerotized cuticles
[Vickers hardness 0.2-0.5 GPa and Young's modulus 1-10 GPa
(Vincent and Wegst, 2004)].
However, obtained hardness values (0.10±0.07 GPa) of the fresh gula
samples are even below this range. This suggests that the present study may
have characterised the cuticle of the insect joint in a true native condition
for the first time.
Comparison with other insects
Other studies on cuticle mechanical properties using indentation have
obtained comparable hardness values on dry cuticle samples of different
insects. Different parts of a dehydrated locust cuticle, measured by means of
a Leitz Miniload hardness tester using a Vickers diamond, had a hardness of
0.24-0.33 GPa (Hillerton et al.,
1982). Dry wing membrane cuticle of the dragonfly Aeshna
cyanea (Odonata, Anisoptera), tested with a nanohardness tester (Hysitron
TriboScope), exhibited hardness of 0.2 GPa
(Kreuz et al., 1999). However,
dry samples of the beetle gula were even harder than any of these cuticles
(H=0.49±0.14 GPa). It is also not surprising that the elastic
modulus values obtained for the dry beetle gula are very high
(E=7.5±1.8 GPa) when compared with those of the dry wing
membrane of the dragonfly Ae. cyanea (E=1.5±0.5 GPa)
(Kreuz et al., 1999). The
reduced modulus for different dehydrated body parts of the dragonfly was
determined from quasistatic nanoindentation experiments (Hysitron Inc.,
Minneapolis, MN, USA;
http://www.hysitron.com/PDF/0501-001.pdf).
The mean values were also much lower (Er=1.5-4.7 GPa) than
for dry samples in the present study.
A recent study on the integument of Drosophila melanogaster during
various developmental stages has been recently performed using a similar
method. The results showed that the thickness of the cuticle and the
development stage of the insect are important factors influencing cuticle
stiffness (Kohane et al.,
2003). The mean Er of 0.41 MPa, 15.43 MPa, and
4.37 MPa were determined by in vivo experiments for the cuticles of
larvae, pupae and adult insects, respectively.
Conclusions and outlook
Our results support the assumption that the head articulation joint
surface, according to its structure, consists of material adapted for
friction- and wear-minimising (fibre orientation in the external layers of the
exocuticle, presence of channels in the cuticle and secretory substances on
the surface) and mechanical properties (hard layer of the epicuticle, with
more compliant underlying layers). Surfaces of engineering bearing systems
show a layered material structure with gradients in mechanical properties
(Barwell, 1979;
Bhushan, 1999). Technical
systems demonstrate also a great difference in the mechanical properties of
materials of both contacting surfaces. It is believed that such a difference
is an adaptation for reduction of friction and wear. Our future investigations
will concentrate on the gula counter surface located in the prothorax, in
order to find a possible correlation between structure and properties of both
counterparts. Friction measurements of both materials using the
microtribotester are currently underway.
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Acknowledgments |
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References |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Andersen, S. O. (1979). Insect cuticle. Annu. Rev. Entomol. 24,29 -61.[CrossRef]
Andersen, S. O., Peter, M. G. and Roepstorff, P. (1996). Cuticular sclerotization in insects. Comp. Biochem. Physiol. 113B,689 -705.[CrossRef]
Arzt, E., Enders, S. and Gorb, S. (2002). Towards a micromechanical understanding of biological surface devices. Z. Metallkunde 93,345 -351.
Barwell, F. T. (1979). Bearing Systems. Oxford: Oxford University Press.
Bennet-Clark, H. C. (1963). The relation between epicuticular folding and the subsequent size of an insect. J. Insect Physiol. 9,43 -46.[CrossRef]
Bhushan, B. (1999). Principles and Applications of Tribology. New York: Wiley & Sons.
Bhushan, B. and Li, X. (2003). Nanomechanical characterization of solid surfaces and thin films. Int. Mater. Rev. 48,125 -164.[CrossRef]
Bueckle, H. (1965). Mikrohaertepruefung und ihre Anwendung, pp.62 -64. Stuttgart: Berliner Union.
Burnett, P. J. and Rickerby, D. S. (1987). The mechanical properties of wear-resistant coatings. I. Modelling of hardness behaviour. Thin Solid Films 148, 41-50.[CrossRef]
Burnett, P. J. and Rickerby, D. S. (1987). The mechanical properties of wear-resistant coatings. II. Experimental studies and interpretation of hardness. Thin Solid Films 148, 51-65.[CrossRef]
De Renobales, M., Nelson, D. R. and Blomquist, G. J. (1991). Cuticular Lipids. In Physiology of the Insect Epidermis (ed. K. Binnington and A. Retnakaran), pp.240 -251. Melbourne: CSIRO.
Enders, S. (2000). Untersuchungen der mechanischen Eigenschaften von spröden Schicht- und Kompaktsystemen durch Deformation kleiner Volumina. Dissertation, Martin-Luther-Universität Halle-Wittenberg, FB Physik.
Enders, S., Barbakadze, N., Gorb, S. N. and Arzt, E. (2004). Exploring biological surfaces by nanoindentation. J. Mater. Res. 19,880 -887.[CrossRef]
Fraenkel, G. and Rudall, K. M. (1940). A study of the physical and chemical properties of the insect cuticle. Proc. R. Soc. Lond. B 129, 1-34.
Gorb, S. N. (1998). The design of the fly adhesive pad: distal tenent setae are adapted to the delivery of an adhesive secretion. Proc. R. Soc. Lond. B 265,747 -752.[CrossRef]
Gorb, S. N. (2001). Attachment Devices of the Insect Cuticle. Dordrecht: Kluwer Academic Publishers.
Gorb, S. N. and Scherge, M. (2000). Biological microtribology: anisotropy in frictional forces of orthopteran attachment pads reflects the ultrastructure of a highly deformable material. Proc. R. Soc. Lond. B 267,1239 -1244.[Medline]
Hay, J. L. and Pharr, G. M. (2000). Instrumented Indentation Testing: ASM Metals Handbook. Materials Park (OH): ASM International.
Hepburn, H. R. and Chandler, H. D. (1976). Material properties of arthropod cuticles: the arthropodial membranes. J. Comp. Physiol. A 109,177 -198.
Hepburn, H. R. and Joffe, I. (1976). On the material properties of insect exoskeletons. In The Insect Integument (ed. H. R. Hepburn), pp.207 -235. Amsterdam: Elsevier.
Hepburn, H. R. and Chandler, D. (1980). Materials testing of arthropod cuticle preparations. In Cuticle Technologies in Arthropods (ed. T. A. Miller), pp.1 -44. New York: Springer-Verlag.
Hillerton, J. E., Reynolds, S. E. and Vincent, J. V.
(1982). On the indentation hardness of the insect cuticle.
J. Exp. Biol. 96,45
-52.
Jensen, M. and Weis-Fogh, T. (1962). Strength and elasticity of locust cuticle. In Biology and Physics of Locust Flight. Philos. Trans. R. Soc. B 245,137 -169.
Jönsson, B. and Hogmark, S. (1984). Hardness measurements of thin films. Thin Solid Films 114,257 -269.[CrossRef]
Kohane, M., Daugela, A., Kutomi, H., Charlson, L., Wyrobek, A. and Wyrobek, J. (2003). Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development. J. Biomed. Mater. Res. A 66,633 -642.[CrossRef][Medline]
Korsunsky, A. M., McGurk, M., Bull, S. J. and Page, T. F. (1998). On the hardness of coated systems. Surf. Coat. Tech. 99,171 -183.[CrossRef]
Kreuz, P., Kesel, A., Kempf, M., Göken, M., Vehoff, H. and Nachtigall, W. (1999). Mechanische Eigenschaften biologischer Materialien am Beispiel Insektenflügel. BIONA Rep. 14,201 -202.
Locke, M. (1964). The structure and formation of the integument in insects. In The Physiology of Insecta (ed. M. Rockstein), pp. 123-213. New York: Academic Press.
McGurk, M. R., Chandler, H. W., Twigg, P. C. and Page, T. F. (1994). Modelling the hardness response of coated systems: the plate bending approach. Surf. Coat. Tech. 68/69,576 -581.[CrossRef]
Neville, A. C. (1975). Biology of the Arthropod Cuticle. Berlin: Springer Verlag.
Noble-Nesbitt, J. (1991). Cuticular permeability and its control. In Physiology of the Insect Epidermis (ed. K. Binnington and A. Retnakaran), pp.240 -251. Melbourne: CSIRO.
Oliver, W. C. and Pharr, G. M. (1992). An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J. Mater. Res. 7,1564 -1583.[CrossRef]
Rother, B. and Jehn, H. A. (1996). Coating and interface characterization by depth-sensing indentation experiments. Surf. Coat. Tech. 85,183 -188.[CrossRef]
Scherge, M., Chakhvorostov, D. and Pöhlmann, K. (2003). Fundamental wear mechanisms of metals. Wear 255,395 -400.[CrossRef]
Shakhvorostov, D., Pöhlmann, K., Scherge, M., Pinto, H., Pyzalla, A. and Enders, S. (2004). Mechanical properties of tribologically modified nanolayers. Tribol. Lubr. Eng. 1,59 -62.
Snodgrass, R. E. (1935). Principles of Insect Morphology. New York, London: McGraw-Hill.
Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium for electron microscopy. J. Ultrastruct. Res. 26,31 -43.[CrossRef][Medline]
Vincent, J. F. V. (1980). Insect cuticle - a paradigm for natural composites. In The Mechanical Properties of Biological Materials. Symp. Soc. Exp. Biol., vol.34 (ed. J. F. V. Vincent and J. D. Currey), pp.183 -210.
Vincent, J. F. V. (1990). Structural Biomaterials. Princeton: Princeton University Press.
Vincent, J. F. V. (2002). Arthropod cuticle - a natural composite shell system. Appl. Sci. Manufact. A 33,1311 -1315.[CrossRef]
Vincent, J. F. V. and Wegst, U. G. K. (2004). Design and mechanical properties of insect cuticle. Arthr. Str. Dev. 33,187 -199.
Wainwright, S. A., Biggs, W. D., Currey, J. D. and Gosline, J. M. (1976). Mechanical design in organisms. Princeton: Princeton University Press.
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