J. exp. Biol. (1982), 96, 45-52 45 With 6 figures Printed in Great Britain ON THE INDENTATION HARDNESS OF INSECT CUTICLE BY J. E. HILLERTON,* S. E. REYNOLDSf AND J. F. V. VINCENT* • Biomechanics Group, Department of Zoology, University of Reading, WhiteknighU, Reading RG6 2AJ, and \ School of Biological Sciences, University of Bath, Claverton Down, Bath BAz 7A Y (Received 21 January 1981) SUMMARY The indentation hardness of locust cuticle was measured using a Vickers diamond applied to the surface of the cuticle. A number of areas of cuticle showed similar hardness values, approximately 24 kg mm~ ! , for the de- hydrated cuticle but the mandibles (298 kg mm 4 ) and the dorsal meso- thorax (33-3 kg mm 4 ) were harder. The mandibles have localized areas of hardness which are related to their function. It is suggested that the hardness of the dorsal thorax may be related to its function as an energy storing structure in flight. The measured hardness of cuticle depends on the hydration of the material but relative differences between cuticles from different parts of the locust persist whether tested fresh or after dehydration. Hardness is correlated with the dihydroxyphenol content of cuticle (unlike stiffness), conversely it is not correlated with the hydrophobicity of the matrix proteins (unlike stiffness). It is emphasized that hardness and stiffness are very different mechanical properties. INTRODUCTION Colloquially, 'hardness' and 'stiffness' are often taken to be synonymous. For the engineer, the two are very different (Vincent & Hillerton, 1979). At the simplest level, stiffness is a measure of resistance to recoverable deformation and may be estimated in a non-destructive test (at least, at small deformations). The stiffness of composite materials, the class of material to which insect cuticle belongs, is well understood in the tensile mode of deformation and partially understood in the com- pressive mode (Harris, 1980). By contrast, the measurement of hardness involves a more complex mode of deformation, which is fairly well understood in ceramics and metals (Kelly, 1973) but which is a very complex and not well undejitood quantity in composites (Vincent, 1980). To measure hardness a body of well-defined geometry (a sphere, cone or pyramid) is pressed into the material under test with a known load for a known time. The size of the indentation which this leaves in the material is a function of its fracture properties and of its viscous and plastic com- ponents. In a ceramic or metal there is little or no viscous, i.e. time-dependent, component to the deformation, and the elastic limit (the deformation at which the
Transcript
J. exp. Biol. (1982), 96, 45-52 4 5With 6 figures
Printed in Great Britain
ON THE INDENTATION HARDNESS OFINSECT CUTICLE
BY J. E. HILLERTON,* S. E. REYNOLDSf AND J. F. V. VINCENT*
• Biomechanics Group, Department of Zoology, University of Reading,WhiteknighU, Reading RG6 2AJ, and
\ School of Biological Sciences, University of Bath,Claverton Down, Bath BAz 7A Y
(Received 21 January 1981)
SUMMARY
The indentation hardness of locust cuticle was measured using a Vickersdiamond applied to the surface of the cuticle. A number of areas of cuticleshowed similar hardness values, approximately 24 kg mm~!, for the de-hydrated cuticle but the mandibles (298 kg mm 4 ) and the dorsal meso-thorax (33-3 kg mm 4 ) were harder. The mandibles have localized areasof hardness which are related to their function. It is suggested that thehardness of the dorsal thorax may be related to its function as an energystoring structure in flight. The measured hardness of cuticle depends onthe hydration of the material but relative differences between cuticles fromdifferent parts of the locust persist whether tested fresh or after dehydration.Hardness is correlated with the dihydroxyphenol content of cuticle (unlikestiffness), conversely it is not correlated with the hydrophobicity of thematrix proteins (unlike stiffness). It is emphasized that hardness and stiffnessare very different mechanical properties.
INTRODUCTION
Colloquially, 'hardness' and 'stiffness' are often taken to be synonymous. For theengineer, the two are very different (Vincent & Hillerton, 1979). At the simplestlevel, stiffness is a measure of resistance to recoverable deformation and may beestimated in a non-destructive test (at least, at small deformations). The stiffness ofcomposite materials, the class of material to which insect cuticle belongs, is wellunderstood in the tensile mode of deformation and partially understood in the com-pressive mode (Harris, 1980). By contrast, the measurement of hardness involvesa more complex mode of deformation, which is fairly well understood in ceramicsand metals (Kelly, 1973) but which is a very complex and not well undejitoodquantity in composites (Vincent, 1980). To measure hardness a body of well-definedgeometry (a sphere, cone or pyramid) is pressed into the material under test witha known load for a known time. The size of the indentation which this leaves in thematerial is a function of its fracture properties and of its viscous and plastic com-ponents. In a ceramic or metal there is little or no viscous, i.e. time-dependent,component to the deformation, and the elastic limit (the deformation at which the
46 J. E. HILLERTON, S. E. REYNOLDS AND J. F. V. VINCENT
material yields) is often less than i-o% strain. Beyond this limit the material deformsplastically, i.e. most of the strain energy is dissipated within the material. On removalof the load the ceramic or metal recoils elastically by only a very small amount andthus the remaining deformation is a function of yield stress and plastic flow. Thisresidual deformation is taken to be an index of hardness.
The response of a composite such as insect cuticle is very different. The yieldstrain is much higher-up to 10%, often more-and the mechanical behaviour ofthe material is more complex since it is viscoelastic (i.e. the time for which thematerial is loaded is important, as is the length of time between removing the indentorand measuring the size of the indentation). Additional complications are that thecuticle is unlikely to be mechanically isotropic and will have complex plasticbehaviour.
Notwithstanding these difficulties this paper reports that the measurement ofhardness of insect cuticle with a micro-hardness indentor (a diamond pyramidmounted on a pneumatic loading device in a microscope objective) is repeatable andyields results which have both mechanical and biological significance.
MATERIALS AND METHODS
Cuticle samples were taken from adult Schistocerca gregaria or, for experimentson mandibles, Locusta migratoria migratorioides. Surface hardness of the cuticlewas measured using one of two instruments which gave comparable results. In allcases a Vickers diamond with an applied load of 5 g was used to produce a pyramidalindentation in the cuticle sample. Preliminary experiments confirmed that thehardness measured is load-dependent, as shown for other biological materials byRyge, Foley & Fairhurst (1961). With the Leitz (Wetzler) Miniload hardness tester,the time of loading was 15s and a further 45 s was allowed after removal of the loadbefore the indentation was measured. With the Vickers Micro-hardness tester(M 12 a), the instructions were followed and the load applied for 30 s and theindentation measured 30 s after removal of the load. The diagonals of the pyramidalindentation were measured. Only indentations with diagonals approximately equalwere scored. Vickers hardness numbers (VHN) were calculated according to theformula
VHN = i854P/d2(kgmm-2),
where P is the load in grams and d is the mean length of the diagonals of the indentationin micrometres.
Flat specimens of cuticle were mounted on a flat brass plate using double-sidedSellotape. Mandibles, which are difficult to mount and keep stable, were supportedby Bfesticine, as recommended by Vickers, on aluminium plates. Fresh specimenswere kept in a moist environment to prevent drying before testing. Other specimenswere dried for 24 h in a vacuum desiccator over phosphorus pentoxide, or in thecase of mandibles they were dried in air at room temperature for 48 h before testing.No results from fresh mandibles were obtained as the residual water in the cuticlefills the indentations before they can be measured.
Prior to photography the leg and mandibles were rinsed with methanol to remova
On the indentation hardness of insect cuticle 47
surface debris. They were then mounted with plasticine and photographed with aTessovar photomicroscope.
Cuticles were extracted with 2:1 (v: v) methanol: chloroform at 60 °C, M-NaOHat 100 °C or M-HC1 at 100 °C for 24 h to determine the stability of the impregnatingphenols.
Hydrophobicity was calculated by the method of Bigelow (1967) from the aminoacid analyses of Schistocerca cuticle by Andersen (1971).
RESULTS AND DISCUSSION
The various areas of Schistocerca cuticle tested showed similar values for hardness(Table 1) except for the mandible and mesothorax which are both harder. Both ofthese structures have particularly specialized functions which might be related tohardness.
The mesothorax is used as a resonant box for the flight system of locusts (Pringle,1957). The stiffness required for the cuticle to act as an effective and elastic store ofstrain energy, without undue deformation, will result in a (notional) minimumvalue for the hardness of stiff cuticle. The high values of hardness observed suggestthat the cuticle has a high elastic limit before plastic deformation occurs (see Fig. 1and its legend). This will allow the cuticle of the mesothorax to be subjected tomore stress without danger of failure and thus to store more strain energy duringeach wing cycle. Increasing the hardness of the cuticle may therefore be a way ofincreasing the efficiency of the click mechanism.
Mandibles have a particular function in the mastication of food and it is notsurprising that 'teeth' are hard (Waters, 1980). The value in Table 1 is an averageover the surface of the mandibles. Further measurements on specific areas of theincisors of Locusta show that hardness is localized and that the specially hardenedareas (A and G in Fig. 2) may be related to the mode of action of the incisor (Hillerton,1980). They are twice as hard as the other areas of the mandibles tested (Table 2).The hardness determined is comparable with that found for Schistocerca mandibles(Table 1). Because areas A and G are hardened they can act as cutting edges for thescissor-like action of the incisors (Hillerton, 1980). They appear to be self-sharpening
Table 1. Hardness (VHN: Vickers hardness numbers) of various regions of dehydratedcuticle from Schistocerca: adult insects, 12 days since ecdysis
(Measured using the Leitz Miniload hardness tester.)
Cuticle
TibiaFemurHead capsuleMandibleDorsal mesothoraxAbdominal tergitesCornea of compound eyeOcellus
J. E. HILLERTON, S. E. REYNOLDS AND J. F. V. VINCENT
la
Strain
Fig. i. Stress-strain curves for two cuticles (a and b) with the same stiffness but differentelastic limits (elj and elt). Cuticle b has the higher elastic limit so it will, for the same force,show less plastic deformation and appear harder. If the elastic limit of b is twice that of a (asillustrated) it will store four times as much strain energy before onset of plastic deformation.The energy stored is given in each case by the area under the curve.
Table z. Hardness of dried mandibles from Locusta, measured usingthe Vickers Micro-hardness tester
Region of mandible(see Fig. z a, b)
Left(A) Cutting edge(F) Sheared face
(B) BaseRight
(G) Cutting edge(C) Trailing edge(D) Sheared face(E) Base
VHN(mean±s.E.)(kg mm-')
36-4±i'3Cannot be measuredbecause of its shape
io-7±o-o
322 ±082O-2±O9i8-2±o-8197 ±08
Table 3. Hardness of cuticle from the metathoracic leg of Schistocerca:see Fig. 2 (c) for the key to locations on the leg
(Measured using the Leitz Miniload hardness tester.)
Cuticle
Femur(a) Ribs(6) Between ribs(c) Head (dark)(d) Head (light)
Fig. 2. Keys to the location of cuticle described in Tables 2 and 3. (a) mandibles of Locustafrom the front (scale 1 mm), (b) mandibles of Locutta from within the mouth (scale 1 mm),(c) metathoracic leg of Schiitocerca. (scale 5 mm).
f. E. HILLERTON, S. E. REYNOLDS AND J. F. V. VINCENT (Facing p. 48)
Journal of Experimental Biology, Vol. 96 Fig-3
Fig. 3. Effect of extracting Locusta mandibles with various solvents. Left mandibles onlyshown. Scale i mm. (a) Untreated mandible. (6) Mandible extracted with methanol: chloro-form for 24 h at 60 °C. Much of the pigment is removed from the supporting areas but thepre-ecdysially sclerotized areas are intact, (c) Mandible extracted with M-HCI at 100 °C for24 h. Most of the mandible is destroyed but the sclerotin of the cutting areas is still intact.
J. E. HILLERTON, S. E. REYNOLDS AND J. F. V. VINCENT
On the indentation hardness of insect cuticle 49
las the softer backing material wears away preferentially with the shearing action ofihe incisors leaving two, hard, angled edges. The cutting areas of the mandibles aresclerotized prior to ecdysi9 and, therefore, must be sclerotized in response to adifferent stimulus from that initiating sclerotization of the rest of the cuticle. Hillerton(1980) showed that the sclerotin of the cutting areas of the mandibles is more resistantto hydrolysis by alkali or acid; in fact the actual cutting surfaces survive M-HC1 at100 °C for 24 h when all material except chitin is dissolved from supporting cuticle(Fig. 3). It is possible, therefore, that a different type of sclerotization occurs in thehard part of the mandible from the post-ecdysial sclerotization which is induced bybursicon (Seligman, 1980).
Mandibles are not the only areas of cuticle sclerotized before ecdysis. Most darkbrown or black areas of locust cuticle are sclerotized at the same time, these are theclaws and the areas adjacent to the leg joints. Some of these pre-ecdysially sclerotizedareas have a similar hardness to the cutting edges of the mandibles (head of femur,articulation of tibia, Table 3). These are structures which have a function immediatelyfollowing ecdysis, although this is not true for mandibles. These areas of cuticleare not expanded following ecdysis. They are deposited, following apolysis, by amuch greater area of epidermis than that which lay immediately under the cor-responding part in the previous instar. They, therefore, seem to be precisely mouldedto their new size. There are several possible reasons for this.
(1) Inflation of a soft structure, when the cuticle is plasticized during/or afterecdysis may disrupt the shape of the structure. A similar idea was advanced byCottrell (1962) to explain pre-ecdysial sclerotization in adult blowflies.
(2) The pre-ecdysial sclerotization and moulding may be necessary because it isdifficult to extract thin, pliant structures like unsclerotized claws and other appendagesfrom a stiff and thick part of the exuvia (no digestion and reabsorption of this partof the exuvia appears to occur) without causing too much damage.
(3) Perhaps for a very thick sclerotized structure like the mandible the cuticle hasto be sclerotized as it is deposited.
(4) If the type of sclerotization is different it might be difficult for the sclerotizingagents to be transported through the cuticle.
(5) Perhaps in these areas of the cuticle the proteins are different so it is difficultto get the sclerotizing agents through the cuticle. Further work is obviously necessaryto explain the need for pre-ecdysial sclerotization.
The hardness values in Tables 1 and 2 are those determined for dried specimens.With many fresh specimens it is difficult to measure the indentation as it fills withfluid (water or possibly labile surface lipids) immediately the indentor is removed.These hardness values are, therefore, rather greater than would be shown by cuticlein vivo. Table 3 shows a comparison of hardness values determined for fresh anddried material taken from areas where indentations could be more easily measured.Every precaution was taken to prevent loss of water from the fresh samples. Theseresults show that the hardness of fresh material is one-third to one-half that of driedmaterial but that the same relative differences exist between areas of differenthardness. This suggests that water has plasticized the cuticle (Vincent, 1980) resultingin a reduction in the stress at which the elastic limit occurs (see legend to Fig. 1) so
J. E. HILLERTON, S. E. REYNOLDS AND J. F. V. VINCENT
30
20
EE$
X10
• I ' . i ' l
I I I I I I I
6 8Days
10 12
Fig. 4. Changes in hardness (VHN: Vickers hardness numbers) with age of dried cuticlefrom the anterior proximal methathoracic tibia of adult SMstocerca (point h in Fig. 2 c).Measured using the Leitz Miniload hardness tester. Vertical bars represent ± i x s.E.
water appears to have an effect on the hardness of cuticle just as it does on thestiffness (Vincent & Hillerton, 1979).
Other factors may also affect the mechanical properties of the cuticle. Incorporationof polymerized phenols has been supposed to account for the changes in the mechanicalproperties of the cuticle following ecdysis (Pryor, 1940). Fig. 4 shows that thehardness of cuticle increases with the age of the locust. The increase, however, isonly about 50% of the initial value, and occurs over 3 days (as shown by Neville,1975) whilst the change in stiffness, measured as the tangent modulus because thematerial is not Hookean, is at least twofold and occurs in hours (Hepburn & Joffe,J974)- The change in hardness does not, therefore, parallel the change in stiffness.Whereas stiffness is by definition, independent of thickness, this may not be the casefor hardness. It is likely that the thicker parts of the cuticle are harder and thathardness increases with growth and time. Another change occurring over a similartime-span is an increase in the dihydroxyphenol content of the cuticle (Andersen,I973)- Fig- 5 shows a direct correlation of hardness with the amount of ketocatecholsextractable from the cuticle. This, in a given location, suggests that the function ofpolymerized phenols is to act as a filler increasing the indentation hardness of thecuticle and they may not have any major effect on the stiffness of cuticle. The fillerpresumably increases the hardness by changing the yield behaviour (see Fig. 1).This is also seen in tanned silks (Brunet & Coles, 1974; Vincent, 1980).
Generally the mechanical properties of composites are closely dependent on theirfibre content. The relationship of fibre content to hardness is poorly understood butpresumably a higher fibre content will increase the hardness if the fibre is stiffer andless ductile than the matrix, as is usually the case for biological composites. Themeasured hardness will similarly vary with the orientation of the fibres and w i ^
E
s
On the indentation hardness of insect cuticle
35 r
30
25
202 3 4 5
Ketocatechol (%)Fig. 5. Correlation between hardness (VHN) and ketocatechol content of cuticle fromvarious parts of the body of Sckutocerca. Data on ketocatechol content from Andersen (1974).Hardness values from Table 1. Vertical bars represent ± 1 xs.E.
fE00
z
36 1-
34
32
30
28
26
24
22 I
1000 1100H*ave
1200
Fig. 6. Hardness (VHN) versus hydrophobicity (HO ave) of the protein matrix of Schistocercacuticle. Hardness values from Table 1. Vertical bars represent ± 1 x s.E.
differences in fibre diameter. Gardiner & Khan (1979) have shown that the mandibleof the Australian locust Chortoicetes terminifera contains fibres with a diameter160-300 nm, much larger than those found, so far, in any other insect cuticle whichare uniformly 2-8 nm in diameter (Neville, Parry & Woodhead-Galloway, 1976). Theselarge fibres may be expected to contribute to the increased hardness and resistanceto wear of the mandibles (C. R. Chaplin, Department of Engineering, University ofReading, personal communication) although large fibres have not, so far, been
52 J. E. HlLLERTON, S. E. REYNOLDS AND J. F. V. VlNCENT
reported in Schistocerca or Locusta mandibles. Neville (1975) suggested that cuticlehardness would be independent of its chitin content; we disagree and consider thata larger proportion of chitin is likely to lead to increased hardness of the cuticle.As yet no data are available to support either hypothesis and the hardness of com-posites is poorly understood.
Andersen (1979) considered that the stiffness of insect cuticle would be dependentto a greater or lesser extent on the interactions between the matrix proteins andattempted to quantify this by determining the hydrophobicity of the matrix. Fig. 6shows no apparent relationship between the hardness of locust cuticle and thehydrophobicity of the matrix in contrast to Andersen's suggestion that stiffness andhydrophobicity might be related. This serves to show that hardness is a complexmechanical property dependent on more than just secondary bond interactionsbetween the components and that the synonymity frequently made between hardnessand stiffness in the cuticle literature is quite spurious.
We thank the Drs H. C. Bennet-Clark, C. R. Chaplin and A. C. Neville for dis-cussions. The research was supported by funds from the Agricultural ResearchCouncil (J.E.H. and J.F.V.V.) and the Science Research Council (S.E.R.).
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ANDERSEN, S. O. (1971). Resilin. Compr. Biochem. 26 C, 633-657, Elsevier: Amsterdam.ANDERSEN, S. O. (1971). Comparison between the sclerotization of adult and larval cuticle in Schistocerca
gregaria. J. Insect Physiol. 19, 1603-1614.ANDERSEN, S. O. (1974). Cuticular sclerotiiation in larval and adult locusts, Schistocerca gregaria.
J. Insect Physiol. ao, 1537-1552.ANDERSEN, S. O. (1979). Biochemistry of insect cuticle. A. Rev. Ent. 34, 29-61.BIGELOW, C. C. (1967). On the average hydrophobicity of proteins and the relation between it and
protein structure. J. theor. Biol. 16, 187-211.BRUNET, P. C. J. & COLES, B. C. (1974). Tanned silks. Proc. R. Soc. Lond. B 187, 133-170.COTTRELL, C. B. (1962). General observations on the imaginal ecdysis of blowflies. Trans. R. ent. Soc.
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75-97-HEPBURN, H. R. & JOFFE, I. (1974). Hardening of locust sclerites. J. Insect Physiol. ao, 631-635.HILLERTON, J. E. (1980). The hardness of locust incisors. Symp. Soc. exp. Biol. 34, 483-484.KELLY, A. (1973). Strong Solids. Oxford: O.U.P.NEVILLE, A. C. (1975). Biology of the Arthropod Cuticle. Berlin: Springer-Veriag.NEVILLE, A. C , PARRY, D. A. D. & WOODHEAD-GALLOWAY, J. (1976). The chitin crystallite in arthropod
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