Some mechanical properties of crossed fibrillar chitin

J. Insect Physiol., 1972, Vol. 18, pp. 815 to 825. Pergomon Press. Printed in Great Britain

SOME MECHANICAL PROPERTIES CROSSED FIBRILLAR CHITIN

OF

H. R. HEPBURN Department

of Physiology, University of the Witwatersrand, (Received

12 October

1971;

revised

Johannesburg,

26 November

South Africa

1971)

Abstract-The sclerites of P. sinuata consist of crossed reticulate layers of chitin fibrils arranged in the preferred orientation together with protein glues. Stretched beetle whole cuticle and chitin obey Hooke’s law in the elastic region. Anisotropic swelling in a sclerite ensures flexibility and prevents sliding in the plane surface. Chitin micelle orientation can be strain-induced in vitro. Although two-phase materials, neither beetle nor locust cuticle meet the requirements of plywood mechanics. INTRODUCTION

LAYERSof crossed fibrillar chitin in cuticle have been known for many years (MEYER, 1842), The relative simplicity of this pattern offers special opportunities for investigating the physical relationships of whole cuticle and its subunits and allows some insight in generalizing concepts of insect skeletal design. The tendency to equate ‘laminate ’ with ‘plywood ’ in cuticle literature opens the question as to whether crossed fibrillar chitin meets the strict criteria of plywood mechanics. Anisotropic swelling of cuticle, though well established (FRAENKEL and RUDALL,1947), likewise requires direct evidence of its mechanical significance. The concept of strain-induced chitin micelle orientation has been discussed (FRAENKELand RUDALL, 1940; NEVILLE, 1967) but remains unresolved. Finally, some of the conflicting design requirements in the intact cuticle are poorly understood and are investigated here. MATERIALS

AND METHODS

The data reported here are derived from the adults of both sexes of the scarabaeid beetle, Pachynoda sinuata. Test strips of frontoclypeus, pronotum, mesosternum, elytron, metathoracic femur, abdominal sternite II, and abdominal tergite III were cut in the longitudinal axis of the animals. Chitin strips of each sample were obtained from fresh strips that were incubated in a 10% papain solution at 37°C for 48 hr ; rinsed in distilled water ; boiled in concentrated potassium hydroxide for 2 hr ; rinsed and stored in double-distilled water until needed. Treated strips were verified as chitinous with the chitosan-iodine colour test (RICHARDS,195 1). Determination of cuticular subunits was made from both fresh strips and chitin strips which were sectioned and then stained in Mallory’s stain. That each layer of 815

816

H. R. HEPBURN

procuticle consists of unidirectional or preferred chitin fibrils was verified in longitudinal and transverse sections of treated mesosternum examined on an electron microscope at x 40,000. Orientations of preferred layers were measured between crossed polarization filters with a 550 pm gypsum compensator. Layer number was derived from counting them as peeled from the strips and from counting layers in sectioned material. The careful fracture technique (NEVILLE, 1970) was found unreliable for deriving the layer number because if any two neighbouring layers break off short, the pseudo-orthogonal pattern remains but a spurious layer number results. Swelling was measured as a shrinkage value by comparing fully hydrated chitin strips before and after drying at 100°C for 26 hr and retaining them over calcium sulphate for 18 hr. Strips of fresh whole cuticle were evaluated in the same way. Tensile tests were performed on wet and dry chitin strips and on untreated fresh and dry strips of whole cuticle using an Instron floor model universal testing instrument. Silver steel micro-grips of 5 mm dia. were fitted to the instrument. All tensile tests were run at a constant crosshead/chart speed ratio of O-5 mm/min to 50 mm/min. Specimens were observed during the tests by means of a reflected light microscope connected to closed-circuit television. Of the 100 specimens selected for tensile testing, 78 were rejected for the following reasons. Pre-test rejections included 50 specimens that exhibited tearing, taper in the strip axis, obvious variation in cross-sectional thickness, or gripfitting damage. Of the 50 specimens actually tested, 28 were rejected for either single or double breaks near the grips, skewed alignment in the grips, jig drag on grip rods, specimen slip in the grips, or noticeable plane perpendicular buckling resulting from improper grip alignment. A total of 22 tests was accepted as yielding rigidly controlled results. Post-test examinations of broken specimens were made with a polarizing microscope. RESULTS AND DISCUSSION Structure of the cuticle

The procuticle consists of chitin fibrils The epicuticle is not considered. arranged in crossed layers (Fig. lA-C). The layer pattern in P. sinuatu is similar to the pseudo-orthogonal arrangement of Heteroptera and Coleoptera in which the preferred layers are separated by an intervening lamella of 90” pitch (NEVILLE, 1970). The term ‘lamina’ is here restricted to histologically distinguishable layers and connotes only the effects of hardening and darkening sequences. For example, a treated mesosternal strip of 15 layers reveals only the chitin layers. However, in untreated fresh cuticle strips these 15 layers occur in four distinctly coloured laminae. The most basal lamina is colourless, followed by amber, brown, and black laminae. This sequence conforms to the pattern: mesocuticle, exocuticle 1, exocuticle 2, and exocuticle 3 of RICHARDS(1967). Mallory’s_stained sections of fresh material confirm this view with one red-staining and three nonstaining layers.

FIG. 1. Mesosternum of P. sinuata. (A) Chitin fibrils; (B) two crossed reticulate layers; (C) procuticular outer layers out of angle phase with lower layers; (D) typical tensile fracture of wet chitin.

layers-

SOMEMECHANICAL PROPERTIES OFCROSSED FIBRILLAR CHKTIN

819

The sclerotization sequence seen as groups of laminae suggests a progressive change in glue types since there is a strong tendency for layers to peel in lam&r groups rather than individually. Four different bonding systems (glue types) have been proposed for sclerotized cuticle (LIPKE and GEOGHEGAN,1971) all of which might be used in the same cuticle. It seems probable that specific kinds of bonding will be found to coincide with each histologically recognizable kind of lamina (HACKMAN,1971). ZELAZNY(1969) has shown that the number of preferred layers varies in different parts of the skeleton of the scarabaeid, @@es r~~~~~os. Sillily, in P. simmta the frontoclypeus averaged 12 layers, pronotum 16, mesosternum 16, elytron 13 in upper surface and 8 in lower surface, metathoracic femur 11, abdominal sternite 12, and abdominal tergite 3. No sexual dimorphism relating to layer number is apparent. The angles resulting from the orientation of preferred layers to the longitudinal axis of the animal alternate at +45” and -45”. Neighbouring layers are 90” apart (Fig. IB). The sequence may begin with either the + 45” or -45” layer; but in each case, the next layer forms the correct angle and maintains the pseudoorthogonal arrangement. The angle sequence is not perfectly in phase throughout the procuticle because the older layers deviate from the O-90” pattern (Fig. 1C). For all samples of fresh and dry cuticle and wet and dry chitin, the three most basal layers are not birefringent; however, if experimentally stretched they become sharply and irreversibly strain birefringent. Consistent with initial lack of optical activity, these basal layers exhibit poorly developed reticular networks. All subsequent layers typically exhibit well-developed fibrils, networks, and strong form birefringence. Pseudo-orthogonal cuticles lack a clockwise-anticlockwise sense since the pattern is continuous across the median axis of the animal. The system is not asymmetrical as suggested by NEVILLE (1970) but one of dysymmetric enarrtiomorphic fibrils wound alternately at 45” to an axis but not plane or centre of symmetry. Modifications of the pseudo-orthogonal pattern include elytral trabeculae which are continuous with the upper and lower elytral surfaces. Trabecular cross-sections consist of concentric rings of fibrils like those of Buprestidae (NEVILLE, 1967). The enigmatic pattern of alternating azan-staining and non-staining bands reported by REUTER(1937) from the elytra of CaZundru result from transecting the reticulate layers of those weevils. In the abdominal tergites of P. sinuata the chitin fibrils are discontinuous at intersegmental folds with only half the layer thickness below the folds as on either side of the folds. Swelled For various cuticles, FRAENKELand RUDALL(1947) have shown that the degree of water swelling is greater perpendicular to the surface than in the plane surface. Test strips of all measured sclerite regions of P. sinuatalikewise swell anisotropically. The chitin strips have a plane surface value of 9 per cent and a plane perpendicular value of 49 per cent. Strips of fresh, untreated cuticle swell plane

H. R. HEPBURN

820

perpendicularly 15 per cent and only 1 per cent in the plane surface. The notable hygroscopicity of chitin is characteristic of natural fibres generally (FREYWYSSLING, 1953). Wetting drastically affects the stress/strain characteristics of bio-materials from cellulose (HEARMON, 1953) to bone (YAMADA, 1970). Cuticle and chitin also behave differently under dry and wet testing conditions (Table 1; Figs. 2, 3). The

TABLE I-MECHANICAL PROPERTIES OF CUTICLE*

Material Wet chitin mesosternum Dry chitin mesosternum Whole fresh mesosternum Whole dry mesosternum Wet chitin abd. tergite Whole fresh locust tibia

Ultimate strength (tensile) (N m-s x 10’)

1941

19.8

5.7

56.4

This paper

9000

90.0

3.6

162.0

This paper

2477

47.0

4.3

101.0

This paper

2277

41 *o

4.0

82.0

This paper

1700

17.3

3.0

26.0

This paper

9413

94.2

2.5

118.0

Fresh resilin dragonfly

300.0

435.0

Absorption energy (J mm3 x 10’)

17.7

Wet chitin purified Dry chitin purified

Extension elastic limit (%)

Modulus elasticity (tensile) (N m-x x loo)

-

93.2

2

2.9

-

Source

JENSENand WEIS-FOCH (1962) THOR and HENDERSON (1940) THOR and HENDERSON (1940) JENSENand WEIS-FOGH (1962)

* All quoted values recalculated into S.I. units. Absorption energy values extrapolated from original data where possible. P. sinuata ultimate tensile strength data based on: wet chitin mesostemum 12= 7, S.E. = 4.2; dry chitin mesostemum 12= 2, S.E. = 30; whole fresh mesosternum n = 5, S.E. = 6.9; whole dry mesosternum n = 6, S.E. = 11; wet chitin abdominal tergite tt = 2, S.E. = 1.4. ‘Die modulus of elasticity, extension, and absorption energy are derivative functions.

usual physical explanation for increased strength on dryness is that strength of cuticle depends upon inter-fibrillar bonding rather than individual fibril strength (RUDALL, 1963) ; but results from P. sinuatu cuticle do not support this view.

SOME

MECHANICAL

PROPERTIES

OF CROSSED

FIBRILLAR

CHITIN

(a) 9-

f

0

Ultimate

22-

tensile

200

600

Strain,

pm/min

.

e

1000

100

0

Strain,

200

300

pm/min

FIG. 2. Stress/strain curves for P. ~~a~~ mesosternal chitin. (a) Wet chitinslips 3 to 3 coincide with lattice slack, gradual plastic deterioration indicates failure of individual fibrils; (b) dry chitin-Hookean, rapid plastic failure.

3c

(0)

b)

Ultimate tensile

!f&F$ strength

“E ,E 2c ; .E? 5 z

% E m

IO

100

Strain,

200

fun/min

300

Id@ Strain,

SO0 pm/min

FIG. 3. Stress/strain curves for P. sinuata whole mesostemum. (a) Fresh cuticleHookean, rapid plastic failure with only one slip ; (b) dry cuticle-Hookean, rapid plastic failure.

821

H. R. HEPBURN Tensile tests of fresh and dry whole cuticle and wet and dry chitin strips of mesosternum were made to determine how tensile strength and elasticity are affected by hydration, protein glues, and layer angle in solid cuticle. Abdominal tergite strips were also tested to assess the role of layer number. All other sclerite regions were rejected because of their undesirable test qualities. Table 1 indicates a fivefold increased strength in chitin on drying. These values agree closely with those of THOR and HENDERSON (1940) for purified chitin. Fresh whole cuticle strength is intermediate between wet and dry chitin. The addition of protein glues to hydrated chitin offsets the limitations of wet chitin alone. This does not imply that the protein fraction has a tensile strength equal to the difference between wet chitin and whole fresh cuticle, but only that in combination the product is materially stronger than wet chitin. Whole fresh mesosternum was found to be stronger than whole dry mesosternum, a notable departure from most biological materials. Differences in elastic extension for cuticular materials are summarized in Table 1 and illustrated in Fig. 4. Strength

6

1

(dragonfly)

0

1

2

3

4

5

6

loge % Elongatii

FIG. 4, Relationship between intrinsic strength and flexibility, hydration and flexibility, and hydration and strength for various cuticular materials.

comparisons between wet mesosternum and wet abdominal tergites indicate that the layer number plays a very little r81e in this regard since they were respectively of 14 and 4 layers. JENSENand WEIS-FOGH (1962) obtained similar results for locust cuticle and concluded that additional deposition of endocuticle only confers greater damping potential. value

SOME MECHANICAL

PROPERTIES

OF CROSSED FIBRILLAR

CHITIN

823

Mechanical behaviour By simultaneously observing the stress/strain print-out and the specimen during the tensile tests, it was possible to correlate the two and construct a picture of the mechanical behaviour of the test specimens. For wet chitin (Fig. 2a) the events are as follows. Slip 1 coincided with a decrease in the layer angle from 90” allowing slight slack. The material remains elastic until it reaches its ultimate strength peak after which the first fibrils fail and bring the material into the plastic region. There is momentary reduction in strain at slip 2 as the fibrils realign and layer angle decreases. A second strength peak nearly as high as ultimate strength is followed by the gradual deterioration of the remaining intact layers as the fibrils realign, slip, and flow. The wet chitin strips begin tearing from one side at 45” to the strip axis (Fig. 1D). The stress/strain curves of fresh and dry whole cuticle and dry chitin (Figs. 2b, 3a-b) show them to be considerably less flexible than wet chitin. Extensive changes in layer angle are not evident in fresh cuticle and the plastic deterioration is rapid. Dry whole cuticle and dry chitin behave similarly. Specimens of fresh cuticle always failed with an audible click but the others did not. The relationships between strength and flexibility of cuticular materials are graphically shown by their absorption energy characteristics (Fig. 4; Table 1). The points in Fig. 4 were derived from strength/extension curves as the areas from zero to ultimate strength and indicate energy relationships for each material shown. Elasticity is inversely proportional to intrinsic strength for all materials. For chitin alone, strength is inversely proportional to hydration. Whole fresh beetle cuticle is intermediate between wet and dry chitin which indicates that although elongation of sclerites is rigidly controlled in the plane surface, required flexibility is ensured from hydration in the plane perpendicular axis. Fresh beetle and locust cuticle show close absorption energy values but highly different tensile and elastic properties thus reflecting different solutions to the strength/flexibility problem. In this connexion, the absorption energy values of an entirely helicoid cuticle would be of interest. The mechanical properties of cuticular subunits vary in isolation and in combination with each other (Table 1; Fig. 4). Hydration confers flexibility at all levels. Against this, there is a corresponding decrease in wet chitin strength. Hydration also decreases brittleness and lowers resistance to twisting, both desirable qualities under dynamic loads. Similarly, protein glues increase brittleness and decrease flexibility. Although NEVILLE (1967) argued ag ainst the effects of anisotropic skeletal strains in orienting chitin micelles in the locust tibia, his view only assumes shear acting on the members. The arrangement in pseudo-orthogonal cuticles consists of +45” and -45” layers. As such, these sclerites simultaneously experience both shear and length change when sheared; twisting and bending under torque. In either case, any applied force will consist of both principal and shear components. In the tensile tests of all samples of P. sinuata mesosternum described above, the three most basal layers begin the strain period as non-birefringent

824

H.

R. HEPBURN

layers devoid of fibril structure and terminate in strain birefringent fibrils, ordered and oriented in the line of the applied force. This result suggests mechanically induced micelle orientation such as FRAENEELand RUDALL(1940) reported from blowfly puparial cuticle. The role of hydration in this process has been partially established by the X-ray diffraction studies of RUDALL(1963). The arrangement of reticular preferred layers (Fig. 1B) in a lattice of laminae is ultimately the mechanism by which sclerite flexibility is maintained. It is the wet lattice which allows for layer angle changes and not the leaf spring mechanism proposed by AHEENS(1930). Previous reports that cuticle does not obey Hooke’s law are based on the results from locust tibia (JENSENand WEIS-FOGH, 1962). In the case of P. sinuata all cuticular materials tested (Figs. 2, 3) are Hookean for most of the elastic region, possibly reflecting basic mechanical differences between helicoid/preferred and only preferred chitins. This beetle cuticle is similar in behaviour to wood samples of Douglas Fir under both wet and dry conditions (BODIG, 1966). In simple plywoods (layers at +45” and -45” to strip axis) the modulus in bending differs from that of compression/tension, ultimate tensile strength varies with layer number, angle, and degree of hydration (HEARMON, 1953). Locust cuticle fails all of these criteria (data from JENSENand WEIS-FOGH, 1962). The bending modulus was not measured for P. sinuatu. There is no clear difference in tensile strength with regard to layer number between wet mesosternal (14) and abdominal tergite (4) chitin. Only differences in hydration meet plywood requirements. It would be of interest to test the beetle Oryctes for progressive change in layer angle. Insect solid cuticle in the natural state was first established as a two-phase material by the elegant work of JENSENand WEIS-FOGH (1962). However, empirical details for a wide variety of materials remain to be gathered. Investigations of the material properties of locust and beetle cuticles indicate that the physical limitations of individual constituents are biologically manipulated in different ways to meet various design requirements. Acknowledgements-I am grateful to the following at the University of the Witwatersrand: T. O’D. DUGCANprovided the use of his Instron instrument for the tensile tests; C. ROSENDORFF and R. J. MCCARTER kindly discussed the manuscript; J. WALKER prepared the illustrations; D. VEENHOF and W. MADDISON constructed the micro-grips. The Tedelex Group (Johannesburg) provided the television equipment. A portion of this work was materially supported by C.S.I.R. Grant M30/71/Pl. REFERENCES AHRENS W. (1930) Uber die Korpergliederung, die Haut und die Tracheenorgane der Termitenkijnigin. 2. Naturw. 64, 449430. BODIGJ. (1966) Stress-strain relationship for wood in transverse compression. J. Materials 1, 645-666. FRAENKEL G. and RUDALL K. M. (1940) A study of the physical and chemical properties of the insect cuticle. PYOC.R. Sot. (B) 129, l-35.

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FRAENKELG. and RUDALLK. M. (1947) The structure of insect cuticles. Proc. R. Sot. (B) 134,111-144. FREY-WYSSLINGA. (1953) Submicroscopic Morphology of Protoplasm. Elsevier, Amsterdam. HACKMANR. H. (1971) Distribution of cystine in a blowfly larval cuticle and stabilization of the cuticle by disulphide bonds. J. Insect PhysioE. 17, 1065-1071. HEARMONR. F. S. (1953) The elastic and plastic properties of natural wood. In MechanicaE Properties of Wood and Paper (Ed. by MEREDITH R.), Chap. 2. North-Holland, Amsterdam. JENSENM. and WEIS-FOGH T. (1962) Biology and physics of locust flight-V. Strength and elasticity of locust cuticle. Phil. Trans. R. Sot. (B) 245, 137-169. LIPKE H. and GEOGHEGANT. (1971) Enzymolysis of sclerotized cuticle from Periplaneta americana and Sarcophaga bullata. J. Insect Physiol. 17, 415-425. MEYER H. (1842) Ueber den Bau der Hornschale der Kafer. Arch. Anat. Physiol. 3, 12-16. NEVILLE A. C. (1967) Chitin orientation in cuticle and its control. A& Insect Physiol. 4, 213-286. NEVILLE A. C. (1970) Cuticle ultrastructure in relation to the whole insect. Sym. R. ent. Sot. Lond. 5, 17-39. RICHARDS A. G. (1951) The Integument of Arthropods. University of Minnesota, Minneapolis. RICHARDSA. G. (1967) Sclerotization and the localization of brown and black colours in insects. Zool. Jb. (Anat.) 84, 25-62. REUTER, E. (1937) Elytren und Alae wahrend der Puppen- und Kaferstadien von Calandra granaria und Calandra oryzae. Zool. Jb. (Anat.) 62, 449-506. RUDALLK. M. (1963) The chitin/protein complexes of insect cuticles. Adw. Insect Physiol. 1, 257-313. THOR C. J. B. and HENDERSON W. F. (1940) The preparation of alkali chitin. Am. Dyestufl Reptr. 29, 461-464 and 489-491. (Cited from JENSENand WEIS-FOGH, 1962.) YAMADAH. (1970) Strength of Biologica Materials. Williams & Wilkins, Baltimore. ZELAZNYB. (1969) Ablagerung und Orienterung der Endocuticulaschichten bei Kaefern. Doctoral Thesis, Frankfurt. (Cited from NEVILLE, 1970.)