A Rubber-Like Protein in Insect Cuticle

From a theoretical point of view the demonstration of a true protein rubber may claim some interest not only because insects make use of it in the form of nearly perfect mechanical springs (Weis-Fogh, 1959) but particularly for two other reasons. First, even the best thermodynamic experiments on elastin are ambiguous as to the rubber-like nature of this protein because elastin shrinks when heated so that the internal energy seems to change a great deal with stretch rather than to remain constant (Meyer & Ferri, 1936 ; Wöhlisch, Weitnauer, Griming & Rohrbach 1943). However, a number of quantitative tests proved beyond doubt that at least one protein, resilin, behaves as a true physical rubber (Weis-Fogh, 1961). These experiments were done on an elastic tendon from dragonflies described for the first time in this paper. Secondly, a true rubber consists of a three-dimensional network of molecular chains which are nearly free of one another, thermally agitated and randomly kinked, but which are fixed in the network by means of a few stable cross-linkages (cf. Treloar, 1958). A protein rubber may then serve as a stable deformable network in cells and tissues and it is possible that the so-called stroma proteins fall into this category although next to nothing is known about their nature.

In the course of an anatomical investigation La Greca (1947) noticed some hyaline, elastic structures in the thoracic skeleton of grasshoppers; they are now known to play an important mechanical role in the flight machinery of locusts because they store kinetic energy from the wings as elastic energy during the upstroke and pay it back during the subsequent downstroke (Weis-Fogh, 1959). So far, they have not been investigated in any detail.

In the desert locust (Schistocerca gregaria Forskål) and in other grasshoppers there are two suitable samples, the prealar arm and the main wing-hinge ligament.

The prealar arm (Fig. 1 A) mediates the anterior suspension of the mesonotum, i.e. the movable plate between the forewings. It is a blunt conical peg about 0.5 mm. long which projects abruptly from a base of hard dark cuticle; at either side of the V-shaped groove it continues as ordinary tough arthrodial membrane (not shown). At the tip, the membrane is thickened to a tough flexible ligament which connects the prealar arm with the first basalar sclerite of the pleuron. A thread can be wound round the ligament and used in pulling experiments when the sclerotized base is embedded in plaster of Paris.

The wing-hinge ligament (Fig. 1B) is a thick complicated cushion of hyaline rubbery cuticle which is inserted between the sclerotized pleural wing process and an equally hard and dark process on the underside of the second axillary wing sclerite (cross-hatched). The cushion represents the hinge proper and there is an exceedingly sharp and abrupt transition between it and the sclerotized parts although, of course, structural continuity is retained between the three elements of the hinge.

The third test piece is a peculiar sausage-like swelling of the tendon for the pleuro-subalar muscle in dragonflies (Fig. 1C; muscles 34, 56; Clark, 1940). Clark (1940) mentions that the tendon feels elastic but the small swelling has escaped notice so far; in Aeshna grandis it is about 0.7 mm. long and 0.15 mm. wide. The entire tendon is built like the other cap tendons of this group, i.e. it is a deep hollow invagination from the dorsal body wall which widens at its ventral end to a cap-like insertion for a wing muscle. For the greater part of its length the tendon is tough and inextensible and only the hyaline swelling, the elastic tendon, is elastic like a rubber band.

Thin threads of nylon can be fastened to the tough parts for stretching experiments. The elastic tendon proper is a thick-walled, nearly cylindrical tube whose bore, here called the central canal, represents the lumen of the air-filled invagination. It has been found in all Odonata hitherto examined (Uropetala carovei Wh., Libellula spp., Orthetrum sp., Sympetrum spp., Aeshna spp., Agrion spp.). Tendons from the metathorax of Aeshna cyanea Müll, and A. grandis L. are almost cylindrical and suited for quantitative studies.

In Fig. 1 the three samples have been placed in saline at pH 7 and are shown unstrained as well as strained to the same extent as in the working animal. Even after great deformation it is characteristic that the elastic cuticle snaps back to the unstrained shape as soon as the load is removed. While solid cuticle breaks when stretched by 1 to 2% (Martin Jensen & Weis-Fogh, 1961), rubber-like cuticle is normally subject to deformations of 20-40%, exceeding 100% in the case of the tendon.

It is easy to remove the thick hypodermal cells and their basement membrane and such stripped samples are transparent like glass and devoid of colour. The refractive index exceeds 1.4 and at pH 7 the water content is 50-60% (drying over P₂O₅, microbalance), in accordance with the swelling experiments illustrated in Fig. 8. Rubber-like cuticle is therefore a rather concentrated material.

The three samples all swell in alkaline buffers and shrink on the acid side of neutrality but they remain rubbery throughout. They become solid and glass-like oftly when dried in air or dehydrated in alcohol stronger than 90%. However, when water is again admitted they re-swell and become rubbery within seconds or minutes. Experiments like those illustrated in Fig. 2 may be repeated indefinitely with the same pieces of elastic tendon ; in 70 % alcohol they are completely rubbery, in 80 % slightly plastic, in 90 % they can be bent like plastic rods and deformed. If made hard in absolute alcohol while being strained to the extreme between a pair of forceps, the pieces quickly ‘thaw’ up in 70% alcohol and completely regain their original shape and size. This happens even after treatment with ordinary fixatives, protein coagulants and tanning agents (see p. 899) and also after embedding in paraffin wax or methacrylate. It is not usual to associate such properties with proteins.

It is, nevertheless, easy to prove that the characteristic elasticity resides with an insoluble protein, as is shown by the following experiments.

After incubation with papain at pH 7 or with other pure proteases (of. p. 13), the prealar arm and the wing-hinge both became transformed into flaccid bags of thin lamellae. Although the external shape was retained, the rigidity and the elasticity had all gone and the cuticle appeared white and opaque due to light scattering by the refractile and glossy lamellae and the water between them. The delicate lamellae were continuous and could be separated by means of fine needles. They continued into the sclerotized cuticle (not affected) and into the membranes so that the pieces remained inextensible when stretched parallel to the lamellae. Similar results were obtained after mild alkaline or acid hydrolysis, such as 1 N-HCI at 65° C. for 5-6 hr. and 0.05 N-HCI or 0.05 N-NaOH for 1 hr. at 125° C. in sealed tubes.

Before hydrolysis slices of the prealar arm and of the wing hinge gave distinct Millon’s and xanthoproteic reactions and stained blue with bromothymol blue followed by 5% acetic acid. After incubation about 80% of the dry matter had disappeared and the tests were negative. It was found that the hydrolysate contained many amino acids but neither sugars nor amino sugars ; less than 2 % of the original dry weight remained in the lamellar residue in the form of amino acids (Bailey & Weis-Fogh, 1961). The elastic material is therefore not a mucopolysaccharide or a glycoprotein, but seems to be a true protein which can be removed from the insoluble lamellae by proteases or mild hydrolysis.

The lamellae consist mainly (or exclusively ?) of chitin. After treatment in saturated KOH at 160° C. for 15 min., all protein and elasticity had gone, but the lamellae were still present as glossy fragile sheets of the same shape and size as in the fresh cuticle. In all respects they reacted like chitosan (cf. Richards, 1951).

Most cuticles contain at least two protein fractions of which one is water-soluble and the other not. Prolonged treatment of thin slices from the prealar arm and the wing hinge with cold or hot (95° C.) buffered water failed to bring any significant amount of substance into solution (weighing). Moreover, a chromatographic analysis of the supernatant revealed that less than 0.3% of the protein goes into solution (Bailey & Weis-Fogh, 1961). Heating in water had no effect upon the elasticity of the three samples and, disregarding the thin epicuticle, there was no trace of lipid.

It is particularly instructive to consider the elastic tendon. After treatment with proteases, or with weak acids or bases, it appeared as an extremely delicate double tube. The central tube corresponded to the epicuticular lining round the central canal ; the cortical tube was flush with the original surface of the stripped swelling and continued at either end as the ‘external’ layer (nearest to the hypoderm) of the tough cuticle. The space between the two tubes was emptied and filled with water and the tubes were so delicate that the structure was virtually deprived of strength in all directions. The intact tendon gave the usual protein reactions and the hydrolysate contained the same amino acids in approximately the same proportions as found in the two former preparations (Bailey & Weis-Fogh, 1961). Obviously, the entire strength and elasticity of the tendon is caused by a protein, both in compression and in extension. The cortical tube gave a faint but typical chitosan reaction, the tough ends and the transitional zone a massive reaction.

We may conclude that, disregarding the thin epicuticle, rubber-like cuticle owes its elasticity to a swollen water-insoluble protein. The only other structural component is sheeted chitin and the two components may be separated simply by means of mild hydrolysis.

The complexity of ordinary solid cuticle is not found in the rubber-like samples and, knowing the arrangement of chitin and protein, it was therefore possible to analyse some fundamental properties of resilin from experiments with intact test pieces.

Fresh or heat-treated rubber-like cuticle became sapphire blue in weak solutions of methylene blue or toluidine blue in dilute neutral buffers, 2-5 mg. dye per 100 ml. 0.05 M buffer. The colour was retained in pure buffer and is associated with the protein and not with the lamellae. This was particularly clear in the thick pad of elastic protein from the wing hinge which is described below and where there is no chitin at all. Toluidine blue stained orthochromatically so that the protein does not contain sulphate or metaphosphate to any appreciable extent (cf. Pearse, 1960). The colour reactions indicate that the isoelectric point is on the acid side of neutrality, in contrast to collagen, and it has been used to find rubber-like cuticles in a variety of other insects.

Fresh slices as well as paraffin sections of fixed rubber-like cuticle (Carnoy, Susa) did not stain with the periodic-acid-Schiff reagent (PAS), in contrast to the neighbouring tough ligaments and the arthrodial and basement membranes (cf. Wigglesworth, 1956). This is consistent with the absence of non-chitinous polysaccharide.

In transverse frozen sections of the prealar arm and its hypodermic cells, fixed in neutral formaldehyde, embedded in gelatin and plasticized in glycerol (cf. p. 12), the chitin lamellae form a concentric system of continuous refractile lines parallel to the hypoderm, each less than 0.2 µ thick. Nearest to the cells they are spaced apart by distances smaller than 1 µ and form a sort of ‘skin’; further inside the interlamellar distance is 2-3 µ. In phase contrast and at the highest optical resolution the interlamellar substance is completely deprived of structure and is transparent like a gel. No trace has been found of pore canals and ducts from glands. In thin paraffin sections (4 µ) of material treated with Carnoy’s and Susa’s fixatives and embedded in balsam, the lamellae are distinct when stained with chlorazol black or light green (Fig. 3 A). The interlamellar substance remains unstained and appears as an amorphous glass, often broken up into flakes by the passage of the knife but with no regular structure. It becomes deep red with Masson’s and Mallory’s triple stains where the green or blue lamellae are hardly visible unless in thin sections (4/x; Fig. 3B). The red-staining material is not seen in the tough ligament nor in the arthrodial membrane but suddenly appears in the prealar arm proper ; it is absent or not visible in the sclerotized part. It is also seen in the ‘skin’ which therefore differs from the remaining part only by a smaller distance between the lamellae. The hypodermal cells stain green for the first 1-2 µ nearest to the cuticle. After hydrolysis only the lamellae are present. The ‘red ‘material is therefore considered identical with the elastic protein. It shows some affinity for Heidenhain’s iron haematoxylin but, again, no microscopical structure.

The swollen protein acts as an elastic glue between the chitin lamellae which it separates by distances one order of magnitude greater than the thickness of thelamellae themselves. Thus the prealar arm is a simple multi-ply structure, and it is understandable that it behaves like a sample of pure rubber provided it is bent normal to the direction of the lamellae, as is the case in Fig. 1A and also in the experiments reviewed below.

In connexion with studies on locust flight, the prealar arm was tested during static and vibrational bending (Martin Jensen & Weis-Fogh, 1961). It turned out that the ‘skin’ had little effect in itself and that the rigidity is not due to turgor pressure but to the elastic properties of the entire structure, i.e. mainly to the protein. The static modulus of elasticity is about 20 kg. cm.^-2 (2.10⁷ dyn. cm.^-2) which is well within the range of true rubbers. Three additional results are also relevant here. After prolonged strain, the prealar arm recovers completely when the load is removed ; the material therefore does not flow. The internal friction is astonishingly small since the dynamic stiffness increases by less than 5 % compared with the static value and since the energy lost as heat per half cycle, the loss factor, amounts to less than 3% of the stored energy. These figures refer to speeds of deformation of the order of six sample lengths per second on the average.

True homogeneous rubbers are optically isotropic when unstrained but become birefringent on deformation. In frozen sections plasticized in glycerol and placed under the polarizing microscope, the protein between the lamellae could be seen to be isotropic provided the lamellae were viewed exactly from the edge. In most places and in thick transverse sections the swollen cuticle is positively birefringent in the direction of the lamellae ; this is to be expected in a body where refractile laminae of chitin are embedded in a less refractile isotropic medium, as in a typical Wiener body. However, the birefringence could be modified and its sign reversed by swelling in alkaline media (pH 12). Since the chitin constrains swelling parallel to but not normal to the lamellae, the protein swells anisotropically and now it becomes positively birefringent in the direction of extension, i.e. normal to the lamellae, to such an extent that this intrinsic birefringence supersedes the structural birefringence and reverses the over-all sign. The changes are fully reversible.

When the prealar arm is mechanically compressed, the birefringence decreases and becomes negative in the direction of compression. A similar strain birefringence is also observed in extension : the structural birefringence was reduced by means of aqueous potassium mercury iodide (Thoulet’s solution), the refractive index of which was adjusted to that of chitin (about 1.54) ; this solution causes the protein to swell and to extend normal to the lamellae, but does not dissolve it.

Mechanically the hinge shows the same elastic recovery and a similar small degree of damping as the prealar arm (Weis-Fogh, unpublished). The major part is also occupied by the same kind of lamellar cuticle (Fig. 4) but there are two differences. For a short length of the ligament, the ventral part is tough, dense and strongly birefringent when unstrained (striped). It contains protein which is not removed by hydrolysis in 1 N-HCI for 6 hr. at 65^µ C. since Millon’s test was positive after hydrolysis; it appears as coarse fibrils densely packed between the chitin lamellae. The more interesting part is a pad of elastic protein up to 100 p, thick and situated between the hypoderm and the lamellar cuticle (white in Fig. 4). It has no microscopic structure whatsoever and is completely isotropic in polarized light. It can be broken up into irregular sharp-edged pieces ; they remain optically isotropic at all degrees of swelling, and since they retain their relative proportions (within the accuracy of measurements, cf. p. 902) they are also mechanically isotropic; at equilibrium, the swelling forces must balance the elastic restoring forces. The swelling is reversible. The pieces exhibit typical strain birefringence, positive in the direction of extension.

Pl. 17, fig. 1 shows the dehydrated mesothoracic tendon in situ (muscle no. 34, Aeshna cyanea, embedded in balsam). The hypoderm is just discernible; a glassy isotropic substance fills the broad space between it and the central canal. The cortical and central tubes are too thin to be seen, the dark outlines being optical artifacts due to refraction. In stained paraffin sections the tough invaginated membrane just dorsal to the swelling (Fig. 5 A) appears like ordinary arthrodial membrane in Periplaneta (Dennell & Malek, 1954) and resembles the tendinous continuation at the ventral end. At the transition between membrane and swelling

(Fig. 5 B), green (Masson) or blue (Mallory) concentric lamellae dominate as in the former section, but an amorphous, strongly red substance makes its first appearance. The thin 0.2 µ pink epicuticle is thrown into wrinkled folds about 5 µ across, as in the membrane. A few microns further down (Fig. 5 C) the folds are absent and the membrane is densely supplied with papillae of a size close to the optical resolving power. The red substance has now spread over the entire cross-section apart from the cortical area and the appearance is like that of the prealar arm. However, still a few sections further down, the wall has attained its full thickness and the entire space between the papillose colourless epicuticle and the thin green cortical tube (0.2 µ or less) consists of a structureless solid stained red with Masson and Mallory and grey with iron haematoxylin. The outer 1.2 µ of the cortical zone is more faintly coloured with the two former stains and darker with iron haemo-toxylin (Fig. 5 D). This must be the elastic protein which appears slightly different only towards the periphery but otherwise as in the locust. The cylindrical belly of the tendon is thus a thick-walled tube of pure protein, suitably anchored at both ends by a chitinous bell-shaped system of lamellae and contained between the delicate cortical and central tubes.

When the elastic tendon was stretched or compressed according to the methods described elsewhere (Weis-Fogh, 1961), the ultimate deformability amounted to 2.5 to 3 times the swollen length in extension (pH 7) and to 0.3 in compression, i.e. ten times variation in length in the direction of the axis. The diameter of the belly decreased uniformly with the degree of stretch both along the axis and in cross-section (cf. Pl. 17, fig. 2). The tensile strength was at least 30 kg. cm.^-2. When the belly broke, the surfaces were oriented almost normal to the axis and had a texture like that of broken flint (conchoidal fracture). After the break the two surfaces of the unstrained parts fitted exactly so that flow was absent even under these extreme conditions. When extended isotonically up to twice its previous length in neutral saline, no flow could be measured after days and even weeks of strain at room temperature; on release the tendon immediately snapped back to its original isotropic length within the degree of accuracy of estimation (0.5%). The only difference was a faint residual birefringence, too small for measurement, next to the central canal after strains lasting for a week or more. It seemed to be connected with permanent deformations of the central tube and not of the rubber. After the break, the central tube sometimes projected from the surface; the cortical tube never came loose. The elastic recovery of the pure protein is therefore complete and instantaneous in the elastic tendon, in contrast to the prealar arm and the wing hinge where it takes some time, probably due to the chitin component (Martin Jensen & Weis-Fogh, 1961).

In extension the elastic modulus was estimated as about 6 kg. cm.^-2, allowance being made for the degree of swelling (cf. Treloar, 1958; Weis-Fogh, 1961). It was of the same magnitude in compression where neither the central nor the cortical tube could contribute to the elastic force. Taken together with the digestion experiments already described, the above results therefore apply to the rubber-like protein itself.

Pl. 17, fig. 2 shows a series of photomicrographs of the metathoracic tendon of Aeshna cyanea in neutral saline placed between crossed Nicols. The belly is completely isotropic at the unstrained length (λ = 1) and becomes increasingly birefringent, positive in the direction of extension, up to the greatest length (λ = 2.48). In the last photograph, the relative darkening is due to the appearance of the first-order red interference colour. A dark band at either end marks the transition between the anchoring zone and the ordinary birefringent tendon. The unstrained belly and pieces cut from it were isotropic in all directions and at all degrees of swelling. The protein is therefore not constrained in any direction by materials behaving differently from itself. In highly swollen tendons (pH 12), there was sometimes a faint longitudinal striation not visible at ordinary degrees of swelling, but since it does not reflect any noticeable differences in strength or density it is justifiable to consider the protein as an isotropic rubber.

Apart from the epicuticle, typical rubber-like cuticle consists of only two components both of which are insoluble and heat stable. The solid part consists of thin (0.2 µ) glossy lamellae of chitin which are spaced apart and glued together by thick continuous layers of an isotropic rubbery protein. In some places the protein is the only component of mechanical significance. Pore canals and other complicated structures or inclusions have never been observed.

The structural, optical and mechanical properties of the protein are basically alike in the three samples and correspond to those of a true rubber. Since, moreover, the reactions to water, dyes and chemicals seem identical, and since it has a unique amino-acid composition the protein component will be called resilin from now on (from Latin resilire, to spring back; Bailey & Weis-Fogh, 1961). It is further characterized in the next section.

Except where stated otherwise, the following tests were performed on slices cut from the three types of rubber-like cuticle just described.

In neutral water and in sealed tubes resilin does not dissolve and is not affected by heating up to 125°C. and it does not begin to disintegrate until between 140 and 150° C.

The following protein coagulants, tanning agents and fixatives all failed to bring about any permanent change from the rubbery to the solid state even after prolonged treatment, i.e. no histological or mechanical ‘stabilization’ is imposed: methanol, ethanol, acetone, 5% acetic acid, 5% HgCl₂, 1.5% K₂Cr₂O₇ dilute as well as 40% formaldehyde, saturated aqueous p-benzoquinone in phosphate buffer at pH 7.6 (cf. Gustavson, 1956), 1 % OsO₄ in neutral veronal buffer for 3 hr., Susa’s, Bouin’s (aqueous and alcoholic), and Carnoy’s solutions.

When treated for 3 days at room temperature in aqueous 1 % CrO₃ with or without addition of sodium chloride and osmium tetroxyde (Flemming’s solution), the strongly oxidizing reagent darkens the elastic tendon and causes it to shrink. The resilin is obviously degraded since it dissolves rapidly at pH 12. In fresh 10 % hypochlorite the entire tendon rapidly dissolves. If left for days in 1 % OsO₄, it goes pitch black by osmium deposits but still shows reversible swelling afterwards, although of reduced amplitude. When fixed according to Palade (1952) or Sjöstrand (1956), resilin attains only a light, smoky colour. It should be stressed that all aqueous solutions quickly and easily penetrate the rubber.

Isolated resilin slowly turns brownish when exposed to atmospheric air but its mechanical properties are not changed.

Some of the above reagents alter the swelling maximum apparently because they block various ionizable side groups (Martin Jensen & Weis-Fogh, 1961). This is, in fact, the only significant change that was observed as a result of treatments which profoundly alter most proteins. The secondary and tertiary structure of resilin must either be extremely stable or of little significance for rubberiness and stability.

Only polar solvents are of interest because non-polar solvents do not seem to penetrate at all. In water-free dioxan, for example, resilin shrinks by dehydration and remains glassy even at elevated temperature. A tendon was stretched in 70% ethanol and dehydrated through the alcohol series. Its dimensions and strain birefringence remained constant in absolute methanol, ethanol, heptan, chloroform, benzene, benzyl alcohol, benzyl benzoate, α-bromonaphthalene, and methylene iodide. There is therefore hardly any chance of finding a non-polar embedding medium for sectioning which can penetrate and thus ‘stabilize ‘resilin and similar proteins.

Some water-free polar solvents plasticize and swell resilin and make it rubbery, but none of them dissolve it. When a fresh piece is placed in glycol or glycerol, water is first removed by an osmotic effect and the sample becomes glassy before it swells again, starting from the outside. The following solvents act as plasticizers : ethylene glycol (slowly at room temperature, quickly at 60° C.), glycerol (starts at 60° C.), glacial formic acid (dissolves Nylon; loosens structure of collagen and keratin and makes them shrink, cf. Lloyd & Garrod, 1946), and concentrated formamide (rubberizes collagen). Several other plasticizers may undoubtedly be found.

Some aqueous solutes are known to break hydrogen bonds in proteins and to dissolve them but these had no effect on resilin : 6 molar urea, saturated guanidine hydrochloride, lithium chloride and iodide in various concentrations and temperatures. Thoulet’s solution causes resilin to swell but does not dissolve it, as mentioned already.

Alkaline thioglycolate reduces the disulphide bonds of cystine and dissolves keratin and so does 6 molar urea plus 1 % thioglycolic acid, but resilin was resistant to these as well as to fresh performic acid which oxidizes the disulphide bridges; Silk fibroin and cellulose are both held together by strong and numerous hydrogen bonds which are broken by alkaline cupric ethylenediamine without damage to the primary backbone (pH 12.4, prepared according to Coleman & Howitt, 1947). This reagent had no effect on resilin or chitin apart from the usual swelling at high pH.

Resilin is thus insoluble in spite of the fact that the molecular units are strongly solvated by water (hydration) and by other polar solvents, even when there is no net charge. It must therefore be cross-linked by very stable bonds or groups.

Fresh diaphanol (Gurr, 50% acetic acid saturated with chlorine dioxide) had an interesting effect. It is known to dissolve quinone-tanned proteins but not chitin in the course of a few days in darkness (cf. Brown, 1950). If a wing hinge such as that in Fig. 1 B is treated with diaphanol, the dark sclerotized cuticle becomes pale like amber in 3 days and is now softer than the rubber-like ligament which appears unchanged. The protein is therefore not cross-linked by quinones ; but in the course of 10 or 12 days it slowly dissolves with the formation of a sticky glue, i.e. it probably breaks up into branched three-dimensional units rather than into linear molecules. The cross-linkages seem more resistant than the peptide backbone.

The insolubility of resilin is in marked contrast to the ease with which it is digested by all the proteolytic enzymes tested so far. Apart from non-crystalline papain at neutral pH and a 1 % solution of the mixed proteolytic enzymes in the saliva of the reduviid bug Platymerus rhadamanthus at 28° C, and pH 7.5 (Soerensen’s buffer, sample supplied by Mr J. S. Edwards, Cambridge), the following crystalline enzymes dissolved pure fresh resilin in the given order of speed; in lamellar cuticle the rate was smaller but eventually all resilin had gone : 1 % porcine pepsin at 38° C. and pH 2.2 (0.1 M Mcllvaine’s buffer; L. Light and Co.), 0.5% elastase from pig pancreas at 37° C. and pH 8.9 (0.1 M Kolthoff’s buffer, sample supplied by Dr M. A. Naughton, Cambridge), 0.5% salt-free chymotrypsin at 37° C. (0.1 M Kolthoff’s buffer; Worthington), 0.5 and 1% trypsin at 38° C. and pH 7.5 (Kolthoff’s buffer ; salt-free trypsin from L. Light and MgSO₄-containing trypsin from Worthington).

Resilin is then easily digested, even by trypsin, and both in acid and alkaline media. The breakdown products are not known but the backbone must have many sites readily accessible to enzymes.

It is well known that the degree of swelling of a polyelectrolyte in water depends both on the solvating power and on the degree of ionization. The swelling increases with the number of ionized sites (cf. polyvinyl phosphate, Katchalsky & Eisenberg, 1950; polyglutamyl-lysyl-cysteine, Tani, Yuki, Sakakibara & Taki, 1953).

In dilute buffers, an amphoteric polyelectrolyte like resilin should therefore exhibit minimum of swelling at the isoelectric point. For this reason and in order to demonstrate the reversibility of the swelling phenomenon and the good correspondence between resilin samples from two different orders of insects (Odonata and Orthoptera), the following experiments were performed. Two almost rectangular pieces were cut from the fresh wing-hinge pad of Schistocerca gregaria, each containing about 2 µ g. of dry matter. Similarly, two cylindrical pieces were prepared from the elastic tendon of Aeshna grandis (deep-frozen). The four pieces were placed on bronze gauze and simultaneously transferred to various dilute buffers (0.01-0.02 M; and 0.1 N-HCI), about 30 min. in each. Three characteristic lengths were measured microscopically as a set for each piece and used to calculate the volume relative to that of the air-dried sample placed in benzene. The accuracy is not great, ± 1-2 % for relative length, so that the difference between the three lengths of one set could only be estimated to within ± 3 % for each pad (found + 4%) and ± 2% for each tendon (found ± 2%). But control observations in polarized light and the observed differences in relative lengths proved that the four samples swelled isotropically throughout the experiments summarized in Fig. 6.

Each point is the average of two samples. The sequence of transfers is indicated by the lower-case letters at the top. Taken together with the smoothness of the curves, the results prove that the swelling of resilin is perfectly reversible, in conformity with the lack of mechanical flow and the chemically stable nature of the crosslinkages already referred to.

The two curves take almost the same course and both the tendon (filled circles) and the pad (dots) seem to have an isoelectric point about pH 4, although they are too flat in this region for accurate estimates. In fact, this flatness explains why the stiffness of the intact prealar arm changed so little from pH 1.8 to pH 5.6 compared with the changes at higher pH (Martin Jensen & Weis-Fogh, 1961).

Resilin from the two sources is then fundamentally alike in physical properties, but the results indicate that the pad may be less solvatable than the tendon; see also the inserted values from glycol and ethanol-water mixtures in Fig. 6B. However, this need not be so because it may only reflect a higher degree of cross-linking in the pad than in the tendon and therefore a higher resistance to swelling in the former, a result in accordance with the differences in elastic modulus.

According to modern views (see, for example, Treloar, 1958), a typical unstrained rubber consists of a three-dimensional network of randomly kinked and randomly oriented long-chain molecules. The actual chemical constitution is immaterial ; the term ‘rubber-like’ describes a specific physical state of matter which is characterized in the following. The junction points of the network are either simple physical entanglements which are liable to flow or stable chemical cross-linkages. The chains must possess many freely rotating links between each pair of junction points and the secondary forces between the chains must be weak so that the average chain is randomly kinked as a result of thermal agitation, i.e. the configurational entropy of the rubber is maximum. The network owes its great deformability to the fact that the probable distance between the end points (junction points) of a randomly kinked chain is only a fraction of the extended chain length. Below a certain temperature, the rubber becomes rigid and solid like glass because the thermal movements are insufficient to overcome secondary intermolecular forces. By solvating the chains (by plasticizers) or by increasing the temperature the material resumes its rubberiness. Such a system is optically and mechanically isotropic in the glassy state, in the rubber-like state and at all degrees of swelling. When strained, the chains are removed from their statistically most probable configuration to which thermal agitation tends to bring them back. At the same time, the rubber becomes birefringent in the direction of the strain provided, of course, that the polarizability is different along the backbone and normal to it. At ordinary temperatures and at degrees of cross-linking compatible with a rubber-like behaviour, both theoretical and experimental estimates of the elastic modulus indicate a range from about 5 to 30 kg. cm.^-2. These figures are two to three orders of magnitude lower than found in most non-rubbery substances.

All the evidence presented here strongly indicate that resilin is a true rubber and nothing speaks against this interpretation. However, its behaviour may result from a random network of protein chains which are regularly coiled and held in the coiled configuration by secondary forces, as is probably the case in keratin swollen with glacial formic acid (Lloyd & Garrod, 1946 ; 1948). The extensibility would then be caused partly by a lengthwise orientation of the network which could account for extensions up to twice the unstrained length and partly by an uncoiling of the chains. The latter mechanism means breaking of the secondary structure of the peptide backbone, and this is highly unlikely because we have seen that resilin is practically unaffected by reagents known to interfere strongly with the secondary structure of proteins (heat, coagulants, tanning agents, fixatives, agents breaking hydrogen bonds). Moreover, the elastic modulus is so low and the concentration of the material so high (40-50 % dry matter at pH 7) that, in the case of uncoiling, the secondary structure of a chain should be broken by forces two magnitudes lower than what is necessary to transform α-keratin into the stretched β-configuration (cf. Bull, 1945). A rigid experimental proof for the rubber-like nature of resilin is provided elsewhere (Weis-Fogh, 1961).

All evidence taken together strongly indicate that there are great physical and chemical similarities between resilin and elastin. Elastin is here considered as the homogeneous protein prepared according to Partridge, Davis & Adair (1955) from the ligamentum nuchae in the form of fragments of rather uniform fibrils, and not as the composite birefringent elastic tissue consisting of elastin, collagen, vitreous fibres and mucopolysaccharides (Hall, Reed & Tunbridge, 1952; Wood, 1954; Cruise, 1957; Romhányi, 1958).

In neutral water elastin and resilin are both rubber-like and swollen with about 50-60% of water, but they become rigid and glass-like on drying (McCartney, 1914). Lloyd & Garrod (1948) claim that elastin swells in methanol and ethanol, but the swelling is insignificant in resilin (Fig. 6 B) which remains rigid at concentrations above 90%. On readmission of water, in aqueous alcohols and in many other polar solvents dried elastin and resilin re-swell isotropically and become rubbery, as in formamide and glacial formic and glacial acetic acid (Lloyd & Garrod, 1948). The isoelectric point of elastin is about pH 4 (Partridge, Davis & Adair, 1955) and, like resilin, it therefore swells in acid as well as in alkaline media. Neither is tanned by chromium salts (Roddy & O’Flaherty, 1939). Both are characteristically insoluble in all solvents and reagents which do not directly attack the peptide backbone (Lloyd & Garrod, 1948), including agents which reduce or oxidise disulphide groups (Wood, 1954); but they are digestible even by trypsin, according to Lloyd & Garrod (1948). They are heat stable and apparently not affected by neutral water up to 150° C.

It is therefore most likely that elastin and resilin share at least two important structural characteristics, a very limited tendency for the backbone to form secondary structures combined with a very stable kind of cross-linkage, the nature of which is unknown in both proteins (cf. Partridge & Davis, 1955). The swelling of resilin is the outcome of forces of solvation, of electrostatic repulsion between ionized groups and of Donnan phenomena.

Apart from these similarities and the large amount of glycyl residues in both, the amino-acid composition of resilin is very different from that of elastin (Bailey & Weis-Fogh, 1961) so that the ideas of Lloyd & Garrod (1948; cf. Kendrew, 1954, p. 948) concerning the conditions necessary for the establishment of rubberiness in proteins must be revised. Resilin is devoid of colour but it is not known whether the yellow pigment in elastin has any real significance or not (cf. Loomeijer, 1958).

Further chemical and physical analyses appear to be much simpler in resilin than in elastin, partly because resilin can be obtained as pure samples with no apparent structural or chemical impurities and partly because Nature has provided a neatly suspended pure sample of convenient shape, the elastic tendon.

It was observed that the large isotropic pad of resilin in the wing hinge of Schistocerca is deposited after the final moult and in the course of the first days of adult life. Since the pad does not contain carbohydrates or amino sugars, it is a particularly favourable object for studies on how the process of cross-linking is brought about and how organisms may synthesize three-dimensional networks. According to Smith (1957), the biosynthesis of elastin is virtually unknown.

The demonstration of a new well-defined protein in insect cuticle is of interest to entomologists, particularly because it is clearly separated from chitin chemically and physically. The optical analysis has been confirmed by electron microscopy to be reported elsewhere. It has led to some new thoughts about the structure and morphogenesis of insect cuticle which are discussed in connexion with mechanical and functional aspects of solid and rubber-like cuticles (Martin Jensen & Weis-Fogh, 1961).

1. A new type of hyaline, colourless cuticle, called rubber-like cuticle, is described and analysed qualitatively with respect to mechanical behaviour, structure and composition. Externally it is covered by ordinary thin epicuticle, but otherwise it represents the simplest type of cuticle known and consists only of thin continuous lamellae of chitin (0.2 µ) separated and glued together by an elastic protein, resilin, not hitherto described. There are only traces of water-soluble substances present and resilin sometimes occurs as pure, hyaline patches more than 100µ thick and suitable for macroscopic experiments.

2. In all physical respects, resilin behaves like a swollen isotropic rubber but the rigid experimental proof is given elsewhere (Weis-Fogh, 1961). An outstanding feature is the complete lack of flow not paralleled by other natural or synthetic rubbers.

3. Resilin resembles elastin but it is devoid of colour and has a different and characteristic amino-acid composition (Bailey & Weis-Fogh, 1961). The nature of the cross-linkages is unknown at present but they are extremely stable, of aco-valent type and different from other known cross-linkages in proteins. This accounts for its insolubility and resistance to all agents which do not break the peptide backbone.

4. Resilin is a structure protein in which the primary chains show little or no tendency to form secondary structures; they are bound together in a uniform three-dimensional network (the tertiary structure) with no potential limits as to size.

It is a pleasure to thank Sir James Gray, F.R.S., and Prof. V. B. Wigglesworth, F.R.S., for their help and hospitality during my years as a Balfour Student in the Department of Zoology, Cambridge. I have also profited much from discussions with Dr L. E. R. Picken, Dr M. G. M. Pryor and Dr O. Sten-Knudsen. The work was supported by grants from the Carlsberg Foundation, The Department of Scientific and Industrial Research and from The Rockefeller Foundation.