722 The Journal of Experimental Biology 209, 722-730 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) N. Barbakadze, S. Enders*, S. Gorb and E. Arzt Evolutionary Biomaterials Group, Department Arzt, Max-Planck-Institute for Metals Research, Heisenbergstr. 3, 70569, Stuttgart, Germany *Author for correspondence (e-mail: [email protected]) Accepted 27 December 2005 Summary Insect exoskeleton (cuticle) has a broad range of layer on the mechanical behavior of the material, the mechanical properties depending on the function of a samples were tested in fresh, dry and chemically treated particular structure of the skeleton. Structure and (lipid extraction in organic solvents) conditions. mechanical properties of the specialised cuticle of insect Nanoindentation results were found to be strongly joints remain largely unknown to date. We used scanning influenced by desiccation but only slightly by lipid (SEM) and transmission electron microscopy (TEM) to extraction. Decreasing water content (~15–20% of the obtain information about the material structure of the cuticle mass) led to an increase in hardness (from 0.1 to gula plate, the head part of the head-to-neck articulation 0.49·GPa) and elastic modulus (from 1.5 to 7.5·GPa). The system in the beetle Pachnoda marginata. The surface of lipid extraction caused a slight further hardening (to this cuticle appears rather smooth in SEM. The fibers of 0.52·GPa) as well as stiffening (to 7.7·GPa) of the material. the exocuticle are partly oriented almost perpendicular to The results are discussed in relation to the mechanical the surface, which is rather unusual for arthropod cuticle. function of the gula plate. Nanoindentation experiments were performed to determine the local mechanical properties (hardness and elastic modulus) of the gula material. To understand the Key words: desiccation, gula plate, insect cuticle, mechanical effect of desiccation and the influence of an outer wax property, nanoindentation. Introduction Arthropod cuticle has a multilayer structure (Neville, 1975; The body of insects is completely covered by cuticle, which Andersen, 1979) (Fig.·1). It typically consists of three main is a multifunctional interface between the animal and the layers: epicuticle, exocuticle and endocuticle. The latter two environment (Hepburn and Joffe, 1976; Gorb, 2001). It serves layers form the procuticle. In some insects, there is a layer of primarily as an exoskeleton that gives the body its shape and mesocuticle located between the endocuticle and the stability. The cuticle is also a barrier against evaporation of exocuticle (Neville, 1975; Andersen, 1979; Noble-Nesbitt, water from the body and hence protects the insect against 1991). The non-chitinous, tanned lipoproteinous epicuticle desiccation (Locke, 1964; De Renobales et al., 1991; Noble- is the outer layer, which is very thin and has a relatively Nesbitt, 1991). high tensile strength (Bennet-Clark, 1963). The surface Like most biological materials, cuticle is a fiber composite of the epicuticle is coated with wax and lipids. The (Hepburn and Chandler, 1980). The fibers mainly consist of exocuticle has a dense chitin–protein structure and may chitin, and the matrix is formed by proteins. Chitin is a natural become hard and stiff due to sclerotization. The endocuticle polymer composed of 300·nm-long and 3·nm-thick nanofibrils is usually the thickest region of the cuticle (Locke, 1964; (Vincent, 1980; Vincent, 2002). Each nanofibril contains ~19 Neville, 1975). molecular chains (Vincent, 1980; Vincent, 2002) running anti- In various animal groups and on different body parts, there parallel to one another (i.e. alpha chitin) (Neville, 1975; Vincent, are different types of cuticle varying in mechanical properties 1980). The protein matrix stabilizes the chitin fibers. It normally from very hard and brittle to soft, ductile and also rubber-like contains some amount of water (in some cuticles it is ~90% of (Jensen and Weis-Fogh, 1962). In many larvae, the cuticle is the protein matrix mass). The function of water in the cuticle is soft and colorless. During sclerotization, a hard and colored largely unknown, but presumably it is separation of the two main integument is formed in most adults (Fraenkel and Rudall, components of the cuticle from each other (Vincent, 1980). 1940). The variation in mechanical properties depends on THE JOURNAL OF EXPERIMENTAL BIOLOGY Mechanical properties of gula plate cuticle 723 Epicuticle the cuticle were studied by means of scanning electron microscopy (SEM). The gula samples were mechanically Exocuticle tested in the fresh, dry and chemically treated conditions, in order to identify the influence of desiccation (dry versus fresh Endocuticle Cuticle conditions) and removal of an outer wax layer (dry versus Procuticle chemically treated conditions). Desiccation rate measurements were also carried out, in order to obtain information about the Epidermal cell layer 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- Fig.·1. Diagram of the multilayered structure of the insect cuticle. The reducing and wear-resistant properties. The following shaded areas represent the single cuticle layers: non-chitinous questions were asked. (1) Do the structure and local epicuticle; procuticle consisting of exocuticle and endocuticle with mechanical properties of the gula show any features of chitin fibers usually oriented parallel to the surface. An epidermal cell layer underlies the endocuticle. 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 fractions of the main constituents (Hepburn and Chandler, hardness and elastic modulus of the gula? 1976). Mechanical properties of the insect cuticle are adapted to the function of particular body parts. Properties of the material of Materials and methods insect joints remain largely unknown. The present Sample preparation investigation focuses on the head articulation system of the Samples were prepared from the gula of the beetle Pachnoda beetle Pachnoda marginata (Fig.·2). Surfaces in this system marginata Drury. The beetles were obtained from a commercial operate in contact and must be resistant to wear and friction supplier (Insektenfarm Walta, Schkeuditz, Germany), kept in and at the same time have to provide high mobility of the joint. the plastic terraria and fed with various fruits. For structural and From studies on technical systems, the structure and the experimental studies, beetles were anesthetized with CO2, their mechanical properties of superficial layers of the material are heads were cut off from the bodies, and the edges of the believed to be very important for its tribological performance posterior openings (foramen occipitale) of the head were freed (Scherge, 2003; Chakhvorostov, 2004). of soft tissues. To prevent specimen desiccation, fresh gula All mechanical investigations were carried out on the head cuticles were tested immediately (within 3–5·min after part of the head articulation called gula, which is normally in dissecting from the body). Dry samples were obtained from contact with the counter surface of the thorax during the head fresh ones by drying in an oven for 24·h at 40°C. For lipid movement. The gula is a median ventral plate of the head, in extraction (chemical treatment), the dry samples were some prognathous insects formed by a sclerotization of the immersed in a solution of chloroform and methanol (2:1) for neck region proximal to the posterior tentorial pits, continuous 50–60·min and finally air-dried. with the postmentum or submentum (Snodgrass, 1935). In the present study, the detailed information about the surface and Scanning electron microscopy internal structure of the gula cuticle was obtained, and The samples were fixed in 2.5% glutaraldehyde in a mechanical properties (hardness and elasticity modulus) of its phosphate buffer (pH 7.3). The specimens were dehydrated in superficial cuticle layers were measured using an ascending series of ethanol and then critical-point dried. nanoindentation. The surface morphology and ultrastructure of 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) A A B a l l and examined in a Hitachi S-800 scanning electron microscope (SEM) at 20·kV. md Gula Transmission electron microscopy Gula For transmission electron microscopy (TEM), fresh gula was fixed for 12·h at 4°C in 2.5% glutaraldehyde Prothorax (in 0.01·mol·l–1 phosphate buffer at pH 7.3) and postfixed for 1·h in 1% osmium tetroxide in phosphate Prothorax buffer at 2°C (Gorb, 1998). After washing in distilled water, the preparations were stained for 1·h at 4°C in Fig.·2. Diagram of the location of the gula surface in the beetle body. 0.1% aqueous uranyl-acetate solution, washed, Parasagittal (A) and frontal (B) virtual sections through the head–neck dehydrated and embedded in a low-viscosity resin articulation. a, anterior direction; l, lateral direction; md, midline. (Spurr, 1969). Ultra-thin sections were picked up on THE JOURNAL OF EXPERIMENTAL BIOLOGY 724 N. Barbakadze and others Force Coil/magnet assembly generation Support springs Indenter tip – type Berkovich Displacement Capacitance gauge measurement Fig.·3. (A) Diagram of the nanoindenter system used (Enders, 2000). The force imposed on the Indenter tip indenter is generated through a coil 13° 25° that sits within a circular magnet. The displacement sensing system consists Lateral motion Pyramid with equilateral of a three-plate (circular disks) stages triangle base capacitor. (B) Berkovich tip: a pyramid with an equilateral triangle as Sample tray the base area. Computer A B copper grid slots coated with formvar film (Plano GmbH, maximum load and the contact area, A, generated during the Wetzlar, Germany), contrasted with uranyl acetate and lead indentation: citrate, and observed in TEM Philips CM10.