Introduction
The arthropod cuticle is one of the most remarkable and versatile biological material [1]. Not only it provides protection to all body parts, but it also forms highly sophisticated appendices, dedicated to specific functions. The cuticle enables basic tasks such as physiological exchanges, sensing and actuation[2–4], walking, climbing and adhesion on different surfaces [5], swimming and flying[6]. Food treatment (e.g. through mandibles and stomach teeth) [7], mechanical[8,9] and optical shielding[10], as well as puncturing and capturing[11] are additional ingenious functions performed with the cuticle. It is remarkable that such multifunctionalitiy is obtained thanks to a tiny and lightweight biological structure, formed by the epidermis, renewed periodically by the moult process, and essentially comprising a thin superficial waterproof layer (the epicuticle) made of a lipid-protein matrix, and a thicker internal fibrous layer (the procuticle), generally subdivided into exo- and endocuticle depending on secretion time [12,13]. The basic building blocks of the procuticle are the chitin-protein microfibers, composed of a crystalline α-chitin core which is coated by a helical sheath of protein units [14–17]. Matrix proteins are also found outside the microfibers when considering higher structural organization. The chitin-protein microfibers interact with a (partially) proteinaceous and hydrated matrix, which features a rich variety of proteins, enabling selective cross-linking[17–19]. The relative fraction of protein and matrix is not well known for the mantis shrimp and may depend on the considered region. Previous data on decalcified crab cuticle indicates that chitin content should range between 50 to 75% dry weight[20]. In areas of the cuticle where hardness and wear resistance are major requirements, the chitin-protein biocomposite is reinforced either by mineral incorporation in the matrix or by sclerotization that cross-link the matrix, sometimes with the help of transition metals (e.g. Mn, Zn, Fe) as seen in insect mandibles[21] and spider fangs [22]. In the procuticle, the chitin-protein microfibers assemble in larger bundles to form fibers and, in turn, the fibers form higher order structural motifs: arrays of locally aligned fibers are assembled into beds, and several beds are stacked into a three-dimensional helicoidal twisted plywood structure, resembling a man-made laminate, but at the nano/micro length scales [23,24] . The regular rotation of the fibers in horizontal beds gives rise to a periodic lamellate aspect, each lamella corresponding to a rotation of 180°. The composition of the matrix, the type of sclerotization, the relative fraction of reinforcing minerals, together with the tunable fiber organization and lamellar thickness are at the basis of the outstanding versatility of the arthropod cuticle and, consequently, they are key elements of the evolutionary success of arthropods in a variety of very dissimilar environments [24,25].
An instructive example of how the cuticle has evolved into highly specialized tools, even within the same organism, is found in the stomatopod crustaceans (or mantis shrimps). During their evolution, these marine animals modified their cuticle for feeding, hunting and defense purposes [9]. Stomatopods are traditionally subdivided into two branches based on the structure of their anterior appendages [26–28]: the well-known “smashers”, that use a hammer-like club to destroy hard-shell preys, and the less-famous “spearers” (Figure 1 A) that have a harpoon-like appendage to impale and grasp their (generally) soft-body preys. Thanks to an efficient “amplification system”, which includes a dedicated area (the saddle) to store and release elastic energy[29], together with an ingenious system of articulation [30], stomatopods can deploy their anterior appendages at impressive velocities (up to 6 m/s for the spearers and to 23 m/s for the smashers) [31]. Repetitive impacts at such velocities require excellent damage tolerance, and previous investigations have revealed that the hammer-club of the smashers shows numerous strategies to cope with damage [28,32–34]. Macroscopically, the size of the club is smaller than the critical radius at which, according to contact mechanics, the damage response switches from quasi-plastic to brittle, thus hampering catastrophic failure [33]. Microscopically, the club presents three different regions solving different mechanical functions: a heavily mineralized impact region characterized by mineral gradients [32,35] and by oriented fluorapatite (FAP) crystals perpendicular to the outer surface to enhance impact resistance; beneath the impact region there is a less mineralized periodic region showing the common helicoidal twisted plywood pattern, which may dissipate possible cracks generated during impact [32]; on the lateral sides, the periodic region is encircled by a striated region with chitin fibers well aligned along a preferential orientation [32]. This belt-like area is believed to provide later confinement to the periodic region, hence preventing high tensile stresses and the associated tensile cracking [28].
Although deployed at smaller velocities, the anterior appendage of the spearer is not less fascinating. It is a biological tool solving multiple functions: its base presents a flat surface used to hammer the opponents and sharing some of the construction strategies seen in the smasher [28,35]. On the top, it is decorated by several spikes (Figure 1B) which, in analogy to man-made harpoons, have to penetrate and grab preys within a fraction of second during a high-speed capturing event [31]. This task requires the combination of conflicting mechanical requirements: the spike must be tough to cope with the initial impact with the victim, it must also be stiff to avoid large deflections while penetrating a moving target and it must be strong to prevent rupture and prey lost. These constrains should come together with additional strategies to retain the prey. By comparing the spike cuticle of spearing limbs with different body parts (i.e., cephalic shield and abdominal tergites) we revealed some compositional and microstructural modifications of the cuticle in the spike. These include: (i) the replacement of the leathery inner epicuticle (commonly having an organic nature) by a highly mineralized exocuticle reinforced by FAP, and (ii) the development of a unidirectional fiber region interrupting the classical helicoidal twisted plywood structure in the less mineralized endocuticle[36]. Here, we investigate the spike in more details and at multiple length scales using micro-computed tomography (micro-CT), backscattered and secondary electron imaging based on scanning electron microscopy (BSE-SEM and SE-SEM), elemental analysis by energy dispersive spectroscopy (EDS), confocal Raman spectroscopy, depth sensing nanoindentation and multimaterial 3D printing. The central aim of our work is to elucidate the structure-function relationship and the mechanical behavior of the spike, highlighting the morphological, compositional, and microstructural adaptations allowing this biological tool, which is built very quickly, to be an effective natural harpoon.
Results and Discussion