Microstructure
The microstructure of the spike, observed with SEM on fractured and polished samples, features four regions with distinct fiber architecture (Figures 2 A, B and summery scheme in I). The first innermost layer, named inner helicoidal region (IHR, Figure 2C), belongs to the endocuticle [36] and displays a twisted plywood organization consisting of numerous lamellae gradually increasing in thickness from about 1.5 to 10 µm when moving outwards. Within each lamella, layered beds of chitin-protein fibers embedded into a protein-mineral matrix are stacked with regular changes in fiber directions between each bed, resulting into stepwise and rough fracture surface (Figure 2F). Pore canals, pervading the cuticle up to the epicuticle, are visible at high magnification SEM and have a characteristic twisted-ribbon shape [44] with unmineralized dark lumens (Figure 2C). They serve for the secretion of the cuticle and perhaps provide supplementary “waterproof” capability, as inferred by their lipid content [36]. In the second layer, referred as the striated region of the endocuticle (STR), the helicoidal pattern is replaced by highly aligned fibers parallel to the long axis of the spike as highlighted by a longitudinally fractured sample showing fiber sheets (oriented vertically in Figure 2G). In this region, the pore canals lose their typical twisted-ribbon aspect and cross perpendicularly the horizontal fiber beds, giving a characteristic striated pattern typically observed in polished (transverse) sections, where the canal lumen appears dark and the mineralized fibrous sheath appears bright (Figure 2D). While the twisted plywood motif is universal in arthropods cuticle, the parallel-fiber organization is very unusual[45]. In stomatopods, striated regions with parallel longitudinal fibers have been found only in specific locations including the dactyl club of the smasher [32], the impact area of the dactyl in the appendage carrying the spikes of the spearer [28] and the back defensive spikes located at the uropods (posterior structures found in crustaceans)[42]. One common feature of those locations is a challenging biomechanical environment, solving critical offensive or defensive tasks. Most interesting, sandwiched between the striated and the external region, there is an additional layer of the endocuticle[36] presenting a twisted plywood organization (Figure 2E), called outer helicoidal region (OHR). This location has two unusual features: it is composed of a very limited number of lamellae, spanning a width of only a few micrometers, and it appears darker under backscattered electron (BSE) imaging, indicating a lower mineral content. Even more, when going from this outer helicoidal region to the exterior of the spike exocuticle, the helicoidal architecture is not lost immediately, but there is a transition region (TR) having high mineral content (arrows in Figure 2E) and characterized by one interface lamella. The last region of the spike cuticle identified here is the external highly mineralized region (HMR), where larger and more isolated fibers achieve a last half rotation within a heavily mineralized matrix before the twisted plywood organization is finally lost towards the spike outer surface [36]. The presence of a continuous twisted plywood at the interface between the highly mineralized region and the less mineralized outer helicoidal region is a remarkable feature of the spike that may enhance the anchoring between such dissimilar layers, as suggested by the rough fracture surface at that location (Figure 2H). A closer examination of fracture surfaces (Figure S4 ) highlights that in this highly mineralized region, crystals form nanometer sized crystallites, in analogy with those found in the dactyl club’s outer layer [35]. The remaining of the highly mineralized region presents a very low fraction of chitin-protein fibers [36], but is still travelled by numerous pore canals, running perpendicular to the interface with the outer helicoidal region (Figure 2E). The spike cross-section is largely dominated by the striated and the inner helicoidal regions: for about two-third of the spike length, 90% of the wall thickness is only due to those two regions with the contribution of the outer helicoidal region and of the highly mineralized region being about 3% and 7%, respectively (Figure S5 ).