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