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\begin{document}

\title{Design Strategies of the Mantis Shrimp Spike: How the Crustacean Cuticle
Became a Remarkable Biological Harpoon?}



\author[1]{Yann Delaunois}%
\author[1]{Alexandra Tits}%
\author[1]{Quentin Grossman}%
\author[1]{Sarah Smeets}%
\author[1]{Cedric Malherbe}%
\author[1]{Gauthier Eppe}%
\author[2]{Harry van Lenthe}%
\author[1]{Davide Ruffoni}%
\author[1]{Philippe Compere}%
\affil[1]{University of Liege}%
\affil[2]{KU Leuven}%


\vspace{-1em}



  
  \date{May 27, 2024}


\begingroup
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\selectlanguage{english}
\begin{abstract}
Spearing mantis shrimps are aggressive crustaceans using specialized
appendages with sharp spikes to capture fishes with a fast movement.
Each spike is a biological tool that have to combine high toughness, as
required by the initial impact with the victim, with high stiffness and
strength, to ensure sufficient penetration while avoid breaking. We
performed a multimodal analysis to uncover the design strategies of this
harpoon based on chitin. We found that the spike is a slightly hooked
hollow beam with the outer surface decorated by serrations and grooves
to enhance cutting and interlocking. The cuticle of the spike resembles
a multilayer composite: an outer heavily mineralized, stiff and hard
region (with average indentation modulus and hardness of 68 and 3 GPa),
providing high resistance to contact stresses, is combined with a less
mineralized region, which occupies a large fraction of the cuticle (up
to 50\%) and features parallel fibers oriented longitudinally, enhancing
stiffness and strength. A central finding of our work is the presence of
a tiny interphase (less than 10 \selectlanguage{greek}μ\selectlanguage{english}m in width) based on helical fibers and
showing a spatial modulation in mechanical properties, which has the
critical task to integrate the stiff but brittle outer layer with the
more compliant highly anisotropic parallel fiber region. We highlighted
the remarkable ability of this helicoidal region to stop
nanoindentation-induced cracks. Using three-dimensional multimaterial
printing to prototype spike-inspired composites, we showed how the
observed construction principles can not only hamper damage propagation
between highly dissimilar layers (resulting in composites with the
helical interphase absorbing 50\% more energy than without it) but can
also enhance resistance to puncture (25\% increase in the force required
to penetrate the composites with a blunt tool). Such findings may
provide guidelines to design lightweight harpoons relying on
environmentally friendly and recyclable building blocks.%
\end{abstract}%



\sloppy


\textbf{Design Strategies of the Mantis Shrimp Spike: How the Crustacean
Cuticle Became a Remarkable Biological Harpoon?}

Yann Delaunois\textsuperscript{1}, Alexandra Tits\textsuperscript{2},
Quentin Grossman\textsuperscript{2}, Sarah Smeets\textsuperscript{1},
C\selectlanguage{ngerman}édric Malherbe\textsuperscript{3}, Gauthier Eppe\textsuperscript{3}, G.
Harry van Lenthe\textsuperscript{4}, Davide
Ruffoni\textsuperscript{2*}and Philippe Compère\textsuperscript{1,5*}

\textsuperscript{1}Laboratory of Functional and Evolutionary Morphology,
FOCUS Research Unit, Department of Biology, Ecology and Evolution,
University of Liège, Liège, Belgium.

\textsuperscript{2}Mechanics of Biological and Bioinspired Materials
Laboratory, Department of Aerospace and Mechanical Engineering,
University of Liège, Liège, Belgium.

\textsuperscript{3}Mass Spectrometry Laboratory, MolSys Research Unit,
Department of Chemistry, University of Liège, Liège, Belgium.

\textsuperscript{4}Department of Mechanical Engineering, KU Leuven,
Leuven, Belgium.

\textsuperscript{5}Center for Applied Research and Education in
Microscopy (CAREM) and Biomaterials Interfaculty Center (CEIB),
University of Liège, Liège, Belgium.

*Equal supervision of the work and corresponding authors.

Corresponding authors:

Davide Ruffoni, druffoni@uliege.be

Department of Aerospace and Mechanical Engineering

University of Liege

Quartier Polytech 1, Allée de la Découverte 9

B-4000 Liège, Belgium

Philippe Compère, pcompere@uliege.be

Department of Biology, Ecology and Evolution,

University of Liège

Quartier Agora, Allée du six Août 15

B-4000 Liège, Belgium

ABSTRACT

Spearing mantis shrimps are aggressive crustaceans using specialized
appendages with sharp spikes to capture fishes with a fast movement.
Each spike is a biological tool that have to combine high toughness, as
required by the initial impact with the victim, with high stiffness and
strength, to ensure sufficient penetration while avoid breaking. We
performed a multimodal analysis to uncover the design strategies of this
harpoon based on chitin. We found that the spike is a slightly hooked
hollow beam with the outer surface decorated by serrations and grooves
to enhance cutting and interlocking. The cuticle of the spike resembles
a multilayer composite: an outer heavily mineralized, stiff and hard
region (with average indentation modulus and hardness of 68 and 3 GPa),
providing high resistance to contact stresses, is combined with a less
mineralized region, which occupies a large fraction of the cuticle (up
to 50\%) and features parallel fibers oriented longitudinally, enhancing
stiffness and strength. A central finding of our work is the presence of
a tiny interphase (less than 10 \selectlanguage{greek}μ\selectlanguage{english}m in width) based on helical fibers and
showing a spatial modulation in mechanical properties, which has the
critical task to integrate the stiff but brittle outer layer with the
more compliant highly anisotropic parallel fiber region. We highlighted
the remarkable ability of this helicoidal region to stop
nanoindentation-induced cracks. Using three-dimensional multimaterial
printing to prototype spike-inspired composites, we showed how the
observed construction principles can not only hamper damage propagation
between highly dissimilar layers (resulting in composites with the
helical interphase absorbing 50\% more energy than without it) but can
also enhance resistance to puncture (25\% increase in the force required
to penetrate the composites with a blunt tool). Such findings may
provide guidelines to design lightweight harpoons relying on
environmentally friendly and recyclable building blocks.

\textbf{Introduction}

The arthropod cuticle is one of the most remarkable and versatile
biological material \textsuperscript{{[}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\textsuperscript{{[}2--4{]}}, walking, climbing and adhesion on
different surfaces \textsuperscript{{[}5{]}}, swimming and
flying\textsuperscript{{[}6{]}}. Food treatment (e.g. through mandibles
and stomach teeth) \textsuperscript{{[}7{]}},
mechanical\textsuperscript{{[}8,9{]}} and optical
shielding\textsuperscript{{[}10{]}}, as well as puncturing and
capturing\textsuperscript{{[}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
\textsuperscript{{[}12,13{]}}. The basic building blocks of the
procuticle are the chitin-protein microfibers, composed of a crystalline
\selectlanguage{greek}α-\selectlanguage{english}chitin core which is coated by a helical sheath of protein units
\textsuperscript{{[}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\textsuperscript{{[}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\textsuperscript{{[}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\textsuperscript{{[}21{]}} and spider fangs
\textsuperscript{{[}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
\textsuperscript{{[}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\selectlanguage{ngerman}°. 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
\textsuperscript{{[}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 \textsuperscript{{[}9{]}}. Stomatopods are
traditionally subdivided into two branches based on the structure of
their anterior appendages \textsuperscript{{[}26--28{]}}: the well-known
``smashers'', that use a hammer-like club to destroy hard-shell preys,
and the less-famous ``spearers'' (\textbf{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\textsuperscript{{[}29{]}}, together with an ingenious system of
articulation \textsuperscript{{[}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) \textsuperscript{{[}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 \textsuperscript{{[}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 \textsuperscript{{[}33{]}}.
Microscopically, the club presents three different regions solving
different mechanical functions: a heavily mineralized impact region
characterized by mineral gradients \textsuperscript{{[}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 \textsuperscript{{[}32{]}}; on the lateral sides, the periodic
region is encircled by a striated region with chitin fibers well aligned
along a preferential orientation \textsuperscript{{[}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 \textsuperscript{{[}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 \textsuperscript{{[}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 \textsuperscript{{[}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: \emph{(i)} the replacement of the leathery
inner epicuticle (commonly having an organic nature) by a highly
mineralized exocuticle reinforced by FAP, and \emph{(ii)} the
development of a unidirectional fiber region interrupting the classical
helicoidal twisted plywood structure in the less mineralized
endocuticle\textsuperscript{{[}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.

\textbf{Results and Discussion}\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Figure 1: Morphology and biomechanics of a spike from the spearing
mantis shrimp \emph{L. maculata}. (A) Photograph of a specimen (scale
bar: 5 cm) and (B) of a raptorial appendage showing, in the white boxes,
the distal spikes characterized here (scale bar: 1 cm). (C) Micro-CT
reconstruction of a distal spike attached to the dactyl. The grey levels
give a qualitative indication of the heterogeneous density of the
cuticle (increasing density from light grey to black). (D) Micro-CT
reconstruction of a single spike showing the gross morphology.
Superimposed to the micro-CT are arrows having length proportional to
the local curvature \selectlanguage{greek}κ \selectlanguage{english}of the spike (a representative arrow indicating a
curvature of 0.14 mm\textsuperscript{-1} is shown at the bottom). The
inserts (yellow and orange boxes) show two typical cross-sections at the
base and near the tip of the spike (scale bars: 200 \selectlanguage{greek}µ\selectlanguage{english}m). By comparing
the two cross-sections, a slight rotation (of about 15 deg) along the
main axis of the spike is evident. (E) Side view of the lateral outer
border of the spike in SE-SEM showing serrations and surface grooves.
(F) Spatial variations of maximum (I\textsubscript{max}) and minimum
(I\textsubscript{min}) second area moments as well as polar moment (J)
of the spike cross-sections along its length.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Morphology and biomechanics of the entire spike}

The raptorial appendage of the spearer zebra mantis
shrimp\emph{Lysiosquillina maculata} (Figure 1A) features 10 spikes of
increasing size, regularly arranged along the dactyl (Figure 1B). The
morphology and the external surface of distal spikes were characterized
by high resolution micro-CT and SEM. Each distal spike is a tapered,
thin and elongated offensive weapon (Figure 1C). In contrast to the
straight needle-like shape common to many man-made
harpoons\textsuperscript{{[}11{]}}, it has a curved form and an
elliptical cross-section (Figure 1D). The local curvature of the spike
varies along its length such that 3 regions can be identified: a
slightly curved zone close to the base, a fairly straight segment in the
middle and a region with the highest curvature before reaching the tip
(Figure 1D, the length of the arrows is proportional to the local
curvature k). This allows the spike to orient its sharp end at an almost
right angle with respect to the dactyl within a short distance, while
avoiding a large angle at the base, which may cause stress localization
upon bending. A curved rather than a straight profile may be linked to
the curved trajectory of the appendage during a strike
\textsuperscript{{[}31{]}}and could as well facilitate the parking of
the spikes within dedicated holes in the propodus at resting position
(\textbf{Figure S1} ). In nature, the shape of puncturing systems is
highly diverse and closely connected with their function
\textsuperscript{{[}11{]}}. A straight offensive tool such as the
harpoon of the cone snail\textsuperscript{{[}37{]}}, the bee stinger
\textsuperscript{{[}38{]}}or the mosquito proboscis
\textsuperscript{{[}39{]}} may be used to maximize penetration depth
\textsuperscript{{[}11{]}}, whereas highly curved devices like
crustaceans claws \textsuperscript{{[}40{]}} or spider fangs
\textsuperscript{{[}41{]}} should enable puncture along different
trajectories and facilitate prey retention\textsuperscript{{[}35,37{]}}.
The unusual form of the spike, featuring both curved and straight zones,
may be a trade-off between large penetration depth and prey grabbing.
Micro-CT imaging of the spike cross-section reveals a thin highly
mineralized cover bordering a less mineralized but much thicker layer
with a central cavity (Figure 1D). This dual mineralization is a
specific reinforcement feature of raptorial appendages used for impaling
or smashing preys as well as for fighting with opponents
\textsuperscript{{[}28,32,34--36{]}}. In general, the electron dense
region is particularly pronounced on the impact surface and much less in
other region of the cuticle\textsuperscript{{[}9,42{]}}. After a heavily
mineralized and sharp tip, characterized by a radius of curvature of
about 20 \selectlanguage{greek}µ\selectlanguage{english}m, which should facilitate puncture into the tough skin of the
preys, the spike exhibits a spatially varying geometry. The
cross-section has an ellipsoidal profile and the cross-sectional area
increases gradually from tip to base, always having a pronounced
eccentricity (\textbf{Figure S2} ). An additional peculiarity is a small
rotation (of about 15\selectlanguage{ngerman}°) of the cross section around the longitudinal
axis of the spike (insert in Figure 1D), resulting in a slightly twisted
shape. From a biomechanical viewpoint, such a slender and eccentric
hollow beam may suffer from low bending and torsional rigidity, thus
questioning the ability to remain straight during a capturing event. We
evaluate how cross sectional properties evolve along the longitudinal
length of the spike from tip to base and we estimate the consequences
for bending and torsional resistance using second area and polar moments
as surrogate for mechanical properties (Figure 1F). Both maximum and
minimum second moments of area (\(I_{\max}\) and
\(I_{\min}\)), corresponding approximately to dorso-ventral and
lateral loading, increase proximally and show a steeper slope starting
at \selectlanguage{english}~90\% of the spike length, where the spike begins to merge with the
appendage. The polar moment \(J\) (a proxy for torsional
rigidity) follows a similar trend, being bounded by\(I_{\min}\)
and\(\ I_{\max}\). Despite a fairly high eccentricity, the ratio
\(I_{\max}/I_{\min}\) suggests only a moderate mechanical anisotropy, with
resistance to dorso-ventral loading (i.e., parallel to the prevalent
hunting movement of the raptorial appendage) being in average 3.3 times
higher than lateral loading. Additionally, the ratio\(I_{\max}/I_{\min}\)
is fairly constant for about 60\% of the spike length. Although
simplified, this analysis suggests that the spike is not only very stiff
along the longitudinal direction but it can also face multiaxial
loading, consistent with the assumed ability of the spearing mantis
shrimps to control and direct the strike\textsuperscript{{[}31{]}}.

The outer surface of the spike is decorated with two reinforcing
serrated ridges, running all the way from tip to base (Figure 1E). Such
``shark fin-shaped'' serrations are part of the highly mineralized
layer; with the exception of a zone close to the tip, they have fairly
constant dimensions (i.e., \(\sim\)100 \selectlanguage{greek}µ\selectlanguage{english}m in height and
\(\sim\)55 \selectlanguage{greek}µ\selectlanguage{english}m in length, \textbf{Figure S3} ) in the first
third of the spike, while their size decreases when approaching the
base, suggesting that only a portion of the spike (closer to the tip) is
engaged to impale preys. Serrations are prominent features in many
diverse biological cutting tools, ranging from teeth to insect
stingers\textsuperscript{{[}43{]}}, and could have a dual function: they
should help to cut through the tissue of the prey by acting as local
stress concentrators and, owning to their specific shape, they may
prevent the prey from slipping off the spikes by interlocking with the
damaged tissue\textsuperscript{{[}11{]}}. In addition to the serrations,
the surface of the spikes features a characteristic ``grooved'' pattern
(Figure 1E and Figure S3). The grooves have a periodic spiral
arrangement, starting and ending at two opposite serrated ridges, and
have dimensions smaller than the serrations (i.e., \textasciitilde{}5 \selectlanguage{greek}µ\selectlanguage{english}m
in height and \textasciitilde{}35 \selectlanguage{greek}µ\selectlanguage{english}m in width, Figure S3). The grooves
should provide the spike surface with a controlled roughness which may
increase frictional forces as an additional strategy at a smaller length
scale to enhance prey retention. From a biological viewpoint, grooves
should reflect the specific growth pattern of the spike outer layer,
regulated by the epidermis cells which are arranged into parallel
arrays.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
crc\tabularnewline
\midrule
\endhead
Figure 2: Microstructure of the spike. (A) Representative polished
transversal section imaged in BSE-SEM accompanied on the left by a
micro-CT reconstruction showing the location of the section. The
multilayered architecture of the cuticle, comprising a highly
mineralized region (HMR), an outer helicoidal region (OHR), a striated
region (STR) and an inner helicoidal region (IHR) is evident. (B) SE-SEM
of a longitudinal fracture offering a complementary view of the 4 layers
accompanied, on the right by, a micro-CT reconstruction showing the
location of the section. Scale bars in (A) and (B) are 200 \selectlanguage{greek}µ\selectlanguage{english}m. (C-E)
Details of the different layers from the polished surface. In (E), the
arrows indicate the transition region (TR) and the dotted lines bound
the OHR. In (D), horizontal parallel chitin-protein fibers are
cross-sectioned and fiber-rich strands (medium dark) alternate with pore
canal lumens (dark) and their densely mineralized sheaths (bright
rod-like features). Twisted ribbon-shaped pore canals with unmineralized
lumen also appear in (C) the IHR and (E) OHR while they are straight and
mineralized in (E) the HMR. Scale bars in (C--E): 10 \selectlanguage{greek}µ\selectlanguage{english}m. (F-H) Close-up
views of the fractured surface of the different layers highlighting: (F)
a rough surface for the IHR in agreement with the twisted plywood
organization; (G) sheets of parallel fibers (oriented vertically in the
figure) in the STR and (H) a rough stepwise fractured surface in
connection with the OHR (bounded by dotted lines), which is in strike
contrast with the smoother surface of the HMR. (I) Schematic summarizing
the main microstructural features of the spike. Scale bars in (F--H): 8
\selectlanguage{greek}µ\selectlanguage{english}m.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Microstructure}

The microstructure of the spike, observed with SEM on fractured and
polished samples, features four regions with distinct fiber architecture
(\textbf{Figures 2} A, B and summery scheme in I). The first innermost
layer, named inner helicoidal region (IHR, Figure 2C), belongs to the
endocuticle \textsuperscript{{[}36{]}} and displays a twisted plywood
organization consisting of numerous lamellae gradually increasing in
thickness from about 1.5 to 10 \selectlanguage{greek}µ\selectlanguage{english}m 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 \textsuperscript{{[}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 \textsuperscript{{[}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\textsuperscript{{[}45{]}}. In stomatopods, striated regions with
parallel longitudinal fibers have been found only in specific locations
including the dactyl club of the smasher \textsuperscript{{[}32{]}}, the
impact area of the dactyl in the appendage carrying the spikes of the
spearer \textsuperscript{{[}28{]}} and the back defensive spikes located
at the uropods (posterior structures found in
crustaceans)\textsuperscript{{[}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\textsuperscript{{[}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 \textsuperscript{{[}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 (\textbf{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
\textsuperscript{{[}35{]}}. The remaining of the highly mineralized
region presents a very low fraction of chitin-protein fibers
\textsuperscript{{[}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
(\textbf{Figure S5} ).\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Figure 3: EDS elemental mappings and profiles (line scans) on (A)
BSE-SEM image of a polished cross-section of the spike cuticle. The
dotted line represents the location where the profile plots in E and F
were taken. (B-D) Elemental mapping of (B) calcium, (C) phosphorus and
(D) carbon. Line scans of selected (E) major and (F) minor elements
along the dotted line in (A) from the out surface towards the epidermis.
IHR: inner helical region; HMR: highly mineralized region; OHR: outer
helical region; STR: striated region. Scale bars: 50\selectlanguage{greek}µ\selectlanguage{english}m.\tabularnewline
\bottomrule
\end{longtable}

\textbf{Composition}

The spike has a spatially tuned and graded chemical composition
featuring 3 different minerals: fluorapatite (FAP), amorphous calcium
phosphate (ACP) and amorphous calcium carbonate (ACC). The minerals are
closely related to the underlying microstructural organization and fiber
architecture. Elemental microanalysis by energy dispersive spectroscopy
of X-rays (EDS) on representative locations highlights the presence of
C, O, P and Ca as major chemical elements and of F, Na and Mg as minor
components \textsuperscript{{[}36{]}}. Those elements are unevenly
distributed in the different regions as shown on a transverse
cross-section (\textbf{Figure 3} A) using 2D elemental mapping (Figures
3B-D) and line scans (Figures 3E, F and \textbf{Figure S6} ). The highly
mineralized region is rich in Ca and P together with F; a complementary
Raman analysis, focusing on the outer part of the spike cuticle
(\textbf{Figure 4} A), shows a fairly sharp phosphate peak centered at
965 cm\textsuperscript{-1} (Figure 4B), indicating that FAP is most
likely the predominant mineral phase of this
region\textsuperscript{{[}7,46{]}}. The sharpness of the peak further
suggests a high degree of crystallinity \textsuperscript{{[}35{]}}.
Confocal Raman imaging reveals interesting spatial heterogeneities
within the highly mineralized region. Firstly, the phosphate band
presents higher intensity at the outer surface as well as in the
transition region close to the individual lamella anchoring the highly
mineralized region to the less mineralized thin outer helicoidal region
(arrows in Figure 4C). This agrees with variations in P concentration
detected by EDS (Figure 3E). The same zone co-localizes with an
increased crystallinity of FAP, estimated by the full width at half
maximum (FWHM) of the phosphate peak (Figure 4D). One additional feature
of the highly mineralized region is a central band having relatively low
phosphate intensity and crystallinity, along with some traces of ACC
(arrows in Figure 4F). Comparison of the phosphate intensity at the 0\selectlanguage{ngerman}°
and 90° polarization angle confirms spatial differences in the
orientation of the mineral phase, with less oriented mineral found in
regions with higher FWHM (Figures 4E and \textbf{Figure S7} ). A similar
tuning of the mineral phases has been reported at the impact region of
the spearing dactyl, with small amounts of sulfate co-localized with the
more crystalline FAP regions \textsuperscript{{[}35{]}}. The presence of
fluorapatite, rather than the more common amorphous calcium carbonate,
is a remarkable compositional modification at the surface of spearing
and smashing appendages of mantis shrimp \textsuperscript{{[}35{]}}. In
decapods crustaceans, a similar modification involving FAP occurs in the
external layer covering the molar area of the crayfish
mandible\textsuperscript{{[}7{]}}. This mineral is used not only to
enhance the biomechanical performance of the cuticle, providing superior
impact and wear resistance, but the presence of fluor decreases the
solubility of apatite, thus improving its chemical stability. At the
interface between the highly mineralized and the outer helicoidal
regions there is an abrupt transition in the mineral from FAP to ACP as
indicated by the sudden shift in phosphate position (Figure 4G). A
central observation of the compositional analysis performed here is that
the thin outer helicoidal region has the lowest mineral content as
inferred by Ca and P concentrations both showing a minimum there (Figure
3E). Moving further towards the interior of the spike, ACP still
predominates within the striated region, whereas in the inner helicoidal
region it is gradually replaced by Mg-rich ACC
\textsuperscript{{[}36{]}}.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Figure 4: Raman spectroscopy of the outer regions of the spike cuticle.
(A) BSE-SEM image highlighting the locations of the Raman spectra and
mapping (scale bar: 10\selectlanguage{greek}µ\selectlanguage{english}m). (B) Raman spectra obtained in different
locations across a straight line from the heavily mineralized outer
surface to the striated region. Three different peaks corresponding to
amorphous calcium phosphate (ACP, 955cm\textsuperscript{-1}),
fluorapatite (FAP, 965 cm\textsuperscript{-1}) and amorphous calcium
carbonate (ACC, 1080 cm\textsuperscript{-1}) are emphasized. (C-G) Raman
mapping of (C) phosphate intensity, (D) ACC/ACP ratio, (E) phosphate
peak position and (F) full width at half maximum (FWHM), (G)
polarization ratio on the phosphate peak (I-0\selectlanguage{ngerman}° / I-90°). (H) Schematic
representation of the main compositional features of the spike (the
underlying predominant fibers arrangement is also
reported).\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Nanoscopic biomechanical properties: indentation modulus,
hardness and damage behavior}The region-dependent chemical composition
and microstructural organization of the spike cuticle are reflected into
spatially varying mechanical properties, as measured by depth sensing
nanoindentation (\textbf{Figure 5} A). The outer FAP-dominated region is
clearly the stiffest and hardest location (indentation modulus of 64.9 ±
3.5 GPa and hardness of 4.0 ± 1.0 GPa). Both mechanical parameters show
a considerable drop (almost a factor of 2 for the modulus and of 5 for
hardness) in the thin outer helicoidal region, which displays the lowest
values of the entire spike, consistent with the low mineral content and
compositional modifications. Moving to the striated region, indentation
modulus and hardness increase and the former also shows slightly higher
values with respect to the inner twisted plywood (Table 1), which is
conceivable considering the highly ordered arrangement of the fibers.
Furthermore, a small negative gradient of about 4 GPa is observed in the
indentation modulus in this location, probably reflecting the
corresponding decrease in Ca content. To better characterize the spatial
tuning in local mechanical behavior from the heavily mineralized to the
parallel fibers region, we performed high resolution nanoindentation
with a sharp cube corner probe (Figures 5B-D and \textbf{Figure S8} ).
The high-resolution maps confirm the unusual drop in modulus within the
outer twisted plywood region, with values slightly below 20 GPa and no
differences between longitudinal and transverse sections, consistent
with the isotropic nature of the twisted plywood organization. In
particular, the transition region (featuring an individual lamella with
fairly high mineral content) has a clear signature in the mechanical
maps (Figures 5C and D) with a modulus of about 40-45 GPa, allowing a
gradual switch from the highly mineralized to the outer helicoidal
region. One additional feature of the highly mineralized region is a
quite heterogeneous mechanical behavior (\textbf{Figure S9} ), likely
reflecting the spatial variations of phosphate intensity and
crystallinity measured by Raman spectroscopy. A similar mechanical
modulation featuring stiffer crystalline domains and less stiff
amorphous islands has been reported for the heavily mineralized impact
region of the spearer appendage \textsuperscript{{[}35{]}}. Although a
thin outer helicoidal region enclosing highly co-aligned fibers has been
found in other biological penetrating tools such as the uropod back
spike of the mantis shrimp \textsuperscript{{[}42{]}} and the spider
fang \textsuperscript{{[}45{]}}, a novel feature observed here is that
this region, sandwiched between the hard outer cover and the stiff
parallel-fiber region, presents substantially lower mechanical
properties (modulus and hardness) than the neighborhood.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Figure 5: Nanomechanical properties of the spike cuticle measured by
depth sensing nanoindentation. (A) Profile plots of indentation modulus
and hardness from the exterior to the interior of the cuticle on a
transverse cross-section (the nanoindentation grid is visible on the
light micrograph). A drop in local mechanical properties within the
outer helicoidal region (OHR) is evident. (B-C) Spatial variation of
indentation modulus, E, measured with higher lateral resolution from the
highly mineralized to the striated region, considering both (B)
transverse and (C) longitudinal cross-sections. (D-E) High resolution
maps of indentation modulus focusing on the transition between HMR and
STR on (D) transverse and (E) longitudinal cross-sections. Probed areas
are highlighted in squares on the light micrographs and lines indicate
the position of the mechanical profiles shown in B and C. HMR: highly
mineralized region; IHR: inner helicoidal region OHR: outer helicoidal
region; STR: striated region and TR: transition region.\tabularnewline
\bottomrule
\end{longtable}

Table 1: Local mechanical properties (indentation modulus E, and
hardness H) of the different regions of the spike cuticle measured with
nanoindentation (with a cube corner probe). HMR: highly mineralized
region, TR: transition region, OHR: outer helical region, STR: striated
region, IHR: inner helicoidal region. Values are reported as
mean\(\pm\) standard deviation.\selectlanguage{english}
\begin{longtable}[]{@{}lll@{}}
\toprule
\textbf{Region} & \textbf{E {[}GPa{]}} & \textbf{H
{[}GPa{]}}\tabularnewline
HMR & 68.33 \selectlanguage{ngerman}± 5.05 & 2.96 \selectlanguage{ngerman}± 0.39\tabularnewline
TR & 42.35 \selectlanguage{ngerman}± 4.88 & 1.51 \selectlanguage{ngerman}± 0.23\tabularnewline
OHR & 21.13 \selectlanguage{ngerman}± 3.49 & 0.62 \selectlanguage{ngerman}± 0.16\tabularnewline
STR & 32.46 \selectlanguage{ngerman}± 3.87 & 0.85 \selectlanguage{ngerman}± 0.08\tabularnewline
IHR & 31.00 \selectlanguage{ngerman}± 3.18 & 0.85 \selectlanguage{ngerman}± 0.09\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

To further elucidate the possible biomechanical role of the outer
helicoidal region, we performed qualitative fracture experiments: high
load indentation was used to induce cracking and the sample surface was
examined in SEM (Figure 6). Indents placed in the striated region (along
the fiber axis, transverse section) require a fairly high load
(\(\sim\)1000 mN) to induce widespread damage: cracks follow a
rather straight path and spread radially in the matrix between fiber
stacks (Figure 6A). A central remark is that all cracks going towards
the highly mineralized region are stopped by the outer helicoidal
arrangement (Figure 6B and \textbf{Figure S10} ). This is consistent
with the extensive delamination-based damage observed in transverse
sections (Figure 2A) and confined within the striated region by the
inner and outer twisted plywood. The damage observed in Figure 2A was
not induced by indentation but, most likely, by sample preparation.
Specifically, sample dehydration and related shrinkage are possible
sources of local cracking. Since mineral is believed to replace water,
the amount of dehydration (and shrinkage) should be higher in STR than
in HMR (which is more mineralized and therefore should contain less
water). At the same time, cracks were not observed in the OHR and IHR,
which have a comparable degree of mineralization than STR but helicoidal
arrangements preventing crack growth. A comparable damage behavior has
been reported for the uropod back spike, which has a similar
multilayered microstructure, but lacking the external highly mineralized
region\textsuperscript{{[}42{]}}. Indents performed in the highly
mineralized region require smaller loads (i.e., 50 - 500 mN) to generate
extensive cracking. In this heterogeneous region, a relevant observation
is that damage is more likely observed far from the transition region,
which is also the most compliant zone of the highly mineralized region.
Indeed, indents placed in the vicinity of this transition region either
do not cause cracks (Figure 6C) or induce damage running parallel to the
transition region and its lamella (Figure 6D). Remarkably, no cracks are
observed crossing the interface between the highly mineralized and the
outer helicoidal region. Typical high-load indentation curves a for the
highly mineralized region (HMR) and for the less mineralized but highly
anisotropic region (STR) are shown in \textbf{Figure S11} . HMR featured
a characteristic pop-in event (highlighted by the arrow) associated with
sudden damage beneath the contact surface. Conversely, the indentation
curve of STR is free from pop-in events, indicating a more progressive
damaging behavior. The tiny dimension of the OHR (less than 10 \selectlanguage{greek}μ\selectlanguage{english}m in
width) precluded a quantitative assessment of the fracture toughness of
this region as previously done for the impact surface of both spearer
and smasher stomatopods \textsuperscript{{[}33,35{]}}. Nevertheless, our
indentation-based fracture study proofs the critical role of the OHR for
decoupling damage mechanisms between HMR and STR regions. A closer
examination of the surface of a fractured spike highlights the damage
behavior of the regions from a different perspective (Figures 6E and F).
There is a clear transition between the rather straight fractured
surface of the highly mineralized region and the much rougher surface at
its interface with the outer helicoidal region. Inside the latter,
individual lamellae can be seen, suggesting a plausible role in crack
deflection and a corresponding switching from a more brittle to a more
ductile damage behavior. Furthermore, between the fiber beds of the
outer helicoidal region, unbroken pore canals are present. As in
decapods crustaceans \textsuperscript{{[}44{]}}, canals of the
stomatopods also contain fibers \textsuperscript{{[}36{]}} that may act
as bridges between the chitin-protein fibers beds, possibly enhancing
fracture resistance \textsuperscript{{[}47{]}}.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Figure 6: Indentation induced damage and fracture behavior in the spike
cuticle. (A-B) SE-SEM images of cracks produced by high-load indentation
at 1000 \selectlanguage{greek}µ\selectlanguage{english}N in the striated region which are stopped at the outer
helicoidal region as seen both at low (A, scale bar: 20\selectlanguage{greek}µ\selectlanguage{english}m) and high (B,
scale bar: 5\selectlanguage{greek}µ\selectlanguage{english}m) high magnification. (C-D) BSE-SEM images of cracks
produced by high-load indentation at 200 \selectlanguage{greek}µ\selectlanguage{english}N in the highly mineralized
region observed at low (C, scale bar: 20\selectlanguage{greek}µ\selectlanguage{english}m) and high (D, scale bar: 5\selectlanguage{greek}µ\selectlanguage{english}m)
magnification. (E-F) SE-SEM images of the interface between the highly
mineralized region and the outer helicoidal region in a fractured sample
showing the deviation of the fracture plane in correspondence of the
outer helicoidal region (E, scale bar: 10\selectlanguage{greek}µ\selectlanguage{english}m). (F) The presence of
vertical chitin-protein fibers belonging to pore canals (arrow heads)
and bridging horizontal fibers beds is highlighted (scale bar: 5\selectlanguage{greek}µ\selectlanguage{english}m).
HMR: highly mineralized region; OHR: outer helicoidal region; STR:
striated region; TR: transition region.\tabularnewline
\bottomrule
\end{longtable}

The importance of the thin helicoidal region for the overall damage
behavior of the spike is further demonstrated using multimaterial 3D
printing (\textbf{Figure 7} ). We designed spike-inspired multilayered
samples with and without a thin twisted plywood (mimicking the outer
helicoidal region) sandwiched between a stiff monolithic but brittle
outer region (mimicking the highly mineralized region) and a more
compliant but anisotropic parallel-fiber region (mimicking the striated
region). We used cuboid samples and performed macroscopic penetration
tests at high strain rate using a blunt tip (Figure 7A). Notched samples
were tested in 3-point-bending up to fracture (Figure 7B). The twisted
plywood region connecting the still/brittle with the
compliant/anisotropic region has a strong impact on the strength, energy
absorption and failure characteristics of the spike-inspired system in
both scenarios. Considering penetration tests, force-displacement curves
show two peaks (Figure 7A): the first one happening just before the
breaking of the stiff outer layer and the second due to damage
propagating within the underlying fibrous and more compliant region
(Figure 7C). Interestingly, the presence of the plywood not only
increases the force needed to propagate the damage in the softer region
(by 40\%) but also the force necessary to break the stiff layer (by
25\%). At the same time, it has no influence on the contact stiffness,
as indicated by the same initial slope of the curves. Concerning the
3-point-bending tests (Figure 7B), in the sample without twisted
plywood, a main crack nucleates from the notch (Figure 7D-I) and
propagates straight in the parallel fibers region with only minimal
delamination (Figure 7D-II). As soon as the crack meets the stiff outer
region, a catastrophic failure is observed and the broken sample
``splashes away'' from the supports (Figure 7D-III). In the second
scenario, the crack also starts from the notch within the parallel
fibers region (Figure 7D-pI) and propagates straight (Figure 7D-pII) up
to the helicoidal region. There, the main crack branches symmetrically
into two smaller cracks which propagate horizontally, causing
delamination. A similar pattern is repeated one more time, while the
crack advances in the helicoidal plywood region (Figure 7D-pIII). Such
crack branching and delamination are important toughening mechanisms
which delay catastrophic failure, increasing by almost 50 \% the energy
absorption of the synthetic sample. Interpreting this behavior in the
context of the spike may suggest that, that although HMR and STR are
well-suited to reinforce the spike under contact forces (HMR) and axial
loading (STR), the combination of these two regions is not efficiently
dealing with damage propagation. The thin plywood region is therefore
required to fulfill this task.\selectlanguage{english}
\begin{longtable}[]{@{}l@{}}
\toprule
Figure 7: Mechanical tests on 3D printed samples mimicking the spike
cuticle. (A) Blunt penetration and (B) 3-point-bending load-displacement
curves of samples with and without the plywood connecting the monolithic
brittle layer to the parallel fiber anisotropic region. Snapshots of
sample deformation and failure behavior during mechanical testing are
given in (C) and (D). Scale bars: 1 cm. * denotes crack deflection.
Details of sample geometry, fiber architecture and testing configuration
are also shown (H= 4 cm, W= 3cm, L= 20cm, h=1.5 cm, s= 16 cm, d= 0.1 cm,
rotation angle between fiber beds \selectlanguage{greek}γ \selectlanguage{english}is 18\selectlanguage{ngerman}°).\tabularnewline
\bottomrule
\end{longtable}\selectlanguage{ngerman}

\textbf{Conclusions}The invention of harpoons is a fundamental milestone
not only in the evolution of natural life but also for
humans\textsuperscript{{[}48{]}}. In Nature, evolution produced a large
variety of harpoons and puncturing devices of increasing complexity,
ranging from the passive and fairly simple cacti
spines\textsuperscript{{[}49{]}}, to the more sophisticated offensive
tools such as spider \textsuperscript{{[}41{]}} and viper
fangs\textsuperscript{{[}50{]}}, scorpion
stinger\textsuperscript{{[}51{]}}, and even including drilling machines
as in mosquito or parasitic wasps \textsuperscript{{[}52,53{]}}.
Particularly fascinating are the chitinous penetrating tools of
arthropods, which often allow piercing through several layers of a
similar material, and possessing multiple modifications to fulfill such
task \textsuperscript{{[}54{]}}. The spikes emerging from the spearing
appendage of the mantis shrimps are biological harpoons deployed very
quickly to impale and grab moving fishes, which have the bad luck to
swim nearby. This is a challenging task requiring the ability not only
to resist the initial impact with the scaly skin of the prey, but also
to penetrate it for several millimeters to avoid that the fish swims
away. Here, we have investigated the spike using imaging and material
characterization techniques, to elucidate the many design strategies of
this biological tool. From the material point of view, the spike cuticle
of stomatopod is a chitin-based multilayered biocomposite, reinforced by
different minerals together with compositional gradients and specific
fiber arrangement\textsuperscript{{[}36{]}}. A hard heavily mineralized
outer shell of crystalline FAP, essentially lacking fibers, is designed
to resist to impact and wear. This exterior cover is combined with inner
fiber-rich regions characterized by lower mineral content and by
amorphous minerals (ACP and ACC). Two distinct fiber architectures are
observed: a unidirectional arrangement bordered by helicoidal twisted
plywood of dissimilar size. The latter is ubiquitous in arthropod
\textsuperscript{{[}12{]}} and presents two main biomechanical
advantages \textsuperscript{{[}45{]}}: starting from a strongly
anisotropic elementary building block (the chitin-protein fiber), it
provides the cuticle with in-plane isotropic mechanical behavior and
high shear stiffness\textsuperscript{{[}41,55{]}}. Moreover, the
helicoidal twisted plywood arrangement has remarkable fracture
resistance obtained by interacting with cracking at different
levels\textsuperscript{{[}56{]}}, essentially enhancing crack driving
force\textsuperscript{{[}57{]}} and reducing strain energy release
rate\textsuperscript{{[}58{]}}. Conversely, the parallel-fiber
organization is less usual \textsuperscript{{[}45{]}} and is used here,
well oriented along the long axis of the spike from tip to base, to
enhance stiffness and bending resistance. The combination of a parallel
fiber with a twisted plywood region is therefore well-suited to support
a complex mechanical environment with axial, bending and torsional
loading. A central finding of our work is that, in the spike cuticle,
the parallel fibers region is ``joined'' to the highly mineralized cover
by a thin fiber-rich twisted plywood region, showing compositional
modifications which make it the most compliant zone of the entire spike.
In the cuticle, plywood (or Bouligand structures) are generally thick
regions occupying a major part of the cuticle width and previous works
have demonstrated the superior fracture toughness of Bouligand structure
\textsuperscript{{[}34,59,60{]}}. The unique feature observed here is
the presence of an extremely thin plywood structure (i.e., less than 10
\selectlanguage{greek}μ\selectlanguage{english}m in width) sandwiched between two very dissimilar regions and allowing
the integration of a highly mineralized brittle outer layer with a less
mineralized but highly anisotropic straight fibers region. Not only the
plywood is very effective to stop crack propagation (both from the
striated region to the hard shell and vice-versa), as demonstrated by
nanoindentation fracture experiment and by 3-point-bending tests on
spike-inspired synthetic systems, but it also increases the force
required to penetrate both the stiff layer and the underlying complaint
fibrous matrix (as assessed on 3D printed replicas). Considering the
attachment of different materials, introducing a more compliant region
at the interface joining two dissimilar components is a construction
principle common to other biological systems\textsuperscript{{[}61{]}}.
Tendon, for example, attaches to bone through a transition zone which is
not only more compliant than bone but also than tendon
\textsuperscript{{[}62{]}}. This region, which co-localizes with the
unraveling and splaying out of tendon fibers into smaller fibrils
\textsuperscript{{[}61{]}} and which is made up of fibrocartilage
\textsuperscript{{[}63{]}}, is believed to protect the attachment region
by reducing stress-concentration, effectively strengthening the
interface \textsuperscript{{[}64{]}}. In analogy with the tendon-bone
attachment, the thin and more compliant twisted plywood region may offer
protection against stress localization at the transition between the
hard and the parallel fibers region, hence increasing the damage
tolerance of the spike. Helix reinforced composites are common in
engineering applications and the construction principles of biological
materials can improve the performance of the synthetic counterparts
\textsuperscript{{[}65{]}}. Focusing on the mantis shrimp dactyl club,
Bouligand and herringbone arrangements as well as nanoscale features of
the impact surface, have inspired the design of impact resistance
man-made composites\textsuperscript{{[}66{]}}. In light of the endless
advancement in nano- and micro-scale manufacturing methods, the
biological tool investigated here could inspire the design of new
synthetic harpoons for example based on environmentally friendly and
recyclable building units as sees in the spike cuticle, with improved
wear resistance and puncture abilities for repeated piercing on
different surfaces.

\textbf{Experimental Section}

\emph{Stomatopods and sample preparation} : The specie of spearing
mantis shrimps used in this study is \emph{Lysiosquillina maculata}
(Fabritius, 1793) also called the striped mantis shrimp (Figure 1A).
Eight living stomatopods from Kendary (Indonesia), ranging from 20 to 40
cm in length, were delivered by Marine Life (Paris, France) and were
kept in captivity in proper tanks at the Functional Morphology Lab
(ULiege). Spikes were harvested from 3 different individuals after
euthanasia, for a total of 9 spikes analyzed with the different analyzed
with the different methods. All specimens were dissected in the
intermolt stage C4, corresponding to entire and mature
cuticle\textsuperscript{{[}67{]}}. Distal spikes were then separated
from each other (Figure 1B). Samples imaged with micro-CT (n = 2) were
directly embedded in epoxy resin to minimize movement artefact. Samples
used for SEM (n = 4), Raman (n = 3) and nanoindentation (n=6) were
dehydrated in a graded series of ethanol bath (50\%, 70\%, 90\%, and
100\%) and embedded in epoxy resin (EpoFix resin Kit, Struers Inc.,
Germany) under vacuum. The resin blocks where then polished (Rotopol-2,
Struers, Germany) with series of silicon carbide disks of decreasing
grain size (P800, P1200, P2400, P4000, Matador, Germany). Final
polishing steps were performed with a diamond spray (DP-Spray P 1 \selectlanguage{greek}μ\selectlanguage{english}m,
Struers, Germany) and an aluminum suspension (Eposil F, 0.1 \selectlanguage{greek}µ\selectlanguage{english}m, ATM,
Germany).

\emph{Micro-CT imaging and processing:} Micro-CT scans of resin embedded
spikes were done at a nominal isotropic voxel size of 2 \selectlanguage{greek}μ\selectlanguage{english}m. The micro-CT
(Skyscan 1272, Bruker, Belgium) was operated at tube voltage of 60 kV
and current of 166 \selectlanguage{greek}μ\selectlanguage{english}A, in combination with a 0.25 mm thick aluminum
filter. The samples were rotated over 180\selectlanguage{ngerman}° with a rotation step of 0.2°
(corresponding to 940 projections), with an exposure time of 3600 ms and
a frame averaging of 4, leading to a scan time of approximately 4 h. A
bigger sample including also the dactyl was scan at a lower resolution
of 15 \selectlanguage{greek}μ\selectlanguage{english}m. Scans were reconstructed using Nrecon (v.1.7.5.2, Skyscan),
and further analyzed with ImageJ (v.1.52a), Matlab (R2018a; The
Mathworks, USA) and CTAn (v.1.19.4.0, Skyscan). Each virtual spike was
first aligned along its principal axes of inertia using BoneJ
(v.1.4.3)\textsuperscript{{[}68{]}}, a module of ImageJ. Images were
binarized using a global threshold based on Otsu's
method\textsuperscript{{[}69{]}} and filtered to extract the biggest
connected component. Geometrical properties of the cross-sections were
measured using the Matlab Image Processing Toolbox. Using the general
formula\(I=\int{z^{2}\text{dA}}\ \) where z is the shortest distance of the area
element dA from a given axis, the planar (\(I_{x}\)
and\(I_{y}\)) and polar (\(J=\ I_{x}+\ I_{y}\ \)) second moments of
area passing through the centroid were computed. Major and minor axis
were deduced by fitting an ellipse having the same second moments of
area as the current cross-section. Eccentricity was computed as the
ratio of the distance between the foci of the ellipse and the length of
its major axis. Principal moments of area about the minor
(\(I_{\min})\)and the major (\(I_{\max})\ \)axis of each
cross-section, corresponding approximately to dorso-ventral and lateral
loadings, were calculated based on Mohr's circle theory. To compute the
curvature of the spike, firstly a centroid profile line was extracted
smoothed and down-sampled, to reduce the noise. At each location along
the profile line, the local radius of curvature was computed by fitting
circles, always considering three neighboring points. The curvature
vector was obtained by dividing the unit vector pointing from the middle
point of the triple to the center of the circle by the radius of
curvature\textsuperscript{{[}70{]}}. A similar procedure was followed to
compute the radius of curvature at the tip.

\emph{Scanning electron microscopy and energy dispersive spectroscopy} :
Observations of polished sections were carried out with an environmental
scanning microscope ESEM-FEG XL-30 (FEI, Nederland) in low vacuum mode
(0.4 Torr) at 20 KeV accelerating voltage. Images were taken using a
back-scattered electron detector. A silicon drift detector of X-rays
(Bruker, USA) with a super-ultra-thin window was used to obtain energy
dispersive spectra and to perform elemental mapping using a Quantax
analyzer and the software Esprit 2.1 (Bruker, USA). To measure the
different elements concentration, the following energy peaks were used:
Carbon K\selectlanguage{greek}α \selectlanguage{english}peak (0.277 keV), Phosphorus K\selectlanguage{greek}α \selectlanguage{english}peak (2.013 keV), Calcium K\selectlanguage{greek}α
\selectlanguage{english}peak (3.690 keV), Magnesium K\selectlanguage{greek}α \selectlanguage{english}peak ( 1.253 keV), Fluorine (K\selectlanguage{greek}α \selectlanguage{english}peak at
0.677 keV) and Sodium (K\selectlanguage{greek}α \selectlanguage{english}peak at 1.041 keV). Fractured samples were
sputter-coated with silver (SCD030, Balzers Liechtenstein) and observed
in high vacuum mode using the same microscope with an acceleration
voltage of 15 kV and a secondary electron detector. Measurements of
grooves and serrations were made using ImageJ
software\textsuperscript{{[}71{]}} on SEM-BSE micrographs of the spike
in longitudinal and transversal sections to quantify their length and
depth.

\emph{Raman spectroscopy} : Polished sections of the spike were analyzed
by Raman spectroscopy with a green DPSS laser (\selectlanguage{greek}λ \selectlanguage{english}= 532nm, LabRAM 300,
HORIBA Jobin Yvon, Japan). Spectra were acquired along straight lines at
several different positions within the highly mineralized region, the
outer helicoidal layer and the striated region of the cuticle. Each
spectrum was acquired for 5 seconds. High resolution Raman mapping was
performed on the outer region of the spike cuticle with the same
microscope by focusing the laser beam through an Olympus MLPlan
objective (NA 0.75), using an integration time of 1 s and resulting into
a nominal pixel size of 500 nm. The mapping was performed with two
different polarization angles (0 and 90 deg). The obtained spectra were
analyzed with Rstudio (Rstudio Team, 2015) to extract the phosphate
intensity (peak intensity in the 952 to 968
cm\textsuperscript{-1}region), the carbonate intensity (peak intensity
in the 1077 to 1083 cm\textsuperscript{-1} region), the phosphate
position (wavelength at which the phosphate \selectlanguage{greek}υ\selectlanguage{english}1 vibration mode is the
highest), the FWHM (width of the phosphate \selectlanguage{greek}υ\selectlanguage{english}1 vibration mode at its half
height) and the polarization ratio (intensity of the 960
cm\textsuperscript{-1} band in 0 / 90 deg polarization).

\emph{Depth sensing nanoindentation} : Nanoindentation tests were
conducted with a TriboIndenter TI-950 (Bruker, USA) on re-polished
surface (the same samples used for SEM were probed), considering both
transversal and longitudinal sections. We first used a Berkovich diamond
tip to perform nanoindentation grids spanning across the different
regions of the spike with indents spaced 10 \selectlanguage{greek}μ\selectlanguage{english}m apart (grid size in
Figure 5A: 32x5 indents). We employed a trapezoidal load-controlled
function (10s-5s-10s for loading, holding, and unloading segments) with
a peak load of 5000 \selectlanguage{greek}µ\selectlanguage{english}N. Higher resolution nanoindentation mapping
covering \(\sim\)45x45 \selectlanguage{greek}μ\selectlanguage{english}m areas was carried out on two
locations at the interface between the highly mineralized region and the
outer helicoidal region after scanning the surfaces with the tip of the
indenter to check the roughness (average RMS roughness in the range
\(\sim\)16-25 nm depending on the location). We used a sharper
cube corner probe and a displacement-controlled indentation (10s-5s-10s)
with a maximum penetration depth of 200 nm, allowing a smaller spacing
of 1.5 \selectlanguage{greek}μ\selectlanguage{english}m between indents \textsuperscript{{[}72{]}}. Both tips were
calibrated in fused quartz. Force-depth curves were analyzed with the
Oliver and Pharr method \textsuperscript{{[}73{]}} to extract the
indentation modulus and hardness. High lateral resolution
nanoindentation data were used to generate 2D maps of the indentation
modulus (Figures 5C and D and S8) and raw data were interpolated with
the MESHGRID function of Matlab. A qualitative high load fracture study
was performed on polished samples with a high load 10 N transducer
(Omniprobe, Bruker, USA) equipped with a Berkovich probe. Several
indentation loads ranging from 50 mN to 1000 mN were used to generate
surface damage in different locations of the spike.

\emph{3D printing and testing:} Spike inspired samples were designed
with IronCAD (2020, USA), fabricated using a 3D multimaterial polyjet
printer (Objet 260, Stratasys, USA) and tested. Specifically, we
performed penetration tests with a blunt tip (5 mm tip radius) and
3-point-bending tests (MTS Criterion C43.304, USA). For the penetration
tests we considered cuboid samples having the following dimensions: 4 cm
x 3 cm x 3 cm (height, width and length). For 3-point-beding, we
fabricated beam-shaped samples, with dimensions: height (H) 4 cm, width
(W) 3 cm and length (L) 20 cm. Each sample was a multilayer composite,
featuring an outer monolithic region (mimicking the heavily mineralized
shell) and inner fiber-reinforced region. For the fiber reinforced part,
two different architectures were considered: \emph{i)} a thick region
composed of unidirectional parallel fibers, all oriented perpendicular
to the longitudinal axis of the sample (mimicking the striated region)
and \emph{ii)} a two-layer system with a thin helicoidal twisted plywood
region (mimicking the outer helicoidal region) sandwiched between the
monolithic part and the unidirectional fibers array. The monolithic
layer as well as the fibers were printed with a rigid glassy polymer
(commercial name Vero White Plus), having Young's modulus at room
temperature in the range of 2-3 GPa \textsuperscript{{[}74{]}}. For the
matrix embedding the fibers we used a rubbery polymer (commercial name
Tango Black Plus) with a tangent modulus at room temperature of about
0.5-1 MPa \textsuperscript{{[}75{]}}. Based on the printer resolution
and considering the finite size (i.e., up to 150 \selectlanguage{greek}μ\selectlanguage{english}m) and the properties
of the interface between fiber and matrix \textsuperscript{{[}76{]}},
the 3D printed fibers had a diameter of 600 \selectlanguage{greek}μ\selectlanguage{english}m. For the region mimicking
the helicoidal region structure, 10 sheets were stacked with a pitch
angle of 18\selectlanguage{ngerman}°. At the bottom surface of the samples, a rigid layer made
up of Vero White Plus was added to prevent excessive sink-in at the
contact point with the supports. A notch (0.15 cm deep) was directly
printed in the samples to trigger check initiation within the parallel
fiber array. Penetration tests were done at 10 mm/min whereas
three-point-bending tests (span length of 16 cm) were conducted at 60
mm/min, considering preload of 5 N and with a 30 kN load cell. During
tests, samples were imaged with a HD camera at 15 fps and movies were
used to analyze deformation mechanisms. Mechanical tests were performed
1 day after printing to have always the same post-curing time.
Fabrication, storage, and testing were done in a room with controlled
humidity and temperature to minimize experimental variability.

\textbf{Acknowledgments}

Y. D. and A.T. are PhD fellow of FRIA (Fund for Research Training in
Industry and Agriculture). The CAREM (Centre for Applied Research and
Education in Microscopy-ULiege) is acknowledged for the microscopy
facilities. We thank Erwan Plougonven from ULiege for the micro-CT scan
of the spike and its base, used to prepare Figure 1C.

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