ConclusionsThe invention of harpoons is a fundamental milestone not only in the
evolution of natural life but also for humans[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[49], to the more sophisticated offensive tools
such as spider [41] and viper fangs[50], scorpion stinger[51], and even including drilling machines as in
mosquito or parasitic wasps [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 [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[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 [12] and presents two
main biomechanical advantages [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[41,55]. Moreover, the helicoidal twisted
plywood arrangement has remarkable fracture resistance obtained by
interacting with cracking at different levels[56], essentially enhancing crack driving force[57] and reducing strain energy release rate[58]. Conversely, the parallel-fiber
organization is less usual [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 [34,59,60]. The
unique feature observed here is the presence of an extremely thin
plywood structure (i.e., less than 10 μm 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[61]. Tendon, for example, attaches to bone
through a transition zone which is not only more compliant than bone
but also than tendon [62]. This region, which
co-localizes with the unraveling and splaying out of tendon fibers
into smaller fibrils [61] and which is made up
of fibrocartilage [63], is believed to protect
the attachment region by reducing stress-concentration, effectively
strengthening the interface [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 [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[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.
Experimental Section
Stomatopods and sample preparation : The specie of spearing mantis
shrimps used in this study is 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[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 μm,
Struers, Germany) and an aluminum suspension (Eposil F, 0.1 µm, ATM,
Germany).
Micro-CT imaging and processing: Micro-CT scans of resin embedded
spikes were done at a nominal isotropic voxel size of 2 μm. The micro-CT
(Skyscan 1272, Bruker, Belgium) was operated at tube voltage of 60 kV
and current of 166 μA, in combination with a 0.25 mm thick aluminum
filter. The samples were rotated over 180° 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 μm. 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)[68], a module of ImageJ. Images were
binarized using a global threshold based on Otsu’s method[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[70]. A similar procedure was followed to compute
the radius of curvature at the tip.
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 Kα peak (0.277 keV), Phosphorus Kα peak (2.013 keV), Calcium Kα
peak (3.690 keV), Magnesium Kα peak ( 1.253 keV), Fluorine (Kα peak at
0.677 keV) and Sodium (Kα 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[71] on SEM-BSE micrographs of the spike in
longitudinal and transversal sections to quantify their length and
depth.
Raman spectroscopy : Polished sections of the spike were analyzed
by Raman spectroscopy with a green DPSS laser (λ = 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-1region), the carbonate intensity (peak intensity in the 1077 to 1083
cm-1 region), the phosphate position (wavelength at
which the phosphate υ1 vibration mode is the highest), the FWHM (width
of the phosphate υ1 vibration mode at its half height) and the
polarization ratio (intensity of the 960 cm-1 band in
0 / 90 deg polarization).
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 μm 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 µN. Higher resolution nanoindentation mapping
covering \(\sim\)45x45 μm 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 μm
between indents [72]. Both tips were calibrated in
fused quartz. Force-depth curves were analyzed with the Oliver and Pharr
method [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.
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: i) a thick region
composed of unidirectional parallel fibers, all oriented perpendicular
to the longitudinal axis of the sample (mimicking the striated region)
and 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 [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 [75]. Based on the printer resolution
and considering the finite size (i.e., up to 150 μm) and the properties
of the interface between fiber and matrix [76],
the 3D printed fibers had a diameter of 600 μm. For the region mimicking
the helicoidal region structure, 10 sheets were stacked with a pitch
angle of 18°. 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.