Microstructure and nanomechanical properties of foreleg surface of the praying mantis ( Mantis religiosa Linnaeus )

Detailed investigations on the microstructure and mechanical properties of the surface of the praying mantis’ foreleg were carried out in this work since the microstructure and mechanical properties of insect organisms surface are well adjusted to their functions. Scanning electron microscopy revealed the surface microstructures of the tibia and femur were composed of squamous fibre and the spine surfaces on these two parts are arris. A nanoindenter was used to investigate the mechanical properties of the praying mantis’s foreleg, including the mean Young’s modulus and hardness of the femur cuticle, tibia cuticle and the apical claw. The test results on these parts were E = 5.533 GPa, H = 0.239 GPa, E = 3.471 GPa, H = 0.186 GPa, E = 2.232 GPa, H = 0.449 GPa, respectively. The variation of mechanical properties agreed with the analysis of microstructure.


Introduction
The morphology and microstructure of the surface of an animal organism provide a significant system for its function, and the functional properties contribute adaptability and longevity [1][2][3].For instance, the material and structural properties of the membranes, together with the venations, of dragonfly [4,5] and cicada [6] wings are associated with the flight of the insects; the beetle cuticle [7,8] efficiently provides a lightweight skeleton for flight and strong and sharp mandibles of the termite [9] support effective chewing.There is a plenty of literature on microstructure and mechanic property of insect organisms.Vincent and Wegst [10] and Chen et al. [11] found the beetle cuticle is a typical natural composite, consisting of arrangements of highly crystalline chitin nanofibres embedded in a matrix of protein, polyphenols and water, and its microstructure has structural characteristics, which is of important significance to strength, stiffness, and fracture toughness.Barbakadze et al. [12] investigated the mechanical properties of the gula plate, the head part of the head-to-neck articulation system in the beetle Pachnoda marginata.The nanoindendation experiments showed that the hardness and elastic modulus of the gula plate in fresh conditions were 0.1 and 1.5 GPa, respectively.Oh et al. [13] explored the structural and mechanical properties of the hind leg joint of katydids and found the microsurface of femur-tibia joint significantly reduced the adhesive forces between the coupling surfaces.Among these organisms, the insect leg has various functions depending on the morphology and microstructure, from crawl [14] to jump [15], from capture [16] to excavation [17], and even perception [18,19].These biological morphologies and surface microstructure can provide valuable information for biological material and bionic engineering [20,21].
Like a predator in the insect world, the praying mantis has a pair of powerful tools, two sharp and strong forelegs.The femur and tibia are both armed with a double row of strong spines along their posterior borders.The spines can be flexed on each other to grasp any object firmly.Additionally, the apical claw in the tibia is falciform that is keen and hard, allowing little chance that the prey has to escape.There has been a little study on the microstructure of the foreleg surface in this fierce creature, except for observations on the praying habit [22,23] and physiology of the praying mantis [24][25][26][27].Mechanical properties of the tibia and femur of praying mantis remain largely unknown.The present investigation focused on the microstructure and the mechanical properties (elastic modulus and hardness) of the sharp spine, tibia cuticle and femur surface, in particular, the differences of mechanical properties among the apical claw, tibia and femur.Twenty-five adult praying mantises (Mantis religiosa Linnaeus) were observed with a scanning electron microscope (SEM).The relevant geometrical and morphological characteristics of the microstructure of the spines, tibia surface and femur surface were measured.The nanomechanical properties were examined by a Nanoindenter.

Sample preparation
The praying mantises, which belong to the species of Mantis religiosa Linnaeus were found in the countryside of Northeast China.
Twenty-five adult praying mantises were prepared and cleaned with distilled water and anaesthetised with ether.Their morphology was then observed with an STJ-30 microscope (Olympus Co., Ltd.Tokyo, Japan).
For microstructure studies, these praying mantises were anaesthetised with ether before the femur and tibia were cut off from the bodies.Each femur and tibia was bisected longitudinally along the middle line between the double spines.Samples were fixed in 2.5% glutaraldehyde for 12 h and dehydrated in an ascending series of ethanol and then critical-point dried.After these preparation processes, the samples were mounted on special holders, sputter-coated with gold-palladium (10 nm thickness), and examined in a cold field scanning electron microscope (JSM-6700F, JEOL Co., Ltd., Tokyo, Japan) at 10 kV.
Since the femur was filled with soft tissues, for analysis of the nanomechanical properties, the outer surface was sliced with a microscope using a surgical blade.To prevent specimen desiccation, fresh tibia and femur were tested immediately (within 15 min after dissecting from the body).Additionally, the specimens were slightly polished before being fixed on the holder with instant glue.

Morphology observation
For morphology measurements, 25 adult praying mantises were observed with a stereoscopic microscope (STJ-30, Olympus Co., Ltd., Tokyo, Japan), as shown in Fig. 1.To minimise the system errors, the length of samples was measured using CAD software in which the reference point can be more correctly found rather than  that using caliper (Fig. 2).The length range of these mantises was 45-65 mm, and the total length of tibia and femur was from 16.52 to 24.74 mm.

Nanoindentation
Nanoindentation is an effective technique for the evaluation of mechanical properties (such as hardness and Young's modulus) for quite a small volume of biological material.During nanoindentation, a geometrically well-defined diamond pyramid applied force on the sample surface and the displacement (indentation) formed in the specimen can be reported simultaneously.The load-displacement curves are used to confirm the hardness and Young's modulus of the tested material.
Based on the nanoindentation theory of materials and Oliver-Pharr method, the initial unloading contact stiffness (S) can be obtained from the slope of the unloading segment of load-displacement curve.The hardness (H ) is defined as the ratio between the maximum load and the contact area (A), generated from the indentation process [12] H = F max /A (1) where k is a geometric constant of the tip, h c is the contact depth that can be described as where 1 is a geometric constant of the indenter.The indentation modulus E r is related to the elastic modulus by where y s and y i are the Poisson ratios of the sample and the indenter, and E S and E i are the elastic modulus of the sample and the indenter, respectively.
The relation between the load-displacement data and the experimentally measured contact stiffness (S) and the contact area (A) is where β is constant related to the tip geometry.In this study, the elastic modulus and hardness of the samples were measured using a nanoindenter (G200, Agilent Technologies, Santa Clara, California, USA) with a Berkovich diamond tip.20 samples  for tibia and femur and 4 indents on each sample.Since the claw tip is too small and unsmooth, only one indent point can be tested successfully.To avoid contaminating the tip of the indenter, it was cleaned with ethanol and recalibrated by testing the reference material (SiO 2 ) every two measurements.The nanoindentation experiments were displacement-controlled and a depth of 1000 nm was applied.In order to minimise the impact of material creep, a holding time of 20 s was set on the peak load.

Microstructure of the tibia and femur surface
The SEM photomicrographs were used to analyse the various microstructures of the surface on tibia, femur, apical claw and other general spines in the two parts.In this work, a voltage of 10 kv was applied and the magnifications ranged from 25 to 2000.The femur surface was quite smooth on amplification of 25× (Fig. 4a).However, the structure of the surface is squamous on 1800× amplification.The size of cells varies, and the side length of the largest squamous cell (d max ) is about 15 μm, the length of the smallest cell (d min ) is about 5 μm.Besides, there is an obvious gap between the two cells, of which width (w) is about 2 μm (Fig. 4b).Fig. 5a shows the tibia surface on 2000× amplification.Tibia surface also has squamous structure, and the size of each cell is close, the side length of the largest squamous cell (l max ) is about 15 μm, length of the smallest cell (l min ) is about 12 μm.The gap width between the two cells is about 0.5 μm.The cell distribution on tibia surface is well-aligned and denser compared with that of the femur.Fig. 5b indicates the microstructure of the cuticle section and internal surfaces: the internal surface is distributed with irregular polygon cells of which length is ∼7 μm, and the profile section shows the tibia cuticle has a layered structure and the thickness of each layer is about 6 μm.Fig. 6 shows the surface structures of the general spine in femur, tibia, apical claw and the longitudinal section of the apical claw.The surfaces on the spines are unsmooth.The fibres distribute like arris in architecture, as shown in Figs.6a-c.

Mechanical properties
To compare and analyse the mechanical properties of the praying mantis's foreleg surface, a nanoindenter was used to test the hardness of the samples.Nanoindentation measurements revealed the differences among the elastic modulus and hardness of the tibia, femur and apical claw.The typical load-displacement curves of these parts are shown in Fig. 7.Because the tip of the apical claw was too small and unsmooth to be indented by the tester, only 1 point which located on the slightly smooth surface of the claw was chosen to represent the general tip surface.The mean displacement and load at max load were concurrently recorded (Table 1).The nanoindentation results of these four different parts were presented in Fig. 8.

Discussion
The mantis has two sharp and strong forelegs armed with a double row of strong spines along their posterior borders to grasp the prey firmly.Additionally, the apical claw in the tibia is falciform and is especially hard and sharp.
Based on the SEM photographs (Figs.4-6), the surface structures of tibia and femur have a distribution of dense squamous cells, which could satisfy the function of stiffness and lightless.Additionally, the microstructures of the general spine and apical claw are rather unsmooth and the fibres display as arris which makes the spine looks like a minaret with lots of arrises.The arris is able to supply the hard and sharp structure to make the spine powerful, and the tibia and femur become a grasp and prey tool.
The hardness variations on the surface of the tibia, femur and apical claw can be noted in the nanoindentation results.The hardness of the tibia surface (0.186 GPa) was slightly lower than that of the femur surface (0.239 GPa) and the apical claw (0.373 GPa).The hardness on the tip of the apical claw was higher (0.449 GPa) than that on the base (0.373 GPa).Like most biological materials, the surface of tibia, femur and spine are viscoelastic material, the creep phenomenon is found in the loaddisplacement curve.In Fig. 7, there are two line segments in the loading curve and unloading curve, which reveals that the creep is going with the nanoindentation in the plastic deformation region.In addition, Young's modulus of femur surface is considerably higher than that of the tibia and the apical claw.It suggests that the femur hold comparatively high elasticity and resilience, and these properties of the tibia are less than that of the femur.Since the main function of the apical claw is for the strong and quick attack, its surface stiffness is dominant, the elasticity is inferior to the other two parts, thus the elasticity modulus gradually reduces along the profile of claw.The value on the tip is 2.94 GPa lower than that on the base.
From the results above, it can be seen that the foreleg shape of the praying mantis highly serves the prey action, and the dense and well-aligned fibres offer excellent mechanical properties to the powerful capture tool-tibia and femur.

Conclusions
The different mechanical properties, elastic modules and hardness of the mantis foreleg surface were caused by various microstructures.The results of our analysis demonstrated that the shape and the structure of the foreleg in praying mantis could well serve the pray action and supply remarkable mechanical surface properties to the powerful capture tool-tibia and femur.

Fig. 6
Fig. 6 SEM photomicrographs of the spines a Femur spine b Tibia spine c Apical claw

Fig. 7
Fig. 7 Typical load-displacement curves for the different part surfaces