The mechanical characterization of the legs, fangs, and

The nanoindentation technique allows researchers to perform local maps of mechanical properties, which are important to understand the mechanics of artificial and natural structures29,30,31,32,33,35,41,42. In this work, the investigation was focused on different elements of the spider’s exoskeleton for which we expect different mechanical properties: legs, prosoma, and fangs. The reason of this can be ascribed to the different cuticular structures present in these parts. Indeed, the spider cuticle is similar to the insects one that consists in various layers (exo-, meso-, and endocuticle)1,43. These are present in different proportions depending on the considered body part of the spider15, and have various level of sclerotization (i.e. mesocuticle is more sclerotized than endocuticle) and microstructural organization, two factors that both define the mechanical properties of the exoskeleton44,45,46.

In general, the reported results are aligned with previous indentation studies on spider fang’s tissue10,11,12,13,14. However, no previous work reports the comparison of mechanical properties of different body parts. This work aims to fill this gap, providing a comparison of the mechanical properties of the sclerotized layer of cuticle in the legs, prosoma, and fangs.

The prosoma exoskeleton has the main functions to shield some vital organs as well as protect the spider from quick dehydration47. Moreover, it is also the tagma responsible for locomotion, feeding, and sensing, since the prosoma’s cuticle is the base for the animal’s haemolymph pressure pump (i.e. the drive for the extension of two major joints of all of their legs and pedipalps)1,3. Legs are crucial for locomotion3 and sensing purposes since they host the major part of mechanical and chemical sensors4. On the other hand, fangs are indispensable to feed, dig, and defend the spider10. Thus, the fangs are the ones exposed to major external stresses, which require a particular chitin structure, consisting in a lamellar architecture of the internal cuticle’s layers, confirmed by the here reported SEM images10. This structural design results in mechanical properties that​ depend on the thickness and orientation of lamellas13. This was confirmed here through nanoindentation, which showed a gradient of Young’s modulus and hardness. In particular, the closer the cuticle’s layers the higher Young’s modulus and hardness were measured. Moreover, as already demonstrated in other studies for other species10,13, the fang’s tip presented higher values of the hardness and Young’s modulus with respect to the base. This is related to the biological function of the tip, i.e. the penetration of surfaces as well as the durability and wear resistance of the tool19. From this point of view, a recent work showed that the actions of penetrating surfaces as well as resisting wear and tear are aided by metals inclusion in the cuticle12. This is in accordance with a previous study, which showed the presence of metal ions in the fangs of the spiders that act as reinforcing elements12,14. Moreover, the different microstructural organization of the cuticle body parts (legs, prosoma, outer layer, and inner layer of the fangs) has been related to the polymorphic forms differentiated into their crystal structure48. Interestingly, the different microstructural organization of the cuticle as well as the gradient of the mechanical properties is found also in beetles49, highlighting the common evolutionary strategies (i.e. microstructural organization) to optimize the biomechanical performances of anatomical parts.

The OL of the fangs was stiffer and harder than IL which is in agreement with the prediction of previous studies13. Interestingly, the metatarsus of the I pair legs’ cuticle was found as stiff as the fangs’ one. The legs of the Theraphosidae host the majority of lyriform organs47, which are sensory systems with the aim to detect vibrations4. These, in different shapes and numbers up to the considered species, deform because of the detected vibration and transmit the relative signal by means of the nerves. The high stiffness of the legs’ cuticle, found in this work, may be thus important to facilitate the transmission of mechanical stimuli50,51, which may depend on both temperature and humidity17, to slit sensilla. Indeed, spider lyriform organs have high mechanical sensitivity, which also depends on their exponential stiffening behaviour, i.e. the relation between the cuticle’s stress and strain follows an exponential curve. This allows the animals to detect a wide range of vibration amplitudes over four orders of magnitude in frequency as low as 0.1 Hz and as high as several kHz15,16,17,18, which is what commonly is present in nature18. For these reasons, the stiffer leg cuticle in Harpactira curvipes may aid the perception of vibrations.

Moreover, we do not exclude that stiff cuticle tissue is beneficial for the spiders’ locomotion. This requires high differences in the pressure applied by haemolymph, which flows inside the legs, on the cuticle3. In this context, a recent work23 shows that spider haemolymph behaves as a shear-thinning non-Newtonian fluid, whose fluid behaviour index is 0.5 (usually < 1 for pseudoplastic fluids, = 1 for Newtonian fluids, and > 1 for dilatant fluids). This means that the higher the shear stress applied on the haemolymph, the lower is its viscosity. During spiders’ legs fast movements, there is a quick increase of pressure in the haemolymph, which results in higher shear stress, induced by the interactions with the cuticles’ walls3. In this way, the stiffer the walls are, the higher is the shear stress on the haemolymph52, which results to be less viscous, flows better in the joints, and facilitates locomotion. Moreover, a more rigid cuticle sustains better the continuous changes in pressure within the legs23,28. These results may be helpful to design bio-inspired hard or hard/soft systems, such as cutting tools or soft actuators, providing an example of how an external rigid layer may help in the development even of soft actuators. In particular, the optimized alternation between soft and rigid tissue can improve actuation mechanisms (e.g. hydraulic) aiding the speed, the force output, the displacement, and the efficiency of artificial joints by localizing the pressure of the actuating fluid preserving hardness28.

Although it is very challenging to have a mechanical characterization of all spiders’ body parts (including microstructures such as hairs, slit sensilla, joints, and so on), this study provides an example of the importance in performing such comparative analysis, since it confirms previous studies’ results and offers a starting point for future discussion.

The role of hydration in affecting nanoindentation results

Nanoindentation experiments were not performed on fresh samples, which were here dissected from animals preserved in ethanol (70%) and kept in the refrigerator (4–8 °C). The hydration status strongly affects the mechanical properties of arthropods cuticle53,54, as deeply investigated for insects in Klocke et al.55. In that study, it is reported a significant reduction of hardness and Young’s modulus (up to a factor of 9 and 7.4 respectively) in the hydrated cuticle with respect to the dry one. Moreover, it is highlighted that in some cases the relative differences between meso/endo cuticle can be reverted if the samples were dried.

To investigate hydrated samples by means of nanoindentation it is mandatory to use specific instruments in which experiments in fluid are allowed. Indeed, the state of hydration must be maintained during the test, and this is not possible with most of the available devices34. Nonetheless, there are instruments and techniques that give the possibility to test samples while fully submerged in fluid56. However, these techniques should also be coupled with a method that takes into account the capillary forces that are known to interfere with sample surface detection57 and that strongly depend on the indenter geometry58. For this reason, to perform such tests, special indenter probes are used for fluid immersed-samples59. Finally, a wet sample may display viscoelastic unloading response when the probe is withdrawn60. In this sense, the well-known and established method developed by Oliver and Pharr36, designed for dry samples, is based on the assumption that the unloading of the sample is elastic. Thus, the calculation of the mechanical properties of hydrated samples should be done also considering their viscoelasticity.

For the sake of clarity, it is important to discuss what are the possible effects of hydration on the results of this work. Klocke et al.55 found that endo/meso cuticle are those mostly affected by water, whereas exocuticle is less affected. Following Barth15 and Foelix1 and looking at the indentation regions of this study, it is possible to say that mostly meso/endo cuticle were here transversally tested55. In this context, if we assume that water affects the mechanical properties at the same level as described in Klocke et al.55, for both the Young’s modulus and the hardness we expect that the difference between dry and wet samples will not be enough to change the trends observed in this work. Indeed, the relative differences between the meso/endo cuticle are 30% and 10% in the dry state for Young’s modulus and hardness respectively, whereas in the wet state such differences are 17% and 100%. These relative differences are not comparable to the ones that we observed among the different regions of the fang (e.g. basal and tip differ for a factor ~ 3, Fig. 5). On the other hand, this cannot be said for the differences between the external and internal layers of the cuticle. Nonetheless, this does not affect the finding of the remarkable stiffness of the metatarsus cuticle, where no gradient was found (Fig. 5).

In finding the best technique to store cuticle samples, Aberle et al.61 suggested that freezing them preserved better their mechanical properties, which is in common with octopuses flesh62 and that has been also recently used for locusts54. On the other hand, they also show that the moduli of the sample preserved in ethanol are lower (up to three times) with respect to the fresh ones. Before being embedded in resin, the samples tested in this work were stored in ethanol, which may have balanced the dehydration effect described earlier55.

When we designed the experiments for this work, the published studies related to spiders were considered. In Tadayon et al.19 the species C. salei was analysed, testing the samples in both dry and wet conditions. This was done using an instrument designed for the purpose that implemented Oliver and Pharr36 method. In that case, the dry samples reported an increase of Young’s modulus and hardness compared to the hydrated ones of about 41% and 35% respectively. These values have been used to estimate the mechanical properties in hydrated state in this study (Figure S5). Nonetheless, they show that the gradients inside the fangs were preserved, which further supports the validity of our study. This is also supported by the studies conducted by Erko et al.14 and Politi et al.10, in which C. salei tissue were preserved in Ethanol 70% and stored at 4–8 °C, and then tested in dry conditions with a Berkovich tip and using Oliver and Pharr36 method, as it has been done in this study.

Thus, the mechanical properties here presented are to be considered as comparative with respect to the literature, and differences with respect to fresh tissue should be expected. We hope that this work will stimulate further discussion on the delicate topic of measuring the mechanical properties of arthropods cuticle, as thoroughly dealt in Stamm et al.20.

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