The ability to climb almost every surface is a big advantage for animals. Therefore, many species convergently evolved different types of adhesive footpads to perform locomotion even on smooth vertical surfaces. One of these types is a soft bubble based smooth adhesive system that has been discovered for example in Orthoptera, Hymenoptera, Solifugae and Scincidae. The second type are fibrillary/hairy, seta based adhesive pads found for example in Dipterans, Coleopterans, Araneae, Gekkonidae and Anolinae (Gorb and Heepe, 2017). Among the animal groups using fibrillary pads, the geckos (Gekkonidae) and spiders (Araneae) developed an exceptionally effective reversible adhesive system for so called “dry adhesion” that works without any sticky fluid involved. In geckos, the dry adhesive system consists of a multitude of keratinous setae that branch at the tips and are arranged in lamella on the toes (Rizzo et al., 2006). In spiders, the dry adhesive system for locomotion is located on the most distal leg parts (pretarsi) and made up of chitinous setae bundled in the so-called scopula claw tuft. From each of these setae, a multitude of microtrichia branch off, which are arranged densely close to the seta tips and face the substrate (Niederegger and Gorb, 2006; Schaber et al., 2019). As in geckos, the terminal contact elements of the setae are flat and thin platelet-shaped spatulae.

Adhesion of the dry attachment systems is enhanced by shearing, which promotes alignment of the spatulae with the substrate surface and increases the contact area for attractive van der Waals forces (Wolff and Gorb, 2013; Flenner et al., 2020). In the gecko, the adhesive force of a single seta strongly depended on its sliding in contact with a substrate. The adhesion force ranged from 0.6 µN without sliding, to 13.6 µN with sliding, and 194 μN at 5 µm sliding and a preload of 15 µN. Assuming that all setae were simultaneously and maximally attached, these data indicated an adhesive force of 100 N of the single gecko foot (Autumn et al., 2000). In the jumping spider Evarcha arcuata, the adhesive force of a single spatula was measured to be 38 nN. The assumption that all spatulae were in contact resulted in an adhesive force that could support 173 times the body weight of the animal (Kesel et al., 2003; Kesel et al., 2004). In the Central American wandering spider species Cupiennius salei, which was also used in the present study, the adhesion of a single leg scopula on glass when shear force was applied was found to be 35 mN, which would suffice to hold an adult female animal on a vertical surface. However, the vertical pull-off force without shear preload was zero (Wohlfart et al., 2014). The arrangement of the spatulae on the setae was previously explained to be most important for the spider’s traction forces on different micro-rough substrates in the species Philodromus dispar (Wolff and Gorb, 2012a).

Up to now, the adhesive forces of the single spider setae that make up the adhesive scopula on the claw tufts of the spider remained unknown. To find out more on the interplay of the adhesive setae with different kinds of substrates, here we examined the morphology of the claw tuft and the setae and measured adhesion forces of individual setae on various characterized substrates with different surface energies and roughness and at different setal contact angles on glass.

The examination of the general morphology of the pretarsus of Cupiennius salei in the present study showed the claws and the two lobed claw tufts of scopula setae. This finding confirms those of previous authors (Wolff and Gorb, 2013; Labarque et al., 2017). It enables the spider to use the specific attachment mechanism dependent on the geometry and the mechanical properties of the substrate. The claws are assumed to enable clamping on soft or very rough surfaces, whereas the adhesive setae are used for adhesion to smoother and hard surfaces (Figures 1B,C). The setae themselves are covered with differently shaped microtrichia. The microtrichia with pointed tips along the shaft can be interpreted as spacers similar to those of other spider species (Eggs et al., 2015) and avoid that the setae to stick to each other. The increased density of the spatula-shaped microtrichia in the tip region and their arrangement toward the substrate make them well suited to make contact with a surface during locomotion. This hierarchical structure of the pretarsal attachment system of spiders is different from the structure of fibrillary adhesive systems of insects, e.g. in beetles and earwigs, which are made up of shorter, unbranched setae on the tarsi (Haas and Gorb, 2004; Gilet et al., 2018).

On the smooth substrates with different surface energy, the setae adhered best to the glass, which also exhibited the highest hydrophilicity and a highest amount the polar shares of surface energy (Figure 5). For adhesion, the polar shares are responsible for hydrogen bonds between two materials. The disperse shares result in attractive van der Waals forces (Autumn et al., 2002). On the epoxy resin and the silicon surface that are only slightly hydrophilic and for which the amount of disperse shares of surface energy are similarly high, the adhesive force of the setae dropped down to approximately two thirds of that on glass. On the hydrophobic and non-polarizable PTFE substrate, the adhesive forces of the setae was lowest and approximately 30% of those on glass.

For the gecko seta, it has been shown that its adhesion purely relies on van der Waals forces, because the adhesive forces were the same on polarizable hydrophilic and hydrophobic substrates and confirmed the model prediction (Autumn et al., 2002). For a single Gecko spatula, however, adhesion increased with increasing relative air humidity and substrates with increasing hydrophilicity (Huber et al., 2005), and the authors suggested monolayers of water being adsorbed between the spatulae and the substrate leading to additional capillary forces. For the spider setae, here we show stronger adhesion on the hydrophilic glass surface with a high polar share of surface energy, which makes the influence of thin layers of water on the adhesion strength likely. For different spider species with scopulae, the capillary forces between the thin water layer on a substrate and the scopula hairs were found to be most important for their adhesion (Homann, 1957). On the polarizable epoxy resin and the silicon substrate, which mainly exhibit disperse shares of surface energy, the adhesion of the spider setae is still fair. Together with the presence of adhesive force on the hydrophobic non-polarizable PTFE substrate, these findings lead to the conclusion that a combination of van der Waals forces and hydrogen bonds between polar substrates and the spatulae is responsible for the adhesion of the attachment setae of Cupiennius salei on substrates with different surface energies. Capillary forces may also play an important role especially at higher relative humidity, because it has been shown that the shear adhesion of the spider Philodromus dispar on an epoxy resin substrate was highest at a relative humidity of 60% (Wolff and Gorb 2012b).

The tests for seta adhesion on differently rough surfaces were performed on the same material of epoxy resin molds to avoid different chemistry of the substrates. The small adhesion forces of the setae to the P0.05 substrate likely result from its rms roughness of 396 nm and the size of the spatulae. This roughness may be too small for the single spatulae to form proper contact. For the reduced adhesion to the SP2 surface, the interplay between the substrate morphology and the seta size likely is the limiting factor. The grains on the SP2 substrate appear spherically shaped and spread on a rather flat surface. For an approaching seta, it is crucial whether it hits a grain and which aspect of the seta tip – the one with or the one without the spatula – gets in contact on top or at the edges of the grain. If the seta tip makes contact with its spatula-free side at the edges of the grains, adhesion forces will be low. It can be assumed that the surface morphology of the SP2 substrate strongly inhibits proper contact making of the spatulae. The statistically significantly increased adhesion value of the setae on the roughest substrate SP3 results from the high adhesion force of the seta 1. Both seta 2 and seta 3 on SP3 do not show values higher than on other substrates. The obvious different adhesion of different setae to the substrates with different roughness indicates that most likely the tip shape and the distribution of the spatulae plays the key role for the attachment of the individual setae. Neighboring setae plucked from the center of the claw tuft scopula showed four different tip shapes and arrangements of the tip microtrichia in four randomly selected setae (Figure 8A).

Shear force applied on an already attached seta, however not sliding the seta, was shown to largely increase the pull-off force from 3.6 µN up to 25 µN by the alignment of a large number of spatulae with a smooth glass surface (Flenner et al., 2020). In the present study, no shear force was applied on the three individual tested setae. Therefore, we measured the result of the arrangement of the spatulae, which spontaneously aligned with the substrate structures upon contact. For such an adhesive behavior, the tip shapes and the arrangement of the microtrichia and spatulae of the seta is crucial for the adhesive force generation. The tip shapes of the three individual tested setae clearly differed (Figures 8B–D), as most likely did the arrangement of the spatulae. As can be seen in Figure 4, adhesion on glass greatly differed at different contact angles of the setae. The mostly higher adhesion forces of seta 1 at the angle of 45° may be the result of the orientation of the spatula-rich side of the seta toward the structural features of the rough substrates. The higher number of the flexible microtrichia and spatulae are more likely to find proper surface features to attach. For seta 2 and seta 3, the angles of 85°, and 88°, respectively, were also selected within ranges of high adhesion for the respective setae. However, their adhesion on differently rough surfaces was low compared to that of seta 1. For seta 2, at the chosen angle of 85°, likely only the microtrichia next to the tip contacted the substrate with their sides. This lead to alignment and adhesion of a smaller number of spatulae and less adhesive force compared to seta 1. Seta 3 showed the lowest adhesion on the differently rough epoxy resin surfaces. Considering the angle of 88° chosen for the measurements and the rounded shape of the tip, it appears likely that seta 3 contacted the surface with the backbone and not with the ventral side, rich in spatula-shaped microtrichia, which resulted in reduced attachment forces.

Even when taking the highest adhesive value of 830 nN into account and assuming that all 18,800 setae (∼2,350 per leg; Wolff and Gorb, 2012c) of the spider were in contact with a substrate, the resulting adhesive force of approximately 16 mN could not support the body weight (∼3.6 g) of an adult female spider. Since Cupiennius salei easily climbs vertical surfaces and is able to walk upside-down also on smooth surfaces, there must be mechanisms of adhesion other than those examined in the single setae in the present study. Interestingly, the adhesive setae are not rigidly fixed in the pretarsal cuticle. In the natural situation, the movability of the setae toward the substrate is strictly limited by a stopper-like structure of the basal hair shaft directly at the insertion of the seta in its socket (Figure 9). This stopper keeps the position of the seta fixed in its position in the socket at dorsad movements of the claw tuft to facilitate pulling-off. On the dorsal (distal) side of the socket, however, there is some space that allows a seta deflection by approximately 6–7° when placed onto a substrate. In our experiments, the setae were well fixed to the stiff silicon cantilevers. In the natural situation, the flexibility of the seta suspension in the pretarsal cuticle may help each individual seta of the claw tuft to find better contact with the structural features of the substrate and consequently a good place to adhere. This movability of the setae also supports their shearing in order to bring more adhesive spatulae in contact with the substrate (see Flenner et al., 2020), which likely resulted in the better adhesion of Cupiennius, when opposing leg pairs were in contact with the ground at pulling-off the whole spider (Wohlfart et al., 2014). Considering the previously measured 25 µN adhesive force of a single attachment seta preloaded in shear, 1,400 setae in contact (which is less than two thirds of the setae of a single leg) would suffice for an adult female Cupiennius salei to adhere on smooth glass. Interestingly, adhesion of the whole scopula claw tuft was zero at vertical pulling-off even after shearing (Wolff and Gorb, 2013; Wohlfart et al., 2014). In the present study the single setae did show adhesive forces at vertical pulling-off on all the different substrates tested. These results indicate that the specific movements of the individual setae of the claw tuft may be most important for making adhesive contact with the substrate.

The results of the present study indicate a distribution of different adhesive tip shapes of the adhesive setae, which likely represents an adaptation of the spider attachment system for efficient interaction with substrates of different roughness. The cuticle of Cupiennius salei has been reported to have an effective Young’s modulus of 18 GPa (Blickhan and Barth, 1985). For the individual seta, the specific structural arrangement of the mechanically stiffer dorsal seta backbone (Schaber et al., 2019) and the softer ventral “brush” of spatula-shaped microtrichia is crucial for adhesion at certain angles with the substrate. It can be assumed that the individual morphology of each individual seta and its position within the scopula orchestration are optimized for maximal adhesion on natural substrates with many different properties as found in the habitat of the spider. The individual variability of the morphology of the spider attachment setae, which is strongly interdigitated with their adhesive performance, has been shown here for the first time. Furthermore, we reveal the importance of examining the relevant different length and force scales for understanding the functionality of specific structures in the entire adhesive system of the spider. This knowledge can lead to new bioinspired materials with outstanding properties such as nanostructured reversible residue-free dry adhesives e.g. based on cellulose nanofibers (Schaber et al., 2018), carbon nanotubes (Bhushan et al., 2008; Schaber et al., 2015a; Schaber et al., 2015b; Su et al., 2020; Yang et al., 2020) or other polymeric materials (Xue et al., 2012; Pattantyus-Abraham et al., 2013; Xue et al., 2013; Borodich and Savencu 2017; Di Tan et al., 2020).