Wetting of the tarsal adhesive fluid determines underwater adhesion in ladybird beetles.

Many insects can climb smooth surfaces using hairy adhesive pads on their legs, mediated by tarsal fluid secretions. It was previously shown that a terrestrial beetle can even adhere and walk underwater. The naturally hydrophobic hairs trap an air bubble around the pads, allowing the hairs to make contact with the substrate as in air. However, it remained unclear to what extent such an air bubble is necessary for underwater adhesion. To investigate the role of the bubble, we measured the adhesive forces in individual legs of live but constrained ladybird beetles underwater in the presence and absence of a trapped bubble and compared these with its adhesion in air. Our experiments revealed that on a hydrophobic substrate, even without a bubble, the pads show adhesion comparable to that in air. On a hydrophilic substrate, underwater adhesion is significantly reduced, with or without a trapped bubble. We modelled the adhesion of a hairy pad using capillary forces. Coherent with our experiments, the model demonstrates that the wetting properties of the tarsal fluid alone can determine the ladybird beetles' adhesion to smooth surfaces in both air and underwater conditions and that an air bubble is not a prerequisite for their underwater adhesion. This study highlights how such a mediating fluid can serve as a potential strategy to achieve underwater adhesion via capillary forces, which could inspire artificial adhesives for underwater applications.

A force-measuring and behaviour-recording system consisting of 24 individual 3D force plates for the study of single limb forces in climbing animals on a quasi-cylindrical tower.

This study describes the design of a new force measuring array with a quasi-cylindrical surface for measuring the 3D ground reaction forces of animals climbing on a surface with high curvature. This force-measuring array was assembled from 24 individual 3D force sensors, each with a resolution at the millinewton (mN) level, which were installed from top to bottom in four columns and six rows, with sensors in neighbouring columns staggered in height. Three cameras were used to simultaneously record the climbing behaviours of animals (in these experiments tree frogs) on the cylinder-like force measuring array. We were thus able to simultaneously record the ground reaction forces of each of the four limbs of tree frogs (here six individuals of the Chinese gliding or flying frog, Rhacophorus dennysi, with forelimb spans in the range 163-201 mm) climbing or descending both smooth and rough surfaces on a quasi-cylindrical structure with an overall diameter of 79 mm. We describe the design and calibration of the individual force sensors, their installation and arrangement on the quasi-cylindrical climbing tower, the recording of ground reaction forces and climbing behaviour, data transformations necessitated by the angular relationship of neighbouring sensors, and data processing using MATLAB scripts. Additionally, we present preliminary data on the use of a clamping grip by climbing frogs and the existence of small pull-off forces that aid toe-pad detachment at the end of each locomotor stance phase.

Innate turning preference of leaf-cutting ants in the absence of external orientation cues.

Most ants use a combination of cues for orientation but how do ants find their way when all external cues are suppressed? Do they walk in a random way or are their movements spatially oriented? Here, we show for the first time that leaf-cutting ants (Acromyrmex lundii) have an innate preference for turning counter-clockwise (left) when external cues are precluded. We demonstrated this by allowing individual ants to run freely on the water surface of a newly developed treadmill. The surface tension supported medium-sized workers but effectively prevented ants from reaching the wall of the vessel, which was important to avoid wall-following behaviour (thigmotaxis). Most ants ran for minutes on the spot but also slowly turned counter-clockwise in the absence of visual cues. Reconstructing the effective path walked revealed a looping pattern which could be interpreted as a search strategy. A similar turning bias was shown for groups of ants in a symmetrical Y-maze where twice as many ants chose the left branch in the absence of optical cues. Wall-following behaviour was tested by inserting a coiled tube before the Y-fork. When ants traversed a left-coiled tube, more ants chose the left box and vice versa. Adding visual cues in the form of vertical black strips either outside the treadmill or on one branch of the Y-maze led to oriented walks towards the strips. It is suggested that both turning bias and wall following are employed as search strategies for an unknown environment which can be overridden by visual cues.

The biomechanics of tree frogs climbing curved surfaces: a gripping problem.

The adhesive mechanisms of climbing animals have become an important research topic because of their biomimetic implications. We examined the climbing abilities of hylid tree frogs on vertical cylinders of differing diameter and surface roughness to investigate the relative roles of adduction forces (gripping) and adhesion. Tree frogs adhere using their toe pads and subarticular tubercles, the adhesive joint being fluid-filled. Our hypothesis was that on an effectively flat surface (adduction forces on the largest 120 mm diameter cylinder were insufficient to allow climbing), adhesion would effectively be the only means by which tree frogs could climb, but on the 44 and 13 mm diameter cylinders, frogs could additionally utilise adduction forces by gripping the cylinder either with their limbs outstretched or by grasping around the cylinder with their digits, respectively. The frogs' performance would also depend on whether the surfaces were smooth (easy to adhere to) or rough (relatively non-adhesive). Our findings showed that climbing performance was highest on the narrowest smooth cylinder. Frogs climbed faster, frequently using a 'walking trot' gait rather than the 'lateral sequence walk' used on other cylinders. Using an optical technique to visualise substrate contact during climbing on smooth surfaces, we also observed an increasing engagement of the subarticular tubercles on the narrower cylinders. Finally, on the rough substrate, frogs were unable to climb the largest diameter cylinder, but were able to climb the narrowest one slowly. These results support our hypotheses and have relevance for the design of climbing robots.

Three-dimensional patterning in biomedicine: Importance and applications in neuropharmacology.

Nature manufactures biological systems in three dimensions with precisely controlled spatiotemporal profiles on hierarchical length and time scales. In this article, we review 3D patterning of biological systems on synthetic platforms for neuropharmacological applications. We briefly describe 3D versus 2D chemical and topographical patterning methods and their limitations. Subsequently, an overview of introducing a third dimension in neuropharmacological research with delineation of chemical and topographical roles is presented. Finally, toward the end of this article, an explanation of how 3D patterning has played a pivotal role in relevant fields of neuropharmacology to understand neurophysiology during development, normal health, and disease conditions is described. The future prospects of organs-on-a--like devices to mimic patterned blood-brain barrier in the context of neurotherapeutic discovery and development for the prioritization of lead candidates, membrane potential, and toxicity testing are also described. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1369-1382, 2018.

Soiled adhesive pads shear clean by slipping: a robust self-cleaning mechanism in climbing beetles.

Animals using adhesive pads to climb smooth surfaces face the problem of keeping their pads clean and functional. Here, a self-cleaning mechanism is proposed whereby soiled feet would slip on the surface due to a lack of adhesion but shed particles in return. Our study offers an in situ quantification of self-cleaning performance in fibrillar adhesives, using the dock beetle as a model organism. After beetles soiled their pads by stepping into patches of spherical beads, we found that their gait was significantly affected. Specifically, soiled pads slipped 10 times further than clean pads, with more particles deposited for longer slips. Like previous studies, we found that particle size affected cleaning performance. Large (45 µm) beads were removed most effectively, followed by medium (10 µm) and small (1 µm). Consistent with our results from climbing beetles, force measurements on freshly severed legs revealed larger detachment forces of medium particles from adhesive pads compared to a flat surface, possibly due to interlocking between fibres. By contrast, dock leaves showed an overall larger affinity to the beads and thus reduced the need for cleaning. Self-cleaning through slippage provides a mechanism robust to particle size and may inspire solutions for artificial adhesives.

The use of clamping grips and friction pads by tree frogs for climbing curved surfaces.

Most studies on the adhesive mechanisms of climbing animals have addressed attachment against flat surfaces, yet many animals can climb highly curved surfaces, like twigs and small branches. Here we investigated whether tree frogs use a clamping grip by recording the ground reaction forces on a cylindrical object with either a smooth or anti-adhesive, rough surface. Furthermore, we measured the contact area of fore and hindlimbs against differently sized transparent cylinders and the forces of individual pads and subarticular tubercles in restrained animals. Our study revealed that frogs use friction and normal forces of roughly a similar magnitude for holding on to cylindrical objects. When challenged with climbing a non-adhesive surface, the compressive forces between opposite legs nearly doubled, indicating a stronger clamping grip. In contrast to climbing flat surfaces, frogs increased the contact area on all limbs by engaging not just adhesive pads but also subarticular tubercles on curved surfaces. Our force measurements showed that tubercles can withstand larger shear stresses than pads. SEM images of tubercles revealed a similar structure to that of toe pads including the presence of nanopillars, though channels surrounding epithelial cells were less pronounced. The tubercles' smaller size, proximal location on the toes and shallow cells make them probably less prone to buckling and thus ideal for gripping curved surfaces.

When the going gets rough - studying the effect of surface roughness on the adhesive abilities of tree frogs.

Tree frogs need to adhere to surfaces of various roughnesses in their natural habitats; these include bark, leaves and rocks. Rough surfaces can alter the effectiveness of their toe pads, due to factors such as a change of real contact area and abrasion of the pad epithelium. Here, we tested the effect of surface roughness on the attachment abilities of the tree frog Litoria caerulea. This was done by testing shear and adhesive forces on artificial surfaces with controlled roughness, both on single toe pads and whole animal scales. It was shown that frogs can stick 2-3 times better on small scale roughnesses (3-6 µm asperities), producing higher adhesive and frictional forces, but relatively poorly on the larger scale roughnesses tested (58.5-562.5 µm asperities). Our experiments suggested that, on such surfaces, the pads secrete insufficient fluid to fill the space under the pad, leaving air pockets that would significantly reduce the Laplace pressure component of capillarity. Therefore, we measured how well the adhesive toe pad would conform to spherical asperities of known sizes using interference reflection microscopy. Based on experiments where the conformation of the pad to individual asperities was examined microscopically, our calculations indicate that the pad epithelium has a low elastic modulus, making it highly deformable.

On Heels and Toes: How Ants Climb with Adhesive Pads and Tarsal Friction Hair Arrays.

Ants are able to climb effortlessly on vertical and inverted smooth surfaces. When climbing, their feet touch the substrate not only with their pretarsal adhesive pads but also with dense arrays of fine hairs on the ventral side of the 3rd and 4th tarsal segments. To understand what role these different attachment structures play during locomotion, we analysed leg kinematics and recorded single-leg ground reaction forces in Weaver ants (Oecophylla smaragdina) climbing vertically on a smooth glass substrate. We found that the ants engaged different attachment structures depending on whether their feet were above or below their Centre of Mass (CoM). Legs above the CoM pulled and engaged the arolia ('toes'), whereas legs below the CoM pushed with the 3rd and 4th tarsomeres ('heels') in surface contact. Legs above the CoM carried a significantly larger proportion of the body weight than legs below the CoM. Force measurements on individual ant tarsi showed that friction increased with normal load as a result of the bending and increasing side contact of the tarsal hairs. On a rough sandpaper substrate, the tarsal hairs generated higher friction forces in the pushing than in the pulling direction, whereas the reverse effect was found on the smooth substrate. When the tarsal hairs were pushed, buckling was observed for forces exceeding the shear forces found in climbing ants. Adhesion forces were small but not negligible, and higher on the smooth substrate. Our results indicate that the dense tarsal hair arrays produce friction forces when pressed against the substrate, and help the ants to push outwards during horizontal and vertical walking.

The role of fore- and hindlimbs during jumping in the Dybowski's frog (Rana dybowskii).

Anurans are well known for their jumping abilities, making use of their strong hindlimbs. In contrast, the function of the forelimbs during take-off has rarely been studied. We measured the ground reaction forces exerted by forelimbs and hindlimbs during short jumps in the Dybowski's frog Rana dybowskii. Take-off occurred in two phases. Phase one (from the initial time until the forelimbs took off), which lasts a relatively long time (63.2 ± 4.1% of the total take-off phase, N = 20), provides sufficient time for the forelimbs to elevate the body to a suitable posture to deliver the best take-off angle. Phase two (from the forelimbs lift-off until hindlimbs lift-off) was dominated by the hindlimbs which provided a constant and fast elevation. The force angle (angle of the resultant vector from fore-aft and normal force components towards the plane of the substrate) of the hindlimbs and body trajectory was variable before the forelimbs lifted off of the substrate and then primarily followed the direction of the line from the foot-substrate point to the center of mass (COM). The preparation angle adopted when the forelimbs lifted off of the substrate was a good predictor of the take-off angle. The total normal force oscillated around body weight (BW) before the forelimb normal force peaked. The BW shifted from the hindlimbs to the forelimbs during the initial phase of take-off. A simple lever model suggests that the forelimbs are responsible for raising the COM, thus influencing the take-off angle in short jumps.

Experimental evidence for friction-enhancing integumentary modifications of chameleons and associated functional and evolutionary implications.

The striking morphological convergence of hair-like integumentary derivatives of lizards and arthropods (spiders and insects) demonstrates the importance of such features for enhancing purchase on the locomotor substrate. These pilose structures are responsible for the unique tractive abilities of these groups of animals, enabling them to move with seeming ease on overhanging and inverted surfaces, and to traverse inclined smooth substrates. Three groups of lizards are well known for bearing adhesion-promoting setae on their digits: geckos, anoles and skinks. Similar features are also found on the ventral subdigital and distal caudal skin of chameleons. These have only recently been described in any detail, and structurally and functionally are much less well understood than are the setae of geckos and anoles. The seta-like structures of chameleons are not branched (a characteristic of many geckos), nor do they terminate in spatulate tips (which is characteristic of geckos, anoles and skinks). They are densely packed and have attenuated blunt, globose tips or broad, blade-like shafts that are flattened for much of their length. Using a force transducer, we tested the hypothesis that these structures enhance friction and demonstrate that the pilose skin has a greater frictional coefficient than does the smooth skin of these animals. Our results are consistent with friction being generated as a result of side contact of the integumentary filaments. We discuss the evolutionary and functional implications of these seta-like structures in comparison with those typical of other lizard groups and with the properties of seta-mimicking synthetic structures.

Sticking under wet conditions: the remarkable attachment abilities of the torrent frog, Staurois guttatus.

Tree frogs climb smooth surfaces utilising capillary forces arising from an air-fluid interface around their toe pads, whereas torrent frogs are able to climb in wet environments near waterfalls where the integrity of the meniscus is at risk. This study compares the adhesive capabilities of a torrent frog to a tree frog, investigating possible adaptations for adhesion under wet conditions. We challenged both frog species to cling to a platform which could be tilted from the horizontal to an upside-down orientation, testing the frogs on different levels of roughness and water flow. On dry, smooth surfaces, both frog species stayed attached to overhanging slopes equally well. In contrast, under both low and high flow rate conditions, the torrent frogs performed significantly better, even adhering under conditions where their toe pads were submerged in water, abolishing the meniscus that underlies capillarity. Using a transparent platform where areas of contact are illuminated, we measured the contact area of frogs during platform rotation under dry conditions. Both frog species not only used the contact area of their pads to adhere, but also large parts of their belly and thigh skin. In the tree frogs, the belly and thighs often detached on steeper slopes, whereas the torrent frogs increased the use of these areas as the slope angle increased. Probing small areas of the different skin parts with a force transducer revealed that forces declined significantly in wet conditions, with only minor differences between the frog species. The superior abilities of the torrent frogs were thus due to the large contact area they used on steep, overhanging surfaces. SEM images revealed slightly elongated cells in the periphery of the toe pads in the torrent frogs, with straightened channels in between them which could facilitate drainage of excess fluid underneath the pad.

Rapid preflexes in smooth adhesive pads of insects prevent sudden detachment.

Many insects possess adhesive organs that can produce extreme attachment forces of more than 100 times body weight but they can rapidly release adhesion to allow locomotion. During walking, weaver ants (Oecophylla smaragdina) use only a fraction of their maximally available contact area, even upside-down on a smooth surface. To test whether the reduced contact area makes the ants more susceptible to sudden and unexpected detachment forces, for example, by rain or wind gusts, we investigated the reaction of untethered ants to rapid horizontal displacements of the substrate. High-speed video recordings revealed that the pad's contact area could more than double within the first millisecond after the perturbation. This contact area expansion is much faster than any neuromuscular reflex and therefore represents a passive 'preflex', resulting from the mechanical properties and geometrical arrangement of the (pre-)tarsus. This preflex reaction protects ants effectively against unexpected detachment, and allows them to use less contact area during locomotion. Contact area expanded most strongly when the substrate displacement generated a pull along the axis of the tarsus, showing that the ants' preflex is direction-dependent. The preflex may be based on the ability of Hymenopteran adhesive pads to unfold when pulled towards the body. We tested Indian stick insects (Carausius morosus), which have smooth pads that lack this motility. Similar to the ants, they showed a rapid and direction-dependent expansion of the contact area mainly in the lateral direction. We propose that the preflex reaction in stick insects is based on the reorientation of internal cuticle fibrils in a constant-volume system, whereas the ants' pad cuticle is probably not a hydrostat, and pad extension is achieved by the arcus, an endoscelerite of the arolium.

Sticking like sticky tape: tree frogs use friction forces to enhance attachment on overhanging surfaces.

To live and clamber about in an arboreal habitat, tree frogs have evolved adhesive pads on their toes. In addition, they often have long and slender legs to facilitate not only long jumps, but also to bridge gaps between leaves when climbing. Both adhesive pads and long limbs are used in conjunction, as we will show in this study. Previous research has shown that tree frogs change from a crouched posture (where the limbs are close to the body) to a sprawled posture with extended limbs when clinging on to steeper inclines such as vertical or overhanging slopes. We investigated this change in posture in White's tree frogs (Litoria caerulea) by challenging the frogs to cling onto a tiltable platform. The platform consisted of an array of 24 three-dimensional force transducers, which allowed us to measure the ground reaction forces of the frogs during a tilt. Starting from a crouched resting position, the normal forces on the forelimbs changed sign and became increasingly negative with increasing slope angle of the platform. At about 106° ± 12°, tilt of the platform the frogs reacted by extending one or two of their limbs outwards. At a steeper angle (131° ± 11°), the frogs spread out all their limbs sideways, with the hindlimbs stretched out to their maximum reach. Although the extension was strongest in the lateral direction, limbs were significantly extended in the fore-aft direction as well. With the extension of the limbs, the lateral forces increased relative to the normal forces. The large contribution of the in-plane forces helped to keep the angle between the force vector and the platform small. The Kendall theory for the peeling of adhesive tape predicts that smaller peel angles lead to higher attachment forces. We compare our data with the predictions of the Kendall model and discuss possible implications of the sliding of the pads on the surface. The forces were indeed much larger for smaller angles and thus can be explained by peeling theory.

Self-cleaning in tree frog toe pads; a mechanism for recovering from contamination without the need for grooming.

Tree frogs use adhesive toe pads for climbing on a variety of surfaces. They rely on wet adhesion, which is aided by the secretion of mucus. In nature, the pads will undoubtedly get contaminated regularly through usage, but appear to maintain their stickiness over time. Here, we show in two experiments that the toe pads of White's tree frogs (Litoria caerulea) quickly recover from contamination through a self-cleaning mechanism. We compared adhesive forces prior to and after contamination of (1) the whole animal on a rotatable platform and (2) individual toe pads in restrained frogs mimicking individual steps using a motorised stage. In both cases, the adhesive forces recovered after a few steps but this took significantly longer in single toe pad experiments from restrained frogs, showing that use of the pads increases recovery. We propose that both shear movements and a 'flushing' effect of the secreted mucus play an important role in shedding particles/contaminants.

Walking on smooth or rough ground: passive control of pretarsal attachment in ants.

The hymenopteran tarsus is equipped with claws and a movable adhesive pad (arolium). Even though both organs are specialised for substrates of different roughness, they are moved by the same muscle, the claw flexor. Here we show that despite this seemingly unfavourable design, the use of arolium and claws can be adjusted according to surface roughness by mechanical control. Tendon pull experiments in ants (Oecophylla smaragdina) revealed that the claw flexor elicits rotary movements around several (pre-) tarsal joints. However, maximum angular change of claws, arolium and fifth tarsomere occurred at different pulling amplitudes, with arolium extension always being the last movement. This effect indicates that arolium use is regulated non-neuronally. Arolium unfolding can be suppressed on rough surfaces, when claw tips interlock and inhibit further contraction of the claw flexor or prevent legs from sliding towards the body. To test whether this hypothesised passive control operates in walking ants, we manipulated ants by clipping claw tips. Consistent with the proposed control mechanism, claw pruning resulted in stronger arolium extension on rough but not on smooth substrates. The control of attachment by the insect claw flexor system demonstrates how mechanical systems in the body periphery can simplify centralised, neuro-muscular feedback control.

Locomotion and adhesion: dynamic control of adhesive surface contact in ants.

Tarsal adhesive pads of insects are highly dynamic organs that play an important role in locomotion. Many insects combine fast running performance with strong resistance to detachment forces. This capacity requires an effective control of attachment forces at the tarsus and pretarsus. Here we investigate mechanisms of attachment control in Asian weaver ants (Oecophylla smaragdina) by measuring the dynamics of the adhesive contact area and the claws during locomotion. O. smaragdina ants walking upside down on a smooth substrate used only a fraction (approx. 14%) of their maximum possible contact area. When these ants were loaded with 30 mg weights (corresponding to approx. 6 times their own body weight), however, they employed much larger (but still submaximal; approx. 60%) contact areas. The increase of contact area was accompanied by a stronger flexion of the claws, which demonstrates the participation of the claw flexor muscle in the control of adhesive contact. However, only part of the contact area dynamics could be explained by the action of the claw flexor. During the stance phase, adhesive contact area changed while the claws remained motionless. Even when corrected for the effects of claw flexion, adhesive contact areas differed by a factor of 2.1 between loaded and unloaded ants. Our findings give evidence that running ants control their adhesive contact area by a combination of active movements of the claw flexor muscle and passive reactions of the mechanical system.