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Anatomic differences on the toe pad epithelial cells of torrent and tree frogs (elongated versus regular geometry) are believed to account for superior ability of torrent frogs to attach to surfaces in the presence of running water. Here, the friction properties of artificial hexagonal arrays of polydimethylsiloxane (PDMS) pillars (elongated and regular) in the presence of water are compared. Elongated pillar patterns show significantly higher friction in a direction perpendicular to the long axis. A low bending stiffness of the pillars and a high edge density of the pattern in the sliding direction are the key design criteria for the enhanced friction. The elongated patterns also favor orientation-dependent friction. These findings have important implications for the development of new reversible adhesives for wet conditions.

Cryo-scanning electron microscopy (cryo-SEM) and atomic force microscopy (AFM) offer new avenues for the study of the morphology of tree frog adhesive toe pads. Using these techniques, we compare toe pad microstructure in two distantly related species of tree frog, Litoria caerulea, White (Hylidae) and Rhacophorus prominanus, Smith (Rhacophoridae), in which the toe pads are considered to be convergent. AFM demonstrates the extraordinary similarity of both surface microstructures (largely hexagonal epithelial cells surrounded by deep channels) and nanostructures (an array of nanopillars, ca. 350 nm in diameter, all with a small dimple at the apex). The cryo-SEM studies examined the distribution of the fibrillar cytoskeleton within the different layers of the stratified toe pad epithelium, demonstrating that the cytoskeletal elements (keratin tonofilaments) that lie at an angle to the surface are relatively poorly developed in L. caerulea, clearly so in comparison to R. prominanus. Cryo-SEM also enabled the visualization of the fluid layer that is critical to a toe pad's adhesive function. This was achieved by examination of the frozen fluid residues left behind after removal of a toe within the cryo-SEM's experimental chamber. Such 'toeprints' demonstrated the presence of a wedge of fluid surrounding each toe pad, as well as fluid filling the channels that surround each epithelial cell. Cryo-SEM was used to examine epithelial cell shape. In a sample of 582 cells, 59.5% were hexagonal, the remainder being mainly pentagonal (23.1%) or heptagonal (16.1%). The distribution of differently-shaped cells was not random, but was not associated with either pad curvature or the distribution of mucous pores that provide fluid for the frogs' wet adhesion mechanism. Our main finding, the great similarity of toe pad structure in these two species, has important implications for biomimetics, for such convergent evolution suggests a good starting point for attempts to develop adhesives that will function in wet conditions.

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.

Elastic, microstructured surfaces (hydrophobic and hydrophilic) mimicking the surface structure of tree-frog toe-pads are fabricated. Their adhesion and friction behaviour in the presence of a liquid layer is evaluated and compared to flat controls. Tree-frog-like patterns are beneficial for wet adhesion only if the liquid does not wet the surface. The situation is different in friction, where the surface structure lead to significantly higher friction forces only if the liquid does wet the surface. Taking into account that tree-frog attachment pads are hydrophilic and that their secretion wets all kind of surfaces, our results indicate that the surface structure in tree-frog toe-pads has been developed for climbing, when shear (friction) forces are involved. These results evidence the benefits and limitations of the surface design (microstructure and hydrophilicity) for adhesion and friction under wet conditions.

SUMMARY Knowledge of both surface structure and physical properties such as stiffness and elasticity are essential to understanding any adhesive system. In this study of an adhesion surface in the tree frog, Litoria caerulea White, a variety of techniques including atomic force microscopy were used to investigate the microstructure and properties of an epithelium that adheres through wet adhesion. Litoria toe pads consist of a hexagonal array of flat-topped epithelial cells, separated by mucus-filled channels. Under an atomic force microscope, this `flat' surface is highly structured at the nanoscale, consisting of a tightly packed array of columnar nanopillars (described as hemidesmosomes by previous authors), 326±84 nm in diameter, each of which possesses a central dimple 8±4 nm in depth. In fixed tissue (transmission electron microscopy), the nanopillars are approximately as tall as they are broad. At the gross anatomical level, larger toe pads may be subdivided into medial and lateral parts by two large grooves. Although the whole toe pad is soft and easily deformable, the epithelium itself has an effective elastic modulus equivalent to silicon rubber (mean Eeff=14.4±20.9 MPa; median Eeff=5.7 MPa), as measured by the atomic force microscope in nanoindentation mode. The functions of these structures are discussed in terms of maximising adhesive and frictional forces by conforming closely to surface irregularities at different length scales and maintaining an extremely thin fluid layer between pad and substrate. The biomimetic implications of these findings are reviewed.

Many species of climbing frog possess large disc-like digital pads, which facilitate adhesion. The consensus of earlier studies is that frogs depend on a wet adhesive mechanism, chiefly influenced by pad area and surface tension properties of the mediating adhesive fluid. The relationship between morphology, size and adhesion is of particular interest for tree frogs, because evolution of adhesive ability has facilitated niche expansion into arboreal habitats. If frogs are geometrically similar, and growth occurs isometrically, then mass will increase at a higher rate than toe-pad area, and an adhesive system directly dependent on area may be adversely affected. We investigated scaling of adhesive ability with ontogeny in seven species of hylid tree frog to test whether the responses to the challenges of maintaining adhesion with growth were sufficient to allow adult frogs to stick as effectively on smooth substrates as juvenile frogs. In all species, mass increased at a lower rate than expected with isometric growth. This was less pronounced in larger species, perhaps owing to the demands of locomotory modes such as jumping. In smaller hylids, this non-isometric increase in mass was sufficient to enable adult frogs to adhere as effectively as juveniles. In larger species, however, the ability of adult frogs to adhere was significantly lower than that of juveniles, despite evidence of increased toe-pad efficiency. Within all species adhesive forces increased at a greater rate than toe-pad area, suggestive of changes in the relative influence of the contributory components of the wet adhesive mechanism with growth.

Toe-pad development in Phyllomedusa trinitatis is described using light microscopy, SEM and TEM. Pad development in forelimbs was slightly in advance of hindlimbs. The first sign of a difference between pad (ventral) skin and dorsal skin occurred at Gosner stage 37 as a convolution of the basal side of the epidermis. By stage 39, pad basal cells were elongated in shape, with highly convoluted basal sides, whereas the dorsal epidermis had a basal layer of cuboidal cells with relatively straight bases. Mucous glands developed from pad epidermis earlier than at the dorsal side, and penetrated deeply into a matrix-rich dermis with a sparse cell population. Specialization of digit tip shape first became apparent at stage 38, with appearance of the distal circumferal groove. Widening of toe tips occurred at stage 39. Surface cells of the digital pads began to be shed by stage 40, in advance of epidermis elsewhere. Specialized pad surface cells were fully developed by stage 46, the end of metamorphic climax. During growth to adulthood, pads increased in overall area, but pad surface cells did not alter their dimensions. However, pad epidermis did become several cell layers thicker, much more so than in most other species described so far. Pad cells also had a surface area higher than in many other described species. The value of developmental studies in aiding our understanding of the multiple origins of adhesive pads in different anuran lineages is discussed.

Reflex leg levation habituates during repeated electrical stimulation of mechanosensory afferents in the dactyl of the fifth walking leg of the crayfish, Procambarus clarkii. This was investigated in decerebrate crayfish, and reproduced in an isolated thoracic ganglion preparation. In vivo, trains of stimuli delivered every 2.5 s produced a gradual decrease in the amplitude of the mechanical response, and a concomitant decrease in the number of impulses per burst in the levator muscle myogram. Near complete recovery occurred after 10 min rest, and transient dishabituation was observed after electrical stimulation of the telson. Less frequent or stronger stimuli led to less rapid habituation. In vitro, the same parametric characteristics of habituation were observed in the levator nerve responses, while the intrinsic variability of the reflex was reduced. The response decrement was shown to be unrelated to changes in the afferent excitation. Evoked polysynaptic excitatory postsynaptic potentials (EPSPs) in levator motorneurons decreased in parallel with the levator neurogram. This decrease was unrelated to any change in the resting membrane potential of the levator motorneurons. Interneurons with habituating EPSPs, antagonistic depressor motorneurons with habituating inhibitory postsynaptic potentials and non-habituating responses in other motorneuronal groups were also found. These findings point to a central locus of habituation upstream from the motorneurons, and offer prospects for a detailed investigation of the mechanisms of habituation in a polysynaptic system.

Tree frogs are remarkable for their capacity to cling to smooth surfaces using large toe pads. The adhesive skin of tree frog toe pads is characterized by peg-studded hexagonal cells separated by deep channels into which mucus glands open. The pads are completely wetted with watery mucus, which led previous authors to suggest that attachment is solely due to capillary and viscous forces generated by the fluid-filled joint between the pad and the substrate. Here, we present evidence from single-toe force measurements, laser tweezer microrheometry of pad mucus and interference reflection microscopy of the contact zone inLitoria caerulea, that tree frog attachment forces are significantly enhanced by close contacts and boundary friction between the pad epidermis and the substrate, facilitated by the highly regular pad microstructure.

Marchand, A. R., W.J.P. Barnes, and D. Cattaert. Primary afferent depolarizations of sensory origin within contact-sensitive mechanoreceptive afferents of a crayfish leg: properties, quantal analysis, and role in presynaptic integration. J. Neurophysiol. 77: 3340–3354, 1997. Recordings from the central branches of single identified dactyl sensory afferent (DSA) neurons in a crayfish in vitro preparation were performed to study modifications of the sensory message occurring before the first central synapse. These afferents comprised hairs and force-sensitive mechanoreceptors with phasic and phasotonic response characteristics in the terminal segment (dactyl) of the crayfish leg. More than one afferent spike size was often observed in intracellular recordings from these afferents, thus indicating the presence of electrical coupling between the central processes of DSA fibers. Additionally, in identified DSA fibers with large spike sizes, primary afferent depolarizations (PADs) of up to 15 mV were observed, which sometimes triggered antidromic spikes in the afferent. Nevertheless, PADs were clearly inhibitory, because they shunted the afferent spikes. They exhibited the following properties. First, each PAD was preceded by an afferent spike from a neighboring hair, indicating that the PADs had a sensory rather than central origin. Second, PADs could follow high frequencies of afferent discharges without failure, a property suggestive of monosynaptic connections, but because PAD latencies varied by ±0.5 ms it is more likely that they were mediated by a disynaptic pathway. Third, although PADs were evoked in an extremely reliable manner, their amplitude varied in a quantal manner. Most unitary PADs were the result of the release of

1. The reflex responses initiated by cuticular stress detector one (CSD1) afferents in anterior levator motor neurnons in the fifth walking leg of crayfish were studied in an in vitro preparation by means of intracellular recordings. 2. The response in the anterior levator reversed with strong mechanical stimulation. Inhibitory responses were correlated with the activation of low-threshold CSD1 units, excitatory responses with the activation of high-threshold CSD1 units. Two opposing reflexes thus originate from the same sense organ at different levels of stimulation. 3. Some low-threshold units were inactive at stimulus strengths that activated the high-threshold units. Other low-threshold units remained active. Their reflex effects were reduced by either pre- or postsynaptic mechanisms. 4. Functionally, this force dependent response reversal could be important in switching from postural/locomotor responses to defensive responses such as limb autotomy.

Cuticular Stress Detector afferents (CSD1 and CSD2) in the 5th walking legs of crayfish (Pacifastacus leniusculus, Procambarus clarkii) have been studied in an in vitro preparation allowing intracellular recordings to be made from the central terminals of primary afferent fibres during mechanical stimulation of the sense organs. Biocytin anterograde fills and transverse sections of the sensory nerves showed CSD1 to comprise fewer and more heterogeneous fibres than CSD2. Lucifer yellow filling of single fibres showed branching patterns compatible with monosynaptic projections to some motorneuronal groups. Whole nerve recordings during sinusoidal or ramp stimuli showed an important contribution from units with phasic properties. The intracellular recordings identified three features unique to CSD1: 1. Many ‘on-off’ units have a phasic response to both increases and decreases of force. 2. Many ‘high threshold’ units respond only to high amplitude vibratory stimuli. 3. A few sensory fibres have a main branch projecting rostrally within the interganglionic connectives, possibly as far as the brain. In vivo recordings of CSD1 activity during forward locomotion on a treadmill showed a discharge occurring in advance of, as well as during, the power stroke. It is therefore suggested that at least some CSD1 fibres encode active as well as passive force during locomotion.

Myograms were recorded from crabs being trained to raise their legs to avoid shocks. Shock avoidance was associated with an increase in the firing frequency of a slow tonic flexor motoneuron, whose firing frequency increased with increased carpopodite flexion. The firing frequency of this motoneuron could be directly altered by means of a computer‐controlled training procedure. When shocks were given whenever the frequency was below a predetermined threshold, significant increases in frequency were obtained compared with control animals that received the same regime of shocks as experimental crabs.

Decerebrate crabs were trained to raise their legs to avoid electric shocks. Compared with yoked controls, experimental crabs received significantly fewer shocks after 6–7 minutes training and also during the first minute of a subsequent testing period when both experimental and control crabs were in a training situation. There was considerable variation in the performance of different crabs. Some learned quickly, some slowly, while others did not learn at all. In some, the raised leg position was maintained without further reinforcement. In others, periods when the animal was receiving few shocks were interrupted by periods when the leg was lowered and several shocks were received. A variety of leg movements were utilised by crabs to avoid shocks, the most common being flexion at the mero‐carpopodite joint.

1. Cine films of fiddler crabs (Uca pugnax) walking sideways on land were analysed frame by frame in order to study the co-ordination of the legs both during normal walking and following amputation (induced autotomy) of one of the 3rd pair of legs. 2. Although the average gait of unoperated animals approximates to an alternating tetrapod rhythm, equivalent to the alternating tripod gait of insects (Figs. 3–7), the stepping order of legs on leading and trailing sides of the animal is different. The dominant sequence on the leading side is 2435 (legs numbered from front to rear), that on the trailing side 2534 (Table 1), the chelae (leg 1) being usually held off the ground. 3. Stepping parameters also change with walking speed (Figs. 8, 10), the most clear-cut of these changes being the reduced relative duration of the power stroke at high walking speeds. At low walking speeds, clear-cut metachronal co-ordination is sometimes seen in trailing legs (Fig. 9), but the data nevertheless do not fit Wilson's 1966 metachronal model for insect locomotion. 4. Amputation of one of the 3rd pair of legs causes adaptive changes in the gait. The most obvious of these changes are that the chelae are now frequently used in walking and legs 4 and 2 on the operated side of the animal now alternate whereas before they moved together (Figs. 11–13, Table 2). Evidence is presented to show that these changes are brought about by the changed sensory input and are not due either to the effects of axon section on motorneurone electrical excitability, or to the animal walking more slowly and using a different but normally occurring gait.

A substance which is extractable with acetone from the marginal ganglia of the jellyfish Aurelia causes acceleration of the rhythm of intact ganglia used as test preparations in concentrations of under 50 ganglia/ml., while higher concentrations cause inhibition. As comparable extracts of non-nervous tissue had no effect, the active substance probably originates from nervous tissue. It is distinct from sub-stances extractable from mesogloea. The RF lies near 0·85 in tert.-butanol-methanol-water (40:50:10 by vol.). The chemical nature of the acceleratory substance is unknown though it is certainly none of the known or suspected neurohumours of other organisms. The active substance has no effect on crustacean or clam hearts or upon crustacean hindgut assay preparations but it does accelerate the rhythm of a hydromedusan Phialidium.

(1969). Corophium volutator—An Intertidal Crustacean Useful for Teaching in Schools and Universities. Journal of Biological Education: Vol. 3, No. 4, pp. 283-298.

The movements of the two eyecups of the crab, Carcinus, have been recorded simultaneously during optokinetic responses. Experiments in which the eyes view different visual stimuli reveal that, at the start of a response, the eyecups have a considerable degree of independence and can even move in opposite directions. As the response progresses, interaction between the eyes increases, until the eyecups move at similar velocities in the direction of the slower of the two visual inputs, or are stationary. Similar interactions between the eyes were observed during memory responses and during the responses to sinusoidal oscillation of the two sets of stripes. Each eye has its own system for converting perceived motion into eyecup movement. These two systems are linked on the afferent rather than the efferent side of the brain. The fast phase of optokinetic nystagmus is governed by the eye whose fast-phase movement occurs away from the midline, and the fast phases of this eyecup lead the other by 30-80 msec. Also, fast phases only occur at their normal frequency when the governing eye can see the stripes.

Electronics in the Life Sciences. By Stephen Young. Pp. 198. (Macmillan: London and Basingstoke, August 1973.) £5.50 boards; £2.75 paper.