How do plant waxes cause flies to slide? Experimental tests of wax-based trapping mechanisms in three pitfall carnivorous plants

The waxy surfaces of three carnivorous plants, Nepenthes ventrata (Nepenthaceae), Brocchinia reducta and Catopsis berteroniana (Bromeliaceae), were compared using scanning electron microscopy (SEM). Their effects on attachment and locomotion of the fly Calliphora vomitoria were studied. The waxy surface of N. ventrata is comprised of a heterogeneous layer from which only platelet-shaped crystalloids could be detached by brushing. In the two bromeliads, the crystalloids are thread-shaped and form a homogenous dense network, which was entirely removable from the epidermis. Experimental data showed that none of the flies was able to walk across any of the waxy surfaces and only a few were able to take off from those surfaces. Both the absence of sites for claw anchorage, especially in N. ventrata, and the wax itself were shown to contribute to the trapping ability of the plants. Only half of the flies quickly recovered their locomotion ability on a glass surface after 20 min of being tested on waxy plant surfaces. SEM observations revealed that the wax of C. berteroniana formed a powder of broken crystals on the tenent setae of the flies' pulvilli. In contrast, the waxes of B. reducta and N. ventrata appeared to have lost their crystal structure in contact with the tenent setae and formed an amorphous substance that adhered setae together. We hypothesize that wax interacts with adhesive fluids secreted by the fly pad and thereby prevents the tenent setae from functioning effectively.     

Tarsal attachment devices of the southern green stink bug Nezara viridula (Heteroptera: Pentatomidae)

Based on analyses with cryo‐scanning and transmission electron microscopy, the present study reports on the morphology and ultrastructure of the attachment structures of the green stinkbug Nezara viridula L. (Heteroptera: Pentatomidae), a cosmopolitan pest of different crops in most areas of the world. In addition, the presence and distribution of large proportions of the elastic protein resilin in these structures was revealed by confocal laser scanning microscopy. The attachment structures of each leg comprise two sclerotised claws, a pair of smooth flexible pulvilli and a hairy adhesive pad located at the ventral side of the basitarsus. No sexual dimorphism is evident. Contact areas of resting individuals on a smooth surface show that N. viridula creates contact to the substrate with the ventral surface of (a) the distal portions of the pulvilli, (b) the setae of the hairy adhesive pad, (c) the two paraempodia representing mechanosensory setae, and (d) the tips of the claws. Each pulvillus is a sac‐like structure formed by complex cuticular layers that vary in their structure and resilin content. The dorsal side consists of sclerotised chitinous material, while the ventral cuticle consists mainly of resilin and shows a very thin epicuticle and a thick exocuticle. The setae of the hairy adhesive pad are pointed and socketed. They exhibit a pronounced longitudinal gradient in the material composition, with large proportions of resilin being present in the setal tips. In most of these setae, especially in those of the distal‐most part of the pad, also a transverse gradient in the material composition is visible.     

The adhesive organ of the blowfly, Calliphora vomitoria: a functional approach (Diptera: Calliphoridae)

The external morphology of the terminal region of the fifth tarsal segment of the blowfly, Calliphora vomitoria (L.) has been studied using light and scanning electron microscopy (S.E.M.). The pulvilli, with their numerous tenent hairs of spatulate form projecting from the ventral surface, are responsible for adhesion to smooth surfaces. The two large claws are believed to be important in clinging to irregularities in surfaces. Two footplates, possibly sensory organs, lie in close association with the base of the large ventral seta, the empodium. Blowflies release a non-volatile lipid secretion on to the spatulate ends of the tenent hairs and this secretion is essential to the adhesion process on smooth surfaces. The force of adhesion has been measured for tethered blowflies on glass using both vertical and lateral pulls; lateral pulls gave much greater forces. It is concluded that surface tension of the lipid secretion under tenent hairs is sufficient to enable successful adhesion to smooth surfaces by blowflies.     

Microstructure of the tarsal adhesive organs in the bark beetle Ips acuminatus,and their implication as external carriers of pathogens

Ips acuminatus is a common group of bark beetles that infest and damage pine and spruce trees. As a part of research for controlling this insect pest, the adhesive organs on the tarsal appendages were examined using field emission scanning electron microscopy (FE-SEM) to reveal the microstructural characteristics of its biological attachment system. In addition, we also demonstrate their ability to act as external carriers of pathogens. This bark beetle has a characteristic attachment apparatus to move both smooth and rough surfaces. The claws are connected with a pretarsal segment, and their apical diverged hooks are developed to hold rough substrates; however, landing on smooth surfaces is achieved by means of three groups of hairy tarsal pads. The adhesive pads are basically composed of the flattened tip setae usually with a spatula-shaped endplate. Although this bark beetle did not have mycangial cavities, yeast-like spores were concentrated at the invaginated surface of legs where cuticular hairs are densely packed. In particular, the base stalk of the adhesive pad had a sufficient space to accept spores during the dynamic movement of tenent setae.     

Structure of the tarsi in some Stenus species (Coleoptera,Staphylinidae): external morphology,ultrastructure, and tarsal secretion

SEM studies show that the differentiation among Stenus species with respect to the formation of the tarsi (wide bilobed vs. slender tarsomeres) takes place with a considerable augmentation of tarsal ventral setae in wide bilobed tarsomeres. The structural diversity of ventral tarsal setae among and within species is discussed with respect to 1) their different roles as mechanosensilla and tenent setae, respectively, and 2) the different selection pressures in terms of adhesive requirements along the longitudinal tarsus axis. The tarsi are provided with four groups of tarsal mechanosensilla, comprising hair and bristle sensilla, campaniform sensilla, and scolopidia. The tarsus wall is supported by an epidermis, which forms three different types of glands pouring their secretion via different exit paths onto the outer cuticle. The organization and ultrastructure of each of these glands is described. Only one (unicellular) gland is directly associated with the ventral tenent setae and is thus considered to form the main part of the adhesive secretion. The beetles appear to release the tarsal secretion through mediation of the tenent setae, which contains a lipid and a proteinaceous fraction. I propose that the secretion is discharged to the outside via a system of very fine pore canals in the wall of the setal shaft. Gas chromatography and infrared spectroscopy revealed that the lipid fraction of the secretion is a mixture of unsaturated fatty acid glycerides and aliphatic hydrocarbons whose spectra are similar to those of extractions of the superficial lipid coating of the body surface.     

Microstructure of pretarsal pulvilli in shield bug Acanthosoma spinicolle (Heteroptera: Acanthosomatidae)

Shield bugs effectively attach themselves on both rough and smooth surfaces, but their advanced biological attachment devices have not been studied closely. Our fine structural examination of the attachment devices in the shield bug A. spinicolle reveals a unique system to achieve extraordinary adhesion that allows vertical climbing. Each appendage has a pair of tarsal claws that attach to rough substrates and a pair of pretarsal pulvilli that attach to smooth surfaces. Similar to other heteropteran insects, the pulvilli of this bug are categorized as a wet adhesion system, which makes use of an adhesive fluid from the pad secretion. However, this deformable pad creates a regular pattern of contact with the mating surface with a compact array of microfolds and setae with filamentous distal protrusions. To date, this distinctive microstructure in pulvilli pads has never been reported. These microstructural characteristics should be further studied to understand biological adhesion as well as create biomimetic applications.     

Show me your tenent setae and I tell you who you are – Telling the story of a neglected character complex with phylogenetic signals using Leiodidae (Coleoptera) as a case study

The tarsal setae in 97 species of Leiodidae and eight outgroups were examined using SEM imaging and dissections. Modified adhesive setae present in males are referred to as “male tenent setae” (MTS). In most cases, dilated tarsomeres were associated with MTS, which were always present on the protarsi and sometimes the mesotarsi. MTS are reported for the first time on the mesotarsi of Leptodirini and on the metatarsi in two genera of Sogdini. Contrary to reports in the literature, the reduction in the number of the MTS bearing mesotarsomeres is considered a derived condition. Both sexes of Leptinus (Platypsyllinae) have modified setae (referred to as tenent setae in the literature), probably related to their specialised association with mammals, and a patch of MTS was recognized for the first time among those modified setae among males. Four main types of MTS are recognised: (1) a plesiomorphic discoidal type that has a shaft with a round cross-section and maintains a similar diameter throughout its length until forming the expanded discoidal terminal plate; (2) a minidiscoidal type, similar to discoidal but with a relatively small terminal plate, found in Cholevinae; (3) a conical type, present in Leiodinae (excluding Estadiini) where the shaft increases in diameter until forming the terminal plate; and (4) a spatulate type, where an even wider terminal plate has a lateral projection, derived from the conical form and synapomorphic for the leiodine tribes Pseudoliodini, Scotocryptini, and possibly Agathidiini.     

Hairy attachment structures in Reduviidae (Cimicomorpha, Heteroptera), with observations on the fossula spongiosa in some other Cimicomorpha

Reduviidae and some other groups of cimicomorphan Heteroptera possess a hairy attachment structure on the apex of the tibia called “fossula spongiosa”. The fossula spongiosa was never studied comparatively across Reduviidae, its fine structure and mode of function is not well documented, and attachment structures in immature stages are virtually unknown. Here, a sample of 171 species of Reduviidae representing 22 subfamilies is examined for presence-absence of the fossula spongiosa on the three pairs of legs. Representatives of 11 of the 22 subfamilies are shown to possess a fossula spongiosa. The fine structure of the fossula spongiosa is examined for a more limited sample of Reduviidae and several Pachynomidae and Nabidae. In addition, scanning micrographs for the fossula spongiosa in other Cimicomorpha are given, among them Anthocoridae, Cimicidae, Microphysidae (first record of a fossula spongiosa), and Thaumastocoridae. The fossula spongiosa in Reduviidae consists of tenent hairs (acanthae) with spatulate or tapering apices interspersed with sensory setae, both of which are embedded in a thick and flexible cuticle, underlain by a hemolymph cavity separated almost entirely from the interior of the remaining tibia by a cuticular invagination. Judging from comparison with non-reduviid Cimicomorpha, this separation of the fossula spongiosa cavity from the tibial interior may be unique to Reduviidae. A simple experiment using live specimens of Platymeris biguttata (Reduviinae) revealed a liquid on the tip of the tenent hairs that might be involved in the attachment of the fossula spongiosa by adhesion mechanisms. The nymphs of Reduviidae whose adults have a fossula spongiosa are here documented for the first time to possess pads of ventrally barbed setae instead of tenent hairs and their tibia lacks the internal cuticular invagination. The nymphal attachment structures seem to rely on increase of friction rather than the adhesion mechanism proposed to be present in the adult. Combined with the tenent setae on the third tarsomere known in some Emesinae and here documented for Saicinae, three types of hairy attachment structures occur on the legs of Reduviidae: tenent hairs (acanthae), which form the fossula spongiosa in many Reduviidae, barbed setae that substitute the fossula in the immatures, and tenent setae on the tarsus which are restricted to only a few taxa.     

Microstructure of the tarsal attachment devices in the earwig Timomenus komarovi

The biological attachment device on the tarsal appendage of the earwig, Timomenus komarovi (Insecta: Dermaptera: Forficulidae) was investigated using field emission scanning electron microscopy to reveal the fine structural characteristics of its biological attachment devices to move on smooth and rough surfaces. They attach to rough substrates using their pretarsal claws; however, attachment to smooth surfaces is achieved by means of two groups of hairy tarsal pads. This biological attachment device consists of fine hairy setae with various contact sizes. Three different groups of tenent setae were distinguished depending on the cuticular substructure of the endplates. Two groups of setae commonly had flattened surfaces, and they were covered with either spoon‐shaped or spatula‐shaped endplates, respectively. While the flattened tip setae were distributed at the central region, the pointed tip setae were characteristically found along the marginal region. There were no obvious gender‐specific differences between fibrillar adhesive pads in this insect mainly because the forceps‐like pincers are used during copulation to grasp the partner.     

The pretarsus of salticid spiders

The pretarsus of Phidippus audax (Hentz) consists of two claws flexibly articulated to a central claw lever which is flanked on either side by a curved plate of tenent setae. The claw apparatus allows for retraction of the claws by means of a dorsal cuticular cable of the pretarsal levator, while extension involves the pull of the pretarsal depressor on a ventral cable attached to the claw lever. A series of slit sensilla are strategically situated on either side of this lever. The anterior and posterior claws of the pretarsus differ in the number and spacing of their constituent teeth. The claw tufts are composed of specialized setae which account for the mechanical traction of the foot-pads. Whorled and filamentous setae of the distal tarsus are associated with the pretarsus. Comparable structures are found on other salticids.     

Insects did it first: a micropatterned adhesive tape for robotic applications

Based on the structural and experimental studies of more than 300 insect species from different lineages, we have developed and characterized a bioinspired polymer material with the ability of multiple glue-free bonding and debonding. The material surface is covered with a pattern of microstructures, which resembles the geometry of tenent hairs previously described from the feet of flies, beetles, earwigs and other insects. The tape with such a microstructure pattern demonstrates at least two times higher pull-off force per unit apparent contact area compared to the flat polymer. Additionally, the tape is less sensitive to contamination by dust particles than a commercially available pressure-sensitive adhesive tape. Even if the 'insect tape' is contaminated, it can be washed with a soap solution in water, in order to completely recover its adhesive properties. We have successfully applied the tape to the 120 g wall-climbing robot Mini-Whegs. Furthermore, the tape can be used for multiple adhering of objects to glass surfaces or as a protective tape for sensitive glass surfaces of optical quality. Another area of potential applications is gripping and manipulation of objects with smooth surfaces.     

Contact behaviour of setal tips in the hairy attachment system of the fly Calliphora vicina (Diptera, Calliphoridae): a cryo-SEM approach

The fly Calliphora vicina (Diptera, Calliphoridae) bears attachment pads (pulvilli) covered with setae on their ventral sides. These structures enable attachment to smooth vertical surfaces and ceilings. The contact between the terminal setal tips (spatulae) and various substrates was visualised using various experimental techniques combined with conventional scanning electron microscopy (SEM) and cryo-SEM. The results show that the setal endplates are highly flexible structures that form contact with the surface by bending their tips in the distal direction. With conventional SEM, a comparison of partly attached endplates with unattached endplates demonstrated the presence of a distinct marginal bulge. As observed with cryo-SEM, the bulge continuously disappeared as a larger area of the endplate came into contact. Two explanations of this result are suggested. First, the volume between the bulge, the mid-part of the endplate and the substrate may be filled with a fluid secretion that is released into the contact area in the endplate region. Second, the flexible central part of the endplate may jump into contact with the substrate during contact formation.     

Contact behaviour of tenent setae in attachment pads of the blowfly Calliphora vicina (Diptera, Calliphoridae)

To enable strong attachment forces between pad and substrata, a high proximity between contacting surfaces is required. One of the mechanisms, which can provide an intimate contact of solids, is a high flexibility of both materials. It has been previously presumed that setae of hairy attachment pads of insects are composed of flexible cuticle, and are able to replicate the surface profile. The aim of this work was to visualise the contact behaviour of the setae by freezing-substitution technique to understand setal mechanics while adhering to a smooth surface. This approach revealed considerable differences in the area of the setal tips between contacting and non-contacting pulvilli. Based on the assumption that setae behave like a spring pushed by the tip, a spring constant of 1.31 N m(-1) was calculated from direct measurements of single setae by atomic force microscopy. In order to explain the relationship between the behaviour of the attachment setae at a microscale and leg movements, high-speed video recordings were made of walking flies. This data show that some proximal movement of the leg is present during contact formation with the substrate.     

Review: mapping proteins localized in adhesive setae of the tokay gecko and their possible influence on the mechanism of adhesion

The digital adhesive pads that allow gecko lizards to climb vertical surfaces result from the modification of the oberhautchen layer of the epidermis in normal scales. This produces sticky filaments of 10–100 μm in length, called setae that are composed of various proteins. The prevalent types, termed corneous beta proteins (CBPs), have a low molecular weight (12–20 kDa) and contain a conserved central region of 34 amino acids with a beta-conformation. This determines their polymerization into long beta-filaments that aggregate into corneous beta-bundles that form the framework of setae. Previous studies showed that the prevalent CBPs in the setae of Gekko gecko are cysteine-rich and are distributed from the base to the tip of adhesive setae, called spatulae. The molecular analysis of these proteins, although the three-dimensional structure remains undetermined, indicates that most of them are charged positively and some contain aromatic amino acids. These characteristics may impede adhesion by causing the setae to stick together but may also potentiate the van der Waals interactions responsible for most of the adhesion process on hydrophobic or hydrophilic substrates. The review stresses that not only the nanostructural shape and the high number of setae present in adhesive pads but also the protein composition of setae influence the strength of adhesion to almost any type of substrate. Therefore, formulation of dry materials mimicking gecko adhesiveness should also consider the chemical nature of the polymers utilized to fabricate the future dry adhesives in order to obtain the highest performance.     

Attachment ability of the beetle Chrysolina fastuosa on various plant surfaces

To study the role of different structures of a plant surface preventing insect attachment, a variety of plant surfaces were screened. Attachment ability of the beetle Chrysolina fastuosa Scop. (Coleoptera, Chrysomelidae) was measured on 99 surfaces among them smooth, hairy, felt-like, waxy, and glandular ones of three plant organs (stems, leaves, fruits) of 83 plant species belonging to 45 families. Insects attached successfully to smooth, hairy, and felt-like substrata. These surface types did not effect the further attachment of C. fastuosa, indicating the adhesive system remained intact after contacting these substrata. However, the beetles could not attach properly to surfaces covered with wax crystalloids or glandular hairs. In most experiments on pruinose plant substrata, no influence of the surfaces on the subsequent attachment ability of insects was observed. Only in one case (the stem of Acer negundo), was such an impairment recorded, but recovery of attachment ability was fast. Crystalloids of this plant species probably temporarily disable function of tenent setae of C. fastuosa. Four hypotheses, explaining anti-adhesive properties of plant surfaces, covered with wax crystalloids are proposed. A plant surface with glandular trichomes disabled the attachment system of the beetle for a long time. Secretions of trichomes probably glue tenent setae together making further attachment impossible.     

Integrative functional morphology of the gekkotan adhesive system (reptilia: gekkota)

Climbing assisted by adhesive subdigital pads in gekkotan lizardshas been the subject of intrigue and study for centuries. Manyhypotheses have been advanced to explain the mechanism of adhesion,and recently this phenomenon has been investigated at the levelof individual setae. The ability to isolate, manipulate andrecord adhesive forces from individual setae has provided newinsights, not only into the mechanism of attachment, but alsointo the physical orientation of these structures necessaryto establish attachment, maximize adhesive force, and effectsubsequent release. This, in turn, has enabled a reassessmentof the overall morphology and mode of operation of the adhesivesystem. Digital hyperextension has often been noted as a behavioralcharacteristic associated with the deployment of the gekkotanadhesive system—this is now understandable in the contextof setal attachment and release kinematics, and in the contextof the evolution of this pattern of digital movement from theprimitive pattern of saurian digital kinematics. The perpendicularand parallel preloads associated with setal attachment are nowreconcilable with other morphological aspects of the gekkotanadhesive system—the lateral digital tendon complex andthe vascular sinus network, respectively. Future investigationsof the integrated adhesive system will help to further elucidatethe interdependence of its structural and functional components.     

Arachnids secrete a fluid over their adhesive pads

Background

Many arachnids possess adhesive pads on their feet that help them climb smooth surfaces and capture prey. Spider and gecko adhesives have converged on a branched, hairy structure, which theoretically allows them to adhere solely by dry (solid-solid) intermolecular interactions. Indeed, the consensus in the literature is that spiders and their smooth-padded relatives, the solifugids, adhere without the aid of a secretion.

Methodology and Principal Findings

We investigated the adhesive contact zone of living spiders, solifugids and mites using interference reflection microscopy, which allows the detection of thin liquid films. Like insects, all the arachnids we studied left behind hydrophobic fluid footprints on glass (mean refractive index: 1.48–1.50; contact angle: 3.7–11.2°). Fluid was not always secreted continuously, suggesting that pads can function in both wet and dry modes. We measured the attachment forces of single adhesive setae from tarantulas (Grammostola rosea) by attaching them to a bending beam with a known spring constant and filming the resulting deflection. Individual spider setae showed a lower static friction at rest (26%±2.8 SE of the peak friction) than single gecko setae (Thecadactylus rapicauda; 96%±1.7 SE). This may be explained by the fact that spider setae continued to release fluid after isolation from the animal, lubricating the contact zone.

Significance

This finding implies that tarsal secretions occur within all major groups of terrestrial arthropods with adhesive pads. The presence of liquid in an adhesive contact zone has important consequences for attachment performance, improving adhesion to rough surfaces and introducing rate-dependent effects. Our results leave geckos and anoles as the only known representatives of truly dry adhesive pads in nature. Engineers seeking biological inspiration for synthetic adhesives should consider whether model species with fluid secretions are appropriate to their design goals.     

Viscoelastic features of adhesive setae of the tokay gecko (<Emphasis Type="Italic">Gekko gecko</Emphasis> L.)

Deformations of particular setae of adhesive toe pad of the tokay gecko were investigated by atomic-force microscopy. The effective elastic modulus of the investigated setae varying within 0.34–19 GPa, a pronounced hysteresis was observed during reversible bending of setae. The hysteresis-related energy losses may be as high as 98% of the total bending work. The pronounced viscous features of the setae contradict the hypothesis of dynamic self-cleaning of the gecko adhesive cover, according to which the setae are considered as absolutely elastic cantilever beams.     

A scanning electron microscope study of tarsal adhesive setae in the Coleoptera

Scanning electron micrographs of the tarsal adhesive setae of 84 species of beetle are described. These show a vast range of setal structure and distribution.     

A whole lamella perspective on the origin of the epidermal free margin of Anolis (Reptilia: Dactyloidae) toe pads

Anoline lamellae terminate in an epidermal free margin carrying the majority of its setae. How the free margin is extruded from the body of the scale is not well understood. Two hypotheses have been advanced to account for it, one advocating distal migration of the outer epidermal layers relative to the body of the lamella, and the other proposing regression of the dermal core. Available evidence provides partial support for both. We assembled a series of specimens of Anolis grahami representing all shedding cycle stages, and prepared histological sections of the toe pads to allow measurement of appropriate lamellar components through the shedding cycle. Through its proliferative phases the lamellae increase markedly in length, with the distance between the distal tip of the dermal core and that of the lamella accounting for most of this, indicating that epidermal extrusion is responsible for production of the new free margin. The dermal core showed no evidence of regression. Concomitant with epidermal extrusion, the lacunar cells on the inner lamellar face hypertrophy and keep pace with the increasing thickness of the outer lamellar face resulting from the lengthening of the replacement setae. The integrated changes observed are consistent with continuity of functioning and alignment of the exposed setal carpet of the outer epidermal generation while ensuring that the new setal carpet is fully aligned and functional immediately after shedding. At shedding the original proportions of the lamellae are restored. Development of the new free margin results from a combination of distal displacement of Oberhäutchen cells along with arrested maturation of the epidermis in this region. Changes in length of the lamellae during the proliferative stages may impact the overall size of the adhesive toe pad, which may have consequences for assessments of the relationship between whole animal clinging ability and adhesive pad area. J. Morphol. 278:360–368, 2017. © 2017 Wiley Periodicals, Inc.