The digging legs of Pachnoda marginata, as follows from the experimental data, are able to some extent to resist external contamination by particles (e.g., soil) with the help of specialized structures organized into a joint cleaning system (Figs. 5 and 6). The functioning of each of these structures acting in concert is discussed in more detail below.
Microsetal pad/membraneous plate
Experimental results indicate that the penetration of contaminants through the dorsal gap is hampered by the presence of the microsetal pad and the membraneous plate (Fig. 5a). The mechanism of functioning of this cleaning system is supposed as follows (Fig. 6a). The particles of the substrate are in contact with the microsetal pad, being located, depending on the size, both among the setae and on the surface of the pad. When extending the tibia, the microsetal pad moves directly under the membraneous plate, while the distance between the tibia and femur in this region is minimal. As a result of the translational movement, the membraneous plate displaces (scrapes) particles from the surface of the pad, the size of which exceeds the size of the gap between the membraneous plate and microsetal pad. Since the orientation of the bristles is co-directed with the movement of tibia during its extension, the setae of the microsetal pad do not impede the movement of the membraneous plate over its surface. In addition, the bristles may reduce the contact area of the particles with the underlying cuticle surface, i.e., they minimize the possibility of stiction by adhesion. It can be assumed that as a result of this, the particles that are on the bristles and are stuck among the setae are unstable and can be easily displaced by the membraneous plate.
Internal cleaning system
Removal of particles that have penetrated into the internal cavity of the joint is necessary to prevent abrasive wear of the surfaces. The particle removal mechanism consists of the contacting surfaces of the joint covered with microprotrusions and works in the interaction as follows (Figs. 5b and 6b). In the internal surface of the joint, particles can penetrate into the TSC, i.e., between the concavity and the femoral condyle (Supplementary Figs. S6j–n and S7j, k), and on the inner surface of the femoral condyle (Supplementary Fig. S6l), that is, between the FC and the TC. The surface of the dorsal part of the femoral condyle (or grater) is covered with microprotrusions oriented co-directionally to the circumference of the condyle, and the surface of the TSC near the outflow canal is covered with microprotrusions oriented towards the canal. When the tibia moves, the grater on the femoral condyle moves within the TSC and slides along the corresponding microprotrusions in the concavity. The particles captured by the microprotrusions of the grater (Supplementary Fig. S6k) are displaced toward the outflow canal, when the tibia is flexing (Fig. 6b). During the reverse movement of the tibia, the particles are retained by the microprotrusions of the TSC and cannot move back to their original position. During the flexing of the tibia, the microprotrusions of the grater displace the particles even further towards the outflow canal. Since all the microprotrusions in the TSC are oriented toward the outflow canal (Fig. 5b), the particles ultimately end up in it and are removed from the inner cavity of the joint. The mechanism of removal of particles from the inner surface of the FC works in the same way. The microprotrusions of the FC and TC are co-directed, and the microprotrusions of the femoral condyle are oriented towards the grater. Particles from the inner surface of the FC are relocated by the movement of the TC (Supplementary Fig. S6n) to the grater, where they are removed in the same way as described above. SEM images of the outflow canal show the presence of microparticles trapped in the lubricant. This could presumably be evidence of one more function of the lubricant being involved in the entrapment of contaminants particles that facilitates their removal21,22.
The ventral surface of the femoral condyle, as well as the space between it and the tibia, is potentially vulnerable to the entry of contaminants. When the tibia is fully flexed, the condyle surface is completely covered by the corresponding part of the tibia and the hairy brush. When the tibia is opened, the surface of the condyle becomes accessible and particles that are not retained by the hairy brush can get on it. The penetration of such particles into the joint can lead to wear on the inner surfaces of the joint (Supplementary Fig. S6j, i, m). The direct participation of the scraper (paired ventro-lateral protrusion of the articular part of the tibia) in cleaning the surface of the femoral condyle is experimentally confirmed here. The edge of the scraper is adjacent to the surface of the condyle and the gap between them is less than 2–3 μm (Fig. 5c). When the tibia is flexing, the edge of the scraper slides over the surface of the condyle and displaces particles from its surface (Fig. 6c). As a result, when the tibia is fully flexed, a clot of displaced particles falls on the surface of the femur, where they are removed by the setae of the hairy brush according to the mechanism described below.
The hairy brush appears to be a barrier against penetration of particles through the ventral gap. As follows from our observations, the hairs fill the space between the femur and tibia as fully as possible at an angle of no more than 90° (Fig. 5d). As shown in our experiments, flexion of the tibia to the femur results in the particle displacement (pushing out) and cleaning of the femoral surface. This is achieved due to several features. (1) The setae are curved and their curvature corresponds to the curvature of the femoral surface. (2) The length of the setae gradually decreases in the distal direction of the tibia. (3) The angle of inclination of the setae changes from approximately straight for the longer setae on the tibial base to the sharp one for the shorter setae situated more distally. Thus, the cleaning mechanism by means of the hairy brush can be described as follows (Fig. 6d). When the tibia starts moving, the longer setae situated closer to the base lie down on the femoral surface and begin to move along it. As the tibia approaches the femur, more and more setae come into contact with the femur surface. In this case, due to the corresponding angle of inclination of the setae, the latter orient parallel to the femur surface. At the same time, the density of the setae increases, and the distance between them decreases. The movement of setae along the surface of the femur leads to the displacement of particles proximally relative to the femur, that is, further from the ventral gap. This is also facilitated by the presence of notches on the surface of setae, whose sharp tips are directed from the base to the apex (Supplementary Fig. S1n). Obviously, the role of the notches is to prevent the movement of particles between the setae. This arrangement of notches on one setal side increases and prevents the movement of particles between the setae. Also, the arrangement of notches promotes pushing the particles out, when the setae move relatively to each other. Some notches trap the particles, whereas the others (from adjacent hairs) scrape them towards the tips of the setae.
Obviously, none of the above mechanisms provide absolute protection against particle penetration. However, there are additional indirect mechanisms that may be of importance during the leg movement, when digging. As indicated above, the maximum dimensions of gaps, in which contaminants can penetrate, appear, when deviating from the optimal angle (about 80–90°). In such positions, especially, when the angle starts to increase by more than 90°, the dorsal gap increases, because the area of the ventral surface of the femoral condyle became not covered by the tibial part. With constant movement of the tibia, especially with greater amplitude, the likelihood of penetration of microparticles is supposedly increases. It could be supposed that, when digging, the beetles try to keep their tibiae in a position of 80–90° relative to the femur and move them minimally during digging. This accomplishes the goal of keeping the gaps as small as possible and minimizing particle penetration. It is also important to note that at this position of the tibia, the hairy brush maximally fills the gap between the tibia and femur. The tibial flexion to an angle greater than 90° leads to an increase of the distance between the setae of the hairy brush and the femur and to a decrease of their effectiveness as a barrier.
Thus, the auto-cleaning system of Pachnoda marginata leg joint is represented by the structural and functional complex of structures, which can be subdivided into four subsystems: (1) microsetal pad/membraneous plate, (2) internal cleaning subsystem, (3) hairy brush, and (4) scraper (Figs. 5 and 6). Two of these subsystems, namely the first and the third ones, not only clean the outer parts of the joint, but also prevent the penetration of particles into the joint cavity, performing a barrier function. The mutual arrangement of surfaces, the structure and orientation of their structural surface elements result in their interaction at every movement of the joint, i.e., flexion-extension of a tibia, which in turn leads to the removal of contaminant particles to the outside of the joint. This cleaning mechanism working exclusively due to the structural and functional organization of the joint and not requiring any special actions is considered here as automatic cleaning system.
The principle of the auto-cleaning system is based on the interaction of the surfaces due to their relative motion and the corresponding structural elements, such as various microprotrusions, setae and scrapers. In this sense, there is an obvious similarity to the active cleaning through grooming. The functioning of the internal cleaning system in the joint (Fig. 6b) described here resembles previously described grooming mechanism of insects, scraping particles with angled bristles11. Another example is cleaning of the antennas (as well as the legs) that can be performed either by the mouthparts or by specialized antennal cleaners11 covered with setae and bristles (by the terminology of Hlavac11). The latter are situated on the forelegs and bear various setae in special depressions on the tibia as for example, inground beetles Carabidae23. In some insect groups, such as Hymenoptera, antenna is cleaned by passing through a modified tibial spur and a notch with a comb of bristles on the first protarsomere16.
It may be supposed that leg joint structures involved in the cleaning mechanism in Pachnoda marginata are the result of specialized adaptation for digging. Thus, the legs of beetles not adapted to burrowing may not have such adaptive structures, as seen by the example of the simple walking legs of darkling beetle Zophobas morio21,22 whose joints can easily be contaminated (Supplementary Fig. S9).
There are only very limited options for cleaning joints through grooming, with significant limitations and low effectiveness, such as accessibility of only the external surface and only for those species, whose body structure and joint mobility allow for it. For instance, in Hymenoptera legs can be groomed by rubbing against each other and principally it cannot be excluded the possibility for these insects to clean external surface of joints in this way followed by observations reported in ref. 16, though it was not specifically emphasized. Obviously, in the case of burrowing insects, such as the model object of this study, any grooming of this kind can be excluded. Nonetheless, it is theoretically possible to groom the dorsal surface of the joint by rubbing it with the tarsus of the legs from the anterior pair, for example, to rub the joint of the hind legs by the tarsus of the middle one, although this type of grooming has never been recorded14. It is also potentially possible to clean the external surface of the femoro-tibial joint by rubbing against the elytra or abdomen, but this is only potentially possible from the side facing the corresponding surface. One way or another, grooming is excluded as a means to clean the inner cavity of the joint.
The auto-cleaning system found in Pachnoda marginata leg joints has a number of features that distinguish it from both passive self-cleaning and active grooming.
Behavioral pattern of movements are not involved as in grooming and there are no restrictions associated with body and limbs shape and 3D joint kinematics.
Cleaning is carried out by the interaction of two surfaces within one organ of the body, but not due to the interaction of different organs of the body.
Cleaning is performed by the mechanical removal of contaminating particles at the movement of the contacting surfaces, but not due to the anti-adhesive properties of the surface alone like in Lotus effect.
The cleaning system is autonomous, i.e., does not require any special actions from the insect and works constantly with every movement of the joint.
The cleaning system works both preventively, impeding the penetration of contaminants and actively, removing particles of contaminant that already have got inside.
The principles of functioning of the auto-cleaning system of joints in digging beetles may be of potential interest for further research in bionics/biomimetics, in particular, in areas related to legged robotics and microelectromechanical devices. The joints and hinges of such devices may be designed in a similar way to those of insects and may also be exposed to the environment. In this regard, similar mechanism of protection from contaminants may be of advantage for them.