Jan Michels

Evidence for a material gradient in the adhesive tarsal setae of the ladybird beetle Coccinella septempunctata

For an insect to be able to efficiently attach to surfaces, the adhesive pads on the distal parts of its legs must establish large contact areas. In case of hairy adhesive pads this requires flexibility of the contact-forming bristles, called adhesive tarsal setae. However, too flexible setae would have a low mechanical stability resulting in a decreased attachment ability of the pads. Here we show that the adhesive tarsal setae of the ladybird beetle Coccinella septempunctata feature pronounced gradients in the material composition and properties along their length. The Youngs modulus ranges from 1.2 MPa at the tips, where we found the incorporation of high proportions of the elastic protein resilin, to 6.8 GPa at the bases of the setae. These gradients likely represent an evolutionary optimization, which increases the performance of the adhesive system by enabling effective adaptation to rough surfaces while simultaneously preventing lateral collapse of the setae.

Detailed three-dimensional visualization of resilin in the exoskeleton of arthropods using confocal laser scanning microscopy

Resilin is a rubber‐like protein found in the exoskeleton of arthropods. It often contributes large proportions to the material of certain structures in movement systems. Accordingly, the knowledge of the presence and distribution of resilin is essential for the understanding of the functional morphology of these systems. Because of its specific autofluorescence, resilin can be effectively visualized using fluorescence microscopy. However, the respective excitation maximum is in the UV range, which is not covered by the lasers available in most of the modern commercial confocal laser scanning microscopes. The goal of this study was to test the potential of confocal laser scanning microscopy (CLSM) in combination with a 405 nm laser to visualize and analyse the presence and distribution of resilin in arthropod exoskeletons. The results clearly show that all resilin‐dominated structures, which were visualized successfully using wide‐field fluorescence microscopy (WFM) and a ‘classical’ UV excitation, could also be visualized efficiently with the proposed CLSM method. Furthermore, with the application of additional laser lines CLSM turned out to be very appropriate for studying differences in the material composition within arthropod exoskeletons in great detail. As CLSM has several advantages over WFM with respect to detailed morphological imaging, the application of the proposed CLSM method may reveal new information about the micromorphology and material composition of resilin‐dominated exoskeleton structures leading to new insights into the functional morphology and biomechanics of arthropods.

Assessment of Congo red as a fluorescence marker for the exoskeleton of small crustaceans and the cuticle of polychaetes

In this study, the potential of the common dye Congo red as a fluorescence marker for chitin in the exoskeleton of small crustaceans and collagen in the polychaete cuticle was tested. The Congo red staining turned out to be rather efficient and yielded intensively fluorescing structures, which made a very detailed visualization by confocal laser scanning microscopy possible. The excellent results are comparable to those described for the utilization of other efficient fluorescence dyes and intense autofluorescence. The application of Congo red is easy, the fluorescence of this dye is very stable, and the excitation maximum of the structures stained with Congo red is in a range, which is covered by the lasers of most of the confocal laser scanning microscopes. These advantageous properties make the fluorescence staining by Congo red a method of choice for the detailed visualization of the external morphology of small crustaceans and polychaetes.

Confocal laser scanning microscopy: using cuticular autofluorescence for high resolution morphological imaging in small crustaceans

The utility of cuticular autofluorescence for the visualization of copepod morphology by means of confocal laser scanning microscopy (CLSM) was examined. Resulting maximum intensity projections give very accurate information on morphology and show even diminutive structures such as small setae in detail. Furthermore, CLSM enables recognition of internal structures and differences in material composition. Optical sections in all layers and along all axes of the specimens can be obtained by CLSM. The facile and rapid preparation method bears no risk of artefacts or damage occurring to the preparations and the visualized specimens can be used for later analyses allowing for the investigation of irreplaceable type specimens or parts of them. These features make CLSM a very effective tool for both taxonomical and ecological studies in small crustaceans; however, the maximum thickness of the specimens is limited to a few hundred micrometers. Three‐dimensional models based on CLSM image stacks allow observation of the preparations from all angles and can permit, improve and speed up studies on functional morphology. The visualization method described has a strong potential to become a future standard technique in aquatic biology due to its advantages over conventional light microscopy and scanning electron microscopy.

Tools for crushing diatoms – opal teeth in copepods feature a rubber-like bearing composed of resilin

Diatoms are generally known for superior mechanical properties of their mineralised shells. Nevertheless, many copepod crustaceans are able to crush such shells using their mandibles. This ability very likely requires feeding tools with specific material compositions and properties. For mandibles of several copepod species silica-containing parts called opal teeth have been described. The present study reveals the existence of complex composite structures, which contain, in addition to silica, the soft and elastic protein resilin and form opal teeth with a rubber-like bearing in the mandibles of the copepod Centropages hamatus. These composite structures likely increase the efficiency of the opal teeth while simultaneously reducing the risk of mechanical damage. They are supposed to have coevolved with the diatom shells in the evolutionary arms race, and their development might have been the basis for the dominance of the copepods within todays marine zooplankton.

Anisotropic Friction of the Ventral Scales in the Snake Lampropeltis getula californiae

Since the ventral body side of snakes is in almost continuous contact with the substrate during undulating locomotion, their skin is presumably adapted to generate high friction for propulsion and low friction to slide along the substrate. In this study, the microstructure of ventral scales was analyzed using scanning electron microscopy, atomic force microscope and confocal laser scanning microscopy. Dynamic friction was investigated by a microtribometer. The ventral scales demonstrated anisotropic frictional properties. To analyze the role of the stiffness of underlying layers on the frictional anisotropy, two different types of scale cushioning (hard and soft) were tested. To estimate frictional forces of the skin surface on rough substrates, additional measurements with a rough surface were performed. Frictional anisotropy for both types of scale cushioning and rough surfaces was revealed. However, for both types of surface roughness, the anisotropy was stronger expressed in the soft-cushioned sample. This effect could be caused by (1) the stronger interaction of the microstructure with the substrate in soft-cushioned samples due to larger real contact area with the substrate and (2) the composite character of the skin of this snake species with embedded, highly ordered fiber-like structures, which may cause anisotropy in material properties.

Functional diversity of resilin in Arthropoda

Summary Resilin is an elastomeric protein typically occurring in exoskeletons of arthropods. It is composed of randomly orientated coiled polypeptide chains that are covalently cross-linked together at regular intervals by the two unusual amino acids dityrosine and trityrosine forming a stable network with a high degree of flexibility and mobility. As a result of its molecular prerequisites, resilin features exceptional rubber-like properties including a relatively low stiffness, a rather pronounced long-range deformability and a nearly perfect elastic recovery. Within the exoskeleton structures, resilin commonly forms composites together with other proteins and/or chitin fibres. In the last decades, numerous exoskeleton structures with large proportions of resilin and various resilin functions have been described. Today, resilin is known to be responsible for the generation of deformability and flexibility in membrane and joint systems, the storage of elastic energy in jumping and catapulting systems, the enhancement of adaptability to uneven surfaces in attachment and prey catching systems, the reduction of fatigue and damage in reproductive, folding and feeding systems and the sealing of wounds in a traumatic reproductive system. In addition, resilin is present in many compound eye lenses and is suggested to be a very suitable material for optical elements because of its transparency and amorphousness. The evolution of this remarkable functional diversity can be assumed to have only been possible because resilin exhibits a unique combination of different outstanding properties.

Reduction of female copulatory damage by resilin represents evidence for tolerance in sexual conflict

Intergenomic evolutionary conflicts increase biological diversity. In sexual conflict, female defence against males is generally assumed to be resistance, which, however, often leads to trait exaggeration but not diversification. Here, we address whether tolerance, a female defence mechanism known from interspecific conflicts, exists in sexual conflict. We examined the traumatic insemination of female bed bugs via cuticle penetration by males, a textbook example of sexual conflict. Confocal laser scanning microscopy revealed large proportions of the soft and elastic protein resilin in the cuticle of the spermalege, the female defence organ. Reduced tissue damage and haemolymph loss were identified as adaptive female benefits from resilin. These did not arise from resistance because microindentation showed that the penetration force necessary to breach the cuticle was significantly lower at the resilin-rich spermalege than at other cuticle sites. Furthermore, a male survival analysis indicated that the spermalege did not impose antagonistic selection on males. Our findings suggest that the specific spermalege material composition evolved to tolerate the traumatic cuticle penetration. They demonstrate the importance of tolerance in sexual conflict and genitalia evolution, extend fundamental coevolution and speciation models and contribute to explaining the evolution of complexity. We propose that tolerance can drive trait diversity.

Functional morphology of the male caudal appendages of the damselfly Ischnura elegans (Zygoptera: Coenagrionidae)

Odonata are usually regarded as one of the most ancient extant lineages of winged insects. Their copulatory apparatus and mating behavior are unique among insects. Male damselflies use their caudal appendages to clasp the females prothorax during both copulation and egg-laying and have a secondary copulatory apparatus for sperm transfer. Knowledge of the functional morphology of the male caudal appendages is the basis for understanding the evolution of these structures in Odonata and respective organs in other insects. However, it is still not exactly known how the zygopteran claspers work. In this study, we applied micro-computed tomography and a variety of microscopy techniques to examine the morphology, surface microstructure, cuticle material composition and muscle topography of the male caudal appendages of Ischnura elegans. The results indicate that the closing of the paraproctal claspers is mainly passive. This indirect closing mechanism is very likely supported by high proportions of the elastic protein resilin present in the cuticle of the paraproctal bases. In addition, the prothoracic morphology of the female plays an important role in the indirect closing of the male claspers. Our data indicate that both structures - the male claspers and the female prothoracic hump - function together like a snap-fastener.

Resilin-like protein in the clamp sclerites of the gill monogenean Diplozoon paradoxum Nordmann, 1832

Resilin is a soft and elastic protein, which is found in many exoskeletal structures of arthropods. Proteins with similar chemical properties have been described for other invertebrates including monogenean fish parasites. However, for the latter taxon no clear microscopic evidence for a resilin-like protein has been shown so far. Here, we present the results of detailed microscopic analyses of the clamp sclerites (attachment devices) of the monogenean Diplozoon paradoxum. Toluidine blue, which is known to stain resilin, selectively stained the material in the clamp sclerites. In addition, when exposed to UV light, this material exhibited an intense blue autofluorescence. The emission spectrum of this autofluorescence has its maximum at 424 nm and is nearly identical to emission spectra of blue autofluorescences observed in 2 well-studied structures containing high proportions of resilin in the exoskeleton of the locust Schistocerca gregaria. The results strongly indicate that the sclerite material of D. paradoxum contains a resilin-like protein. The presence of such a protein likely enhances the attachment efficiency of the clamp sclerites and increases their lifetime.