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.     

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.     

Sequence of Age-Associated Changes to the Mouse Neuromuscular Junction and the Protective Effects of Voluntary Exercise

Loss of connections between motor neurons and skeletal muscle fibers contribute to motor impairment in old age, but the sequence of age-associated changes that precede loss of the neuromuscular synapse remains uncertain. Here we determine changes in the size of neuromuscular synapses within the tibialis anterior muscle across the life span of C57BL/6J mice. Immunofluorescence, confocal microscopy and morphometry were used to measure the area occupied by nerve terminal synaptophysin staining and postsynaptic acetylcholine receptors at motor endplates of 2, 14, 19, 22, 25 and 28month old mice. The key findings were: 1) At middle age (14-months) endplate acetylcholine receptors occupied 238±11 µm² and nerve terminal synaptophysin 168±14 µm² (mean ± SEM). 2) Between 14-months and 19-months (onset of old age) the area occupied by postsynaptic acetylcholine receptors declined 30%. At many endplates the large acetylcholine receptor plaque became fragmented into multiple smaller acetylcholine receptor clusters. 3) Between 19- and 25-months, the fraction of endplate acetylcholine receptors covered by synaptophysin fell 21%. By 28-months, half of the endplates imaged retained ≤50 µm² area of synaptophysin staining. 4) Within aged muscles, the degree to which an endplate remained covered by synaptophysin did not depend upon the total area of acetylcholine receptors, nor upon the number of discrete receptor clusters. 5) Voluntary wheel-running exercise, beginning late in middle-age, prevented much of the age-associated loss of nerve terminal synaptophysin. In summary, a decline in the area of endplate acetylcholine receptor clusters at the onset of old age was followed by loss of nerve terminal synaptophysin from the endplate. Voluntary running exercise, begun late in middle age, substantially inhibited the loss of nerve terminal from aging motor endplates.     

Attachment force of the beetle Cryptolaemus montrouzieri (Coleoptera, Coccinellidae) on leaflet surfaces of mutants of the pea Pisum sativum (Fabaceae) with regular and reduced wax coverage

This study represents an investigation of surface-related plant–insect interactions. Surface micro-morphology of leaflets in pea (Pisum sativum) with wild-type crystalline surface waxes (waxy) and with reduced crystalline surface waxes (glossy) caused by a mutation (wel) were studied using various microscopy techniques. The free surface energy of these plant surfaces was estimated using contact angles of droplets of three different liquids. The morphological study of the attachment system in the ladybird beetle Cryptolaemus montrouzieri was combined with measurements of attachment (traction) forces, generated by beetles on these plant substrates. Differences were found in wax crystal shape, dimensions, and density between the adaxial and abaxial surfaces of waxy and glossy plants. The crystalline wax was not completely eliminated in the glossy plant: it was only slightly reduced on the adaxial side and underwent greater changes on the abaxial side. The free surface energy for both surfaces of both pea types was rather low with strongly predominating dispersion component. Insects generated low traction forces on all intact plant surfaces studied, except the abaxial surface of the glossy plant, on which the force was greater. After being treated with chloroform, all the surfaces allowed much higher traction forces. It is demonstrated that the difference in the crystal length and density of the epicuticular wax coverage within the observed range did not influence wettability of surfaces, but affected insect attachment. The reduction in insect attachment force on plant surfaces, covered with the crystalline wax, is explained by the decrease of the real contact area between setal tips of beetles and the substrate. Handling editor: Lars Chittka.     

Subdigital and subcaudal microornamentation in chamaeleonidae—A comparative study

Locomotion on horizontal and vertical substrates requires effective attachment systems. In three clades of arboreal and rupicolous Iguanidae, Gekkota and Scincidae adhesive systems consisting of microscopic hair‐like structures (setae) have been evolved independently. Also the substrate contacting sites on toes and tails of chameleons (Chamaeleonidae) are covered with setae. In the present comparative scanning electron microscopy study, we show that representatives from the chamaeleonid genera Calumma, Chamaeleo, Furcifer, and Trioceros feature highly developed setae that are species‐specific and similar on their feet and tail. These 10 μm long, unbranched setae rather resemble those in anoline and scincid lizards than the larger and branched setae of certain gecko species. In contrast to the thin triangular tips of other lizards, all examined species of the genera Furcifer and Calumma and one of the five examined species of the genus Trioceros have spatulate tips. All other examined species of genera Trioceros and Chamaeleo bear setae with narrowed, fibrous tips. Unlike the setae of other lizards, chamaeleonid setal tips do not show any orientation along the axis of the toes, but they are flexible to bend in any direction. With these differences, the chameleon's unique microstructures on the zygodactylous feet and prehensile tail rather increase friction for arboreal locomotion than being a shear‐induced adhesive system as setal pads of other lizards. J. Morphol., 2013. © 2013 Wiley Periodicals, Inc.     

Between a rock and a soft place: microtopography of the locomotor substrate and the morphology of the setal fields of Namibian day geckos (Gekkota: Gekkonidae: Rhoptropus)

Many species of gekkotans possess adhesive subdigital pads that allow them to adhere to, and move on, a wide variety of surfaces. The natural surfaces exploited by these lizards may be rough, undulant and unpredictable and therefore likely provide only limited, patchy areas for adhesive contact. Here, we examine the microtopography of rock surfaces used by seven species of Rhoptropus and compare this to several rough and smooth artificial surfaces employed in previous studies of gekkotan adhesion. These data are considered in relation to the form, configuration, compliance and functional morphology of the setal fields of these species. Our results demonstrate that natural rock surfaces are rough and unpredictable at the scale of the setal arrays, with equal amounts of variation existing within and between the various types of rock surfaces examined. Such surfaces differ from smooth and rough artificial surfaces in the proportion of surface area available for attachment and the relative predictability of surface undulance. Generally, setal field characteristics of individual species are not relatable to specific substrates, but instead are configured to allow for sufficient attachment to a wide variety of unpredictable surfaces. Our findings provide insight into the evolution and microanatomy of the adhesive system of gekkotan lizards and its adaptive relationship to topographically unpredictable surfaces.     

Friction and adhesion in the tarsal and metatarsal scopulae of spiders

Friction and adhesion forces of the ventral surface of tarsi and metatarsi were measured in the bird spider Aphonopelma seemanni (Theraphosidae) and the hunting spider Cupiennius salei (Ctenidae). Adhesion measurements revealed no detectable attractive forces when the ventral surfaces of the leg segments were loaded and unloaded against the flat smooth glass surface. Strong friction anisotropy was observed: friction was considerably higher during sliding in the distal direction. Such anisotropy is explained by an anisotropic arrangement of microtrichia on setae: only the setal surface facing in the distal direction of the leg is covered by the microtrichia with spatula-like tips. When the leg is pushed, the spatula-shaped tips of microtrichia contact the substrate, whereas, when the leg is pulled over a surface, setae bend in the opposite direction and contact the substrate with their spatulae-lacking sides. In an additional series of experiments, it was shown that desiccation has an effect on the friction force. Presumably, drying of the legs results in reduction of the flexibility of the setae, microtrichia, spatulae, and underlying cuticle; this diminishes the ability to establish proper contact with the substrate and thus reduces the contact forces.     

Colonization of substrates: Vendian sedentary benthos

Studies of fossils sampled from a single fossiliferous layer (monotopic series) and a summary of previous data on taphonomy and morphology of Vendian macroorganisms suggest that most strategies of attachment of sedentary benthos were formed in the Vendian. These included: (1) free living with possible organic gluing; (2) fixation on the substrate using sucker-like structures and various incrustations; (3) fixation of the basal parts by shallow submergence in the sediment; (4) anchoring into the sediment with discoidal and rhizoid-like holdfasts; and (5) partial submergence and infaunal lifestyle. The most widespread fixation in the Vendian was the attachment by discoid organs, which can be interpreted as symbiotrophic structures. Symbiotrophy in Vendian organisms submerged in the anoxic sediment can apparently be supported by indirect evidence: structural diversity of basal organs, their complex morphology, large area of contact with substrate, unlimited isometric growth, predominant fossilization of attachment discs; inconsistent systematization of organisms.     

Scanning electron microscopy preparation methods: their influence on the morphology and fibril formation in Pseudomonas fragi (ATCC 4973)

The use of cryo-scanning electron microscopy (cryo-SEM) in the study of the morphology of Pseudomonas fragi (ATCC 4973) revealed gross differences when compared to material prepared using conventional chemical methods. Changes associated with each step of the chemical preparatory procedure were monitored by cryo-SEM. It appeared that fibril formation was associated with the ethanol dehydration stage of the chemical preparation method and was not an attachment feature of these cells.     

Combination of microscopic techniques reveals a comprehensive visual impression of biofilm structure and composition

Bacterial biofilms are imaged by various kinds of microscopy including confocal laser scanning microscopy (CLSM) and scanning electron microscopy (SEM). One limitation of CLSM is its restricted magnification, which is resolved by the use of SEM that provides high-magnification spatial images of how the single bacteria are located and interact within the biofilm. However, conventional SEM is limited by the requirement of dehydration of the samples during preparation. As biofilms consist mainly of water, the specimen dehydration might alter its morphology. High magnification yet authentic images are important to understand the physiology of biofilms. We compared conventional SEM, Focused Ion Beam (FIB)-SEM and CLSM with SEM techniques [cryo-SEM and environmental-SEM (ESEM)] that do not require dehydration. In the case of cryo-SEM, the biofilm is not dehydrated but kept frozen to obtain high-magnification images closer to the native state of the sample. Using the ESEM technique, no preparation is needed. Applying these methods to biofilms of Pseudomonas aeruginosa showed us that the dehydration of biofilms substantially influences its appearance and that a more authentic biofilm image emerges when combining all methods.     

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.     

Attachment of acetylcholinesterase to structures of the motor endplate

Summary The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.     

Attachment of acetylcholinesterase to structures of the motor endplate.

The kinetics of AChE solubilization from intact motor endplates of mouse diaphragm, by collagenase, papain and hyaluronidase, was studied in parallel with the ultrastructural localization of AChE in treated neuromuscular junctions. Hyaluronidase did not solubilize more AChE from isolated motor endplate regions than Ringer's solution itself. Residual AChE activity could be demonstrated histochemically in motor endplates even after the plateau of solubilization by collagenase or papain was reached. Less than 35% of junctional AChE is left after collagenase, and less than 20% after papain treatment, as estimated by the percentage of AChE activity left in the isolated endplate region of the diaphragm after protease treatment. Cytochemically, both proteases had a similar effect on postsynaptic AChE. Residual AChE activity was distributed randomly, adhering to the sarcolemma of junctional clefts. Presynaptic AChE localized in the gap between axon terminal and Schwann cell appears to be resistant to collagenase but not to papain treatment. The mode of AChE attachment or the composition of the intercellular material in this gap may differ from that of the primary and secondary clefts.     

Development of rat soleus endplate membrane following denervation at birth

Rat soleus endplates develop some of their characteristic features before birth and others after birth. Specializations appearing before birth include a localized cluster of acetylcholine receptors (AChRs), an accumulation of acetylcholinesterase (AChE) in the synaptic basal lamina, and a cluster of nuclei near the endplate membrane. In contrast, postsynaptic membrane folds are elaborated during the first three weeks after birth. We denervated soleus muscles on postnatal day 1, before folds had appeared, and followed the subsequent development of endplate regions with light and electron microscopy. We found that the denervated endplates initiated fold formation on schedule and maintained their accumulations of AChRs, AChE, and endplate nuclei. However, the endplates stopped fold formation prematurely and eventually lost their rudimentary folds. At about the same time, the junctional AChR clusters were joined by ectopic patches of AChRs. The former endplate regions also became unusually elongated, possibly as a consequence of the lack of membrane folds. Apparently, endplate membranes have only a limited capacity for further development in the absence of both the nerve and muscle activity.     

Surface engineering of poly(DL-lactide) via electrostatic self-assembly of extracellular matrix-like molecules

We report the development of new biomacromolecule coatings on biodegradable biomaterials based on electrostatic assembly of extracellular matrix-like molecules. Poly(ethylene imine) (PEI) was employed to engineer poly(dl-lactide) (PDL-LA) substrate to obtain a stable positively charged surface. An extracellular matrix- (ECM-) like biomacromolecule, gelatin, was selected as the polyelectrolyte to deposit on the activated PDL-LA substrate via the electrostatic assemble technique. The extracellular matrix-like multilayer on the PDL-LA substrate was investigated by attenuated total reflection (ATR-FTIR), X-ray photoelectron spectrscopy (XPS), contact angle, and atomic force microscopy (AFM). The gradual buildup of the protein layer was investigated by UV-vis spectra, and it was further given a quantitative analysis of the protein layer on the PDL-LA substrate via the radioiodination technique. The stability of the protein layer under aqueous condition was also tested by the radiolabeling method. Chondrocyte was selected as the model system for testing the cell behavior and morphology on modified PDL-LA substrates. The chondrocyte test about cell attachment, proliferation, cell activity and cell morphology by SEM, and confocal laser scanning microscopy (CLSM) investigation on extracellular matrix-like multilayer modified PDL-LA substrate was shown to promote chondrocyte attachment and growth. Comparing conventional coating methods, polyelectrolyte multiplayers are easy and stable to prepare. It may be a good choice for the modification of 3-D scaffolds used in tissue engineering. These very flexible systems allow broad medical applications for drug delivery and tissue engineering.     

Heterogeneity of neuromuscular junctions in striated muscle of human esophagus demonstrated by triple staining for the vesicular acetylcholine transporter, α-bungarotoxin, and acetylcholinesterase

During studies on enteric co-innervation in the human esophagus, we found that not all acetylcholinesterase (AChE)-positive motor endplates stained for α-bungarotoxin (α-BT) and the vesicular acetylcholine transporter (VAChT), respectively. Therefore, we probed for differences in neuromuscular junctions in human esophagus by using triple staining for VAChT, α-BT, and AChE followed by qualitative and quantitative analysis. To exclude that the results were caused by processing artifacts, we additionally examined the influence of a number of factors including post-mortem changes and the type and duration of fixation on the staining results. Four types of neuromuscular junction could be distinguished in human esophagus: type I with VAChT-positive and type II with VAChT-negative nerve terminals on a α-BT-positive and AChE-positive endplate area, type III with VAChT-positive nerve terminals on a α-BT-negative but AChE-positive endplate area, and type IV with VAChT-negative nerve terminals on a α-BT-negative but AChE-positive endplate area. On average, 32% of evaluated AChE-positive motor endplates were type I, 6% type II, 24% type III, and 38% type IV. Based on these results, we suggest that, in human esophagus, (1) the most reliable method for staining motor endplates is presently AChE histochemistry, (2) α-BT-sensitive and α-BT-resistant nicotinic acetylcholine receptors exist in neuromuscular junctions, and (3) different types of VAChT or transport mechanisms for acetylcholine probably exist in neuromuscular junctions.This study was supported by the “Johannes und Frieda Marohn-Stiftung”, Erlangen.     

Origin and pathway of the epidermal secretion in the damselfly head-arresting system (Insecta: Odonata)

In damselflies, the arrester system is responsible for an additional attachment of the head to the neck. It consists of a pair of mobile postcervical sclerites (SPC) covered by microtrichia. In their lateral position, SPCs can fixate the head on fields of microtrichia on the back surface of the head. The intact surface of microtrichia of the SPC is usually covered by a lipid-containing secretion. The present study provides ultrastructural data on the secretory epidermis and pore channels adapted to transport the secretion to the cuticle surface. 1) Shock-frozen preparations of the contact area show that microtrichia of co-opted surfaces do not interlock with each other. When co-opted fields are pressed to each other, deformations of flexible microtrichia of the SPC result in an increase of contact area between corresponding structures and consequently in an increase of frictional forces. 2) In the area of the SPC, only electron-lucent vesicles have been found. Histochemical procedures revealed that the material stored in vesicles and liberated on the external surface of the SPC is presumably non-volatile lipid. 3) Only a small number of large pore channels reach the surface of a microtrichium. Most of them terminate with tiny terminal channels, whose diameter is approximately six to twenty times smaller than the diameter of structures of secretory blooms occurring in SEM in shock-frozen and air-dried preparations. The terminal channels do not penetrate the epicuticular layer. It seems that the secretion reaches the epicuticle through terminal channels and diffuses through the epicuticle without any channel structures.     

New observations on the substructure of the active zone of brain synapses and motor endplates

This study offers a new concept on the origin and function of the hitherto enigmatic presynaptic dense projections (dps) of neurons and motor endplates. After a deuterium oxide-albumin pretreatment (da), brain tissue and motor endplate of rat and frog reveal an intricate association of smooth endoplasmic reticulum (ser), microtubules (mts) and synaptic vesicles (sv) at the presynaptic grid-active zone of synapses. The ser entwines the mts, which are clothed in svs, and impinges directly onto the presynaptic membrane as sacs or 'tubular-fibrillar' extensions. Since no dps are seen in these sections, whereas they do occur in conventionally processed material (i.e. without da pretreatment), it is suggested that the dps of conventional material may, in part, originate from improperly fixed ser at these points. Thus for the first time we demonstrate an in vivo system of ser which, because its 'finger' processes come into intimate contact with the presynaptic membrane, may be implicated in Ca2+ ion translocation, presumably out of the presynaptic bulb. Since no such tubular ser has been demonstrated in what are claimed to be sophisticated techniques (i.e. high-speed slam-freezing-freeze substitution) the actual sophistication of such methods is questioned.     

NMR structures of loop B RNAs from the stem-loop IV domain of the enterovirus internal ribosome entry site: a single C to U substitution drastically changes the shape and flexibility of RNA

The 5'-untranslated region of positive-strand RNA viruses harbors many cis-acting RNA structural elements that are important for various viral processes such as replication, translation, and packaging of new virions. Among these is loop B RNA of the stem-loop IV domain within the internal ribosomal entry site (IRES) of enteroviruses, including Poliovirus type 1 (PV1). Studies on PV1 have shown that specific recognition of loop B by the first KH (hnRNP K homology) domain of cellular poly(rC)-binding protein 2 (PCBP2) is essential for efficient translation of the viral mRNA. Here we report the NMR solution structures of two representative sequence variants of enteroviral loop B RNA. The two RNA variants differ at only one position (C vs U) within a six-nucleotide asymmetric internal loop sequence that is the binding site for the PCBP2 KH1 domain. Surprisingly, the two RNAs are drastically different in the overall shape and local dynamics of the bulge region. The RNA with the 5'-AUCCCU bulge sequence adopts an overall L shape. Its bulge nucleotides, especially the last four, are highly flexible and not very well defined by NMR. The RNA with the 5'-AUUCCU bulge sequence adopts an overall U shape, and its bulge sequence exhibits only limited flexibility. A detailed analysis of the two RNA structures and their dynamic properties, as well as available sequence data and known KH domain-RNA complex structures, not only provides insights into how loop B RNA might be recognized by the PCBP2 KH1 domain but also suggests a possible correlation between structural flexibility and pre-existing structural features for protein recognition.     

The Fine Structure of Trichobothria in Moss Mites with Special Emphasis on Acrogalumna longipluma(Berlese, 1904) (Oribatida,Acari, Arachnida)

Using SEM and TEM techniques, the trichobothria of Acrogalumna longipluma(Galumnidae, Pterogasterina) were studied in relation to a variety of other moss mites. Each sensillum is composed of a mostly solid bothridial seta and a very complex socket, the bothridium. The setal base bends sharply as it passes through the six chambers of the bothridium, which are arranged in an S-shaped configuration. The proximal end of the seta is an elongated oval and inserted into a thin socket membrane provided with radiating suspension fibres. This peculiar shape of the setal base proper and probably also the existence of connecting pieces are assumed to provide directionality to the sensillum. Two differently shaped tubular bodies are found under the setal base, of which only one is in contact with the seta. The tubular bodies are surrounded by peculiar ‘dense tubes’, i.e. derivatives of the dendritic sheath. The rather thick, outer dendritic segments curve through an extensive receptor lymph cavity and terminate with ciliary regions. The inner dendritic segments are only short. Perikarya and axons as well as the enveloping cells do not show any peculiarities. The trichobothria of moss mites most probably represent vibration receptors reacting to substrate and/or air-borne stimuli. The variety of shapes and the complexity, which is not found to this extent in any other arthropod group, are discussed in relation to ecophysiological demands.