Proteomic investigation of the blue mussel larval shell organic matrix.

Shell matrix proteins (SMPs) are occluded within molluscan shells and are fundamental to the biological control over mineralization. While many studies have been performed on adult SMPs, those of larval stages remain largely undescribed. Therefore, this study aimed to characterize the larval shell proteome of the blue mussel for the first time and to compare it to adult mussel shell proteomes. Following development of a method for cleaning larval shells of tissue contaminants, 49 SMPs were identified using shotgun proteomics. Twenty-one proteins were independently identified in all samples indicating that they form a subset of the core larval shell proteome. These included: the blue mussel shell protein, a peroxidase domain-containing sequence, a laminin G domain-containing sequence, a ZIP domain-containing sequence and a ferric-chelate reductase 1-like sequence. Additional SMP domains identified were: fibronectin type III, BPTI/Kunitz, chitin-binding type 3, thyroglobulin and EF-hand. While key predictable molluscan shell matrix functions are identified, 67% of sequences remain unknown or uncharacterized, indicating that this shell proteome is unique to mussel larvae. Specifically, comparison with adult mytilids reveals that nine domains are exclusive to the larval shell proteome and only four domains are conserved among species and developmental stages. Thus, strong species-specific and ontogenetic variation exists in shell proteome composition.

Endocuticle sclerotisation increases the mechanical stability of cuticle.

The cuticle plays an important role in the evolutionary success of insects. Many studies on insect cuticles have reported a soft, resilin-rich endocuticle. However, a recent study indicated the presence of a sclerotised endocuticle in the weevil Pachyrhynchus sarcitis kotoensis, which contradicts former knowledge. To understand the degree of sclerotisation in the endocuticle of the weevil and its potential function, we first examined the endocuticle by microscopic and staining techniques. We next performed mechanical tests to measure the material properties of the endocuticle, and numerical simulations to predict the structural effect of the sclerotisation. Our results provide the first evidence of the existence of a sclerotised endocuticle and its remarkable function in improving the mechanical stability of the cuticle. This study highlights the finding of a high degree of sclerotisation in the stiff endocuticle of the weevil, especially the matrix surrounding the fibres. This novel case brings new understanding of cuticle properties and gives promising insights into biomaterial design.

The unusual tracheal system within the wing membrane of a dragonfly.

Some consider that the first winged insects had living tissue inside the wing membrane, resembling larval gills or developing wing pads. However, throughout the developmental process of the wing membrane of modern insects, cells and tracheoles in the lumen between dorsal and ventral cuticle disappear and both cuticles become fused. This process results in the rather thin rigid stable structure of the membrane. The herewith described remarkable case of the dragonfly Zenithoptera lanei shows that in some highly specialized wings, the membrane can still be supplemented by tracheae. Such a characteristic of the wing membrane presumably represents a strong specialization for the synthesis of melanin-filled nanolayers of the cuticle, nanospheres inside the wing membrane and complex arrangement of wax crystals on the membrane surface, all responsible for unique structural coloration.

Ultrastructure of dragonfly wing veins: composite structure of fibrous material supplemented by resilin.

Dragonflies count among the most skilful of the flying insects. Their exceptional aerodynamic performance has been the subject of various studies. Morphological and kinematic investigations have showed that dragonfly wings, though being rather stiff, are able to undergo passive deformation during flight, thereby improving the aerodynamic performance. Resilin, a rubber-like protein, has been suggested to be a key component in insect wing flexibility and deformation in response to aerodynamic loads, and has been reported in various arthropod locomotor systems. It has already been found in wing vein joints, connecting longitudinal veins to cross veins, and was shown to endow the dragonfly wing with chordwise flexibility, thereby most likely influencing the dragonfly's flight performance. The present study revealed that resilin is not only present in wing vein joints, but also in the internal cuticle layers of veins in wings of Sympetrum vulgatum (SV) and Matrona basilaris basilaris (MBB). Combined with other structural features of wing veins, such as number and thickness of cuticle layers, material composition, and cross-sectional shape, resilin most probably has an effect on the vein's material properties and the degree of elastic deformations. In order to elucidate the wing vein ultrastructure and the exact localisation of resilin in the internal layers of the vein cuticle, the approaches of bright-field light microscopy, wide-field fluorescence microscopy, confocal laser-scanning microscopy, scanning electron microscopy and transmission electron microscopy were combined. Wing veins were shown to consist of up to six different cuticle layers and a single row of underlying epidermal cells. In wing veins of MBB, the latter are densely packed with light-scattering spheres, previously shown to produce structural colours in the form of quasiordered arrays. Longitudinal and cross veins differ significantly in relative thickness of exo- and endocuticle, with cross veins showing a much thicker exocuticle. The presence of resilin in the unsclerotised endocuticle suggests its contribution to an increased energy storage and material flexibility, thus to the prevention of vein damage. This is especially important in the highly stressed longitudinal veins, which have much lower possibility to yield to applied loads with the aid of vein joints, as the cross veins do. These results may be relevant not only for biologists, but may also contribute to optimise the design of micro-air vehicles.

Adhesive pad differentiation in Drosophila melanogaster depends on the Polycomb group gene Su(z)2.

The ability of many insects to walk on vertical smooth surfaces such as glass or even on the ceiling has fascinated biologists for a long time, and has led to the discovery of highly specialized adhesive organs located at the distal end of the animals' legs. So far, research has primarily focused on structural and ultrastructural investigations leading to a deeper understanding of adhesive organ functionality and to the development of new bioinspired materials. Genetic approaches, e.g. the analysis of mutants, to achieve a better understanding of adhesive organ differentiation have not been used so far. Here, we describe the first Drosophila melanogaster mutant that develops malformed adhesive organs, resulting in a complete loss of climbing ability on vertical smooth surfaces. Interestingly, these mutants fail to make close contact between the setal tips and the smooth surface, a crucial condition for wet adhesion mediated by capillary forces. Instead, these flies walk solely on their claws. Moreover, we were able to show that the mutation is caused by a P-element insertion into the Su(z)2 gene locus. Remobilization of the P-element restores climbing ability. Furthermore, we provide evidence that the P-element insertion results in an artificial Su(z)2 transcript, which most likely causes a gain-of-function mutation. We presume that this transcript causes deregulation of yet unknown target genes involved in pulvilli differentiation. Our results nicely demonstrate that the genetically treatable model organism Drosophila is highly suitable for future investigations on adhesive organ differentiation.

Variable assessment of wing colouration in aerial contests of the red-winged damselfly Mnesarete pudica (Zygoptera, Calopterygidae).

Wing pigmentation is a trait that predicts the outcome of male contests in some damselflies. Thus, it is reasonable to suppose that males would have the ability to assess wing pigmentation and adjust investment in a fight according to the costs that the rival may potentially impose. Males of the damselfly Mnesarete pudica exhibit red-coloured wings and complex courtship behaviour and engage in striking male-male fights. In this study, we investigated male assessment behaviour during aerial contests. Theory suggests that the relationship between male resource-holding potential (RHP) and contest duration describes the kind of assessment adopted by males: self-assessment, opponent-only assessment or mutual assessment. A recent theory also suggests that weak and strong males exhibit variations in the assessment strategies adopted. We estimated male RHP through male body size and wing colouration (i.e. pigmentation, wing reflectance spectra and transmission spectra) and studied the relationship between male RHP and contest duration from video-documented behavioural observations of naturally occurring individual contests in the field. The results showed that males with more opaque wings and larger red spots were more likely to win contests. The relationships between RHP and contest durations partly supported the self-assessment and the mutual assessment models. We then experimentally augmented the pigmented area of the wings, in order to evaluate whether strong and weak males assess rivals' RHP through wing pigmentation. Our experimental manipulation, however, clearly demonstrated that strong males assess rivals' wing pigmentation. We finally suggest that there is a variation in the assessment strategy adopted by males.

Tanning of the tarsal and mandibular cuticle in adult Anax imperator (Insecta: Odonata) during the emergence sequence.

The arthropod cuticle offers strength, protection, and lightweight. Due to its limit in expandability, arthropods have to moult periodically to grow. While moulting is beneficial in terms of parasite or toxin control, growth and adaptation to environmental conditions, it costs energy and leaves the soft animal's body vulnerable to injuries and desiccation directly after ecdysis. To investigate the temporal change in sclerotization and pigmentation during and after ecdysis, we combined macrophotography, confocal laser scanning microscopy, scanning electron microscopy and histological sectioning. We analysed the tarsal and mandibular cuticle of the blue emperor dragonfly to compare the progress of tanning for structures that are functionally involved during emergence (tarsus/tarsal claws) with structures whose functionality is required much later (mandibles). Our results show that: (i) the tanning of the tarsal and mandibular cuticle increases during emergence; (ii) the tarsal cuticle tans faster than the mandibular cuticle; (iii) the mandibles tan faster on the aboral than on the oral side; and (iv) both the exo- and the endocuticle are tanned. The change in the cuticle composition of the tarsal and mandibular cuticle reflects the demand for higher mechanical stability of these body parts when holding on to the substrate during emergence and during first walking or hunting attempts.

Coupling wings with movable hooks - resilin in the wing-interlocking structures of honeybees.

Hymenoptera are characterised by the presence of one forewing pair and one hindwing pair. The two wings of each body side are coupled to each other during flight making the morphologically four-winged insects functionally two-winged. This coupling is formed by a row of hook-like structures, called hamuli, that are located at the leading edge of the hindwing and interlock with a thickened and recurved margin present at the trailing edge of the forewing. In this study, autofluorescence analyses performed with confocal laser scanning microscopy revealed differences in the exoskeleton material composition of the interlocking structures. While the wing veins and the recurved margin are strongly sclerotised and chitinous, the wing membranes mainly contain the elastomeric protein resilin. The hamuli are composed of sclerotised chitinous material, and each hamulus base is surrounded by and embedded in material that features large proportions of resilin and is located in strongly sclerotised socket-like wing vein structures. This exoskeleton organisation likely allows movements of the hamuli and, in combination with the exoskeleton material gradients visualized in the other interlocking structures, is assumed to guarantee an effective wing coupling and to simultaneously decrease the risk of wear and damage under mechanical loads occurring in flight, coupling and decoupling situations.

Ultrastructure of spider thread anchorages.

Spiders attach silken threads to substrates by means of glue-coated nanofibers (piriform silk), spun into disc-like structures. The organization and ultrastructure of this nano-composite silk are largely unknown, despite their implications for the biomechanical function and material properties of thread anchorages. In this work, the ultrastructure of silken attachment discs was studied in representatives of four spider families with Transmission Electron Microscopy to facilitate a mechanistic understanding of piriform silk function across spiders. Based on previous findings from comparative studies of piriform silk gland morphology, we hypothesized that the fibre-glue proportion of piriform silk differs in different spiders, while the composition of fibre and glue fractions is consistent. Results confirmed large differences in the relative proportion of glue with low amounts in the orb weaver Nephila senegalensis (Araneidae) and the hunting spider Cupiennius salei (Ctenidae), larger amounts in the cobweb spider Parasteatoda tepidariorum (Theridiidae) and a complete reduction of the fibrous component in the haplogyne spider Pholcus phalangioides (Pholcidae). We rejected our hypothesis that glue ultrastructure is consistent. The glue is a colloid with polymeric and fluid fractions that strongly differ in proportions and assembly. We further confirmed that in all species studied both dragline and piriform silk fibres do not make contact with the environmental substrate. Instead, adhesion is established by a thin dense skin layer of the piriform glue. These results advance our understanding of piriform silk function and the interspecific variation of its properties, which is significant for spider biology, web function and the bioengineering of silk.

Structural coloration predicts the outcome of male contests in the Amazonian damselfly Chalcopteryx scintillans (Odonata: Polythoridae).

Iridescence is an optical effect that produces angle dependent coloration in animals. Recently, studies have attempted to unveil structures behind such elaborated visual signals and associated behaviors in Odonata. Here, we studied males of the Amazonian damselfly Chalcopteryx scintillans, which have hindwings that exhibit pronounced iridescence. This optical feature is used by the damselflies for intra-specific communication during territorial fights and courtship. The main question we addressed was whether male wing structural coloration may predict the outcome of male-male contests. We also studied the wing ultrastructure, in order to reveal the mechanisms that are responsible for wing coloration. Using various microscopal and spectroscopal techniques, we demonstrate that hindwing coloration is derived from two main effects: (1) light interference in the cuticle multilayer and (2) a specific angle dependent light scattering and antireflective properties of the epicuticular wax coverage. The results of our field experiment show that wing pigmentation and the hue of the dorsal surface of the hindwings is correlated with the outcome of territorial contests. This is one of the first studies showing that structural coloration derived from multilayer interference may influence the outcome of intrasexual agonistic interactions. This indicates that multicomponent structural coloration in visually guided insects may be under selective forces of male-male competition for resources and females.

Adjustment of triple shellac coating for precise release of bioactive substances with different physico-chemical properties in the ileocolonic region.

Formulations for the controlled release of substances in the human terminal ileum and colon are essential to target the gut microbiome and its interactions with the intestinal mucosa. In contrast to pharmaceutical enteric coatings, reliable food-grade alternatives are still scarce. Shellac coatings have been used for various active ingredients, but their stability is affected by the physicochemical properties of the encapsulated substances. It is well known, that shellac release can be modulated by an acidic subcoating. Here, we hypothesized that a triple shellac coating with an adjusted intermediate coating (acidic or alkaline) can be effectively used to counteract the differences in pH value of various encapsulated substances, allowing a precise targeting of the desired release pH value. First, the system was tested with riboflavin 5'-monophosphate sodium salt dihydrate (RMSD) as a characteristic model substance. Secondly, it was transferred to nicotinic acid (NA) and nicotinamide (NAM) as bioactive compounds with different physio-chemical properties: NAM, an alkaline crystalline and highly water-soluble substance, led to a premature release from conventional shellac microcapsules, whereas RMSD and NA with their medium solubility and neutral to acidic pH properties delayed the shellac dissolution. A precise modulation of the release profile of each substance was possible by the addition of different intermediate subcoatings: an acidic layer with citric acid counteracted the premature release of the alkaline and highly soluble NAM. In contrast, an alkaline sodium bicarbonate intermediate subcoating enhanced shellac swelling and delayed the release of NA and RMSD. In conclusion, the novel triple-layer shellac coating provides a much higher adaptability and reliability for nutritional formulations aiming at a targeted release in the ileocolonic region.

Targeted Microbiome Intervention by Microencapsulated Delayed-Release Niacin Beneficially Affects Insulin Sensitivity in Humans.

OBJECTIVE: Gut microbiota represent a potential novel target for future prediabetes and type 2 diabetes therapies. In that respect, niacin has been shown to beneficially affect the host-microbiome interaction in rodent models. RESEARCH DESIGN AND METHODS: We characterized more than 500 human subjects with different metabolic phenotypes regarding their niacin (nicotinic acid [NA] and nicotinamide [NAM]) status and their gut microbiome. In addition, NA and NAM delayed-release microcapsules were engineered and examined in vitro and in vivo in two human intervention studies (bioavailability study and proof-of-concept/safety study). RESULTS: We found a reduced α-diversity and Bacteroidetes abundance in the microbiome of obese human subjects associated with a low dietary niacin intake. We therefore developed delayed-release microcapsules targeting the ileocolonic region to deliver increasing amounts of NA and NAM to the microbiome while preventing systemic resorption to avoid negative side effects (e.g., facial flushing). In vitro studies on these delayed-release microcapsules revealed stable conditions at pH 1.4, 4.5, and 6.8, followed by release of the compounds at pH 7.4, simulating the ileocolonic region. In humans in vivo, gut-targeted delayed-release NA but not NAM produced a significant increase in the abundance of Bacteroidetes. In the absence of systemic side effects, these favorable microbiome changes induced by microencapsulated delayed-release NA were associated with an improvement of biomarkers for systemic insulin sensitivity and metabolic inflammation. CONCLUSION: Targeted microbiome intervention by delayed-release NA might represent a future therapeutic option for prediabetes and type 2 diabetes.

Functional morphology and evolution of the hyper-elongated intromittent organ in Cassida leaf beetles (Coleoptera: Chrysomelidae: Cassidinae).

The peculiar phenomenon of hyper-elongation of intromittent organs is well known in a number of insect groups. However, the unresolved questions of how and why such a phenomenon originated independently many times continue to attract biologists' attention. To be able to detect the evolutionary driving mechanisms that enabled insects to repeatedly acquire such a peculiarity, first of all the structural key features and the mechanics of these organs have to be determined. In the present study, the morphology of the reproductive organs of two species of the beetle genus Cassida, with a special focus on the male structures, was scrutinised in detail during copulation and at rest using different microscopy techniques. We found that the hyper-elongated structure of the intromittent organ, called flagellum, is part of the male ejaculatory duct. When the flagellum is inserted into the female spermathecal duct, longitudinal muscles of the ejaculatory duct, but not the flagellum, are shortened. These results strongly suggest that the contraction of the longitudinal muscles of the ejaculatory duct causes propulsion of the flagellum into the highly spiralled spermathecal duct of the female. The tip of the cuticular flagellum is curled up, which can suggest that its physical properties differ from those of the rest of the flagellum. Considering the preceding modelling studies, this property aids the flagellum in penetrating within the highly spiralled and very variable female duct. Based on our morphological results and in comparison with the morphology of intromittent organs of other beetles, we discuss the evolutionary origin of the hyper-elongation in the Cassida species and propose a hypothesis that explains the independent origin of the hyper-elongation of intromittent organs.

The influence of the topography and physico-chemical properties of the cuticle surface on the wettability and adhesive properties of the elytra of the dung beetle Geotrupes stercorarius (Coleoptera, Scarabaeidae).

The elytral surface of dung beetles is generally accepted to be self-cleaning due to its anti-adhesive properties. In this article, the wettability and adhesive properties of elytral surface (intact and treated with Acetone and Ethanol) of the beetle Geotrupes stercorarius were characterized. Since these properties are influenced by the surface structure and chemistry, the micro- and nanostructure of the elytra were observed using scanning electron microscopy and the surface roughness were estimated with white light interferometery, whereas the water contact angle and adhesion force of the elytra were evaluated using contact angle measurement device and force transducer, respectively. The results show that the microstructure of the elytra changed greatly after different chemical treatments and the changes in wettability were mainly related to the changes of elytra roughness after treatments. The differences in adhesion force between differently treated elytra and wet sand indicate that the molecular polarity of the elytral surface is decisive for its anti-adhesive (self-cleaning) property.

Functional diversity of resilin in Arthropoda.

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.

Mechanism of the wing colouration in the dragonfly Zenithoptera lanei (Odonata: Libellulidae) and its role in intraspecific communication.

Zenithoptera dragonflies are known for their remarkable bluish colouration on their wings and unique male behaviour of folding and unfolding their wings while perching. However, nothing is known about the optical properties of such colouration and its structural and functional background. In this paper, we aimed to study the relationship between the wing membrane ultrastructure, surface microstructure and colour spectra of male wings in Zenithoptera lanei and test the hypothesis that colouration functions as a signal in territorial fights between males. The results show that the specific wing colouration derives from interference in alternating layers of melanized and unmelanized cuticle in the wing membrane, combined with diffuse scattering in two different layers of wax crystals on the dorsal wing surface, one lower layer of long filaments, and one upper layer of leaf-shaped crystals. The results also show that the thicker wax coverage of the dorsal surface of the wings results in increased brightness and reduced chroma. In the field experiments, we have demonstrated that there is a reduction of aggressive reactions of rivals towards individuals with experimentally reduced amount of blue wing colouration.

Morphological studies of the toe pads of the rock frog, Staurois parvus (family: Ranidae) and their relevance to the development of new biomimetically inspired reversible adhesives.

The morphology of the toe epithelium of the rock frog, Staurois parvus (Family Ranidae), was investigated using a variety of microscopical techniques. The toe pad epithelium is stratified (four to five cell layers), the apical parts of the cells of the outermost layer being separated by fluid-filled channels. The surface of these cells is covered by a dense array of nanopillars, which also cover the surface of subarticular tubercles and unspecialized ventral epithelium of the toes, but not the dorsal epithelium. The apical portions of the outer two layers contain fibrils that originate from the nanopillars and are oriented approximately normal to the surface. This structure is similar to the pad structure of tree frogs of the families Hylidae and Rhacophoridae, indicating evolutionary convergence and a common evolutionary design for reversible attachment in climbing frogs. The main adaptation to the torrent habitat seems to be the straightness of the channels crossing the toe pad, which will assist in drainage of excess water. The presence of nanopillar arrays on all ventral surfaces of the toes resembles that on clingfish suckers and may be a specific adaptation for underwater adhesion and friction. The relevance of these findings to the development of new biomimetically inspired reversible adhesives is discussed.

Hairy suckers: the surface microstructure and its possible functional significance in the Octopus vulgaris sucker.

Octopus suckers are able to attach to any smooth surface and many rough surfaces. Here, we have discovered that the sucker surface, which has been hypothesised to be responsible for sealing the orifice during adhesion, is not smooth as previously assumed, but is completely covered by a dense network of hair-like micro-outgrowths. This finding is particularly important because it provides another demonstration of the role of hair-structures in a sealing mechanism in water, similar to that previously described for clingfish and abalones. Moreover, the discovered hairs may provide an additional adhesive mechanism that works in concert with suction. The discovered surface structures might be potentially interesting for biomimetics of novel technical suction cups with improved adhesion capabilities on non-smooth surfaces.

Air/water interfacial formation of freestanding, stimuli-responsive, self-healing catecholamine Janus-faced microfilms.

A catecholamine freestanding film is discovered to be spontaneously formed at the air-water interface, and the film has unique properties of robust surface adhesiveness, self-healing, and stimuli-responsive properties. The interfacial film-producing procedure is a simple single step containing polyamines and catechol(amine)s. It is found that oxygen-rich regions existing at an air-water interface greatly accelerate the catecholamine crosslinking reaction.