Cuticle Material Research Articles

Mechanical and elemental characterization of ant mandibles: consequences for bite mechanics.

Mandible morphology has an essential role in biting performance, but the mandible cuticle can have regional differences in its mechanical properties. The effects of such a heterogeneous distribution of cuticle material properties in the mandible responses to biting loading are still poorly explored in chewing insects. Here, we tested the mechanical properties of mandibles of the ant species Formica cunicularia by nanoindentation and investigated the effects of the cuticular variation in Young's modulus (E) under bite loading with finite-element analysis (FEA). The masticatory margin of the mandible, which interacts with the food, was the hardest and stiffest region. To unravel the origins of the mechanical property gradients, we characterized the elemental composition by energy-dispersive X-ray spectroscopy. The masticatory margin possessed high proportions of Cu and Zn. When incorporated into the FEA, variation in E effectively changed mandible stress patterns, leading to a relatively higher concentration of stresses in the stiffer mandibular regions and leaving the softer mandible blade with relatively lower stress. Our results demonstrated the relevance of cuticle E heterogeneity in mandibles under bite loading, suggesting that the accumulation of transition metals such as Cu and Zn has a relevant correlation with the mechanical characteristics in F. cunicularia mandibles.

An ABCB transporter regulates anisotropic cell expansion via cuticle deposition in the moss Physcomitrium patens.

Anisotropic cell expansion is crucial for the morphogenesis of land plants, as cell migration is restricted by the rigid cell wall. The anisotropy of cell expansion is regulated by mechanisms acting on the deposition or modification of cell wall polysaccharides. Besides the polysaccharide components in the cell wall, a layer of hydrophobic cuticle covers the outer cell wall and is subjected to tensile stress that mechanically restricts cell expansion. However, the molecular machinery that deposits cuticle materials in the appropriate spatiotemporal manner to accommodate cell and tissue expansion remains elusive. Here, we report that PpABCB14, an ATP-binding cassette transporter in the moss Physcomitrium patens, regulates the anisotropy of cell expansion. PpABCB14 localized to expanding regions of leaf cells. Deletion of PpABCB14 resulted in impaired anisotropic cell expansion. Unexpectedly, the cuticle proper was reduced in the mutants, and the cuticular lipid components decreased. Moreover, induced PpABCB14 expression resulted in deformed leaf cells with increased cuticle lipid accumulation on the cell surface. Taken together, PpABCB14 regulates the anisotropy of cell expansion via cuticle deposition, revealing a regulatory mechanism for cell expansion in addition to the mechanisms acting on cell wall polysaccharides.

A stress-response-related inter-compartmental signalling pathway regulates embryonic cuticle integrity in Arabidopsis.

The embryonic cuticle is necessary for normal seed development and seedling establishment in Arabidopsis. Although mutants with defective embryonic cuticles have been identified, neither the deposition of cuticle material, nor its regulation, has been described during embryogenesis. Here we use electron microscopy, cuticle staining and permeability assays to show that cuticle deposition initiates de novo in patches on globular embryos. By combining these techniques with genetics and gene expression analysis, we show that successful patch coalescence to form a continuous cuticle requires a signalling involving the endosperm-specific subtilisin protease ALE1 and the receptor kinases GSO1 and GSO2, which are expressed in the developing embryonic epidermis. Transcriptome analysis shows that this pathway regulates stress-related gene expression in seeds. Consistent with these findings we show genetically, and through activity analysis, that the stress-associated MPK6 protein acts downstream of GSO1 and GSO2 in the developing embryo. We propose that a stress-related signalling pathway has been hijacked in some angiosperm seeds through the recruitment of endosperm-specific components. Our work reveals the presence of an inter-compartmental dialogue between the endosperm and embryo that ensures the formation of an intact and functional cuticle around the developing embryo through an “auto-immune” type interaction.

Friction properties of the head articulation in the beetle Pachnoda marginata (Coleoptera, Scarabaeidae)

The head articulation of the beetle Pachnoda marginata is a micro-tribological system which consists of the ventral convex structure called gula and the corresponding concave surface of the prothorax. The surfaces of both parts are in contact and are expected to be optimised for friction reduction. The relationship between structure, mechanical properties of the cuticle material and friction properties of this micro-tribological system are investigated. The surface and material structure of the gula and its prothoracic counterpart were studied in fractured pieces of the cuticle using scanning electron microscopy (SEM). Friction force measurements and contact area estimations between the gula and a glass plate were carried out for different normal loads (0.1–10.0 mN) using two different microtribometers. The tribological behavior of the gula cuticle was studied on the (1) fresh, (2) dry, and (3) dry chemically (chloroform-methanol) treated samples. The dry samples exhibited a considerably rougher surface compared to the fresh ones. Furthermore, the chemical treatment led to some decrease in surface roughness. The fresh gula cuticle had the largest contact area and the highest friction coefficient. The drying out of the cuticle led to a decrease in both the contact area and friction coefficient. The friction coefficient was the lowest in the chemically treated gula, although the contact area was larger than in the dry condition. The tribological results were partly explained by direct measurements of the contact area fitted by the Hertz and JKR contact models.

Heme peroxidase HPX-2 protects Caenorhabditis elegans from pathogens.

Heme-containing peroxidases are important components of innate immunity. Many of them functionally associate with NADPH oxidase (NOX)/dual oxidase (DUOX) enzymes by using the hydrogen peroxide they generate in downstream reactions. Caenorhabditis elegans encodes for several heme peroxidases, and in a previous study we identified the ShkT-containing peroxidase, SKPO-1, as necessary for pathogen resistance. Here, we demonstrated that another peroxidase, HPX-2 (Heme-PeroXidase 2), is required for resistance against some, but not all pathogens. Tissue specific RNA interference (RNAi) revealed that HPX-2 functionally localizes to the hypodermis of the worm. In congruence with this observation, hpx-2 mutant animals possessed a weaker cuticle structure, indicated by higher permeability to a DNA dye, but exhibited no obvious morphological defects. In addition, fluorescent labeling of HPX-2 revealed its expression in the pharynx, an organ in which BLI-3 is also present. Interestingly, loss of HPX-2 increased intestinal colonization of E. faecalis, suggesting its role in the pharynx may limit intestinal colonization. Moreover, disruption of a catalytic residue in the peroxidase domain of HPX-2 resulted in decreased survival on E. faecalis, indicating its peroxidase activity is required for pathogen resistance. Finally, RNA-seq analysis of an hpx-2 mutant revealed changes in genes encoding for cuticle structural components under the non-pathogenic conditions. Under pathogenic conditions, genes involved in infection response were differentially regulated to a greater degree, likely due to increased microbial burden. In conclusion, the characterization of the heme-peroxidase, HPX-2, revealed that it contributes to C. elegans pathogen resistance through a role in generating cuticle material in the hypodermis and pharynx.

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 female's 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.

Antimicrobial activity in the cuticle of the American lobster, Homarus americanus

American lobster, Homarus americanus, continues to be an ecologically and socioeconomically important species despite a severe decline in catches from Southern New England and Long Island Sound (USA) and a high prevalence of epizootic shell disease in these populations. A better understanding of lobster immune defenses remains necessary. Cuticle material collected from Long Island Sound lobsters was found to be active against a broad spectrum of bacteria, including Gram-negative and -positive species. The antimicrobial activity was characterized by boiling, muffling, and size fractioning. Boiling did not significantly reduce activity, while muffling did have a significant effect, suggesting that the active component is organic and heat stable. Size fractioning with 3 and 10 kDa filters did not significantly affect activity. Fast protein liquid chromatography fractions were also tested for antimicrobial activity, and fractions exhibiting protein peaks remained active. MALDI mass spectrometry revealed peptide peaks at 1.6, 2.8, 4.6, and 5.6 kDa. The data presented suggest that one or several antimicrobial peptides contribute to antimicrobial activity present in the American lobster cuticle.

Molecular cloning of a novel subtilisin-like protease (Pr1A) gene from the biocontrol fungus Isaria farinosa

Fungal proteases, such as subtilisin-like serine protease (Pr1) and trypsin-like serine protease (Pr2), play an important role in penetration through the host cuticle in entomopathogenic fungi, including Isaria farinosa. In the present study, one gene of I. farinosa subtilisin-like protease (Ifa-pr1A), showing high homology to a Beauveria bassiana cuticle-degrading protease, was amplified by rapid amplification of cDNA ends PCR. The resulting full-length cDNA displayed an open reading frame (ORF) of 960 bp, encoding a protein of 319 amino acids. The Ifa-Pr1A protein was expressed in Escherichia coli to verify its protease activity. The recombinant Ifa-Pr1A protein exhibited high enzymatic activity according to the enzyme assay using a synthetic substrate. The expression profiles of Ifa-pr1A and Ifa-pr1H (another subtilisin-like protease gene from I. Farinosa) were analyzed at different induction times by adding cuticle material to the media. Quantitative reverse transcriptase PCR (RT-PCR) analysis revealed that Ifa-pr1A transcripts increased 58,800-fold at 12 h post-induction, whereas Ifa-pr1H transcripts peaked at 12 h with only an 11-fold increase, indicating that Ifa-pr1A may be a major gene of cuticle-degrading proteases in I. farinosa. Furthermore, Ifa-pr1A gene expression under in vivo conditions showed a clear upward trend during the early stages of the infection process. These results suggest that Pr1A cloned from I. farinosa is a potential virulence factor for the development of engineered biopesticides.

Chitosan‐Fibroin Laminates: Unexpected Strength and Toughness in Chitosan‐Fibroin Laminates Inspired by Insect Cuticle (Adv. Mater. 4/2012)

Javier Fernandez and Donald Ingber demonstrate on page 480 that chitosan-fibroin laminates inspired by natural insect cuticles have unexpected strength and toughness. The inside cover image shows a scaled replica of a Thesoneura Americana hind wing made of Shrilk, which is a synthetic insect cuticle material composed of a laminate of chitosan and fibroin. Thesoneura is a member of the Palepdictyoptera family that is believed to be one of the first primitive flying insects. This insect became extinct about 300 million years ago; the shape was reproduced from fossil records and the vein pattern was reproduced from a grasshopper hind wing.

From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in Insects

One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects.

Diversity of the Cuticle Layer of Avian Eggshells

The structure and mineral composition of eggshell cuticles were studied in 5 species of birds. The approximate thickness of the cuticle layer at the top of shell columns was about 1μm in the Red Junglefowl (Gallus gallus), about 130μm in the White Pelican (Pelecanus onocrotalus), about 10μm in the Japanese quail (Coturnix japonica), about 110μm in the Greater Flamingo (Phoenicopterus ruber roseus) and about 45μm in the Humboldt Penguin (Spheniscus humboldti). The matrix of the cuticle layer decalcified with EDTA was composed of vesicles in a variety of sizes in all birds. Major elements in cuticle materials detected by X-ray microanalysis were O, C, Ca and P, and their percentage numbers of atoms decreased in this order. The concentration of P was significantly higher in the cuticles of the quail, flamingo, and penguin than in those of the junglefowl and pelican. Ca mapping on electron-microscopic images showed strong signals in the shell layer and weaker ones in the cuticle layer, whereas P mapping showed that signals were mostly confined in the cuticle layer. X-ray diffraction analyses on the inside of the shell layer showed a profile of calcite crystals of calcium carbonates in all birds. In the cuticle materials, the profile was of calcite in the junglefowl, and a mixture of calcite and vaterite in the pelican. The profiles in cuticle materials of the quail, flamingo and penguin showed no specific signals, indicating that mineral compounds are amorphous in these forms. It was suggested that the diversity of mineral structures in the cuticle layer is caused by the presence of phosphorous, in addition to the structure of the cuticle matrix.

Revealing the Design Principles of High‐Performance Biological Composites Using Ab initio and Multiscale Simulations: The Example of Lobster Cuticle

Natural materials are hierarchically structured nanocomposites. A bottom-up multiscale approach to model the mechanical response of the chitin-based mineralized cuticle material of Homarus americanus is presented, by combining quantum-mechanical ab initio calculations with hierarchical homogenization. The simulations show how the mechanical properties are transferred from the atomic scale through a sequence of specifically designed microstructures to realize optimal stiffness.

Predator‐induced spine length and exocuticle thickness in Leucorrhinia dubia (Insecta: Odonata): a simple physiological trade‐off?

Abstract.1. Morphological defence structures evolve against predators but are costly to the individual, and are induced only when required. A well‐studied example is the development of longer abdominal spines in dragonfly larvae in the presence of fish. Numerous attempts to discover trade‐offs between spine size and behaviour, development time or body size have, however, produced little evidence.2. We considered a physiological trade‐off. Spines consist of cuticle and using material to build longer structures may result in less material remaining elsewhere. We therefore measured exocuticle thickness at nine locations on Leucorrhinia dubia larvae from habitats with and without fish.3. Our results show a significant effect of the interaction between fish presence and spine length on head and fore leg exocuticle thickness. Relative thickness increased with relative length of lateral spine 9 in the absence of fish, whereas no such relationship existed with fish. Hence, synthesis and secretion of cuticle material occur as a trade‐off when larvae react to fish presence.4. We assume the mechanism to be a selective synthesis of material with different responses in different parts of the larval body. These findings offer a new angle to the fish/spine trade off debate.

Cuticle Formation in Quail Eggs

The present study was conducted to determine both the site at which cuticle materials are produced and the critical period for their production in the oviductal uterus of the Japanese quail, Coturnix japonica. An antiserum was produced against the 32-kDa band in electrophoretic profiles of cuticle materials obtained from eggshells decalcified with EDTA. Immunofluorescence and immunoelectron microscopic observations revealed that the 32-kDa protein was synthesized in luminal ciliated epithelial cells of the uterus until 21 h after the previous oviposition (the first phase) and then secreted during the 4 h before the next oviposition (the second phase). Scanning electron microscopic observations revealed that 10-microm-wide posts appear on the surface of the luminal epithella during the first phase, and that they disappear during the second phase. During the second phase, air canals are formed in the eggshell by the retreat of the posts, and a cuticle layer forms on the eggshell. Our results indicate that the cuticle may function as a lubricant that facilitates egg rotation in the uterus.

Cuticle differentiation during Drosophila embryogenesis

The constitutive criterion for the evolutionary successful clade of ecdysozoans is a protective exoskeleton. In insects the exoskeleton, the so-called cuticle consists of three functional layers, the waterproof envelope, the proteinaceous epicuticle and the chitinous procuticle that are produced as an extracellular matrix by the underlying epidermal cells. Here, we present our electron-microscopic study of cuticle differentiation during embryogenesis in the fruit fly Drosophila melanogaster. We conclude that cuticle differentiation in the Drosophila embryo occurs in three phases. In the first phase, the layers are established. Interestingly, we find that establishment of the layers occurs partially simultaneously rather than in a strict sequential manner as previously proposed. In the second phase the cuticle thickens. Finally, in the third phase, when secretion of cuticle material has ceased, the chitin laminae acquire their typical orientation, and the epicuticle of the denticles and the head skeleton darken. Our work will help to understand the phenotypes of embryos mutant for genes encoding essential cuticle factors, in turn revealing mechanisms of cuticle differentiation.

Involvement of chitin in exoskeleton morphogenesis inDrosophila melanogaster

Exoskeletons stabilize cell, tissue, and body morphology in many living organisms including fungi, plants, and arthropods. In insects, the exoskeleton, the cuticle, is produced by epidermal cells as a protein extracellular matrix containing lipids and the polysaccharide chitin, and its formation requires coordinated synthesis, distribution, and modification of these components. Eventually, the stepwise secretion and sorting of the cuticle material results in a layered structure comprising the envelope, the proteinaceous epicuticle, and the chitinous procuticle. To study the role of chitin during cuticle development, we analyzed the consequences of chitin absence in the embryo of Drosophila melanogaster caused by mutations in the Chitin Synthase-1 (CS-1) gene, called krotzkopf verkehrt (kkv). Our histological data confirm that chitin is essential for procuticle integrity and further demonstrate that an intact procuticle is important to assemble and to stabilize the chitin-less epicuticle. Moreover, the phenotype of CS-1/kkv mutant embryos indicates that chitin is required to attach the cuticle to the epidermal cells, thereby maintaining epidermal morphology. Finally, sclerotization and pigmentation, which are the last steps in cuticle differentiation, are impaired in tissues lacking CS-1/kkv function, suggesting that proper cuticle structure is crucial for the activity of the underlying enzymes.

Determination of the cuticle index of the scales of the iridescent butterfly Morpho menelaus

Butterflies of the Morphidae family probably constitute the most popular example of iridescent colours in nature. They present, many fundamental interests in biomimetic processes and can be used as models or sources of inspiration for many industrial applications. The microstructures of the scales are responsible for the coloration, with a major part attributed to interference effects. Their actual optical properties have been described in detail, but the refraction index of the cuticular material is not known with precision. In this paper, we investigate the absolute diffuse reflectivity of the dorsal side of the wings of the species Morpho menelaus. An effective medium theory applied to a multilayer film permits the determination of the anisotropic index of refraction of the cuticle material in the 350–800 nm range.

Quantified interference and diffraction in single Morpho butterfly scales

Brilliant iridescent colouring in male butterflies enables long–range conspecific communication and it has long been accepted that microstructures, rather than pigments, are responsible for this coloration. Few studies, however, explicitly relate the intra–scale microstructures to overall butterfly visibility, both in terms of reflected and transmitted intensities and viewing angles. Using a focused–laser technique, we investigated the absolute reflectivity and transmissivity associated with the single–scale microstructures of two species of Morpho butterfly and the mechanisms behind their remarkable wide–angle visibility. Measurements indicate that certain Morpho microstructures reflect up to 75% of the incident blue light over an angle range of greater than 100° in one plane and 15° in the other. We show that incorporation of a second layer of more transparent scales, above a layer of highly iridescent scales, leads to very strong diffraction, and we suggest this effect acts to increase further the angle range over which incident light is reflected. Measurements using index-matching techniques yield the complex refractive index of the cuticle material comprising the single–scale microstructure to be n = (1.56+0.01) + (0.06 ±0.01)i. This figure is required for theoretical modelling of such microstructure systems.

Cuticle ultrastructure of the cheirolepidiaceous conifer Hirmeriella muensteri (Schenk) Jung

A study of well preserved fossil cuticle material of Hirmeriella muensteri from the Liassic of Franconia (Germany) was carried out using transmission electron microscopy (TEM), as well as scanning electron microscopy (SEM) and light microscopy (LM). The ultrastructure of normal epidermal cells is described as well as that of the stomata with a differentiation into guard and subsidiary cells. The ultrastructural details allow not only a discussion on the taxonomic position of the species, but also on the structure/function relationships, xeromorphism and the ability of the guard cells, and to a lesser degree of the subsidiary cells, to change in shape and volume for opening and closing.

Relationship between Function and Mechanical Properties of the Pleopods of Isopod Crustaceans

In the isopod crustaceans Idotea wosnesenskii and I. resecata, the first three pairs of abdominal appendages (pleopods) are locomotory, and the remaining two pairs are respiratory. We investigated the mechanical and morphometric properties of the pleopods in order to study constraints that may influence this division of labor. Measurements of the flexural (bending) stiffness of the pleopods demonstrated that the podites of the first and second pairs of pleopods were generally an order of magnitude stiffer than the fourth and fifth pleopods; the third pleopods were variable in stiffness. Anterior pleopods tend to be slightly thicker and have thicker cuticles, but these morphological differences do not entirely account for differences in flexural stiffness among pleopods; there must also be differences in modulus of elasticity (E). Estimates of E based on these stiffness data yield values in the middle of the range typical of arthropod cuticles. The differences in cuticle thickness and material properties between the anterior and posterior pleopods may help explain why the swimming pleopods do not make a significant contribution to gas exchange. Additional key words: cuticle, gills, stiffness, swimming In this study, our goal has been to investigate the physical and mechanical constraints on the closelylinked swimming and respiratory systems in isopod crustaceans. Much recent research has shown that the mechanical properties of an organism or its parts can have a major influence on the organism's way of life. The mechanical properties encompass both the geometric (morphological) features and the material properties of the structure, and either or both can play a role. For example, Koehl (1977a,b) showed that species of sea anemones from high-current and low-current areas have body walls with essentially the same material properties, but their different geometries give them different mechanical properties. Telewski and Jaffe (1986), comparing the bending resistance of pine trees from sheltered and windy areas, also found differences in mechanical properties due mostly to geometric differences. In contrast, Charters et al. (1969) found differences in both material and geometric properties between specimens of Eisenia (an upright alga) from sheltered and high-current environments. Components of an organism commonly have mula Corresponding author: Dept. of Entomology, 2045 Haworth Hall, University of Kansas, Lawrence, KS 66045-2106, USA. tiple functions, both primary and secondary. In geckos, the outer layer of skin is loosely attached and has numerous zones of weakness where it can easily be torn (Bauer et al. 1989); when grasped by a predator, large areas of skin can be sloughed off, giving the lizard a b tter chance of escaping. Thus, a predator-escape function has been added to the skin's primary function as an integumentary barrier. Similarly, in isopod crustaceans, the abdominal appendages, or pleopods, are the primary site of gas exchange (McLaughlin 1980; Schram 1986) and ion exchange (Holliday 1988). Many aquatic isopod groups have independently evolved a secondary function for pleopods in which they are used as paddles for swimming (Schram 1986). Early morphological studies (reviewed by McLaughlin 1980) suggested that such swimming isopods, rather than using all pleopods for both functions, display a division of labor among the pleopods. Recent experiments have confirmed that the anterior pleopods are used for swimming and the posterior pleopods act as gills (Alexander 1988; Alexander & Chen 1989). In the present study, we investigated what mechanical requirements of paddles might prevent them from acting as gills, and what physical requirements of gills might prevent them from paddling. Our approach was to measure the flexural stiffness (resistance to lengthThis content downloaded from 207.46.13.52 on Mon, 24 Oct 2016 04:13:10 UTC All use subject to http://about.jstor.org/terms Alexander, Blodig, & Hsieh