The microfiber texture in a specialized plastic cuticle area within a sensillum field on the cockroach maxillary palp as revealed by freeze fracturing

A field of sensilla extends across the ventral surface of the terminal segment of the maxillary palps of Periplaneta americana. The sensilla project from a sheet of pliable cuticle. Ultrathin sections of the cuticle in this area reveal a clear-cut parabolic microfiber pattern. Microfibers can also be seen from freeze fracture faces running parallel to the cuticular surface. These microfibers have a diameter of 80 A and may consist of chitin crystallites surrounded by a matrix coat. The number of straight parallel microfibers visible in a fracture face increases the more closely parallel to the surface the fracture runs. This result suggests a helicoidal texture, as the model of Bouligand would demand. The layer-to-layer rotational displacement of the microfibers is about 12 degrees. This texture can be regarded as typical for flexible cuticles in general. Other structural properties such as the continuation of the epicuticular dense layer into deeper cuticular layers around the enveloping cells of sensilla can be interpreted as specializations connected with the function of the sensillum field.     

The structure of the cuticle of the shore-crab Carcinus maenas (L.)

The cuticle of Carcinus has been critically examined by light microscopy and found to present features differing from the model proposed by Bouligand (1965), elaborated by Neville and his collaborators (see references), and re-stated by Bouligand (1971). The laminae have the appearance of discrete and separable sheets connected by interlaminar fibres, and between them long unorientated macrofibres pursue a sinuous course and pass from one interlaminar zone to another. Together with the interlaminar connecting fibres the macrofibres appear to account for the plumose appearance of the interlaminar zones.     

A single spiral artefact in Arthropod cuticle

Spirals are often seen in sections transverse to the axes of bumped structures in arthropod cuticle. (Sections through arthropod cornea or exocones yield excellent examples.) As arthropod cuticle has a helicoidal architecture (Bouligand, 1965), it might be expected that the spirals are a simple consequence of that structure. According to a symmetry argument, the spirals thus predicted must be double spirals. In contrast, the observed spirals are usually single. We propose that the single spirals result from an interaction between the microtome knife and the cuticle architecture. The direction of knife travel defines an orientation within the cuticle, subverting the symmetry arguments that require double spirals. Bouligand (1972) presented a model for the interaction of the knife with the cuticle. However, we offer arguments and observations which show that Bouligand's model is incorrect. We argue from detailed observations of the single spiral that it is indeed a knifing artifact and that its explanation probably lies within a certain class of models. Two related models based on relative movements of cuticle components are examined via computer techniques.     

Fine structure of the chitin-protein system in the crab cuticle

The fine structure of the organic matrix of the shore crab cuticle (Carcinus maenas L.), observed in transmission electron microscopy, reveals three different levels of organization of the chitin—protein complex. The highest level corresponds to the ‘twisted plywood’ organization described by Bouligand (1972). Horizontal microfibrils, parallel to the cuticle plane, rotate progressively from one level to another. When viewed in oblique section this structure gives superimposed series of nested arcs, visible in light microscopy or at the lowest magnifications of the electron microscope, in all the chitin-protein layers. At the highest magnifications of the electron microscope and with the best resolution, when the ultrathin sections are exactly transverse to the microfibril, a constant pattern can be observed which consists of rods transparent to electrons, which are embedded in an electron-opaque matrix. In cross-section, these rods often form more or less hexagonal arrays. We call a microfibril one rod and the adjacent opaque material, and question the usual interpretation of the microfibril molecular structure. Between these two levels of organization, there is an intermediate level, which corresponds to the grouping of microfibrils. Microfibrils form a dense structure, with few free spaces in the membranous layer, the deepest and non-calcified layer of the cuticle. In other parts of the cuticle, microfibrils are grouped into fibrils of various diameters or form a reticulate structure, the free spaces of the organic matrix being occupied by the mineral.     

The trichurid egg-shell: Evidence in support of the Bouligand hypothesis of helicoidal architecture

Electron microscopy of thin sections and freeze etch replicas of the eggs of the nematodes Trichuris suis and T. muris is used to provide evidence in support of the Bouligand hypothesis of helicoidal architecture. The evidence presented is as follows:

1. 1. The specific objections to the Bouligand model raised by Dennell (1974) and Dalingwater (1975b) are answered by reference to a pyramid of helicoidal tissue in which the corners are blunt.

2. 2. Sections cut normal to the plane of the laminae do not show parabolic patterning. Parabolae appear if the section is tilted—their direction depending upon the direction of tilting.

3. 3. Freeze etching allows the direct visualization of helicoidal architecture. Fibres are parallel within any one lamina but the fibre direction rotates by an angle of 9 ° in successive laminae. Parabolic arcs are made up of short lengths of straight fibres—curved fibres were not observed.

Planes of sectioning producing single and double spiral artifacts are described and the formation of these artifacts discussed. The sense of rotation of the helicoid is shown to be asymmetrical about any mid-plane through the egg.     

Theory of microtomy artefacts in arthropod cuticle

Arthropod cuticles observed in section generally present alternating clear and dark bands. These have often been interpreted in terms of superimposed layers of different structure or composition. It has been shown, however that this material is homogeneous, and is formed by a twisted arrangement of microfibrils. The dense bands correspond actually to a microtomy artefact and they form dark single spirals in certain distorted areas of the cuticle. A model was proposed, involving the interaction between knife motion and microfibrils; it will be referred to as the stepped model, since the proposed mechanism results in the formation of steps at the surface of sections, on both faces. These steps are limited by structures resembling crests or cliffs, whose regular distribution produces alternating thick and thin bands in the section. This explains the observed contrast (Bouligand, (1972)). Two very interesting models were proposed later (Gordon and Winfree, (1978)) and are referred to as the cos F model and the sand model, but steps and crests are absent in these models. However, Giraud-Guille ((1986)), has shown very clearly the existence of these crests, which seem to be quite essential in this microtomy artefact. To clarify the debate, the texts defining the initial and the two new models are reproduced here and the difficulties encountered by each model are discussed. A mathematical formulation of this artefact is presented in an appendix to the present article; this leads to a more complete discussion of the possible models. Other factors are also taken into consideration: microfibril orientation and staining. The main factor of contrasts is undoubtedly the variation in thickness over a single section, as proposed in the stepped model.     

The structure of the cuticle and sheath of the infective juvenile of Trichostrongylus colubriformis

The infective third-stage juvenile of Trichostrongylus colubriformis is surrounded by its own cuticle as well as the incompletely moulted cuticle of the second-stage juvenile, which is referred to as the sheath. The sheath comprises an outer epicuticle, an amorphous cortical zone, a fibrous basal zone and an inner electron-dense layer. The basal zone of the sheath consists of three layers of fibres; the fibres are parallel within each layer, but the fibre direction of the middle layer is at an angle to that of the inner and outer layers. The cuticle comprises a complex outer epicuticle, an amorphous cortical zone and a striated basal zone. The lateral alae of the cuticle and the sheath are aligned and overlie the lateral hypodermal cords. The lateral alae of the sheath consist of two wing-like expansions of the cortical zone with associated specializations of the inner electron-dense layer which form a groove. The cuticular lateral alae consist of two tube-like expansions of the cortical zone. The lateral alar complex of the cuticle and the sheath may maximise locomotory efficiency and prevent rotation of the juvenile within the sheath.     

The extracellular matrix of the cuticle of Gordius panigettensis (Gordioiidae,Nematomorpha): observations by TEM,SEM and AFM

The cuticle of Gordius panigettensis (Sciacchitano, 1955) was studied by scanning electron microscopy (SEM), transmission electron microscopy (TEM) and atomic force microscopy (AFM). The cuticle is composed of 30-50 compact layers. The number of the layers is higher in the central part of the animal's body and decreases at the extremities. Each layer is composed of parallel tightly packed fibres approximately 640 nm in diameter and of indefinite length. The fibres run strictly parallel within each layer, while in adjoining layers they run at a variable angle from 45 degrees in the central body to 90 degrees in the extremities. Each fibre shows a barely detectable filamentous inner structure and is enveloped in a thin highly regular net formed by hexagonal meshes. Our results suggested that these fibres should be proteinaceous although non-collagenous. Thinner radial fibres run among the large fibres and across all the layers and span the whole thickness of the cuticle from the epithelial layer located deep underneath the large fibres up to the epicuticle on the external surface of the animal.     

Observations on the integument of the water mite Arrenurus major (Acari: Parasitengona)

A study of the integument of the aquatic mite Arrenurus major Marshall is presented. When the cuticle is examined with the unaided eye and the light microscope, it appears to possess numerous tiny pits. However, scanning electron micrographs of the cuticle reveal that it is a solid surface with topographical sculpturing of the epicuticle, indicating that the “pits” are an internal phenomenon. In cuticle which has been sectioned, areas devoid of cuticular material beneath the thin exocuticle are revealed. These areas are the pits which are goblet-shaped. The integument consists of five major strata. These are from the outside to the inside: (1) a superficial layer with a maximum observed thickness of 725 Å, (2) an epicuticle with a thickness of about 900 Å and composed of at least four sublayers, (3) an exocuticle with a thickness of about 1.5 Å. Fibers of the exocuticle are arranged in a Bouligand pattern and exhibit a regularly occurring discontinuity with a spacing of 200 Å. (4) An endocuticle ranging from 15 to 20 μ in thickness. The endocuticle is characterized by bandings which superficially resemble the lamellae of insects but are not homologous, microfibers which exhibit a preferred orientation, and the presence of the pits; and (5) an epidermis lying beneath the endocuticle and extending into the pits. Pore canals are present only in the exocuticle and have their origin at the apices of the pits. The pore canals contain a central filament, and a plug is present just beneath the epicuticle.     

The fibrous structure of coelacanth scales: A twisted ‘Plywood’

Isopedin is a network of collagen bundles present in the scales of most fishes. The scales of coelacanths show a remarkable three-dimensional arrangement of this network which is similar to a regularly twisted plywood. The successive fibrous layers cross at an angle which differs slightly from a right angle. It results that the whole system is twisted. The progressive rotation of the fibril direction is right-handed. Certain preferential orientations of fibrils have been observed, namely parallel to the growth rings. Such arrangements also exist in the embryonic cornea of birds and in the cuticle of certain insects, but do not present such an extensive and regular development.     

The L3 to L4 molt of Brugia malayi: real time visualization by video microscopy

Brugia malayi and other filarial parasites have been studied in great detail, especially in the context of human disease. In common with other nematodes, these organisms molt 4 times in their life cycles, but details of this process have not been described. We have recently developed an in vitro culture system that supports the L3 to L4 molt at high efficiency. This has permitted us to visualize, for the first time, details of this molt using real-time video microscopy. Molting is preceded by a phase of altered motility during which the larva exhibits contractile, coiling movements. The earliest evidence of ecdysis is a clearing at one end, more frequently caudal, caused by the larva retracting from that end. A cleavage develops in the cuticle near the head end, forming a rostral cap, which is continuous with the pharyngeal cuticle. Simultaneously, it retracts out of the cuticle using coiling and writhing movements. This process takes 5 to 10 min. Finally, it retracts out of the cap and extrudes the pharyngeal cuticle. Detachment of the pharyngeal cuticle is the final event in the process and continues up to an hour after the rest of the cuticle has been shed.     

Ellipticity of He-Ne-laser linearly polarized radiation reflected by leaves of plants and leaf cuticle characteristics

The elliptical polarization of He-Ne laser radiation reflected by plant leaves (Begonia Fista and Hedera Helix) was investigated. It was supposed that the elliptic polarization component of reflected radiation is caused by the acts of total reflection of incident radiation on cuticle inhomogeneities. By using the model of the phase-shifting ability of a scattering layer, the cuticle refractive index and the mean angle of inclination of cuticle roughness microfacets to leaf surface was evaluated.     

Viscoelastic nature of isolated tomato (Solanum lycopersicum) fruit cuticles: a mathematical model

Viscoelastic behaviour of isolated tomato fruit cuticle (CM) is well known and extensively described. Temperature and hydration conditions modify the mechanical properties of CM. Mechanical data from previous transient‐creep analysis developed in tomato fruit cuticle under different temperature and hydration conditions have been used to propose a rheological model that describes the viscoelastic nature of CM. As a composite material, the biomechanical behaviour of the plant cuticle will depend not only on the mechanical characteristics of the individual components by themselves but also on the sum of them. Based on this previous information, we proposed a two‐element model to describe the experimental behaviour: an elastic hookean element connected in parallel to a viscous element or Voigt element that will describe the mechanical behaviour of the isolated CM and cutin under the studied conditions. The main parameters of the model, E₁ and E₂ will reflect the elastic and viscoelastic behaviour of the cuticle. Relationship between these physical parameters and the change in CM properties were discussed in order to elucidate the rheological processes taking place in CM. This model describes both the influence of temperature and hydration and the behaviour of the isolated cutin and the inferred contribution of the cuticle fraction of polysaccharides when the whole cuticle is tested.     

An improved water content model for Periplaneta cuticle: Effects of epidermis removal and cuticle damage

All previously reported integumental permeabilities to water for Periplaneta at 20°C have been shown to be overestimates by at least an order of magnitude. Cuticle damage, apparently due to mechanical contact, is a common occurrence in cockroach cultures. Damage is increased by handling during experimental manipulation. Repair of the damage is readily observed in isolated individuals as cuticle permeability declines over a few days. Repaired cuticle permeability is not humidity dependent, but damage affecting a few per cent of the cuticle area, renders it so. Cuticle-water contents are slightly influenced by sex related size differences. Cuticle-water contents are unaffected by variations in ambient activity. Epidermal removal initiates a drop in cuticle-water content, apparently not involving active processes. Cuticle-water contents can be accurately predicted in any ambient vapour pressure using a two-layered model combining equilibrium water contents and component permeabilities. Incorporating a parallel unprotected endocuticle pathway into the model successfully simulates the observed humidity dependence of damaged permeabilities. Epidermal permeability cannot be less than 2× 10⁻⁵ cm.s⁻¹; 2000 times more permeable than the overlying cuticle.     

A theoretical approach to the relationship between wettability and surface microstructures of epidermal cells and structured cuticles of flower petals

Background and Aims The epidermal surface of a flower petal is composed of convex cells covered with a structured cuticle, and the roughness of the surface is related to the wettability of the petal. If the surface remains wet for an excessive amount of time the attractiveness of the petal to floral visitors may be impaired, and adhesion of pathogens may be promoted. However, it remains unclear how the epidermal cells and structured cuticle contribute to surface wettability of a petal.Methods By considering the additive effects of the epidermal cells and structured cuticle on petal wettability, a thermodynamic model was developed to predict the wetting mode and contact angle of a water droplet at a minimum free energy. Quantitative relationships between petal wettability and the geometries of the epidermal cells and the structured cuticle were then estimated. Measurements of contact angles and anatomical traits of petals were made on seven herbaceous species commonly found in alpine habitats in eastern Nepal, and the measured wettability values were compared with those predicted by the model using the measured geometries of the epidermal cells and structured cuticles.Key Results The model indicated that surface wettability depends on the height and interval between cuticular steps, and on a height-to-width ratio for epidermal cells if a thick hydrophobic cuticle layer covers the surface. For a petal epidermis consisting of lenticular cells, a repellent surface results when the cuticular step height is greater than 0·85 µm and the height-to-width ratio of the epidermal cells is greater than 0·3. For an epidermis consisting of papillate cells, a height-to-width ratio of greater than 1·1 produces a repellent surface. In contrast, if the surface is covered with a thin cuticle layer, the petal is highly wettable (hydrophilic) irrespective of the roughness of the surface. These predictions were supported by the measurements of petal wettability made on flowers of alpine species.Conclusions The results indicate that surface roughness caused by epidermal cells and a structured cuticle produces a wide range of petal wettability, and that this can be successfully modelled using a thermodynamic approach.     

Early Development of the Secretory Cavity of Peltate Glands in Humulus lupulus L. (Cannabaceae)

Early development of the secretory cavity of chemically fixed peltate glands in Humulus lupulus L. showed secretions with different densities, light, gray and dark, in the cytoplasm of disc cells and in the periplasmic space adjacent to the developing secretory cavity. Secretions were detected in the disc cell wall and subsequently in the developing secretory cavity under the subcuticular wall of the sheath. Light and gray secretions in the cavity possessed a membrane-like surface feature. Secretions were in contact with the irregular inner surface of the cuticle. Secretions contributed to the thickening of the cuticle, whereas the membrane-like surface feature contributed to a network of Cannabis striae distributed throughout the cuticle. This study supports an early development and organization of the secretory cavity in H. lupulus, parallel to those in Cannabis, and may represent common features for lipophilic glands in angiosperms.     

Effects of benzoylphenylurea on chitin synthesis and orientation in the cuticle of the Drosophila larva

Chitin is an essential constituent of the insect exoskeleton, the cuticle, which is an extracellular matrix (ECM) covering the animal. It is produced by the glycosyltransferase chitin synthase at the apical plasma membrane of epidermal and tracheal cells. To fulfil its role in cuticle elasticity and stiffness it associates with proteins, thereby adopting a stereotypic arrangement of helicoidally stacked sheets, which run parallel to the surface of the animal. One approach to understand the mechanisms of chitin synthesis and organisation is to dissect these processes genetically. However, since only a few genes coding for factors involved in chitin synthesis and organisation have been identified to date using the model arthropod Drosophila melanogaster insight arising from mutant analysis is rather limited. To collect new data on the role of chitin during insect cuticle differentiation, we have analysed the effects of chitin synthesis inhibitors on Drosophila embryogenesis. For this purpose, we have chosen the benzoylphenylurea diflubenzuron and lufenuron that are widely used as insect growth regulators. Our data allow mainly two important conclusions. First, correct organisation of chitin seems to directly depend on the amount of chitin synthesised. Second, chitin synthesis and organisation are cell-autonomous processes as insecticide-treated larvae display a mosaic of cuticle defects. As benzoylphenylurea are used not only as insecticides but also as anti-diabetic drugs, the study of their impact on Drosophila cuticle differentiation may be fruitful for understanding their mode of action on a cellular pathway that is seemingly conserved between vertebrates and invertebrates.     

Floral nectary ultrastructure of Prunus persica (L.) Batch cv. Forastero (Newcomer), an Argentine peach

 Flowers of Prunus persica (L.) Batch. cv. Forastero have an orange toral nectary. The nectariferous tissue was formed by densely packed parenchyma cells (secretory cells) and an epidermis with hairs and modified stomata. The epidermal cells were highly vacuolated with a striated cuticle. The ultrastructure of these cells contained a cytoplasm with endoplasmic reticulum, plastids, mitochondria and dictyosomes. Sub-epidermal cells were barely vacuolated and their ultrastructure was similar to that of the epidermal cells. Differences were observed only in the endoplasmic reticulum, which is organized in a parallel configuration. Plasmodesmata were found between adjacent secretory cells and between secretory and epidermal cells. An electron dense secretion occurred in the intercellular spaces and between the external tangential wall and the cuticle of the epidermal cells. According to the ultrastructural observations, the sugar solution could be passed through the symplast or the apoplast. The nectar could be exuded from the stomata and the micro-channels of the cuticle covering the epidermal cells. Received July 7, 2002; accepted September 24, 2002 Published online: June 2, 2003     

GLANDULAR CUTICLE FORMATION IN CANNABIS (CANNABACEAE)

Formation of the cuticle from components of the secretory cavity and subcuticular wall was studied by transmission electron microscopy of glandular trichomes of Cannabis prepared by high pressure cryofixation-cryosubstitution. Secretory vesicles in the secretory cavity resembled those localized in the subcuticular wall as well as the vesicle-related material associated with the irregular inner surface of the cuticle and appeared to provide precursors for thickening of the cuticle. Some contiguous vesicles in the secretory cavity and subcuticular wall lacked a surface feature at their point of contact, supporting an interpretation of vesicle fusion. Fibrillar matrix from the secretory cavity contributed fibrillar matrix to the subcuticular wall, and persisted as residual fibrillar matrix associated with secretory materials coalesced to the thickened inner surface of the cuticle. Elongated fibrils arranged in uniformly spaced parallel pairs contributed to the organization of fibrillar matrix in the subcuticular wall. Striae were evident in the outer portion of the cuticle, and appeared to represent sites of degraded residual fibrillar matrix associated with secretory materials coalesced to the inner cuticular surface. This study supports an interpretation that contents of secretory vesicles from the secretory cavity contribute to formation of glandular cuticle.     

Structure of the Cuticle of Ceramonema carinatum (Chromadorida: Ceramonenatidae)

The cuticle of Ceramonema carinatum (Chromadorida: Ceramonematidae) is described and illustrated from scanning and transmission electron microscopy. Each of ca. 200 annules is composed of a single ring with eight external flat faces (plates), which are divided by longitudinal ridges formed by pairs of parallel upstanding vanes. Vanes and plates overlap those of the adjacent annules. Longitudinal ridges extend from the cephalic capsule to the tail spike. On the cephalic capsule a simple ridge extends each of the eight ridges to a position just anterior to the amphid. Cuticular plates are formed from the electron-dense cortical layer and contain lacunae filled with fine fibrils. The vanes are denser, with laminations on a central core. In the annular grooves between the plates there is an electron-lucent layer, which it is suggested, by comparison with other nematodes, is the basal layer. An epicuticle overlies the cortical plates, the vanes, and the interannular lucent layer. Cuficular structure is compared with that of other Ceramonematidae and related nematodes.