Endocuticle Layer Research Articles

The ultrastructural development and 3D reconstruction of the transparent carapace of the ostracod Skogsbergia lerneri

The Skogsbergia lerneri is a marine ostracod which possesses a carapace that is both protective and transparent. Since development of this carapace and how it is maintained in the adult is not known, the aim of this investigation was to carry out an in-depth ultrastructural study of the ostracod carapace at different developmental stages. Standard transmission electron microscopy and novel serial block face scanning electron microscopy (SBF-SEM) were undertaken to discern carapace ultrastructure in both two and three dimensions. Analysis revealed a carapace consisting of the same basic layer structure as other myodocopid ostracods, namely an epicuticle, exocuticle, endocuticle and membranous layer, but with a thinner adult carapace of mean thickness of 19.2 ± 1.78 µm, n = 5. The carapace layers, except for instar 1 ostracods, had similar relative proportions throughout development. The endocuticle and membranous layer thickened through advancing developmental stages due to an increase in calcified crystalline polyhedrons and a greater number of chitinous lamellae in the membranous layer. Crystalline polyhedron dimensions were significantly smaller near the boundary with the membranous layer. The borders between the carapace layers were indistinct; SBF-SEM revealed an abundance of epicuticle projections into the exocuticle and apparent gradual merging at the boundary of the exocuticle and the endocuticle. Here, we discuss how the S. lerneri carapace layer structure has evolved to serve a specific mechanical function, allowing surface protection and rigidity. In addition, we suggest that the lack of pigment and graduated layer boundaries contribute to the transparency of the carapace.

Structural Changes and Mechanical Resistance of Claws and Denticles in Coconut Crabs of Different Sizes.

Simple SummaryUnderstanding the diverse mechanisms by which organisms achieve exceptionally high mechanical properties may enable the development of unique, biologically inspired materials. We assessed the microstructure, composition, and mechanical resistance of the pinching side with denticles and of the outer side without denticles in robust claws of coconut crabs with body weight (BW) of 300 g to 1650 g. Surprisingly, they were independent of BW except for low hardness near the surface of the denticles of a small crab of 300 g. Although the microstructure of the denticles was clearly different from that of the exocuticle, their mechanical resistance indicated the same maximum value. The denticle can be regarded as a bulge of the cuticle without phosphorus. The design principles found in the exoskeleton provided promising opportunities for the research and development of novel structural materials.The exoskeleton of the pinching side of claws with denticles and of the outer side without them on the coconut crab, Birgus latro, which is a rare organism, were studied using a materials science approach. The mechanical resistance of three claws of different sizes was investigated along the exoskeleton thickness from the outer surface to the inner surface, and the results were compared, including the contribution of the microstructure and chemical compositions. Mechanical properties, hardness (H) and stiffness (Er), were probed through nanoindentation tests. The results showed the H, Er, microstructures, and chemical components of the exocuticle and endocuticle layers were almost the same, in a BW range of 300 g to 1650 g. At the same time, the H and Er near the surface of the denticles of a small coconut crab of 300 g were lower than those of other large coconut crabs. The microstructure of the denticles was clearly different from that of the exocuticle, but the maximum mechanical properties near their surface indicated almost the same values, Hmax = 4 GPa and Er(max) = 70 GPa, regardless of being on the pinching side or the outer side. A denticle can be regarded as a bulge of the cuticle without phosphorus and with high magnesium. The results provided novel information that expanded our knowledge about the claw microstructure of coconut crabs with different body sizes, and may be used in further studies

Endocuticle is involved in caste differentiation of the lower termite.

Caste differentiation in termites is one of the most conspicuous examples of facultative polyphenism in animals. It is clear that specific cuticular formation occurs in hard exocuticles during caste differentiation. However, the developmental pattern of the soft endocuticle in the differentiation pathways of castes is unknown. To reveal whether the endocuticle is involved in caste differentiation, we compared the exocuticle and endocuticle thickness of individuals in 2 pathways (nymph line and worker line) of caste differentiation in the termite Reticulitermes aculabialis. The endocuticle protein genes were identified by transcriptome analysis and the expression patterns of these genes were confirmed in caste differentiation. We found that the endocuticle structure showed dynamic changes in 2 pathways, and the first difference in endocuticle structure occurred after larvae differentiation bifurcated into workers and nymphs. The thinning of the endocuticle was a significant event from nymphs developing into alates with the thickest exocuticle and thinnest endocuticle. The thickest endocuticle layers were found in the heads of the workers and the ultrastructure of the endocuticle in the heads was more complex than that in the thorax–abdomens. Six endocuticle protein genes were identified and annotated as endocuticle structural glycoproteins SgAbd-2, SgAbd-9, and Abd-5. The expression levels of endocuticle protein genes changed dramatically during caste development and the expression levels in neotenic reproductives (secondary reproductives) were significantly higher than those in alates (primary reproductives). These results reveal the roles of endocuticles in caste differentiation and adaptation to the environment.

Nanoscale Molecular Characterization of Hair Cuticle Cells Using Integrated Atomic Force Microscopy-Infrared Laser Spectroscopy.

The hair cuticle provides significant protection from external sources, as well as giving rise to many of its bulk properties, e.g., friction, shine, etc. that are important in many industries. In this work, atomic force microscopy-infrared spectroscopy (AFM-IR) has been used to investigate the nanometer-scale topography and chemical structure of human hair cuticles in two spectral regions. AFM-IR combines atomic force microscopy with a tunable infrared laser and circumvents the diffraction limit that has impaired traditional infrared spectroscopy, facilitating surface-selective spectroscopy at ultra-spatial resolution. This high resolution was exploited to probe the protein secondary structures and lipid content, as well as specific amino acid residues, e.g., cystine, within individual cuticle cells. Characterization across the top of individual cells showed large inhomogeneity in protein and lipid contributions that suggested significant changes to physical properties on approaching the hair edge. Additionally, the exposed layered sub-structure of individual cuticle cells allowed their chemical compositions to be assessed. The variation of protein, lipid, and cystine composition in the observed layers, as well as the measured dimensions of each, correspond closely to that of the epicuticle, A-layer, exocuticle, and endocuticle layers of the cuticle cell sub-structure, confirming previous findings, and demonstrate the potential of AFM-IR for nanoscale chemical characterization within biological substrates.

Gene functions in adult cuticle pigmentation of the yellow mealworm, Tenebrio molitor

In many arthropod species including insects, the cuticle tanning pathway for both pigmentation and sclerotization begins with tyrosine and is responsible for production of both melanin- and quinoid-type pigments, some of which are major pigments for body coloration. In this study we identified and cloned cDNAs of the yellow mealworm, Tenebrio molitor, encoding seven key enzymes involved in this pathway including tyrosine hydroxylase (TmTH), DOPA decarboxylase (TmDDC), laccase 2 (TmLac2), Yellow-y (TmY-y), arylalkylamine N-acetyltransferase (TmAANAT1), aspartate 1-decarboxylase (TmADC) and N-β-alanyldopamine synthase (Tmebony). Expression profiles of these genes during development were analyzed by real-time PCR, revealing development-specific patterns of expression. Loss of function mediated by RNAi of either 1) TmTH or TmLac2, 2) TmDDC or TmY-y, and 3) TmAANAT1, TmADC or Tmebony resulted in pale/white, light yellow/brown and dark/black adult body coloration, respectively. In addition, there are three distinct layer/regional pigmentation differences in rigid types of adult cuticle, a brownish outer exocuticle (EX), a dark pigmented middle mesocuticle (ME) and a transparent inner endocuticle (EN). Decreases in pigmentation of the EX and/or ME layers were observed after RNAi of TmDDC or TmY-y. In TmADC- or Tmebony-deficient adults, a darker pigmented EX layer was observed. In TmAANAT1-deficient adults, trabeculae formed between the dorsal and ventral elytral cuticles as well as the transparent EN layer became highly pigmented. These results demonstrate that knocking down the level of gene expression of specific enzymes of this tyrosine metabolic pathway leads to abnormal pigmentation in individual layers and substructure of the rigid adult exoskeleton of T. molitor.

Comparative transcriptomic analysis and endocuticular protein gene expression of alate adults, workers and soldiers of the termite Reticulitermes aculabialis

BackgroundThe insect cuticle is mainly composed of exocuticle and endocuticle layers that consist of a large number of structural proteins. The thickness and synthesis of the exocuticle depend on different castes that perform various functions in alates, workers and soldiers. However, it is not clear whether the soft endocuticle is involved in the division of labour in termite colonies. To reveal the structural characteristics of the endocuticle in different castes, we investigated the thickness of endocuticle layers in alates, workers and soldiers of the termite Reticulitermes aculabialis, and then we sequenced their transcriptome and detected the endocuticle protein genes. The differential expression levels of the endocuticular protein genes were confirmed in the three castes.ResultsWe found that there was a great difference in the thickness of the endocuticle among the alates, soldiers and workers. The thickest endocuticle layers were found in the heads of the workers 7.88 ± 1.67 μm. The endocuticle layer in the head of the workers was approximately three-fold and nine-fold thicker than that in the heads of soldiers and alates, respectively. The thinnest endocuticle layers occurred in the head, thorax and abdomen of alates, which were 0.86 ± 0.15, 0.76 ± 0.24 and 0.52 ± 0.17 μm thick, respectively, and had no significant differences. A total of 43,531,650 clean sequencing reads was obtained, and 89,475 unigenes were assembled. Of the 70 identified cuticular protein genes, 10 endocuticular genes that belong to the RR-1 family were selected. qRT-PCR analysis of the five endocuticular genes (SgAbd-2, SgAbd-9, Abd-5, SgAbd-2-like and Abd-4-like) revealed that the endocuticle genes were more highly expressed in workers than in soldiers and alates.ConclusionThese results suggest that SgAbd and Abd are the key components of the endocuticle. We infer that the thicker endocuticle in workers is helpful for them to perform their functions against environmental stress.

Recognizing incipient epizootic shell disease lesions in the carapace of the American lobster, Homarus americanus

Causal factors leading to epizootic shell disease (ESD) lesions in American lobster, Homarus americanus H. Milne-Edwards, 1837, are not well understood. We explore the structural and physiological bases for development of ESD from preclinical stages invisible to unaided eye to early visible stages. We present a lobster shell model, which develops structural functional vulnerability and suggests plausible routes to ESD. Medallions of carapace cuticle were obtained from carapace fixed with protocols to minimize movement of mineral and macromolecular components. Rapid processing of medallions was used to encourage large sample sizes compatible with environmental surveys. One- and two-dimensional analytic maps of polished sections of the cuticle, obtained with an electron microprobe, described the composite mineral and polymeric structures. Micro-Raman spectroscopy was used to identify bond properties of phosphates and carbonates, as well as signatures of organic structures. The frequency and properties of structures identified can be monitored through the lobster molting cycle using a high throughput application of micro-computed tomography (μCT). We observed density differences in the calcite layer, exocuticle, and endocuticle, and the frequency and structure of CaCO3 structures in the endocuticle and membranous layer of carapace cuticle during chosen stages of the molting cycle. The correlative microscopy and μCT of shell structures provides improved understanding of the lobster cuticle structure. Detailed structural differences quantified through development and under different environmental conditions can provide insight into causes and vulnerabilities associated with ESD.

Both stiff and compliant: morphological and biomechanical adaptations of stick insect antennae for tactile exploration.

Active tactile exploration behaviour is constrained to a large extent by the morphological and biomechanical properties of the animal's somatosensory system. In the model organism Carausius morosus, the main tactile sensory organs are long, thin, seemingly delicate, but very robust antennae. Previous studies have shown that these antennae are compliant under contact, yet stiff enough to maintain a straight shape during active exploration. Overcritical damping of the flagellum, on the other hand, allows for a rapid return to the straight shape after release of contact. Which roles do the morphological and biomechanical adaptations of the flagellum play in determining these special mechanical properties? To investigate this question, we used a combination of biomechanical experiments and numerical modelling. A set of four finite-element (FE) model variants was derived to investigate the effect of the distinct geometrical and material properties of the flagellum on its static (bending) and dynamic (damping) characteristics. The results of our numerical simulations show that the tapered shape of the flagellum had the strongest influence on its static biomechanical behaviour. The annulated structure and thickness gradient affected the deformability of the flagellum to a lesser degree. The inner endocuticle layer of the flagellum was confirmed to be essential for explaining the strongly damped return behaviour of the antenna. By highlighting the significance of two out of the four main structural features of the insect flagellum, our study provides a basis for mechanical design of biomimetic touch sensors tuned to become maximally flexible while quickly resuming a straight shape after contact.

Cuticular protein defective Bamboo mutant of Bombyx mori is sensitive to environmental stresses

Insect cuticle acts as a primary protective barrier against environment stresses that may directly impact the insect body. Here, we report the mechanical defense function of a structural cuticular protein, BmorCPH24, to environmental stresses using a silkworm Bamboo (Bo) mutant with this gene mutation. Ultraviolet (UV) irradiation and topical application of an acetone insecticide were used as environmental stresses to determine the differences in susceptibility between Bo and wild-type larvae. UV irradiation resulted in a sunburn phenotype in the Bo strains earlier than the wild-type indicating the sensitivity of Bo. Higher malondialdehyde (MDA) content and a lower survival ratio were also observed in the Bo strains. Treatment with deltamethrin revealed that Bo larvae were more sensitive to insecticides than the wild-type. Furthermore, cuticle analysis by microsection revealed thinner cuticle and a significant decrease in the endocuticle layer (∼64.0%) in Bo. These results suggest that BmorCPH24 mutation can lead to deficiency in resources required to construct the cuticle in Bo resulting in thin cuticle and reduced resistance to UV and insecticides. These results provide us new insight into the role of structural cuticular proteins in insect cuticle against environment stresses.

Expression profiles of cuticular protein genes in wing tissues during pupal to adult stages and the deduced adult cuticular structure of Bombyx mori

We aimed to clarify the regulation of cuticular protein (CP) gene expression and the resulting insect cuticular layers by comparing the expression pattern of CP genes and related ecdysone-responsive transcription factor (ERTF) genes, the coding amino acid sequences of CP genes, and histological observation. The expression of CP and ERTF genes during pupal and adult stages was examined via qPCR. The number of CP genes expressed during pupal and adult stages decreased as compared to that during prepupal to pupation stages, particularly in CPRs. The peaks of transcripts were observed at P5, P6, P9, A0, and A1. The order of the ERTF and CP genes expression resembled that at prepupal and pupation stages, suggesting the relatedness of ERTFs with the same CP genes at both stages. Moreover, the order of expression of CP genes resembled that in prepupal to pupation stages, by which we presumed the spaces of CPs in the epicuticle, outer-exocuticle, inner-exocuticle, endocuticle layer.

Microstructure and mechanical properties of rostrum in Cyrtotrachelus longimanus (Coleoptera: Curculionidae)

ABSTRACTThe microstructure, composition and mechanical properties of the rostrum in Cyrtotrachelus longimanus (JHC Fabre) were studied utilizing light, fluorescent, scanning electron microscopy (SEM) and energy-dispersive spectroscopy. SEM images show the morphological characteristics of rostrum’s cross section; it is a typical lightweight multilayer structure – one rigid exocuticle layer and dense endocuticle layers, which construct unevenly overlapping fiber structures. The composition analysis of the rostrum shows that it is mainly composed of C, H, N, O, as well as some metal elements and microelements, such as Mg, Si, Zn, Ca and Na, which contribute to its mechanical performance. The mechanical properties of the rostrum were tested by the electronic universal testing machine, which shows it has high-specific strength and is almost the same as that of the stainless steel. The results may provide a biological template to inspire biomimetic lightweight structure design.

Site-specific characterization of beetle horn shell with micromechanical bending test in focused ion beam system.

This paper proposed an in-situ manipulation/test method in focused ion beam for characterizing micromechanical properties of beetle horn shell. The challenge in precise and accurate fabrication for the samples with complex geometry was overcome by using nano-manipulators having multi-degree of freedom and a micro-gripper. With the aid of this specially designed test platform, bending tests were carried out on cantilever-shaped samples prepared by focused ion beam milling. Structural differences between exocuticle and endocuticle layers of beetle horn shell were explored and the results provided insight into the structural advantages of each biocomposite structure.

3D-Xray-tomography of American lobster shell-structure. An overview

A total inventory of density defined objects of American lobster cuticle was obtained by using high resolution 3D Xray tomography, micro-computed-tomography. Through this relatively unbiased sampling approach several new objects were discovered in intermolt cuticle of the lobster carapace. Using free and open-source software the outlines of density-defined objects were obtained and their locations in 3D space calculated allowing population parameters about these objects to be determined. Nearest neighbor distances between objects allowed interpretations of structural relationships of and between objects. Several organule types are recognized by their structural outlines and density signatures. A hierarchy of organule types and distribution suggests they develop during sequential molts. New objects (stalactites, Bouligand spirals, and basal granules) are described as mineral structures with well defined morphological character, allowing them to be recognized by their descriptive names and distributions. The three new cuticular objects appear to represent forms of calcium carbonate deposition in the sequential layers of exocuticle, endocuticle and membranous layer and may provide signatures of underlying epidermal cells.

Effect of microstructure on the mechanical and damping behaviour of dragonfly wing veins.

Insect wing veins are biological composites of chitin and protein arranged in a complex lamellar configuration. Although these hierarchical structures are found in many ‘venous wings' of insects, very little is known about their physical and mechanical characteristics. For the first time, we carried out a systematic comparative study to gain a better understanding of the influence of microstructure on the mechanical characteristics and damping behaviour of the veins. Morphological data have been used to develop a series of three-dimensional numerical models with different material properties and geometries. Finite-element analysis has been employed to simulate the mechanical response of the models under different loading conditions. The modelling strategy used in this study enabled us to determine the effects selectively induced by resilin, friction between layers, shape of the cross section, material composition and layered structure on the stiffness and damping characteristics of wing veins. Numerical simulations suggest that although the presence of the resilin-dominated endocuticle layer results in a much higher flexibility of wing veins, the dumbbell-shaped cross section increases their bending rigidity. Our study further shows that the rubber-like cuticle, friction between layers and material gradient-based design contribute to the higher damping capacity of veins. The results of this study can serve as a reference for the design of novel bioinspired composite structures.

Micromorphology of the elytral cuticle of beetles, with an emphasis on weevils (Coleoptera: Curculionoidea)

The elytral cuticle of 40 beetle species, comprising 14 weevils (Curculionoidea) and 26 representatives of other taxa, is examined. All weevils and 18 other species have an endocuticle with prominent macrofibers, which corresponds to a modified pseudo-orthogonal cuticle. Angles between successive layers of macrofibers range between 30° and 90°, but are constantly less than 60° in weevils. In all Curculionoidea, as well as in one buprestid and one erotylid species exo- and endocuticle are densely interlocked. In the weevil Sitophilus granarius, transmission electron microscopy revealed vertical microfibrils extending from the exocuticle between the macrofibers of the underlaying endocuticle. Vertical microfibrils connecting successive macrofiber layers of the endocuticle were observed in S. granarius and Trigonopterus nasutus.Distinct cuticular characters are traced on a beetle phylogeny: the angles between unidirectional endocuticle layers; the presence and the shape of endocuticular macrofibers; and the interlocking of exo- and endocuticle. While character traits seem to be more or less randomly distributed among Coleoptera, the Curculionoidea have a uniform groundplan: The “weevil-specific” combination of characters includes 1) interlocking of exo- and endocuticle, 2) an endocuticle with distinct ovoid macrofibers embedded in a matrix and 3) comparatively small angles between successive endocuticular layers. Thus, phylogenetic constraints appear equally important to functional factors in the construction of the weevil elytron.

Direct age determination by growth band counts of three commercially important crustacean species in Chile

Management of commercially important crustacean stocks has been hindered by the inability to directly determine individual age. A lack of comprehensive age information leads to poor understanding of life history schedules, difficulty in estimation of parameters necessary for modeling population dynamics, and uncertainty in the determination of appropriate harvest levels. This study applied a novel direct age determination method based on band counts to three Chilean crustacean species: red squat lobster (Pleuroncodes monodon), yellow squat lobster (Curvimunida johni), and nylon shrimp (Heterocarpus reedi) for the first time. The growth bands were observed and counted in the endocuticle layer in thin longitudinal sections of the meso-cardiac ossicles of the gastric mills in the two squat lobster species and in the eyestalks of the nylon shrimp. Comparison of growth band counts with independent estimates of age strongly suggests that the bands form annually, thus providing a direct and accurate method of age determination in all of the species examined. Sex-specific growth curves were produced and demonstrated that the females grow at a slower rate than the males in the three species. Size and age-at-sexual maturity were also estimated. Application of this technique to known-age individuals will be necessary to validate band counts as indicators of chronological age.

Effect of pH on growth and survival in the freshwater crayfishAustropotamobius pallipes

Conservation of endangered British crayfish (Austropotamobius pallipes) involves translocation to isolated waterbodies (Ark Sites) and reintroductions from hatchery stock. The species is restricted in the wild to relatively alkaline waterbodies. The optimum pH conditions that maximise growth and survival are unknown. This information would benefit the selection of Ark Sites and hatchery water chemistry. Here we measure growth and survival of juvenile A. pallipes in the laboratory using three pH levels from within the species' natural range. Survival at pH 8.6 was high (94%), whereas that at lower pH levels was low (pH 6.5, 25%; pH 7.1, 34%). Growth (moult increment and frequency) was also higher at pH 8.6. Survival was also lower for Thelohania contejeani-infected animals, with a trend towards higher mortality in infected individuals at lower pH. Electron microscopy revealed a lower cuticle thickness at pH 6.5 than higher pH which was associated with an apparent increase in the number of endocuticle layers. We therefore recommend that high pH (ca. 8.6) be used as a target for Ark Site selection and hatchery water chemistry in order to maximise survival and growth. (

Micro-tensile Testing of the Lightweight Laminated Structures of Beetle Elytra Cuticle

Quantitative measurements of the mechanical properties of insect cuticle are a useful tool in the development of bio-mimetic materials suitable for industrial products. In this study, a micro-tensile tester was used to investigate the mechanical properties of elytra cuticle of the dung beetle (Copris ochus Motschulsky). Micro tensile testing show that: yield strength (Fs) =17.12±3.55N, Maximum tensile (Fb) =14.74±4.11N, yield strength ( ) =1.4±0.15GPa, tensile strength ( )=1.2±0.21GPa, elastic modulus(E) =14.56±4.20GPa, plastic index(δ) =0.241±0.10. Tensile elongation of the specimens was between 12.1-36.3%. Our results demonstrate that the elytra possess ductile material characteristics. Field emission scanning electron microscopy (FESEM) was used to investigate the detailed structure of the elytra cross section in both the transverse and longitudinal directions. In the transverse direction, the fibers of the deeper layers of the endocuticle are orientated in a constant, rotated angle with neighboring fibers rotated in relation to the each other in the same direction. The fibers in the longitudinal direction show that the epicuticle, exocuticle and endocuticle layers clearly create a parallel hierarchical structure. We believe this is a result of the composite effect of the hierarchical structure. Finally, we developed a laminated model based on the parameters provided by tensile testing, FESEM imaging and nanoindentation measurements, and compared the results of the model to our experimental results. Key words: nanoindentation; micro-tensile testing; coupled analysis; laminated structure

FINE STRUCTURE AND MINERALIZATION OF THE GASTRIC MILL IN THE CRAYFISH PROCAMBARUS CLARKII DURING INTERMOLT STAGE

Abstract This paper aims to elucidate the structure and characteristic mineralization of the gastric mill cuticle in the crayfish Procambarus clarkii (Girard) using light and transmission electron microscopy. Structure and mineralization of the lateral teeth of the gastric mill in the intermolt stage are documented. The lateral tooth is composed of two types: incisor-like teeth and molar-like teeth. The tooth structure is divided into three regions: “large-cusp region” is the protuberance region in the incisor-like teeth, “small-cusp region” is the protuberance region in the molar-like teeth, and “marginal region” is the surrounding region. The cuticle is divided into epicuticle, exocuticle, endocuticle, and membranous layer. The cuticle of the large-cusp region lacks epicuticle and endocuticle, while the cuticle of the small-cusp region lacks the epicuticle. The cuticle of the marginal region is composed of epicuticle, exocuticle, endocuticle, and membranous layer. Ultrastructurally, the exocuticle of th...

Cuticle cell defects in lamellar ichthyosis hair and anomalous hair shaft syndromes visualized after detergent extraction.

The biochemical bases for fragility in most of the rare brittle hair shaft syndromes are unknown. The hypothesis being investigated in several syndromes is that the hair cuticle cells show defects in cross-linked protein features. Since transglutaminases stabilize protein structures by cross-linking them, hair from autosomal recessive lamellar ichthyosis patients lacking keratinocyte transglutaminase was examined to find whether this enzyme participates in hair shaft stabilization. Hair shaft samples from patients afflicted with lamellar ichthyosis or several brittle hair syndromes were examined ultrastructurally by transmission electron microscopy after vigorous extraction with detergent and reducing agent to reveal cross-linked protein features. In hair cuticle cells from three patients with lamellar ichthyosis the marginal band (A layer) was present but nonuniform and subject to breakage, while in a fourth sample it was missing altogether. The exocuticle appeared less dense than in normal hair, consistent with extensive protein loss during detergent extraction. In cuticle cells from trichothiodystrophy hair, the exocuticle layer was essentially fully extractable in one sample, while in two others (from siblings) the exocuticle appeared less dense and the A layer was absent or greatly reduced in thickness. A sample of proximal trichorrhexis nodosa also displayed defects in cuticle cells, in which the endocuticle layer appeared subject to rupture. The outer cuticle cells in monilethrix hair displayed a thinning of the A layer and less dense exocuticle, while the cortex exhibited regions lacking remnant cell borders. Pili annulati hair displayed large gaps in the cortex, presumably reflecting the air-filled cavities characteristic of this syndrome, and wavy borders of some cuticle cells. Observations with autosomal recessive lamellar ichthyosis hair indicate that keratinocyte transglutaminase has a major role in maturation of the cuticle but appears unnecessary for stabilization of cell borders in the cortex. Defective cross-linking was also evident in cuticle cells of trichothiodystrophy and monilethrix.