The structure and formation of the cuticulin layer in the epicuticle of an insect, Calpodes ethlius (Lepidoptera, Hesperiidae)

The cuticulin layer is defined as the dense lamina (120–175 Å thick in Calpodes larvae, depending upon the stage) forming the outer part of the epicuticle in insects. It completely invests an insect except for the gut and the openings of some sense organs. It is the first layer to be secreted during the formation of new cuticle. The formation of the cuticulin membrane may be a useful model for studying the origin of membranes in general. It arises as a triple layer de novo and is not a modified plasma membrane. Growth is by accretion at the edges of patches of cuticulin which increase in area until they cover the new surface. The triple layer (i.e. three dense laminae) may develop striations about 30 Å apart transverse to the membrane, which perhaps form a sieve allowing small molecules to pass while protecting the cell from enzymes in the molting fluid. A similar porous structure persists in the tracheoles. After the resorption of molting fluid the triple layered structure again becomes obvious and the outermost layer separates from the other two to become what may be the surface lipid monolayer. The surface patterns in cuticle of various sorts probably arise by buckling of the cuticulin layer as it increases in surface area.     

The hormonal coordination of cuticulin deposition and morphogenesis in Drosophila imaginal discs in vivo and in vitro

Cuticulin is the first layer of the insect cuticle to be deposited and is laid down as a continuous inelastic sheet over the apical surface of cuticle-secreting cells. During metamorphosis in Drosophila melanogaster, imaginal discs deposit the cuticulin layer of the pupal cuticle between 3 and 7 hr after puparium formation. This is a period of rapid morphogenesis involving cell shape changes and cell rearrangements. We have examined cuticulin deposition in vivo and in vitro with a view to understanding the coordination of cuticulin deposition with morphogenesis. We find that the optimum hormonal regimen (of the steroid hormone, 20-hydroxyecdysone) for the completion of both morphogenesis and cuticulin deposition in vitro parallels the changes in hormone titer observed in vivo. We also find that cuticulin is deposited last over cell boundaries, thereby allowing cell rearrangements to occur as cuticulin is laid down. We have identified in vitro conditions under which cuticulin deposition is completed precociously, inhibiting further morphogenesis. Cytochalasin B and colchicine do not inhibit cuticulin deposition and we therefore conclude that an intact cytoskeleton is not necessary for secretion of this extracellular structure. Finally, we present a preliminary protocol for the partial purification of cuticulin synthesized in vitro by mass isolated discs.     

The properties of the lining membrane of the insect tracheal system

In the surface layer of the lining cuticle of the tracheae of adult Calliphora there is no sign of any waterproofing layer of cuticulin (sclerotin + lipid) as seen in the surface of the general body cuticle. In a few insects: Calliphora adult thorax, Rhodnius adult tracheae serving the ovary, Periplaneta abdominal tracheae, it has been possible to introduce silver hydroxide solution into the lumen of tracheae in the living insect. In each case the silver hydroxide reacted at room temperature with the argentaffin structures in the cuticle, as happens in the soft surface cuticle of Rhodnius larva before moulting or after gentle abrasion. In the thorax of Calliphora the taenidia of the tracheae are stiffened by argentaffin cuticulin. but immediately upon entering the cleft in the flight muscle the taenidia disappear and are replaced by simple folds, so that no stiff taenidia enter the muscle and there is no argentffin material deeper in the flight muscle system.     

Fine structure and morphogenesis of the sclerite epicuticle in the Atlantic shore crab Carcinus maenas

Three basic sublayers are identified in the epicuticle of the mineralised sclerites of the Atlantic shore crab Carcinus maenas (Crustacea, Decapoda): the surface coat, the cuticulin layer, and the inner epicuticle. Their morphogenesis and subsequent changes are described throughout the moulting cycle in the normal cuticle and the cuticular structures, namely the sensory bristles and epicuticular spines. At first, the cuticulin layer begins to form just after apolysis. This layer is built directly over the plasma membrane and immediately appears as a membrane-like structure 40 nm thick, composed of five symmetrically arranged laminae: two inner electron-lucent leaflets sandwiched between two thick electron-dense leaflets and separated by a thin dense median stratum. Elaboration of the inner epicuticle below the cuticulin layer is thought to occur via an intussusceptive process involving the pore canal cell extensions as transport routes. The inner epicuticle is made of vertically oriented microfibres embedded in an electron-dense matrix material. During the second half of the premoult period, the surface coat is deposited on the upper side of the cuticulin layer.     

The fine structure and deposition of the larval cuticle of the sheep blowfly (Lucilia Cuprina)

The cuticle of Lucilia is composed of an untanned endocuticle and a complex epicuticle of four layers, superficial layer, outer epicuticle, cuticulin and dense layer. The outer epicuticle and attached epicuticular filaments are resistant to acid hydrolysis. During deposition of the cuticle of each larval instar, the cuticulin and dense layers are formed first, followed by the outer epicuticle, which appears to be laid down by secretions from the epidermis passing through the cuticulin via epicuticular filaments. The outer epicuticle is found in the position normally occupied by the wax layer of other insect species.     

Fine structure of the cuticle of the black widow spider with reference to surface lipids

The surface and transverse sections of the cephalothorax, abdomen, and walking leg cuticle of the black widow spider, Latrodectus hesperus, were examined by scanning and transmission electron microscopy. Cuticle that was untreated prior to normal EM preparative procedures was compared with cuticle subjected to lipid solvents and/or concentrated alkali. The surface of untreated dorsal cephalothorax cuticle contained droplets and a lipid film that obscured fine surface detail. Immersing the cuticle in chloroform: methanol removed the droplets and lipid film, exposing previously covered openings to dermal gland ducts. An epicuticle, exocuticle, and endocuticle were present in all transverse sections of cuticle as was a complex system of pore and wax canals that connected the epidermis with the cuticle surface. The epicuticle of the walking leg was composed of three sublayers: outer membrane, outer epicuticle, and the dense homogeneous layer. A cuticulin layer was not observed. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax/pore canals.     

Design of an insect cuticle associated with osmoregulation: the porous plates of chloride cells in a mayfly nymph

In mayfly nymphs of the genus Coloburiscoides, cell complexes with an osmoregulatory function (so-called chloride cells) are found in the integuments of the oral gills, the abdominal gills and gill filaments, the coxae and the thoracic sternites. The cuticle overlying each cell complex is a rigid circular plate which is known to be porous to colloidal lanthanum suspensions. The present study shows that the plate is composed only of the cuticulin and dense layers of the epicuticle. Both layers have substructures built of subunits on almost perfect hexagonal lattices. The lattice spacings are 53 and 9.5 nm for the dense layer and the cuticulin layer respectively. During moulting the apical plasma membrane of the chloride cell remains adpressed to the old porous plate. The new porous plate is formed from a new chloride cell which intrudes from the base of the integument. Throughout the moult small pores persist in the new and otherwise continuous cuticle to allow continuity of the cytoplasm of the apical and basal portions of the old chloride cell. It is thought that this phenomenon allows osmoregulatory function of the chloride cell complex to be maintained during the moult.     

Structure of the cuticle of the common house cricket with reference to the location of lipids

Cuticle segments from the thorax, abdomen, and jumping legs of the house cricket. Acheta domesticus, were examined using histological techniques for light microscopy, scanning and transmission electron microscopy, and direct examination of frozen-fractured cuticle. The surface of untreated cuticle is covered by a lipid film which obscures fine surface detail. Standard EM preparative procedures, as well as washing the cuticle with ethanol before examination, remove this film exposing previously covered openings to dermal gland ducts and wax canals. An epicuticle, exocuticle, mesocuticle, endocuticle, and a deposition layer were present in all transverse sections of cuticle. Light microscopy showed that the exocuticle and mesocuticle are heavily impregnated with lipids, whereas there is little lipid associated with the endocuticle. Frozen-fractured cuticle clearly shows the ‘plywood’ structure of the meso- and endocuticle, while the exocuticle fractures as if it were a solid sheet. The epicuticle is composed of a dense homogeneous layer, cuticulin, outer epicuticle, and the outer membrane. Superficial wax was detected only in cuticle samples prepared using vinylcyclohexane dioxide as a polar dehydrant. The results were used to construct a comprehensive model of the cuticle of A. domesticus.     

Cuticle formation in the embryo of Drosophila melanogaster

In Drosophila melanogaster embryos cuticle formation occurs between 12 and 16 hours of development at 25°C. The formation of the cuticulin and the protein epicuticular layers is simultaneous in the hypoderm, the tracheoblasts, and the fore- and hindgut cells. The cuticulin forms as a dual lamina, aggregating from granules secreted by the hypodermal cells. This is followed by the formation of a granular protein epicuticle and finally by the secretion of a mixed fibrous and granular endocuticle. All secretory cells are relatively simple in their ultrastructure. The secretory process is a membrane phenomenon, occurring at the tips of hypodermal microvillae on cells at the surface of the embryo and on those hypodermal cells lining the lumen of the fore- and hindgut. It also occurs along the entire surface of the tracheoblast lumen as well as on the outer surface of those cells which form exoskeletal chitinous setae. The process involves a specialization of the plasma membrane with the formation of secretory granules intracellularly beneath the membrane and the extrusion of these granules through the membrane to the outside where final cuticle formation occurs.     

Structure and lipid distribution of polyenoic very-long-chain fatty acids in the brain of peroxisome-deficient patients (Zellweger syndrome).

The polyenoic fatty acids with carbon chain lengths from 26 to 38 (very-long-chain fatty acids, VLCFA) previously detected in abnormal amounts in Zellweger syndrome brain have been shown to be n-6 derivatives and therefore probably derived by chain elongation of shorter-chain n-6 fatty acids such as linoleic acid and arachidonic acid. Polyenoic VLCFA are also present in Zellweger syndrome liver, but this tissue differs significantly from brain in that the saturated and mono-unsaturated derivatives are the major VLCFA. Zellweger syndrome brain polyenoic VLCFA are present in the neutral lipids predominantly in cholesterol esters, with smaller amounts in the non-esterified fatty acid and triacylglycerol fractions. These fatty acids are barely detectable in any of the major phospholipids, but are present in significant amounts in an unidentified minor phospholipid. The polyenoic VLCFA composition of this lipid differs markedly from that observed for all other lipids, as it contains high proportions of pentaenoic and hexaenoic fatty acids with 34, 36 and 38 carbon atoms. A polar lipid with the chromatographic properties in normal brain contains similar fatty acids. It is postulated that the polyenoic VLCFA may play an important role in normal brain and accumulate in Zellweger syndrome brain because of a deficiency in the peroxisomal beta-oxidation pathway, although a possible peroxisomal role in the control of carbon-chain elongation cannot be discounted.     

Studies on the host/parasite interface during the development of a pentastomid arthropod parasite in rodent intermediate hosts, with observations on protective surface membranes

The changing structure of the cuticle of the arthropod pentastomid parasite Porocephalus crotali, during growth to the infective stage in mouse and rattlesnake hosts, is described. The outermost cuticulin layer of the cuticle in instars II-VI is elevated to form a dense mat of epicuticular hairs. Since the VI larval cuticle is retained by the infective (VII) nymph as a protective sheath, effectively all stages in mice present a hairy surface to the host and this may constitute a physical barrier to inflammatory cells. The entire surface is overlain by a triple-track 'unit' membrane whose biophysical properties resemble those of a conventional plasma membrane, and there is evidence to suggest that this membrane is susceptible to immune attack. Under natural circumstances, epicuticular hairs entrap secretion, delivered to the cuticle via innumerable minute ducts which communicate with tegumental secretory cells termed subparietal cells (SPC). SPC synthesize lamellate droplets which unfold on the cuticle to constitute a layer of protective polymorphic vesicles. By contrast, infective nymphs in snakes possess a smooth cuticle and SPC membranous secretion is stacked over the entire surface, in sheets up to 20 deep. The function of the lipid and protein components of SPC secretion is discussed.     

Fine structure of the epicuticle of the desert scorpion, Hadrurus arizonensis, with reference to location of lipids.

The surface and transverse sections of the epicuticle of the desert scorpion, Hadrurus arizonensis, were examined by scanning and transmission electron microscopy, respectively. Sclerite cuticle that was untreated prior to normal EM preparative procedures was compared to cuticle subjected to lipid solvents, high temperature, and concentrated alkali. Surface morphology of untreated intersegmental cuticle was also examined. The epicuticle is composed of four sublayers: outer membrane, outer epicuticle, cuticulin, and the dense homogeneous layer. Lipid solvents did not significantly alter the morphology of any of these layers or the contents of the wax canals that penetrate the cuticulin layer even though the solvents effectively remove lipids from the epicuticle for chemical analysis. The surface of the sclerite cuticle contains amorphous particles, crystalline projections, and scattered openings to dermal gland ducts. Perforations that correspond to the opening of wax canals were faintly visible after extraction of surface waxes and clearly visible after KOH treatment. No openings to dermal gland ducts or wax canals were observed in untreated intersegmental cuticle. However, wax canals are likely obscured by surface waxes similar to those present in sclerite cuticle.     

Fine structure of the intersegmental membrane glands of the sixth abdominal sternum of female Nomia melanderi (Hymenoptera,Apoidea)

The fine structure of the intersegmental glands of the sixth abdominal sternum in 1-week old females of Nomia melanderi is presented. The plasma membrane of the secretory cell is unfolded in many places and is covered by a basement membrane. The microvillous surface is invaginated to form a rather long sinuous cavity. The endoplasm is almost entirely filled by secretory granules. Many secretory granules are located close to the inner surface of the invaginated plasma membrane. The invagination contains a porous ductule, apparently of cuticulin origin, that is connected directly with the inner layer of the transport duct of the duct-forming cell. This type of arrangement allows the direct flow of the secretory substance to the outside in a continuous way. The cylindrical duct-forming cell, besides having typical cell organelles, contains a cuticular transport duct. This duct is composed of a thin cuticulin layer surrounded by a rather thick epicuticular one. The results suggest that the secretory cell has two secretory cycles. The first occurs while the gland is differentiating (at the pupal stage) and is involved in secretion of the cuticulin that forms the porous ductule. The second cycle, which starts by the beginning of nesting, is involved in the secretion of a substance that is carried to the outside via the transport duct of the duct-forming cell.     

Apolysis and the turnover of plasma membrane plaques during cuticle formation in an insect.

The apical plasma membranes of Calpodes epidermal cells have small fattened areas or plaques with an extra density upon their cytoplasmic face. The plaques are typically at the tips of microvilli. The are present during the deposition of fibrous cuticle and the cuticulin layer. Since the plaques are close (less than 15nm) to the sites where these kinds of cuticle first appear, they are presumed to have a role in their synthesis and/or deposition and orientation. When fifth stage larval cuticle deposition ceases prior to pupation, the plaques are lost as the area of the apical plasma membrane is reduced. The plaques pass from the surface into pinocytosis vesicles and multivesicular bodies where they are presumably digested. The loss of plaques occurs as the blood level of moulting hormone reaches a peak at the critical period after which the prothoracic glands are no longer needed for pupation. Apolysis or separation of the epidermis from the old cuticle is the stage when plaques are absent, the old ones have been lost but the new ones have yet to form. After the critical period, the epidermis prepared for pupation with a phase of elevated RNA synthesis at the end of which plaques and microvilli reform in time to secrete the new cuticulin layer and later the fibrous cuticle of the pharate pupa. There is a new generation of plaques for each moult and succeeding intermoult and each generation is involved in two kinds of cuticle deposition before involution and redifferentiation.     

Cuticulogenesis correlated with ultrastructural changes in oenocytes and epidermal cells in the late cockroach embryo

During late embryogenesis in a cockroach, the epidermal cells secrete two cuticles: the embryonic cuticle and the pharate first larval cuticle. Late embryogenesis begins with the deposition of the cuticulin layer of the embryonic cuticle. The embryonic cuticle is an atypical one. It remains relatively thin and a well lamellated endocuticle is usually lacking. After general apolysis of the embryonic cuticle the epidermis secretes the epicuticle of the first larval cuticle and, subsequently, a typical lamellate procuticle. During the penultimate phase of late embryogenesis (i.e. before general apolysis) the epidermis becomes larvally committed. Some epidermal cells start to differentiate into specialized structures of the dermal glands, whereas the differentiated oenocytes appear to have acquired some stability. Nevertheless, shortly before general apolysis some oenocytes display signs of an increased alteration of the SER. When general apolysis occurs, the oenocytes contain a well-developed SER. The whole of the oenocyte population is programmed to regress after epicuticle deposition of the first larval cuticle. The correlation of oenocyte regression with available data on cuticulogenesis, ecdysteroid titres and cuticular lipid synthesis is discussed.     

Histology of Harder's gland of the rockhopper penguin, Eudyptes crestatus

The Harderian gland of the rockhopper penguin, Eudyptes crestatus , lies in the orbit ventral and anteromedial to the eyeball. It is classed as a type II avian Harderian gland as it is similar to that of the duck, differing only in the possession of acini and small amounts of lipid in the epithelial cells of the tubules. Histologically it is a lobulated, compound tubulo-acinar structure surrounded by a thin connective tissue capsule. Plasma cells and Mott cells are present.     

The structure and development of the hairpencil glands in males of the queen butterfly, Danaus gilippus berenice

Queen butterflies do not mate until the male has brushed the tufts of his scented, abdominal ”?hairpencils? over the female's head and antennae. The trichogen cells located at the base of each hairpencil are secretory. Presumably, these cells produce the sex pheromone necessary for mating. The liquid secretion must move from a central, microvillus-lined vesicle through the cuticle of the hairs to coat numerous, free, cuticular ?dust”? particles which adhere to the hairs' surface. The dust carries the secretion to or near the female's antennae. In the pupal stage the dust particles develop as outpocketings of the hair epicuticle. An amorphous matrix, probably protein epicuticle, is deposited in the outpocketings between the cuticulin layer and plasma membrane of the hair. Before the butterfly emerges from the pupa the matrix becomes enclosed by cuticulin, and the particles pinch off from the hair.     

The structure of an epidermal cell during the development of the protein epicuticle and the uptake of molting fluid in an insect

A structure for a generalized insect epidermal cell during the formation of the epicuticle is proposed, based on studies of several different epidermal cell types. The protein epicuticle is defined as the dense homogeneous layer below the cuticulin. The formation of the protein epicuticle involves secretory vesicles arising in Golgi complexes, and marks an interlude in the involvement in cuticle formation of plasma membrane plaques. The plaques are concerned in cuticulin formation before and in fibrous cuticle formation after the deposition of the protein epicuticle. The epidermis is characterized by the possession of a cytoskeleton of microtubules and a matrix of microfibers. In the elongated cells forming bristles and spines, the microfibers are often oriented in bundles with an axial banding which repeats every 120 Å. The microtubules are also arranged in columns with a trigonal packing and center to center spacing of about 800 Å. These cytoskeletal structures separate the other organelles into channels which may restrict the pathways open for the movement of secretory and pinocytotic vesicles. The protein epicuticle arises from the secretory vesicles which discharge at the apical surface. The contents disperse and reaggregate below the cuticulin. The Golgi complexes in the basal and central regions have many secretory vesicles and a small saccular component, differing from those nearer the apex which are smaller and have fenestrated saccules. The small coated vesicles (800 Å in diameter) associated with both sorts of complex, probably move to the apical and basal faces of the cell where they may give rise to the large coated vesicles (2000 Å in diameter) inserted in the plasma membrane. Pinocytosis occurs from both apical and basal faces but most lytic activity is in the apical region. Plant peroxidase injected into the haemocoel is taken up basally and transported to the apical MVBs. The large coated vesicles on the apical face may be concerned in the control of the extracellular subcuticular environment. They appear to fill up and detach, fusing to become the apical MVBs.     

The ABC transporter Snu and the extracellular protein Snsl cooperate in the formation of the lipid-based inward and outward barrier in the skin of Drosophila

Lipids in extracellular matrices (ECM) contribute to barrier function and stability of epithelial tissues such as the pulmonary alveoli and the skin. In insects, skin waterproofness depends on the outermost layer of the extracellular cuticle termed envelope that contains cuticulin, an unidentified water-repellent complex molecule composed of proteins, lipids and catecholamines. Based on live-imaging analyses of fruit fly larvae, we find that initially envelope units are assembled within putative vesicles harbouring the ABC transporter Snu and the extracellular protein Snsl. In a second step, the content of these vesicles is distributed to cuticular lipid-transporting nanotubes named pore canals and to the cuticle surface in dependence of Snu function. Consistently, the surface of snu and snsl mutant larvae is depleted from lipids and cuticulin. By consequence, these animals suffer uncontrolled water loss and penetration of xenobiotics. Our data allude to a two-step model of envelope i.e. barrier formation. The proposed mechanism in principle parallels the events occurring during differentiation of the lipid-based ECM by keratinocytes in the vertebrate skin suggesting establishment of analogous mechanisms of skin barrier formation in vertebrates and invertebrates.     

The use of a fluorescent dye, Nile red, to evaluate the lipid content of single mammalian oocytes

This study aimed to investigate the use of Nile red, a fluorescent dye specific for intracellular lipid droplets, to quantify the lipid content of single mammalian oocytes. It was hypothesized that a higher amount of lipid present in lipid droplets in an oocyte would result in a higher amount of emitted fluorescent light. Following fixation and subsequent staining of denuded oocytes, the fluorescence of the whole oocyte was visualized by fluorescence microscopy and quantified with a photometer and photomultiplier connected to the microscope. The peak of fluorescence was observed in the yellow spectrum (590 nm) and the fluorescence was restricted to the lipid droplets corresponding to apolar lipids. Nile red concentrations ranging from 0.1 to 10 microg/ml yielded similar results. After fixation, a minimum of 2 h staining was necessary to reach maximal fluorescence which remained stable for several hours. The position of the microscopic focus within the oocyte had no influence on the amount of measured fluorescence. Successive measurements of the same oocyte yielded very similar results indicating the repeatability of the method. Finally, the technique was validated by comparing the lipid content of bovine, porcine and murine immature oocytes, which are known to contain different amounts of lipids. After staining, the fluorescence of murine oocytes was 2.8-fold lower than the fluorescence of bovine oocytes which in turn were 2.4 times less fluorescent than porcine oocytes. Based on this study, it can be said that this rather fast and easy technique allows for the relative quantification of the lipid content (present in the lipid droplets) of one single oocyte. The different amounts of emitted fluorescent light in bovine, porcine and murine oocytes correlated with the known lipid contents in these three species. This technique could be used to compare the lipid content of oocytes originating from different donors, from different sized follicles or cultured in various conditions.