The transfer of lipid in insects from the epidermal cells to the cuticle

The structure of the pore canals and the tubular filaments they contain are described in a series of insects and types of cuticle. In all these cuticles the tubular filaments arise from the plasma membrane of the epidermal cells and they contain argentaffin material, regarded as sclerotin precursors, and lipid-staining material, regarded as wax precursors. These materials are transferred to the inner epicuticle and are exuded over the surface of the outer epicuticle to form the waterproofing layer as described in the preceding paper. They are also transported to those parts of the endocuticle destined to form hard exocuticle. There are no terminations of tubular filaments in the soft cuticle of Manduca larva, in the soft expanding cuticle of Rhodnius, and in the non-sclerotized post-ecdysial endocuticle of Tenebrio. Apis. etc. In the puparium of Calliphora lipid appears to be added by the epidermal cells directly and not by way of tubular filaments. It is confirmed that lipid is a component of sclerotized cuticle.     

Sclerotin and lipid in the waterproofing of the insect cuticle

In all the cuticles studied waterproofing is effected by extracuticular material, a mixture of sclerotin precursors and lipids, exuded from the tubular filaments of the pore canals. In Rhodnius larval abdomen it is a layer of thickness similar to the outer epicuticle, believed to be composed of 'sclerotin' and wax, in Schistocerca larval sternal cuticle and in Carausius sternal cuticle it is similar. In Tenebrio adult sternal cuticle of the abdomen, in both the extracuticular exudation and the contents of the distal endings of the tubular filaments, the wax component is obscured by hard 'sclerotin'. In Manduca larva a very thin layer of 'sclerotin' and wax is covered by an irregular wax layer, average 0.75 micron, twice the thickness of the inner epicuticle. In Periplaneta and Blattella the abdominal cuticle is covered by a soft waxy layer, often about 1 micron thick, which is mixed with argentaffin material. Below this is a very thin waterproof layer of wax and 'sclerotin' continuous with the contents of the tubular filaments, which is readily removed by adsorptive dusts. In Apis adult abdominal terga free wax plus sclerotin precursors form a thin layer which is known to be removed by adsorptive dusts. In Calliphora larva there is a very thin layer of the usual mixed wax and sclerotin and below this a thick (0.5 micron) layer, lipid staining and strongly osmiophil, likewise extracuticular and exuded from the epicuticular channels. This material (which is often called 'outer epicuticle') has the same staining and resistance properties as the true outer epicuticle on which it rests. In the abdomen of Calliphora adult the waterproofing wax-sclerotin mixture forms a thin layer over the entire cuticle including the surface of the microtrichia. There is also a thin detachable layer of free wax on the surface.     

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.     

The distribution of phenoloxidases and polyphenols during cuticle formation

The distribution of phenoloxidase and polyphenols have been studied during cuticle formation at the 4th to 5th molt in Colpodes ethius. Cuticular phenoloxidases arise in the epidermis in cisternae of the rough endoplasmic reticulum, pass through the Golgi complex and are transported to the apical face in secretory vesicles. From the cuticular environment some enzyme is pinocytosed and broken down in the apical multivesicular bodies. Phenoloxidase and polyphenols are present during the formation of the cuticulin layer which also reacts as if it were at least partly composed of a phenoloxidase. The rest of the epicuticle incorporates phenoloxidase as it is deposited, particularly that over the dorsal tubercles which later melanize. Polyphenols do not appear until shortly before ecdysis. They are associated with the epicuticular filaments in both epicuticle and presumptive epocuticle. It is proposed that the epicuticular filaments may arise as liquid crystals with a protein component which becomes stabilized like the rest of the cuticle. These structures could provide a channel for the movement of both lipids and quinones to the surface. Phenoloxidases may pass through fibrous cuticle to be deposited as part of the epicuticle but are incorporated in fibrous cuticle scheduled for sclerotization. The time of stabilization is determined by the availability of polyphenols.     

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.     

Ultrastructural and histochemical investigations of peripatus integument

The integument of Euperipatoides leukarti (Onychophora) has been studied by SEM and TEM-histochemical techniques. The thin (2-3 mu) cuticle is essentially arthropodan in design, comprising a thin, lamellate outer epicuticle, a homogeneous inner epicuticle, and an extensive fibrous procuticle. Five tanned lipoprotein lamellae constitute the outer epicuticle, the innermost showing a cross-striated pattern and the outermost also containing carbohydrates. The inner epicuticle contains untanned lipoproteins. The chitin-protein composite procuticle reveals a fibrous ultrastructure but no trace of helicoids. Distally it may be tanned, producing a sclerotin layer (exocuticle), pronounced in the sensory setae, claws and mandibles. Penetrating the epicuticle and extending into the procuticle are numerous, minute pore canals through which mobilised procuticle is resorbed prior to ecdysis. Beneath the procuticle is a monolayered epidermis traversed by tonofibrils and containing dense pigment granules. The membrane junctions are strongly convoluted and include an apical zonula adherens, septate desmosomes and a proximal lymph space. The epidermis is underlain by a thick (5-25mu) basement membrane of helicoidally organised collagen fibres. Functional and phylogetic implications of the integumental structure are discussed. Arthropod affinities are clear and the Onychophora should be included in that phylum if it is to be retained in modern taxonomic nomenclature.     

Histochemical and biochemical investigations concerning the function of larval oenocytes of Tenebrio molitor L. (Coleoptera,Insecta)

Summary Larval oenocytes of Tenebrio molitor were investigated histochemically. In contrast to the lipid droplets of the fat body, they did not stain with Sudan black. A positive reaction for lipoproteins appeared only after destructive oxidation with sodium hypochlorite. These lipoproteins are the remnants of degenerated membranes, as revealed by ultrastructural analysis. Polyphenols could be identified in the exocuticle of exuvia, and in the newly formed procuticle. Endocuticle, epidermis and oenocytes showed no staining reaction. In oenocytes a great amount of lipase is also present which could be detected with several Tweens as substrates. The significance of these lipases remains unclear, since only few glycerides are synthesized in the cells, as shown below. They may play a role in the extended membrane turnover observed in this cell type. In vitro incubation of oenocytes of the larval generation demonstrated that ¹⁴C-labeled acetate was only incorporated into the paraffin fraction. A negligible amount of the label was found in glycerides; wax esters were free of label. Larval epidermis is also capable of paraffin formation, but only to a small degree. Oenocytes of the imaginal generation located between the sternal epidermis cells of pupae and adults do not synthesize paraffins, but other more polar compounds not yet identified. Labeled waxes in cuticular lipids were detected only when ¹⁴C-acetate was injected into whole larvae, and the lipids extracted some hours later. Autoradiographs demonstrated that ¹⁴C-acetate was intensively incorporated into larval oenocytes, the rate varying in different cells. Incorporation into the epicuticle, probably into the wax layer, was clearly shown. Cuticulin and dense layer do not show an intensive label. The lamellated cuticle also seems to be impregnated with acetate derivatives.Supported by the Deutsche Forschungsgemeinschaft     

Temperature and the transpiration of water through the insect cuticle

The effervescence that occurs when an insect is immersed in a mixture of liquid paraffin and butanol indicates that the waterproofing barrier may be very close to the surface, that this barrier varies greatly in permeability in different areas, and that it is readily permeable in the newly moulted insect, but becomes far more resistant as the cuticle hardens. At the height of the effervescence the streams of expanding vesicles, that come from the surface, arise from fixed points-which suggests that the water is escaping from the terminations of the tubular filaments. It is shown that if the intact insect is warmed in a solution of ammoniacal silver at a succession of rising temperatures the argentaffin component of the extracuticular waterproofing layer is usually the first location of silver reduction, to be followed by the contents of the epicuticular channels piercing the outer epicuticle, the substance of the inner epicuticle, and the distal portions of the pore canals. The temperature at which this silver staining increases most rapidly parallels (approximately) the temperature at which transpiration in dry air begins to rise most steeply. It is inferred that both responses are probably dependent on the same temperature effects in the waterproofing lipids.     

Autoradiographic study of protein synthesis in abdominal tissues of Glossina austeni

Protein synthesis at various times during the pregnancy cycle of G. austeni was determined by autoradiographic measurement of the incorporation of H³-leucine and H³-tyrosine into the cells of the fat body, oenocytes, milk gland and epidermis. The rate of utilization of these molecules is such that the labelled pool in the haemolymph is depleted before 0.5 hr after injection. The incorporation of both amino acids into fat body and oenocytes is high at eclosion and just after larviposition, with the incorporation of tyrosine by the oenocytes being much higher than that in the fat body. The same pattern of incorporation is observed in the epidermal cells. Label also appears in the endocuticle during the first 10 days of adult life. Except during the first 4 days following emergence, the incorporation of the two amino acids into the milk gland is very high, with periods of less intense protein synthesis at about the time of larviposition. The milk gland represents a highly efficient secretory system, with a t₅₀ of less than 30 min.     

The plastid in Plasmodium falciparum asexual blood stages: a three-dimensional ultrastructural analysis

The plastid in Plasmodium falciparum asexual stages is a tubular structure measuring about 0.5 micron x 0.15 micron in the merozoite, and 1.6 x 0.35 microns in trophozoites. Each parasite contains a single plastid until this organelle replicates in late schizonts. The plastid always adheres to the (single) mitochondrion, along its whole length in merozoites and early rings, but only at one end in later stages. Regions of the plastid are also closely related to the pigment vacuole, nuclear membrane and endoplasmic reticulum. In merozoites the plastid is anchored to a band of 2-3 subpellicular microtubules. Reconstructions show the plastid wall is characteristically three membranes thick, with regions of additional, complex membranes. These include inner and outer membrane complexes. The inner complex in the interior lumen is probably a rolled invagination of the plastid's inner membrane. The outer complex lies between the outer and middle wall membranes. The interior matrix contains ribosome-like granules and a network of fine branched filaments. Merozoites of P. berghei and P. knowlesi possess plastids similar in structure to those of P. falciparum. A model is proposed for the transfer of membrane lipid from the plastid to other organelles in the parasite.     

Die Feinstruktur des Integumentes und der Muskelansatzstellen vonEchiniscoides sigismundi (Heterotardigrada)

The structure of the integument and the muscle attachments of the marine heterotardigradeE. sigismundi (M. Schultze) was studied by electron microscopy. The cuticle consists of several layers: an outer tripartite (or multilayered) epicuticle, perhaps with an outermost coat; a homogeneous inner epicuticle; a trilaminated layer; an intracuticle; and a fibrous procuticle. These features resemble the cuticle described in Eutardigrada; in contrast, areas on the legs and near the claws, with an outer multilayered epicuticle and a striated layer (inner epicuticle), are — as far as investigated — more similar to the cuticle in Heterotardigrada. The epidermis consists of a single cell layer without glands. The muscle attachments are in line with the general pattern described in the eutardigradeMacrobiotus hufelandi and in Arthropoda.     

Ultrastructure of the embryonic snake skin and putative role of histidine in the differentiation of the shedding complex.

The morphogenesis and ultrastructure of the epidermis of snake embryos were studied at progressive stages of development through hatching to determine the time and modality of differentiation of the shedding complex. Scales form as symmetric epidermal bumps that become slanted and eventually very overlapped. During the asymmetrization of the bumps, the basal cells of the forming outer surface of the scale become columnar, as in an epidermal placode, and accumulate glycogen. Small dermal condensations are sometimes seen and probably represent primordia of the axial dense dermis of the growing tip of scales. Deep, dense, and superficial loose dermal regions are formed when the epidermis is bilayered (periderm and basal epidermis) and undifferentiated. Glycogen and lipids decrease from basal cells to differentiating suprabasal cells. On the outer scale surface, beneath the peridermis, a layer containing dense granules and sparse 25-30-nm thick coarse filaments is formed. The underlying clear layer does not contain keratohyalin-like granules but has a rich cytoskeleton of intermediate filaments. Small denticles are formed and they interdigitate with the oberhautchen spinulae formed underneath. On the inner scale surface the clear layer contains dense granules, coarse filaments, and does not form denticles with the aspinulated oberhautchen. On the inner side surface the oberhautchen only forms occasional spinulae. The sloughing of the periderm and embryonic epidermis takes place in ovo 5-6 days before hatching. There follow beta-, mesos-, and alpha-layers, not yet mature before hatching. No resting period is present but a new generation is immediately produced so that at 6-10 h posthatching an inner generation and a new shedding complex are forming beneath the outer generation. The first shedding complex differentiates 10-11 days before hatching. In hatchlings 6-10 h old, tritiated histidine is taken up in the epidermis 4 h after injection and is found mainly in the shedding complex, especially in the apposed membranes of the clear layer and oberhautchen cells. This indicates that a histidine-rich protein is produced in preparation for shedding, as previously seen in lizard epidermis. The second shedding (first posthatching) takes place at 7-9 days posthatching. It is suggested that the shedding complex in lepidosaurian reptiles has evolved after the production of a histidine-rich protein and of a beta-keratin layer beneath the former alpha-layer.     

Procuticle proteins and chitin-like material in the inner epicuticle of the Drosophila pupal cuticle

The inner (protein) epicuticle of the pupal cuticle of Drosophila is shown to contain at least two hydrophobic proteins (19 and 21 kD) that are also present in the outer procuticle lamellae. An N-acetylglucosamine-containing carbohydrate is also present in the inner epicuticle. This represents the first attempt to characterize the non-lipid components of an insect epicuticle.     

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.     

Haemocytes and basement membrane formation in Rhodnius

During the period between feeding and ecdysis in the fourth instar larva of Rhodnius the basement membrane below the epidermis of the abdomen increases about threefold in thickness. This increase takes place during the time, about 5 to 7 days after feeding, when the plasmatocytes settle and flatten in great numbers on the inner surface of the membrane.By a process beginning with pinocytosis the plasmatocytes accumulate their characteristic periodic acid Schiff (PAS)-positive inclusions, often with rod-like or tubular contents. These inclusions are discharged and spread out on the surface of the basement membrane. The inclusions and the membrane have the same staining properties and contain protein, carbohydrate, and lipid. Similar material is slowly deposited in less compact form between the plasmatocytes which encapsulate foreign bodies.The oenocytoids (granular cells) discharge the electron dense contents of their inclusions into the haemolymph rather later. The subsequent fate of this material is not known.     

The ultrastructure of the oenocytes in the molt/intermolt cycle of an insect

The structure and development of the permanent oenocytes of Calpodes ethlius (Lepidoptera, Hesperiidae) are described. There are three sorts of oenocyte. The permanent oenocytes are arranged ventral to the last two pairs of spiracles on abdominal segments 7 and 8 in four clusters of about 45 cells each. The molt cycle oenocytes are ventral to the other spiracles and only enlarge at molting. The subdermal oenocytes differentiate from the epidermis in large numbers shortly before pupation. The permanent oenocytes are large polyploid cells characterized by a cytoplasm of densely packed smooth tubular endoplasmic reticulum, and a plasma membrane invaginated in a meshwork of tubes ending in a reticular layer about 12 micro below the surface. There are two sorts of Golgi complex, one small and of conventional form, the other composed of clouds of microvesicles. 'Dense bodies', believed to belong to the microbody class of organelles, arise directly from the STER. There is a variety of membranous and 'crystalline' inclusions. The formation of isolation membranes from the tubular endoplasmic reticulum, and the origin of isolation bodies and autophagic vacuoles are described. Some autophagy takes place at all times in the molt/intermolt cycle, but there are phases of massive autophagy before the 4th-5th molt and the 5th-pupal molt. These phases coincide with pinocytosis of blood proteins and overlap with or are followed by phases of nuclear replication, RNA synthesis (ribosomes) and ER regeneration. Nuclear blebbing occurs before pupation. The morphology of the oenocytes is most like that of vertebrate cells engaged in steroid hormone synthesis. It is pointed out that the oenocytes rather than the prothoracic glands could be the source of ecdysone and the stimulus for molting.     

Apple procyanidins decrease cholesterol esterification and lipoprotein secretion in Caco-2/TC7 enterocytes

Decrease of plasma lipid levels by polyphenols was linked to impairment of hepatic lipoprotein secretion. However, the intestine is the first epithelium that faces dietary compounds, and it contributes to lipid homeostasis by secreting triglyceride-rich lipoproteins during the postprandial state. The purpose of this study was to examine the effect of apple and wine polyphenol extracts on lipoprotein synthesis and secretion in human Caco-2/TC7 enterocytes apically supplied with complex lipid micelles. Our results clearly demonstrate that apple, but not wine, polyphenol extract dose-dependently decreases the esterification of cholesterol and the enterocyte secretion of lipoproteins. Apple polyphenols decrease apolipoprotein B (apoB) secretion by inhibiting apoB synthesis without increasing the degradation of the newly synthesized protein. Under our conditions, cholesterol uptake, apoB mRNA, and microsomal triglyceride protein activity were not modified by apple polyphenols. The main monomers present in our mixture did not interfere with the intestinal lipid metabolism. By contrast, apple procyanidins reproduced the inhibition of both cholesteryl ester synthesis and lipoprotein secretion. Overall, our results are compatible with a mechanism of action of polyphenols resulting in impaired lipid availability that could induce the inhibition of intestinal lipoprotein secretion and contribute to the hypolipidemic effect of these compounds in vivo.     

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.     

Design of the labial cuticle in Cenocorixa bifida Hung. (Hemiptera: Corixidae) with reference to ionic transport

The surface topography and ultrastructure of the labial cuticle of Cenocorixa bifida were examined by scanning and transmission electron microscopy. The dorsal wall of the labium consists of seven sclerotized transverse bars each displaying two rows of semicircular grooves and pores. The cuticle is about 20 microm thick and is composed of epicuticle and lamellate exocuticle and endocuticle, the latter separated from the underlying epidermis by subcuticle containing amorphous material. The epicuticle is subdivided into an electron-dense very thin outer epicuticle and a homogenous thick inner epicuticle, which is penetrated by grooves. The exocuticle is filled with electron-dense blocks of material, which may provide mechanical support to the labial wall. The elongate epidermal cells display extensive infoldings of the apical plasma membrane (facing the cuticle) and contain abundant mitochondria in the cytoplasm. The presence of deep epicuticular grooves and pores in the thin labial cuticle and extensive apical membrane infolding and abundant mitochondria in the epidermal cells suggest that the labium in C. bifida is the site of osmoregulatory ionic uptake.     

Etudes sur l'épicuticule des insectes

Résumé L'épicuticule de l'adulte de Tenebrio molitor est composée de deux couches distinctes dénommées épicuticule externe et épicuticule interne. L'épicuticule externe est la première couche cuticulaire sécrétée sous forme de petites plaques s'agrandissant par leurs bords pour recouvrir toute la surface cellulaire. Au moment de sa sécrétion, cette couche est formée de quatre lames denses A, B, C1 et C2. La lame B, très fine, disparaît par la suite et les lames C1 et C2 deviennent très nettes. L'épicuticule externe de l'adulte est donc formée de trois lames denses séparées par deux lames claires.L'épicuticule interne est formée de lames superposées denses et claires de structure complexe, qui sont masquées pendant la sécrétion des premières couches de cuticule lamellée (procuticule). Cette structure correspond à un arrangement moléculaire hautement organisé.La forme de la surface cuticulaire des sternites est déterminée par la forme de la surface cellulaire avant le dépôt de l'épicuticule.

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The development of the epicuticle in the adult Tenebrio molitor L.

Summary The epicuticle of adult Tenebrio consists of two distinct layers named outer and inner epicuticle.The outer epicuticle is the first cuticular layer to be deposited in form of small patches on top of the microvilli. These initial patches are composed of four dense laminae (A, B, C1 and C2) separated by three light spaces. The outer epicuticle grows by densification of diffuse material at the edges of the patches until the entire area is covered.The thickness of outer epicuticle remains constant (175 Å) during the development of the pharate adult, lamina B however rapidly disappears. Thus, the adult outer epicuticle is fivelayered (three dense laminae: A, C1 and C2).After being deposited, the inner epicuticle shows a complex laminar structure interpreted to represent a highly organized molecular system. The laminae are masked during the formation of the first procuticle lamellae.During the deposition of the epicuticle, lamina A is covered by a component of the moulting fluid, forming an irregular dense layer which disappears after the resorption of this fluid. Perhaps this layer protects the new epicuticle from lytic enzymes of the moulting fluid.In adult animals, there is an additional superficial layer, the signification of which is not clear. The possibility of remains of cement or wax is discussed.The development of the surface patterns of the sternal and pleural cuticle is determined before the epicuticle formation by the shape of the epidermal surface. The rate of outer epicuticle deposition appears to depend on the size of the microvilli: epicuticle deposition seems to proceed faster over high microvilli.

Nous tenons à remercier notre directeur de recherche, le Professeur Noirot pour ses encouragements et ses conseils et Madame Curie pour son aide technique efficace.