Ultrastructure, development, and homology of insect embryonic cuticles

Ultrastructure and deposition of the cuticles secreted by embryos representing eight insect orders were examined by transmission and scanning electron microscopy. Embryos of the apterygote silverfish Thermobia domestica deposit two embryonic cuticles. Deposition of the first (EC1) is initiated at the beginning of appendage development when the intercalary segment and the neural groove are clearly visible. This cuticle lacks surface microsculpture and consists of an outer epicuticle and an underlying fibrous layer, thought to represent procuticle. At the time of dorsal closure, deposition of a second embryonic cuticle (EC2) begins; this bears sensilla and functions in the first instar larva. In representative embryos of seven pterygote orders (Ephemeroptera, Odonata, Plecoptera, Neuroptera, Coleoptera, Lepidoptera, and Mecoptera), three cuticles were found to be secreted. The first cuticle in pterygotes is homologous to EC1 of T. domestica, but consists solely of outer epicuticle. EC2, the "prolarval cuticle," bears a characteristic surface microsculpture in embryos of some species and egg-teeth and other hatching devices, and consists of outer and inner epicuticles and a more or less reduced procuticle. EC2 is reduced in the embryos of derived endopterygotes, where a procuticle is lacking and the inner epicuticle is reduced. After hatching, when EC2 is shed, the first instar larva is covered by a third embryonic cuticle (EC3), whose deposition was initiated while the insect was still within the egg. Presence of only two embryonic cuticles in cyclorrhaphous flies is due to the total loss of prolarval cuticle. Investigated exopterygote and endopterygote insects excluding flies thus deposit three embryonic cuticles, and their juveniles (exopterygote "nymphs"; endopterygote "larvae") seem to hatch at equivalent stages of development. Differences between the modes of cuticulogenesis in silverfish and pterygote embryos suggest that the apterygote first larval instar was embryonized and became a fully embryonic prolarva in pterygotes.     

A proposed solution to a fine-structural puzzle: The organization of gill cuticle in a crayfish (Panulirus)

Crayfish gill cuticle is approximately 2 μm thick and comprises an epicuticle and an endocuticle, which is subdivided into outer and inner layers. Sections demonstrate indistinct lamellae in the outer endocuticle and vertically striated lamellae in the inner endocuticle. Microfibrils cannot be seen in sections. Difficulties in interpretation of the fibrous architecture of the cuticle from thin sections have been overcome by examining tilted series of micrographs of sections and also by making freeze-fracture replicas of the cuticle, which reveal the microfibrils clearly. A model for the endocuticle based on a helicoidal configuration of microfibrillar laminae is proposed and the vertically striated structures seen in sections of the outer layer are accounted for by including regular rows of particles oriented perpendicular to microfibrils. The model is compared with cuticles and coverings reported from other invertebrates.     

Ultrastructure and cuticle formation of the carapace in the myodocopan ostracod exemplified by Euphilomedes japonica (Crustacea: Ostracoda)

The ultrastructure and formation of the cuticle of a myodocopan ostracod, Euphilomedes japonica, are investigated utilizing scanning and transmission electron microscopy. The outer lamella cuticle consists of four layers; epicuticle, exocuticle, endocuticle, and membranous layer like in the cuticle of other arthropods. The exocuticle and endocuticle are well-calcified and the organic matrix develops within the both cuticles. The outermost layer of new cuticle (epicuticle) is secreted first and the inner layers (exocuticle, endocuticle and membranous layer) are added proximally in the pre-, and postmoult stages. The calcification takes place in the whole area of carapace at the same time together with the synthesis of organic matrix within the endocuticle. This study demonstrates that the ultrastructure and formation of the cuticle in myodocopans are different from those in podocopans, and that the myodocopan carapaces have achieved a structural diversity for adaptation to different lifestyles.     

CUTICLES FROM CHONDRUS CRISPUS (RHODOPHYTA)1

A simple, nondestructive physical process was developed for routinely isolating the outermost layers from female, male, and sporophyte fronds of Chondrus crispus Stack-house. Yields of pure cuticles from apical segments ranged from 0.74 to 2.35% on a dry weight basis after 5–7 d of culture. These undegraded cuticles were examined by electron microscopy (scanning and transmission electron microscopy), spectroscopy (infrared and X-ray), and chemical means. Cuticles isolated from female or male fronds were characterized by parallel arrays of electron-dense lamellae (typically 6–14) alternating with more electron-transparent regions. The thickness and uniformity of these lamellae provide the physical basis for the iridescence characteristic of C. crispus fronds. Sporophyte fronds are not iridescent. This phenomenon may be explained by the fewer electron-dense cuticular lamellae (usually three to seven) and the fact that these lamellae anastomose freely to form a thin cuticle with a highly irregular substructure. Elements detected by X-ray analysis, in addition to carbon and oxygen, included Mg, Br, S, and Ca in both gametophyte and sporophyte cuticles. Major features of FTIR spectra of all cuticles were absorbances due to proteins. A strong band, indicative of sulfate ester, occurred near 1250 cm^?1 in all cuticle preparations. Gametophyte, but not sporophyte, cuticles absorbed at 935, 846, and 800 cm^?1 consistent with the presence of kappa and/or iota carrageenan. Amino acid analyses showed that sporophyte and gametophyte cuticles were generally similar in gross composition. All contained proline as the principal residue together with significant amounts of cysteine, methionine, and lysine. Protein contents calculated from these analyses ranged from 37.6 to 44.4% on a dry weight basis as compared to 51.5–56.7% calculated from total nitrogen values. Up to 75% of the cuticle mass was solubilized by sodium dodecyl sulfate-β-mercaptoethanol. Three similar migrating bands were seen in female and male cuticle extracts on sodium dodecyl sulfate–polyacrylamide gel electrophoresis; however, none of the three weaker bands from sporophyte cuticles comigrated with those from gametophytes. Chloroform-methanol extraction removed < 3.3% of the cuticle mass, suggesting that lipids were minor components.     

Cuticle secretion during larval growth in Drosophila melanogaster

The moulting cycle and growth of the larval integument of Drosophila melanogaster has been studied by light and electron microscopy. Growth during the first, second and third larval instars is accompanied by 3.0-, 3.4- and 3.7-fold increases in surface area, respectively. Growth in surface area occurs continuously during the larval stages, with no detectable relationship to the moulting cycle. Measurements of the thickness of the cuticular layers show that the endocuticle grows in thickness by apposition and in surface area by stretching. The pre-apolytic epicuticle remains at fairly constant thickness during the increase in surface area, indicating that it grows by intussusception of new components. Post-apolytic epicuticle becomes thinner and increases in surface area by stretching. The epicuticle and pre-ecdysial endocuticle are traversed by filaments, but these do not penetrate the endocuticle secreted after ecdysis. We suggest that the filaments transport breakdown products from the old cuticle inward to the epidermis for reutilization. The growth and deposition of cuticle in two larval growth mutants, lethal (2) giant larvae and Chubby Tubby, involves mechanisms similar to those found in wild-type larvae, but in Chubby Tubby the endocuticle contains inclusions which are ultrastructurally similar to dense epicuticle.     

The formation of the epicuticle and associated structures inOniscus asellus (Crustacea,Isopoda)

Summary The integument of the woodlouse,Oniscus asellus, consists of a two-layered epicuticle, a largely lamellate procuticle — itself divided into two regions (pre-and postecdysial cuticles), and the epidermis. At the initiation of new cuticle production the epidermal cells become vacuolated and retract away from the cuticle. Apolysis occurs immediately after the cessation of postecdysial cuticle production. The formation of the epicuticle is unique among the arthropods since material aggregates along the distal epidermal membrane. By indenting, doubling back on itself, and incorporating septa, the epicuticle forms surface structures such as plaques and tricorns.The innervation, and so the receptive function of the tricorns is confirmed, but since there is no connection between the old and new receptors during premoult, sensory information from these exoreceptors must be severely curtailed. This may explain the biphasic moult in all isopods since it ensures that only half the body experiences this sensory deprivation at any one time. In terrestrial species there is the additional advantage of restricting the area of permeable new cuticle. The frequency of moulting may be due to the need to renew disrupted receptor surfaces.Tricorns do not appear to be the mechanoreceptors involved in the marked thigmotactic response of woodlice since they do not have the typical internal structure of such receptors; rather, the dendrite —which extends into the lumen of the tricorn —is protected from deformation by the previously unreported combination of a dendritic sheath and a cuticular tube. The modality of tricorns is possibly one of hygro-perception. One of the behavioural responses of woodlice to desiccation is aggregation. The numerical distribution of tricorns over the body surface is admirably suited to assist in the formation and maintenance of such aggregates during desiccation and to their observed dispersal when the relative humidity rises.     

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.     

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.     

The Fine Structure of the Integument of Free-Living and Parasitic Copepods. A Review

A perusal of the literature on copepod cuticles has been made, and results of the investigation of six species made by the author are included in this review. The integument of copepods is of the arthropod type. Pore canals and other structures traversing the cuticle, common in most arthropods, are not always present in free-living and some parasitic copepods. In parasitic forms, with advanced morphological changes, the cuticle is generally very thin and the epicuticle in many species forms external microvilli-like structures. In the copepods hitherto investigated the epicuticle is probably the sole layer present in the cuticle. Some copepods show specialized regions of the cuticular surface, the function of which still remains obscure. Integumental organs and integumental structures are numerous and variable. The association of bacteria with the cuticle has been observed in many species. The structure of the integument of parasitic species lacking an alimentary tube and in close contact with the host tissue or hemocoelic cavity supports the idea that the integument could be the obligatory site of nutrient uptake. In spite of the relatively few species of copepods that have been investigated, a remarkable variation of cuticular fine structure has been revealed.     

Structure of the cuticle of the alpine weta,Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae)

Sclerotized cuticle segments from the thorax, dorsal abdomen, and ventral abdomen of the alpine, weta Hemideina maori (Saussure) (Orthoptera: Stenopelmatidae) were examined by light microscopy and by scanning and transmission electron microscopy. An epicuticle, exocuticle (outer and inner), mesocuticle, endocuticle, and deposition layer are present in transverse sections. The epicuticle is further composed of a cuticulin layer and inner epicuticle, the latter being finely laminated and containing narrow wax canals that terminate below the cuticle surface. Openings to dermal gland ducts are visible on the surface as are large setae and smaller sensory pegs. Frozen fractured cuticle reveals the presence of horizontal ducts or channels that run laterally within the cuticle. The structure of weta cuticle is compared with that of the common house cricket and arthropods in general.     

The effects of DOPA decarboxylase inhibitors on the permeability and ultrastructure of the larval cuticle of the Australian sheep blowfly, Lucilia cuprina

Larvae of Lucilia cuprina, fed toxic levels of α-methyl DOPA (or other DOPA decarboxylase inhibitors) during the first or second instar, die at the completion of the next moult, soon after exposing their new cuticles. In electron micrographs of newly synthesised cuticle from these treated larvae, the ultrastructure of the lipid-rich outer epicuticle layer appears to be abnormal. This newly formed cuticle of the treated larvae is apparently defective in its role as a water permeability barrier (compared with that of normal larvae), since it permits the free movement of water in both directions. Thus, treated larvae die most probably as a direct result of dehydration. Larvae fed toxic levels of α-methyl DOPA can be rescued from death by simultaneously adding N-acetyldopamine (the cuticular sclerotizing agent) to the food. The rescued larvae are apparently normal in all respects. This suggests that sclerotization is required for the formation of a normal outer epicuticle. Diflubenzuron, which is known to inhibit chitin deposition in the cuticles of a number of different species of insect, also apparently affects chitin deposition in the larval cuticle of L. cuprina. Thus, in electron micrographs of cuticle from larvae fed toxic levels of diflubenzuron the ultrastructure of the chitin-containing endocuticle layer appears to be abnormal.     

Ultrastructure of the Cuticle of Loricifera and Demonstration of Chitin using Gold-Labelled Wheat Germ Agglutinin

The pharyngeal and lorical cuticles of adult and larval Loricifera were investigated by transmission electron microscopy. LR White sections of larval and adult Loricifera were labelled with the lectin wheat germ agglutinin (WGA) conjugated to colloidal gold. The pharyngeal cuticle of Nanaloricus mysticus exhibits a multilaminate epicuticle and an amorphous basal layer with osmiophilic fibres. The lorical cuticle consists of an osmiophilic or trilaminate epicuticle, one to three amorphous layer(s), and a basal fibrous layer which is strongly labelled by the lectin-gold conjugate. Chitinase treatment or competitive inhibition with N-, N '-, N "-triacetylchitotriose exclude labelling almost completely, whereas competitive inhibition with N -acetyl-D-glucosamine does not affect labelling intensity. The binding of WGA in connection with competition experiments indicates the presence of chitin in the fibrous layer. In most areas of a section, three amorphous layers extend below the epicuticle of the Nanaloricidae. Only in favourably orientated sections can all three "amorphous" layers be seen to be formed by stacks of lamellae. Modified articulation sites with bundles of osmiophilic longitudinal fibres and an osmiophilic plate (Nanaloricidae only) occur in adult Loricifera, but not in the larval stages. The ultrastructure of the lorical cuticle of the Loricifera resembles that of other Nemathelminthes (= Aschelminthes). The morphology of the articulation sites and the number of lorical plates seem to differ between the Loricifera and Priapulida. Therefore, it is currently not possible to conclude whether the lorica of the Loricifera and Priapulida are homologous structures. © 1997 The Royal Swedish Academy of Sciences. Published by Elsevier Science Ltd.     

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.     

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.     

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.     

Ultrastructure and Radiolabelling of Leaf Cuticles from Ivy (Hedera helixL.) Plantsin vitroand duringex vitroAcclimatization

Cuticle ultrastructure and radiolabelling of isolated cuticlesafter incorporation of [¹⁴C] acetate in foliar discs were investigatedwith ivy plants grownin vitrothenex vitro. Results show an increasein thickness, mass and wax content, between young and expandedleaves, for bothin vitroandex vitrocuticles. The cuticle ofinvitrounexpanded leaves was very thin and only constituted alamellate zone. The ultrastructure ofin vitroyoung and expandedleaf cuticles showed characteristics similar toin situcuticles.The thickness of the lamellate zone remained fairly constantand represented 33% of the cuticle thickness in young leaves,but only 11.4% in expanded leaves. The number of lamellar unitsdecreased from 14 to nine between these two growth stages. Themain difference between young leaves developedin vitroorex vitrowasa thinner lamellate zone forex vitrocuticles. However, theselatter cuticles had an intermediary zone between the lamellateand reticulate zones. The cuticle thickness of expanded leaveswas greater forin vitrocuticles suggesting a temporary decreasein cuticle biosynthesis after transfer of the plant fromin vitrotoexvitro.Results from cuticle radiolabelling show higher radioactivityincorporation in cuticles isolated from leaves developedex vitrocomparedtoin vitro. This radiolabelling was particularly marked forexvitroyoung leaf cuticles and depended on the duration of theexvitrogrowth period revealing a progressive activation of cuticlebiosynthesis in response to new environmental conditions. Hedera helix; ivy leaf cuticle; in vitroplants; electron microscopy; radiolabelling; isolated cuticles     

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.     

Fine structure of the cuticle of the desert scorpion, Hadrurus arizonensis.

The structure of the sclerite and intersegmental cuticle of the opithosoma of the desert scorpion, Hadrurus arizonensis, has been examined by transmission electron microscopy. The sclerite cuticle contains a four-layered epicuticle, a hyaline exocuticle, an inner exocuticle and an endocuticle. The outer part of the hyaline exocuticle and the whole of the inner exocuticle are constructed of helicoidally arranged planes of microfibrils. Within the endocuticle, the overall architecture is not helicoidal as previously assumed, but consists of bundles of microfibrils oriented horizontally and vertically. Microbibrils of the inner exocuticle and the endocutile are seen as simple unstained rods, but those of the hyaline exocuticle are electron dense rods with an unstained central core. The intersegmental cuticle contains a four-layered epicuticle and a procuticle. In detail, its fine structure differs in most respects from that of the sclerite cuticle. Electron microscopy reveals that hyaline exocuticle, previously assumed to be continuous from sclerite to intersegmental membrane, is absent in the latter.     

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.     

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.