The pupal cuticle of Drosophila: differential ultrastructural immunolocalization of cuticle proteins

Precise ultrastructural localization of Drosophila melanogaster pupal cuticle proteins (PCPs) was achieved by the immunogold labeling of frozen thin sections. PCPs were found in lamellate cuticle and intracellular vesicles but, curiously, were absent from the assembly zone of the cuticle. Antibodies that distinguish between the two classes of PCPs--low molecular weight (L-PCPs) and high molecular weight (H-PCPs)--revealed that the morphologically distinct outer lamellae contained L-PCPs and the inner lamellae contained H-PCPs. The sharp boundary between these two antigenic domains coincides with the transition from the outer to the inner lamellae, which in turn is correlated with the cessation of L-PCP synthesis and the initiation of H-PCP synthesis in response to 20-hydroxyecdysone (Doctor, J., D. Fristrom, and J.W. Fristrom, 1985, J. Cell Biol. 101:189-200). Hence, differences in protein composition are associated with differences in lamellar morphology.     

Drosophila Chitinase 2 is expressed in chitin producing organs for cuticle formation

The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects.     

Ultrastructure of the integument during moulting of the quiescent tritonymphal instar of trombiculid mite Hirsutiella zachvatkini (Acariformes: Trombiculidae)

The ultrastructure of the integument of the quiescent reduced tritonymph of the trombiculid mite Hirsutiella zachvatkini (Schluger) was investigated by means of transmission electron microscopy. Mites were investigated daily during the 14–16 day tritonymphal period (imagochrysalis). This period includes the deutonymphal moult (1–3 days), the quiescent tritonymph period (2–4 days), and the tritonymphal moult into the adult mite (6–10 days). A distinct recognizable feature of the tritonymphal moulting cycle is a sequence of events independent of precise time intervals. This process involves partial destruction and reorganization of the hypodermis of the previous instar, and formation of a new hypodermis of the subsequent instar from islands of rudimentary hypodermal cells. The integument of the reduced tritonymph differs greatly from that of both larva and active deutonymph and adult. It consists of a simply organized hypodermal layer of varying thickness and a thick clear poorly lamellate cuticle with curved pore canals, and lacking setae. The epicuticle is very thin and without a clear protein layer. The tritonymphal instar as such with its own cuticle situated near the hypodermis is encased within the detached covering of the previous active deutonymph, and may be considered a calyptostasic and entirely pharate instar. There is a tendency for reduced tritonymphal stage to be eliminated from ontogenesis and this stage is not homologous to the pupa of insects.     

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     

La formation des canaux cuticulaires chez l'adulte de Tenebrio molitor L. étude ultrastructurale et remarques histochimiques

The sclerotized cuticle of adult Tenebrio shows (1) an exocuticle composed of rotating lamellate layers and of columns of cuticular material, the fibres of which run perpendicularly through the lamellae, (2) an endocuticle composed of layers with preferred orientation. In the exocuticle, the pore canals are numerous and run along the columns; they do not rotate with the lamellate layers. They show several filaments some of which leave the canals and form a dense intracuticular network. In the last layers of exocuticle, the pericolumnar canals fuse and form large endocuticular canals which rotate in phase with the cuticular fibres. The formation of columns and canals is in relation with cellular expansions which penetrate into the cuticle during cuticle deposition. Exocuticular columns seem characteristic of highly sclerotized cuticles and the intracuticular filaments may have a role in the transport of sclerotisation precursors.     

Two sets of interacting collagens form functionally distinct substructures within a Caenorhabditis elegans extracellular matrix

A ubiquitous feature of collagens is protein interaction, the trimerization of monomers to form a triple helix followed by higher order interactions during the formation of the mature extracellular matrix. The Caenorhabditis elegans cuticle is a complex extracellular matrix consisting predominantly of cuticle collagens, which are encoded by a family of approximately 154 genes. We identify two discrete interacting sets of collagens and show that they form functionally distinct matrix substructures. We show that mutation in or RNA-mediated interference of a gene encoding a collagen belonging to one interacting set affects the assembly of other members of that set, but not those belonging to the other set. During cuticle synthesis, the collagen genes are expressed in a distinct temporal series, which we hypothesize exists to facilitate partner finding and the formation of appropriate interactions between encoded collagens. Consistent with this hypothesis, we find for the two identified interacting sets that the individual members of each set are temporally coexpressed, whereas the two sets are expressed approximately 2 h apart during matrix synthesis.     

Ultrastructural changes in the cuticle of the sheep blowfly, Lucilia, induced by certain insecticides and biological inhibitors

The ultrastructural effects on larval cuticle of Lucilia cuprina of two inhibitors of chitin synthesis, diflubenzuron and polyoxin D and an inhibitor of dihydrofolate reductase, aminopterin, are compared with those of the insecticide, cyromazine. Diflubenzuron and polyoxin D both prevent the formation of a normal lamellate appearance in procuticle and interfere with deposition of epicuticle. Aminopterin and cyromazine cause necrotic lesions in the cuticle which, in the case of cyromazine, are contiguous with invasive processes of epidermal cells. There is an accumulation of electron-dense granules in some epidermal cells in larvae poisoned with aminopterin or cyromazine. Aminopterin has a more drastic cytotoxic effect than cyromazine and it also interferes with the formation of epicuticle. The lesions produced by cyromazine treatment are not mimicked precisely by any of the other chemicals. However, there is closer accord between the effects of cyromazine and aminopterin than between cyromazine and the inhibitors of chitin formation.     

Fine structural observations on the epidermis

Summary In species of Apium, Eryngium and Humulus, the cuticular membrane of the petiole could be resolved into two parts, of which the inner one appeared amorphous and after staining appeared to be penetrated by an electron-dense reticulum, whereas the outer layer showed a lamellate structure consisting of electron-dense and electron-transparent plates, 50–80 Å in thickness. These layers are considered to correspond with the cuticular layer and the cuticle proper, respectively. In species of Abutilon and Rumex the cuticle proper did not exhibit the lamellate structure. In the leaves of Eryngium the outer lamellated structure was present in the cuticle of both young and mature leaves. Both the lamellate and non-lamellate types of the cuticle proper increased in thickness with age of the specimen. The results are discussed in relation to earlier investigations.     

The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. II. Scale-forming and socket-forming cells

In wings of the giant silkmoth, Hyalophora cecropia, scale-forming and socket-forming cells are first observed on day four of pharate adult development. Scale-forming cells appear synthetically active when they are first observed, for their basal region is filled with huge stacks of polyribosome-studed lamellate endoplasmic reticulum, numerous Golgi complexes containing secretory vesicles and many elongated mitochondria. During later development, some of these organelles diminish in number. Neck and scale regions are predominantly filled with longitudinally oriented microtubules and microfibril bundles, suggesting that their function is one of transport rather than synthesis. The scales originate as finger-like projections of the cell body. They subsequently elongate, flatten out and deposit a cuticle which has a surface differing somewhat from that in other lepidopterans. It consists of longitudinal ridges (1.8-2.4 μ apart), transverse ribs (0.6-1.0 μ apart) and microribs (0.10-0.13 μ apart). Socket-forming cells produce a socket around the neck region of each scale-forming cell. The socket differentiates into several morphologically distinct regions: an apical fibrillar region, a ribosomal region, a mitochondrial-microtubular region and a basal fibrillar region. The absence of polyribosome-studded lamellae of endoplasmic reticulum and Golgi complexes suggests that its primary function is not biosynthesis but support and protection of the scale.     

Fine structure of the chorion of a moth, hyalophora cecropia

The chorion of the moth Hyalophora cecropia has been found to consist primarily of a complex of proteins rendered insoluble by disulfide and hydrogen bonds. The bulk of the chorion is lamellate, and as in many cuticles each lamella contains a regular helicoidal series of microfibrils. The lamellar organization is disrupted in a central zone containing narrow irregular channels, and elsewhere the mature chorion is traversed by wide cylindrical channels. These open to the outer chorion surface indirectly via a porous trabeculate zone, and are distinct from the channels leading to the aeropyles, described elsewhere, in lacking cytoplasmic processes during their formation.     

Cuticular morphogenesis during continuous growth of the final instar larva of a moth

The larvae of the tobacco hornworm, Manduca sexta, grow continuously. During the feeding period of the fifth larval instar their weight increases ten-fold (ca. 1·2–12 g) accompanied by a four-fold expansion of the surface area of the abdominal cuticle. We have found that this cuticle contains structures which facilitate its expansion. Folds in the epicuticle (papillae) flatten as the cuticle expands. The endocuticle, in contrast, does not unfold but rather is plastically deformed. This plastic deformation is assisted by vertical structures in the cuticle (cuticular columns) which are more easily deformed than the surrounding lamellate cuticle. The head capsule cuticle, which does not expand as the larva grows, lacks papillae and cuticular columns. Thus, these are specialized structures that are reserved for cuticle that must expand as the larva grows.     

PORE CANALS AND RELATED STRUCTURES IN INSECT CUTICLE

The fine structure and the distribution of an esterase have been studied in the cuticle of Galleria larvae, Tenebrio larvae and pupae, and in the wax-secreting cuticle of the honey bee, and compared with those in the cuticle of the caterpillar of Calpodes. In Galleria and Tenebrio the pore canals are spaces passing through the lamellate endocuticle from the epithelium to the epicuticle. They contain a filament from the cells which may be concerned in their formation. The shape of the pore canal is probably determined by the orientation of the fibres making up the lamellae in the endocuticle and is not a regular helix. The pore canals also contain numerous filaments of another sort which pass on through the epicuticle and are believed to be the origin of the surface wax. They are particularly abundant in the pore canals of the honey bee wax-secreting cuticle and extend into the cell in long pockets surrounded by an envelope of the plasma membrane. The esterase is probably concerned with the final stage of wax synthesis, for its distribution is similar to that of the lipid filaments.     

Ultrastructure of Porocephalus crotali (Pentastomida) cuticle with phylogenetic implications.

The cuticle of P. crotali is pro-arthropodan, composed of an epi-, exo-, and endocuticle. The exo- and endocuticles are separated by a 600-A intermediate cuticular zone. The epicuticle is homogeneous and varies from 100 to 350 A in thickness. The exocuticle varies from 2 to eight mu in thickness and is divided into superficial and deep exocuticular zones. The endocuticle is lamellate and varies from 8 to 30 mu in thickness. Lamellae result from ordered parabolic orientations of 40-A chitin fibrils. Underlying cells lack a basement membrane. Subcuticular muscle cells insert tonofibrils directly into the adjacent endocuticle. No apodemes or apophyses occur.     

Ultrastructure of cuticle deposited inPlodia interpunctella wing discs after variousβ-ecdysone treatments in vitro

Summary Wing discs of the Indian meal moth may be cultured for extended periods in vitro. The discs produced a tanned cuticle after continuous incubation with -ecdysone in medium conditioned with fat body or after a 24-h pulse incubation with -ecdysone in plain medium. We investigated the ultrastructure of the cuticle deposited by such discs. We found that the treatment that produced the most complete cuticle in vitro was the 24-h pulse of hormone. We observed that cuticle formation in vitro was not all-or-none. Depending on culture conditions, discs produced cuticulin only, complete epicuticle, epicuticle plus diffuse endocuticle, epicuticle plus lamellate endocuticle, or even multiple layers of cuticle. The ultrastructural evidence suggests that continuous incubation with -ecdysone in plain medium does not always inhibit cuticle formationper se, but does prevent tanning of the partially formed cuticle.     

Fine structural aspects of secretory processes in a pentastomid arthropod parasite in its mouse and rattlesnake hosts

The histology and development of three extensive glands in the porocephalid pentastomid Porocephalus crotali is described by light and electron microscopy, during growth of the parasite to an infective stage in the tissues of mouse; the infective stage in rattlesnake definitive hosts is also included. These glands elaborate excretory/secretory components which are channelled, via chitin-lined efferent ductules, on to the parasite cuticle. Hook and frontal glands are relatively compact, and within each gland ductules serving individual secretory lobules collect into common ducts which discharge over each of the four hooks, or at the anterior margin of the cephalothorax respectively. Subparietal gland cell lobules, composed of two large and two small secretory cells, are distributed under the cuticle and each is served by a single efferent ductule; these erupt over the entire cuticle. The large cells in subparietal glands secrete lamellate droplets which coat the cuticle with thin layers. Identical cells are found in hook and frontal glands, in addition to to three morphologically distinct types of protein secretory cell. Preliminary data on the composition and immunological properties of the various secretory products are presented.     

The ultrastructure of developing wings in the giant silkmoth, Hyalophora cecropia. I. Generalized epidermal cells

The ultrastructure of wing epidermis of the giant silkmoth, Hyalophora cecropia, was studied during pupal diapause and the first half of development to the adult. In diapause, the generalized epidermal cells are characterized by many free ribosomes, some vesicles and small lamellae of rough endoplasmic reticulum, some scattered short mitochondria and a few small Golgi complexes. During the early states of post-diapause development, before and after the time of apolysis (separation of the epidermis from the overlying cuticle), there is a marked increase in structures often associated with synthetic functions, such as polyribosomes, lamellate rough endoplasmic reticulum and Golgi complexes. On day five of post-apolysis development, just after the appearance of scale-forming and socket-forming cells, the generalized epidermal cells lay down the cuticulin layer of the adult cuticle. At this stage and later, the polyribosomes and lamellate rough endoplasmic reticulum decrease in abundance. Cell nuclei show three phases of temporary transition from predominantly lobed to predominantly round profile, which correspond to periods of reported DNA synthesis. Throughout this developmental process, therefore, there is good correlation of fine structure with changes in macromolecular synthesis recorded elsewhere.     

Ultrastructural observations on the cuticular envelope in salt glands ofFrankenia pauciflora

Summary During five developmental stages in differentiation of salt glands in leaves ofFrankenia pauciflora, details of the deposition of incrusting material in the cuticular envelope,i.e., hydrophobic suberin and/or cutin, have been observed by means of transmission electron microscopy.Around each transfusion area in the cuticular envelope a conspicuous lamellate ring structure is found. Thin 3.5 nm lamellae associated with the plasmalemma around each ring structure appear to participate in the formation of tripartite structures showing a reversed contrast compared to the plasmalemma. Below the ring structure the cuticular envelope is divided into an outer fibrillar and an inner amorphous zone. A gradual transition between the tripartite lamellae of the ring structure and the amorphous material of the envelope is evident. Very dense material present between the lamellae appear to accumulate in the transition zone. The results are discussed in relation to basic structural features of various incrustations of lipid nature.     

Development of plant cuticles: fine structure and cutin composition of Clivia miniata Reg. leaves

The fine structure and monomeric composition of the ester-cutin fraction (susceptible to BF₃/CH₃OH transesterification) of the adaxial leaf cuticle of Clivia miniata Reg. were studied in relation to leaf and cuticle development. Clivia leaves grow at their base such that cuticle and tissues increase in age from the base to the tip. The zone of maximum growth (cell expansion) was located between 1 and 4 cm from the base. During cell expansion, the projected surface area of the upper epidermal cells increased by a factor of nine. In the growth region the cuticle consists mainly of a polylamellate cuticle proper of 100–250 nm thickness. After cell expansion has ceased both the outer epidermal wall and the cuticle increase in thickness. Thickening of the cuticle is accomplished by interposition of a cuticular layer between the cuticle proper and the cell wall. The cuticular layer exhibits a reticulate fine structure and contributes most of the total mass of the cuticle at positions above 6 cm from the leaf base. The composition of ester cutin changed with the age of cuticles. In depolymerisates from young cuticles, 26 different monomers could be detected whereas in older ones their number decreased to 13. At all developmental stages, 9,16-/10,16-dihydroxyhexadecanoic acid (positional isomers not separated), 18-hydroxy-9-octadecenoic acid, 9,10,18-trihydroxyoctadecanoic acid and 9,10-epoxy-18-hydroxyoctadecanoic acid were most frequent with the epoxy alkanoic acid clearly predominating (47% at 16 cm). The results are discussed as to (i) the age dependence of cutin composition, (ii) the relationship between fine structure and composition, (iii) the composition of the cuticle proper, the cuticular layer and the non-depolymerizable cutin fraction, and (iv) the polymeric structure of cutin.Abbreviations CL cuticular layer - CP cuticle proper - MX cutin polymer matrix     

A novel zinc-carboxypeptidase SURO-1 regulates cuticle formation and body morphogenesis in Caenorhabditis elegans

Cuticle formation and molting are critical for the development of Caenorhabditis elegans. To understand cuticle formation more clearly, we screened for suppressors in transgenic worms that expressed dominant ROL-6 collagen proteins. The suro-1 mutant, which is mild dumpy, exhibited a different ROL-6::GFP localization pattern compared to other Dpy mutants. We identified mutations in three suro-1 mutants, and found that suro-1 (ORF R11A5.7) encodes a putative zinc-carboxypeptidase homologue. The expression of this enzyme in the hypodermis and the genetic interactions between this enzyme and other collagen-modifying enzyme mutants suggest a regulatory role in collagen processing and cuticle organization for this novel carboxypeptidase. These findings aid our understanding of cuticle formation during worm development.     

Morphological Alterations of Metarhizium anisopliae During Penetration of Boophilus microplus Ticks

Chronological histological alterations of Metarhizium anisopliae during interaction with the cattle tick Boophilus microplus were investigated by light and scanning electron microscopy. M. anisopliae invades B. microplus by a process which involves adhesion of conidia to the cuticle, conidia germination, formation of appressoria and penetration through the cuticle. Twenty-four hours post-infection conidia are adhered and germination starts on the surface of the tick. At this time, the conidia differentiate to form appressoria exerting mechanical pressure and trigger hydrolytic enzyme secretion leading to penetration. Massive penetration is observed 72 h post-inoculation, and after 96 h, the hyphae start to emerge from the cuticle surface to form conidia. The intense invasion of adjacent tissues by hyphae was observed by light microscopy, confirming the ability of M. anisopliae to produce significant morphological alterations in the cuticle, and its infective effectiveness in B. microplus.