The ‘Embryonic moults’ of the milkweed bug as seen by the S.E.M.

Deposition, detachment and removal of the three embryonic cuticles are studied. The menbrane-like cuticle 1 covers the embryo during katatrepsis and ‘disappears’ thereafter. Cuticle 2 deposition starts shortly before dorsal closure. Its apolysis is accompanied by contractions of the embryo. Ecdysis of cuticle 2 takes place during hatching. Only cuticle 3 (= first larval cuticle) shows differentiations like sensilla and cornea. Peaks of ecdysteroid (and probably JH) titre are observed during apolysis of cuticle 1 and cuticle 2 (Dorn, 1981). Transition from ectoderm to epidermis proper takes place shortly before and during onset of cuticle 2 synthesis.     

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.     

Exoskeletal cuticle differentiation during intramarsupial development of Porcellio scaber (Crustacea: Isopoda)

Exoskeletal crustacean cuticle is a calcified apical extracellular matrix of epidermal cells, illustrating the chitin-based organic scaffold for biomineralization. Studies of cuticle formation during molting reveal significant dynamics and complexity of the assembly processes, while cuticle formation during embryogenesis is poorly investigated. This study reveals in the terrestrial isopod Porcellio scaber, the ultrastructural organization of the differentiating precuticular matrices and exoskeletal cuticles during embryonic and larval intramarsupial development. The composition of the epidermal matrices was obtained by WGA lectin labelling and EDXS analysis. At least two precuticular matrices, consisting of loosely arranged material with overlying electron dense lamina, are secreted by the epidermis in the mid-stage embryo. The prehatching embryo is the earliest developmental stage with a cuticular matrix consisting of an epicuticle and a procuticle, displaying WGA binding and forming cuticular scales. In newly hatched marsupial larva manca, a new cuticle is formed and calcium sequestration in the cuticle is evident. Progression of larval development leads to the cuticle thickening, structural differentiation of cuticular layers and prominent cuticle calcification. Morphological characteristics of exoskeleton renewal in marsupial manca are described. Elaborated cuticle in marsupial larvae indicates the importance of the exoskeleton in protection and support of the larval body in the marsupium and during the release of larvae in the external environment.     

Developmental changes of the antenna and its neurons in the silkworm,Bombyx mori,with special regard to larval-pupal transformation

The larval antenna of Bombyx mori has 13 sensilla and about 52 sensory neurons in its distal portion. The axons form two nerve cords which unite in the cranial hemocoel to supply the brain as the olfactory nerve. The antennal imaginal disc, which is a thick pseudostratified epithelium continuous with the antennal epidermis, thickens markedly during the 5th instar by rapid cell proliferation. At the prepupal stage cell proliferation ceases and the disc everts to form a large pupal antenna. Simultaneously, an extensive cell rearrangement occurs in the antennal epidermis and the disc tissue becomes much thinner because of the abrupt expansion of antennal surface area. The two larval nerve cords thin down markedly by degeneration of axons, but they do not disintegrate totally even after the onset of pupation. The epidermis of the larval antenna forms the distal portion of the pupal antenna, while the imaginal disc forms the more basal portion. Development to the adult antenna occurs almost immediately after the onset of pupation; many adult neurons appear in the simple epidermis facing toward the thick outer side of the newly formed pupal cuticle. By 12 hours after the onset of pupation, these neurons align themselves in many transverse rows which are the first sign of the adult antennal configuration. Addition of these neuronal axons to the once-thinned nerve cords causes resumed thickening of the cords during the first 24 hours and thereafter. Differentiation of adult sensilla begins in the next 24 hours and is almost completed at the third day of pupation, which requires a total of 10 days.     

Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus)

Summary Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.     

Expression and hormonal control of a new larval cuticular multigene family at the onset of metamorphosis of the tobacco hornworm

The pattern of cuticular protein synthesis by the epidermis of the tobacco hornworm larva changes during the final day of feeding, leading to an alteration in cuticular structure and a stiffening of the cuticle. We have isolated a small multigene family which codes for at least three of the new cuticular proteins made at this time. The five genes which were isolated from this family map to two different genomic regions. Sequencing shows that one of the genes is 1.9 kb and consists of three exons coding for a 12.2-kDa acidic (pI = 5.26) protein that is predominantly hydrophilic. The deduced amino acid sequence shows regions of similarity to proteins from flexible lepidopteran cuticles and from Drosophila larval and pupal cuticles, but not to proteins found in highly sclerotized cuticles. This gene family is first expressed late on the penultimate day (Day 2) of feeding in the final larval instar and ceases expression 2 days later when metamorphosis begins. In situ hybridization shows that this gene family is expressed in all the epidermal cells of Day 3 larvae except the bristle cells and those at the muscle attachment site. Expression can be induced in Day 1 epidermis by exposure to 50 ng/ml 20-hydroxyecdysone in vitro, but only if juvenile hormone is absent. Its developmental expression, tissue specificity, and hormonal regulation strongly suggest that this multigene family is involved in the structural changes that occur in the larval cuticle just prior to the onset of metamorphosis.     

Development of the nervous system in the brittle star Amphipholis kochii

There are several studies of neural development in various echinoderms, but few on ophiuroids, which develop indirectly via the production of pluteus larvae, as do echinoids. To determine the extent of similarity of neuroanatomy and neural development in the ophiuroids with other echinoderm larvae, we investigated the development of the nervous system in the brittle star Amphipholis kochii (Echinodermata: Ophiuroidea) by immunohistochemistry. Immunoreactive cells first appeared bilaterally in the animal pole at the late gastrula stage, and there was little migration of the neural precursors during A. kochii ontogeny, as is also the case in echinoids and holothuroids. On the other hand, neural specification in the presumptive ciliary band near the base of the arms does occur in ophiuroid larvae and is a feature they share with echinoids and ophiuroids. The ophiopluteus larval nervous system is similar to that of auricularia larvae on the whole, including the lack of a fine network of neurites in the epidermis and the presence of neural connections across the oral epidermis. Ophioplutei possess a pair of bilateral apical organs that differ from those of echinoid echinoplutei in terms of relative position. They also possess coiled cilia, which may possess a sensory function, but in the same location as the serotonergic apical ganglia. These coiled cilia are thought to be a derived structure in pluteus-like larvae. Our results suggest that the neural specification in the animal plate in ophiuroids, holothuroids, and echinoids is a plesiomorphic feature of the Ambulacraria, whereas neural specification at the base of the larval arms may be a more derived state restricted to pluteus-like larvae.     

Blastodermic cuticles of a springtail,Tomocerus ishibashii Yosii (Collembola : Tomoceridae)

The formation and structure of the blastodermic cuticles of a springtail, Tomocerus ishibashii Yosii (Collembola : Tomoceridae) are described together with the change of egg membrane. The blastodermic cuticles of the Collembola are 2-layered, and formed in the early stages of the embryonic development, preceding the differentiation of germ band. The first blastodermic cuticle is thicker (about 0.8-1.5 μm in thickness) and its surface is provided with complex structures, whereas the second one is thinner (about 0.2-0.4 μm in thickness) and smooth. About 3 days after oviposition, the chorion (about 2 μm in thickness) splits into 2 and the first blastodermic cuticle, provided with many projections and 4 large spines appear on the surface of the egg. Three types of projections are distinguished: button-, cone- and seta-like structures. The halves of the ruptured chorion are attached to the first blastodermic cuticle on both sides below the spines, and no projections are found in the regions concealed by the ruptured chorion. The projections of the first blastodermic cuticle are formed by cellular protrusions of the blastoderm. The conspicuous large spines on the first blastodermic cuticle are formed by the evaginations of the blastoderm. Tendrils of the primary dorsal organ run between the first and second blastodermic cuticles.     

The cuticle of Caenorhabditis elegans. II. Stage-specific changes in ultrastructure and protein composition during postembryonic development

The cuticle of the free-living nematode Caenorhabditis elegans is a proteinaceous extracellular structure that is replaced at each of four postembryonic molts by the underlying hypodermis. The cuticles of the adult and three juvenile stages (L1, Dauer larva, L4) have been compared ultrastructurally and biochemically. Each cuticle has an annulated surface and comprises two main layers, an inner basal layer and an outer cortical layer. The adult cuticle has an additional clear layer which separates the basal and cortical layers and is traversed by regularly arranged columns of electron-dense material. The fine structure of the cortical layer is similar in cuticles from different stages while that of the basal layer is stage specific. Purified cuticles were obtained by sonication and treatment with sodium dodecyl sulfate (SDS) and their component proteins solubilized with a sulfhydryl reducing agent. The degree of cuticle solubility is stage specific and the insoluble structures for each cuticle were localized by electron microscopy. Analysis of ³⁵S-labeled soluble cuticle proteins by SDS-polyacrylamide gel electrophoresis yields unique banding patterns for each stage. Most proteins are of high molecular weight (100–200 K) and are restricted to particular stages. Sixteen of the nineteen major proteins characterized are specifically degraded by bacterial collagenase. The results indicate that the different molts are not reiterative, but require the integration of both unique and shared gene functions. The potential use of stage-specific cuticle differences to identify and characterize regulatory genes controlling cuticle-type switching during development is discussed.     

The cuticle proteins of Drosophila melanogaster: stage specificity

Five stage-specific cuticles are produced during the development of Drosophila. Urea-soluble proteins were extracted from each developmental stage and compared by gel electrophoresis. Proteins from first and second instar cuticle are identical except for minor differences in two proteins. Each subsequent stage, third instar, pupa, and adult, has a unique set of cuticle proteins. Qualitative changes within stages are seen in proteins from third instar and adult cuticle. Third instar cuticle proteins can be divided into “early” [proteins 2a, 3, 4, 5, 7, and 8] and “late” [proteins 2 and 1] groups. Adult cuticle proteins change in relative amounts during pharate adult development and change mobility at eclosion. The lower abdominal pupal cuticle lacks a protein found in the pupal cuticle covering the head and thorax. Cuticle proteins from each stage are immunologically related. Nonetheless, electrophoretic variants of three larval proteins do not affect any major changes in the electrophoretic mobility of proteins from other stages. We propose that each stage (except first and second instar) has proteins encoded by discrete genes.     

Identification of an asparagine amidohydrolase from the filarial parasite Dirofilaria immitis

The nematode cuticle is a complex extracellular structure which is secreted by an underlying syncytium of hypodermal cells. Recent studies have demonstrated that the cuticle of parasitic nematodes is a dynamic structure with important absorptive, secretory, and enzymatic activities. In addition, the cuticle serves as a protective barrier against the host. A 48-h third stage larval Dirofilaria immitis cDNA library was immunoscreened with sera raised against larval cuticles. One clone, L3MC4 that reacted strongly with the anti-cuticle antisera was sequenced. The composite cDNA sequence comprises 2073 bp coding for a full-length protein of 590 amino acids. GenBank analysis showed that DiAsp had significant similarity to a Caenorhabditis elegans gene-product (54% identity) and to other asparaginases at the amino acid level. Escherichia coli-expressed recombinant DiAsp (rDiAsp) catalysed the hydrolysis of asparagine to aspartate and ammonia. Antibodies raised against D. immitis larval cuticles reacted with rDiAsp in immunoblots. This is the first report of identification of a cDNA clone encoding an asparaginase enzyme from a parasitic nematode.     

Developmentally and regionally regulated participation of epidermal cells in the formation of collagen lamella of anuran tadpole skin

We investigated the cellular mechanism of formation of subepidermal thick bundles of collagen (collagen lamella) during larval development of the bullfrog, Rana catesbeiana, using cDNA of alpha1(I) collagen as a probe. The originally bilayered larval epidermis contains basal skein cells and apical cells, and the collagen lamella is directly attached to the basement membrane. The basal skein cells above the collagen lamella and fibroblasts beneath it intensively expressed the alpha1(I) gene. As the skin developed, suprabasal skein cells ceased expression of the gene. Concomitantly, the fibroblasts started to outwardly migrate, penetrated into the lamella and formed connective tissue between the epidermis and the lamella. These fibroblasts intensively expressed the gene. As the connective tissue developed, the basal skein cells ceased to express the gene and were replaced by larval basal cells that did not express the gene. These dynamic changes took place first in a lateral region of the body skin and proceeded to all other regions except the tail. Isolated cultured skein cells expressed the gene and extracellularly deposited its protein as the type I collagen fibrils. Thus, it is concluded that anuran larval epidermal cells can autonomously and intrinsically synthesize type I collagen.     

Pupal cuticle formation by Manduca sexta epidermis in vitro: Patterns of ecdysone sensitivity

During the larval-pupal transformation, various regions of the epidermis of Manduca sexta larvae have previously been found to require different lengths of exposure to the prothoracic glands in order to form pupal cuticle. To distinguish between requirements for differing threshold concentrations of ecdysone and those for differing durations of exposure to ecdysone, wandering stage larval epidermis was cultured in Grace's medium. When most of the thick larval cuticle was removed, the epidermis responded to concentrations of β-ecdysone of 1.0 μ/ml or greater for 4 days by forming cysts which later formed tanned pupal cuticle. No fat body or protein supplement was required. When the larval integument was explanted intact, similar requirements for cuticle formation and for tanning were found. All regions of the fifth abdominal segment required similar concentrations of β-ecdysone (0.4–0.6 μg/ml) for 4 days for 50% to form pupal cuticle, but gin trap epidermis required the least exposure to a threshold concentration of ecdysone (1.5 days in 0.9 μg/ml). The anterior dorsal intersegmental region required about 0.5 day longer, followed by the posterior intersegmental and the dorsal intrasegmental regions. Thus, the duration of exposure seemed more important. About 1 day longer of exposure to ecdysone was required for subsequent tanning of the new cuticle than for cuticle formation, yet tanning of the cuticle did not occur with prolonged exposure to ecdysone.     

An ultrastructural study of the larval integument of the midge,Chironomus riparius meigen (Diptera: Chironomidae)

Summary The larval integument of the midge, Chironomus riparius Mg., is unusually thin although it conforms with the normal insect pattern. The cuticle of the post-cephalic segments is about 3 m thick and overlies an epidermis which has an irregular basal plasma membrane resulting in spaces occurring between it and the basement membrane. The ventral tubuli have a similar epidermis but the cuticle is somewhat thinner. The anal papillae have the thinnest cuticular covering with a uniquely folded epicuticle of variable thickness, and their epidermis has the characteristics of a transporting epithelium. No evidence of pore canals could be found in the cuticle of any part except the head capsule which has a remarkably smooth epicuticle and a distinct layer which may represent the exocuticle. There are no spaces between the basement membrane and basal plasma membrane of the epidermis in the head. Ultrastructural evidence would suggest that gaseous exchange can occur across most of the post-cephalic integument.The author is indebted to Mrs. L. Rolph and Mr. R.L. Jones for their technical assistance     

Correlations between epidermal DNA synthesis and haemolymph ecdysteroid titre during the last larval instar of the tobacco hornworm, Manduca sexta

During the fifth larval instar of Manduca sexta the commitment of the epidermis to the synthesis of pupal cuticle is presumably affected by a small increase in ecdysteroid titre when juvenile hormone levels are minimal. Two sequential rounds of DNA synthesis without an intervening mitosis occur at about this time, resulting in polyploidy of the epidermis. There is a definite temporal correlation between the first peak of ecdysone and the second round of DNA synthesis and indirect evidence has been presented which suggests that this small increase in ecdysteroid titre actually initiates the second period of DNA synthesis. Further, it appears that large doses of ecdysteroids do not elicit the same response as smaller doses at a specific developmental stage, indicating that the different physiological effects of ecdysteroids (reprogramming and apolysis) may be dependent upon the relative concentration of the hormone. Following mitosis which takes place on approximately day 6 of the last instar, the epidermis undergoes apolysis and secretes pupal cuticle, expressing the commitment made 4.5 days earlier. These results support the ‘quantal mitosis’ theory of cytodifferentiation since the covert differentiative event occurs during a period of DNA synthesis and since mitosis precedes the expression of that event.     

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.     

Transverse cuticular structures arise above actin filament bundles in the epidermis of Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae)

Rhodaminyl phalloin labelling of larval epidermal cells in Calpodes ethlius (Stöll) (Lepidoptera : Hesperiidae) shows dorsal areas with apical bundles of F-actin. The bundles are present only during the first 36 hr of the 5th stadium. Most cells have only one or 2, rarely 3, 4 or 5. The bundles extend into the overlying cuticle as the cores of large helical microvilli that continue on as transverse cuticle components, resembling very large helical pore canals. The transverse structures are like those seen in extensible insect cuticles that may allow cuticular stretching during larval growth. Neither the bundles nor the transverse structures are easily resolvable by conventional stains for LM or EM. The results suggest that transverse fibrillar structures may be a more common component of soft cuticles than has been generally realized.     

Development and ultrastructure of the rigid dorsal and flexible ventral cuticles of the elytron of the red flour beetle,Tribolium castaneum

Insect exoskeletons are composed of the cuticle, a biomaterial primarily formed from the linear and relatively rigid polysaccharide, chitin, and structural proteins. This extracellular material serves both as a skin and skeleton, protecting insects from environmental stresses and mechanical damage. Despite its rather limited compositional palette, cuticles in different anatomical regions or developmental stages exhibit remarkably diverse physicochemical and mechanical properties because of differences in chemical composition, molecular interactions and morphological architecture of the various layers and sublayers throughout the cuticle including the envelope, epicuticle and procuticle (exocuticle and endocuticle). Even though the ultrastructure of the arthropod cuticle has been studied rather extensively, its temporal developmental pattern, in particular, the synchronous development of the functional layers in different cuticles during a molt, is not well understood. The beetle elytron, which is a highly modified and sclerotized forewing, offers excellent advantages for such a study because it can be easily isolated at precise time points during development. In this study, we describe the morphogenesis of the dorsal and ventral cuticles of the elytron of the red flour beetle, Tribolium castaneum, during the period from the 0 d-old pupa to the 9 d-old adult. The deposition of exocuticle and mesocuticle is substantially different in the two cuticles. The dorsal cuticle is four-fold thicker than the ventral. Unlike the ventral cuticle, the dorsal contains a thicker exocuticle consisting of a large number of horizontal laminae and vertical pore canals with pore canal fibers and rib-like veins and bristles as well as a mesocuticle, lying right above the enodcuticle. The degree of sclerotization appears to be much greater in the dorsal cuticle. All of these differences result in a relatively thick and tanned rigid dorsal cuticle and a much thinner and less pigmented membrane-like ventral cuticle.     

FURTHER STUDY RE-EXAMINING WHETHER LARVAL EPIDERMIS CAN OMIT THE SECRETION OF A PUPAL CUTICLE, USING THE SILKWORM, BOMBYX MORI

When larval tissue is exposed to a hormonal milieu lacking juvenile hormone, adult characters appear directly, omitting the pupal stage, in some insects but not in others, including Bombyx mori. An attempt was made to induce omission of pupal characters in this species by varying the stage of the larval epidermis to be tested. Pieces of larval integument taken from fourth- and fifth-instar larvae of various stages were transplanted to developing adults. Although the number of cuticle layers and the types of cuticle produced differed depending on the age of the donors, none of the pieces omitted secreting the pupal cuticle. It is concluded that the larval epidermis cannot omit secreting pupal cuticle, and that a transition of tissue competence may play an important part in the sequential appearance of larval, pupal, and adult characters.     

杏花蜜腺的发育解剖学研究

杏（Armeniaca vulgaris Lam.）花蜜腺杯状,位于凹形的花托内壁上,属花托蜜腺,由分泌表皮和产蜜组织组成。其分泌表皮具特化的角质层,气孔器下陷并失去关闭功能。液泡呈有规律的变化,淀粉粒的动态成较明显。分泌表皮中的淀粉粒逐渐减少,在杏花的初放期巳基本上完全消失,而产蜜组织中的淀粉粒积累却在杏花的初放期达到高潮。分泌表皮和产蜜组织中的蛋白质含量都较丰富,且在整个发育过程中基本保持恒定,泌蜜方式为表皮角质层和气孔。