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.     

Embryonic integument and "molts" in Manduca sexta (Insecta,Lepidoptera)

In Manduca sexta the germ band is formed 12 h post-oviposition (p.o.) (=10% development completed) and is located above the yolk at the egg surface. The cells show a polar organization. They are engaged in the uptake and degradation of yolk globules, pinched off from the yolk cells. This process can be observed in the integumental cells during the first growth phase of the embryo that lasts until "katatrepsis," an embryonic movement that takes place at 40% development completed. At 37% development completed, the ectoderm deposits a thin membrane at its apical surface, the first embryonic membrane, which detaches immediately before katatrepsis. The second period of embryonic growth--from katatrepsis to 84 h p.o. (70% development completed)--starts with the deposition of a second embryonic membrane that is somewhat thicker than the first one and shows a trilaminar, cuticulin-like structure. Whereas the apical cell surface is largely smooth during the deposition of the first embryonic membrane, it forms microvilli during deposition of the second one. At the same time, uptake of formed yolk material ceases and the epidermal cells now contain clusters of mitochondria below the apical surface. Rough endoplasmic reticulum (RER) increases in the perinuclear region. The second embryonic membrane detaches about 63 h p.o. At 69 h p.o., a new generation of microvilli forms and islands of a typical cuticulin layer indicate the onset of the deposition of the larval cuticle. The third growth phase is characterized by a steady increase in the embryo length, the deposition of the larval procuticle, and by cuticular tanning at about 100 h p.o. Beginning at that stage, electron-lucent vesicles aggregate below the epidermal surface and are apparently released below the larval cuticle. Manduca sexta is the first holometabolous insect in which the deposition of embryonic membranes and cuticles has been examined by electron microscopy. In correspondence with hemimetabolous insects, the embryo of M. sexta secretes three covers at approximately the same developmental stage. A marked difference: the second embryonic cover, which in Hemimetabola clearly exhibits a cuticular organization, has instead a membranous, cuticulin-like structure. We see the difference as the result of an evolutionary reductional process promoted by the redundancy of embryonic covers in the egg shell. Embryonic "molts" also occur in noninsect arthropods; their phylogenetical aspects are discussed.     

The transition from planktotrophy to lecithotrophy in larvae of Lower Palaeozoic Rhynchonelliform brachiopods

Criteria are established for defining the presence of protegula formed on embryonic or larval mantle in representative genera of Lower Palaeozoic Obolellata, Strophomenata and Rhynchonellata. Width was used to define protegular type. Taxa with only an embryonic protegulum are inferred to have had lecithotrophic larvae while taxa with a larval protegulum or an embryonic protegulum surrounded by a larval protegulum are inferred to have had planktotrophic larvae. All or most of the taxa examined in the Obolellata, the Strophomenata and the orders Protorothida and Orthida in the Rhynchonellata had planktotrophic larvae. In the Pentamerida a minority of genera had only a larval, or an embryonic and a larval protegulum while a majority had protegular widths indicating lecithotrophy. In the orders Rhynchonellida, Atrypida, Athyrida and Spiriferida derived from the Pentamerida (with the exception of one species in the Atrypida) a number of the genera had protegular widths indicating lecithotrophy. It is suggested that the onset of lecithotrophy in the Pentamerida was associated with a developmental innovation in which the mantle lobe of the larva was reflected over the apical lobe during the process of metamorphosis. This evolutionary innovation probably occurred during the late Cambrian or early Ordovician and was subsequently inherited during the process of cladogenesis.     

The morphology and affinity of anabaritids revealed by their internal molds from the Cambrian Fortunian,southern Shaanxi,China

Anabaritids are a group of tubular skeletal fossils known from the lower Cambrian worldwide. Many previously collected specimens of anabaritids lack both the apical and apertural regions, and thus their complete morphology and growth patterns are not fully understood. Here we describe a number of internal molds of Anabarites from the lower Cambrian (Fortunian Stage) Zhangjiagou Lagerstätte, southern Shaanxi Province. Our new specimens preserved the apex, the aperture with three lobes, and three longitudinal grooves initiating apically rather than adapically as previously reported. This work enriches the morphology of anabaritids, casting doubt on the previous proposition that anabaritids originated from a cylindrical tubular ancestor. It is proposed here that a triradial symmetry may have been established in very early ontogenetic stages of some anabaritids; furthermore, the aperture with three lobes resembles those of olivooids and thus supports a possible close relationship between anabaritids and olivooid scyphozoans.     

Morphology and ultrastructure of the esophagus during the ontogeny of the spider crab Maja brachydactyla (Decapoda,Brachyura, Majidae)

The esophagus of the eucrustaceans is known as a short tube that connects the mouth with the stomach but has generally received little attention by the carcinologists, especially during the larval stages. By this reason, the present study is focused on the morphology and ultrastructure of the esophagus in the brachyuran Maja brachydactyla during the larval development and adult stage. The esophagus shows internally four longitudinal folds. The simple columnar epithelium is covered by a thick cuticle. The epithelial cells of the adults are intensively interdigitated and show abundant apical mitochondria and bundles of filamentous structures. The cuticle surface has microspines and mutually exclusive pores. Three muscle layers surrounded by the connective tissue are reported: circular muscles forming a broad continuous band, longitudinal muscle bundles adjacent to the circular muscles, and dilator muscles crossing the connective tissue vertically toward the epithelium. The connective tissue has rosette glands. The esophagus of the larvae have epithelial cells with big vesicles but poorly developed interdigitations and filamentous structures, the cuticle is formed by a procuticle without differentiated exocuticle and endocuticle, the connective layer is thin and the rosette glands are absent. The observed features can be explained by his role in the swallowing of the food.     

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.     

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. Here, we demonstrate that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. We found that Doc expression was restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevented the lateral extension and deepening of these folds at the larval stage and delayed wing disc bending in the early pupal stage. Ectopic Doc expression was sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression expressed Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins were depleted. Overexpression of Doc along the blade/hinge folds caused precocious wing disc bending, which could be suppressed by co-expressing MMP2RNAi.     

Formation of the hindgut cuticular lining during embryonic development of Porcellio scaber (Crustacea,Isopoda)

The hindgut and foregut in terrestrial isopod crustaceans are ectodermal parts of the digestive system and are lined by cuticle, an apical extracellular matrix secreted by epithelial cells. Morphogenesis of the digestive system was reported in previous studies, but differentiation of the gut cuticle was not followed in detail. This study is focused on ultrastructural analyses of hindgut apical matrices and cuticle in selected intramarsupial developmental stages of the terrestrial isopod Porcellio scaber in comparison to adult animals to obtain data on the hindgut cuticular lining differentiation. Our results show that in late embryos of stages 16 and 18 the apical matrix in the hindgut consists of loose material overlaid by a thin intensely ruffled electron dense lamina facing the lumen. The ultrastructural resemblance to the embryonic epidermal matrices described in several arthropods suggests a common principle in chitinous matrix differentiation. The hindgut matrix in the prehatching embryo of stage 19 shows characteristics of the hindgut cuticle, specifically alignment to the apical epithelial surface and a prominent electron dense layer of epicuticle. In the preceding embryonic stage – stage 18 – an electron dense lamina, closely apposed to the apical cell membrane, is evident and is considered as the first epicuticle formation. In marsupial mancae the advanced features of the hindgut cuticle and epithelium are evident: a more prominent epicuticular layer, formation of cuticular spines and an extensive apical labyrinth. In comparison to the hindgut cuticle of adults, the hindgut cuticle of marsupial manca and in particular the electron dense epicuticular layer are much thinner and the difference between cuticle architecture in the anterior chamber and in the papillate region is not yet distinguishable. Differences from the hindgut cuticle in adults imply not fully developed structure and function of the hindgut cuticle in marsupial manca, possibly related also to different environments, as mancae develop in marsupial fluid. Bacteria, evenly distributed within the homogenous electron dense material in the hindgut lumen, were observed only in one specimen of early marsupial manca. The morphological features of gut cuticle renewal are evident in the late marsupial mancae, and are similar to those observed in the exoskeleton.     

Soldier differentiation during embryogenesis of a social aphid, Pseudoregma bambucicola

To understand the developmental process of aphid soldier differentiation, we investigated the morphological characters of normal nymphs, soldier nymphs and developing embryos of Pseudoregma bambucicola. Results of morphometric analyses showed that normal and soldier nymphs formed discrete clusters on the basis of several morphological characters, although a small number of intermediate individuals, termed ‘intercaste nymphs’, were present. In late embryonic stages, normal and soldier embryos were morphologically distinguishable. The earlier the embryonic stage, the smaller the morphological differences between them. In early embryos less than 1000 µm in length, normal and soldier embryos were not morphologically distinguishable, suggesting that the onset of soldier differentiation occurs at an early embryonic stage. Throughout embryonic development, morphological differentiation of the soldier caste proceeded gradually. Notably, several morphological characters of soldiers grew remarkably upon larviposition. Observation of embryonic leg cuticle revealed a characteristic folding structure, indicating that some morphological traits of the soldier are exaggerated upon larviposition through expansion of the folded cuticle. We suggest that morphological differentiation of the soldier caste in P. bambucicola comprises two phases: gradual growth during embryogenesis and rapid growth upon larviposition.     

Moving on hairy surfaces: modifications of Gratiana spadicea larval legs to attach on its host plant Solanum sisymbriifolium

Both larvae and adults of Gratiana spadicea (Klug) (Coleoptera: Chrysomelidae: Cassidinae) feed exclusively on leaves of Solanum sisymbriifolium Lamarck (Solanaceae), which have simple and stellate trichomes. The simple trichomes can be non-glandular or glandular. The stellate trichomes present a long central ray that secretes a viscous exudate when broken. Trichome effects on movement of G. spadicea larvae were evaluated in the laboratory. Larval speed on intact petioles of S. sisymbriifolium was compared to those where the exudates and/or stellate trichomes were removed. Exudates had no effect on larval speed. Stellate trichomes mechanically slowed down first instar movement. Gratiana spadicea larval legs have a modified distal portion, the tarsungulus, whose rounded aperture shape matches that of the cylindrical pointed rays of S. sisymbriifolium stellate trichomes. The first three instars anchor the tarsungulus to the trichome rays, and get both attachment to the leaf surface and support to body impulsion. Morphological comparisons showed that the legs in the first larval instars are shorter than the central ray of the stellate trichome, so larvae have to walk above them. Fifth instar larvae have longer legs and walk by inserting the sharp tip of their tarsungulus directly into the leaf epidermis. The dimensions of the tarsungulus aperture vary in such a way that it can clasp every thickness of trichome ray for any larval instar. Contrary to other tarsungulus portions and larval body features, where growth is allometric, growth of the tarsungulus aperture is isometric throughout the larval stage. Thus, it is suggested that this G. spadicea leg structure is adapted for moving on hairy leaf surface of S. sisymbriifolium.     

Description of the early ontogenic part of the Tentaculitids, with implications for classification

Ontogenic development and classification of tentaculitids at high systematic levels are reevaluated in the light of new findings on shell structure and morphology of larval parts, and these features are here regarded as being of primary importance for taxonomy. Class Tentaculita Bouček, 1964 is subdivided into two subclasses, of which subclass Chionioconarida Farsan, 1994 is distinguished by a tubular larval process closed at the apex and covered with microrings. The process is differentiated into a prolarval, metalarval and epilarval part, of which the latter coincides with metamorphosis. Morphology of the larval parts suggests that metamorphosis proceeds in two different manners, giving rise to superorders within this subclass. Within superorder Trompetoconarida Farsan, 1994 a bilaterally symmetrical larval cone develops with an aperture oblique to the long axis of the conch; following metamorphosis the conch becomes radially symmetrical and the aperture perpendicular to the axis; secondary shell, septa and pseudopunctae develop in the adult phase, and the structure of the shell is lamellar. In contrast, within the second superorder, Lirioconarida Farsan, 1994 the epilarval tube develops into a larval bulb with no changes in symmetry and position of the aperture; secondary shell, septa and pseudopuncta are absent. The microstructure of the shell is lamellar in the larval part whereas in postlarval parts it is either sigmoidal or lamellar. The subclass Dacryoconarida Fisher, 1962 possesses a subspherical, tear- or drop-like embryonic chamber which may have a caudal process. The microstructure of the embryonic chamber is variable within this group, being lamellar in some taxa whereas in others, a single layer of shell is present. The postembryonic parts of the lamellar forms possess nacreous or sigmoidal structures.     

Cuticle morphogenesis in crustacean embryonic and postembryonic stages

The crustacean cuticle is a chitin-based extracellular matrix, produced in general by epidermal cells and ectodermally derived epithelial cells of the digestive tract. Cuticle morphogenesis is an integrative part of embryonic and postembryonic development and it was studied in several groups of crustaceans, but mainly with a focus on one selected aspect of morphogenesis. Early studies were focused mainly on in vivo or histological observations of embryonic or larval molt cycles and more recently, some ultrastructural studies of the cuticle differentiation during development were performed. The aim of this paper is to review data on exoskeletal and gut cuticle formation during embryonic and postembryonic development in crustaceans, obtained in different developmental stages of different species and to bring together and discuss different aspects of cuticle morphogenesis, namely data on the morphology, ultrastructure, composition, connections to muscles and molt cycles in relation to cuticle differentiation. Based on the comparative evaluation of microscopic analyses of cuticle in crustacean embryonic and postembryonic stages, common principles of cuticle morphogenesis during development are discussed. Additional studies are suggested to further clarify this topic and to connect the new knowledge to related fields.     

Molecular cloning and characterization of a cDNA encoding a novel cuticle protein in the silkworm,Bombyx mori

We have cloned the full length of a novel cDNA named Bombyx mori cuticle protein that contains an AlaAlaProAla/Val-repeat (BMCPA) from a cDNA library of integument in the larval silkworm. Both a typical tandem repeat (A-A-P-A/V) for cuticle protein and a unique tandem repeat with Ser, Ala, Gly, Pro, Val, Tyr and Thr were observed in the predicted amino acid sequence of the cDNA encoding BMCPA. Approximately 80% of the amino acids in BMCPA were composed of Ser, Ala, Gly, Pro, Val and Tyr. Northern-hybridization analysis indicated that BMCPA mRNA is expressed only in the larval epidermis and that the expression pattern of the BMCPA gene in the developmental stage was observed mainly at the larval stage. We propose BMCPA may be a novel component of cuticle, and may play an important role in the integument of the larval silkworm.     

Cytoskeletal F-actin patterns in whole-mounted insect Malpighian tubules.

The distribution of actin filaments in Malpighian tubules of the fleshfly Sarcophaga bullata (Parker) was investigated before and after metamorphosis by means of the rhodamine phalloidin staining method. The numerous primary cells show a pattern of thick basal actin bundles resembling stress fibres of cultured cells, while the apical microvillar zone shows a bright and homogeneous labelling. The less abundant stellate cells contain no such basal actin bundles and their apical microvillar zone gets only faintly stained. Late larval stages display fingerlike infoldings and an increased actin filament concentration at the apical membrane of the stellate cells. During metamorphosis the Malpighian tubules dedifferentiate and eventually redifferentiate to give rise to adult tubules resembling larval ones. The different types of actin filament organisation in the primary and stellate cells of the Malpighian tubules are discussed.     

Ultrastructural studies on the early cuticular metamorphosis of adult female Lernaeocera branchialis (L.) (Copepoda,Pennellidae)

After its final moult and fertilization an adult female of the marine fish-parasitic copepod, Lernaeocera branchialis, begins an extensive metamorphosis. This commences while the parasite is still on the flounder intermediate host and is completed once the female has established itself on the whiting final host. One early component of the metamorphosis is a considerable elongation of the parasite's abdominal region. S.e.m. and t.e.m. studies have revealed that part of the mechanism of the elongation consists of a straightening out of a highly folded abdominal cuticle. Before fertilization, the epicuticle and outer procuticular layers of this integument are thrown into a series of transverse, 4–6 µm deep pleats or folds with a density of 1–1.2 folds/µm of abdominal length. Straightening these folds can generate an approximately 6-fold length increase. The folds are already present beneath the female chalimus IV cuticle when the epidermis of this development stage starts to secrete the adult cuticle. Immediately before the final moult, the adult cuticle is super-folded with the whole cuticle displaying second-order folds, 8–10 µm deep.The capacity of Lernaeocera to engage in extensive cuticular modifications without recourse to a moult is compared with similar abilities shown by some insect species.     

Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans

Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions and associated cytoskeletal elements, which provides prominent mechanical attachment of the epidermis to the cuticle and transmits muscle tensions to the exoskeleton. This linkage involves anchoring of the complex extracellular matrix composing the cuticle to the apical membrane of tendon cells and linking of tendon cells to muscles basally. The ultrastructural arhitecture of these attachment complexes during molting is an important issue in relation to integument integrity maintenance in the course of cuticle replacement and in relation to movement ability. The aim of this work was to determine the ultrastructural organization of exoskeleton - muscles attachment complexes in the molting terrestrial isopod crustaceans, in the stage when integumental epithelium is covered by both, the newly forming cuticle and the old detached cuticle. We show that the old exoskeleton is extensively mechanically connected to the underlying epithelium in the regions of muscle attachment sites by massive arrays of fibers in adult premolt Ligia italica and in prehatching embryos and premolt marsupial mancas of Porcellio scaber. Fibers expand from the tendon cells, traverse the new cuticle and ecdysal space and protrude into the distal layers of the detached cuticle. They likely serve as final anchoring sites before exuviation and may be involved in animal movements in this stage. Tendon cells in the prehatching embryo and in marsupial mancas display a substantial apicobasally oriented transcellular arrays of microtubules, evidently engaged in myotendinous junctions and in apical anchoring of the cuticular matrix. The structural framework of musculoskeletal linkage is basically established in described intramarsupial developmental stages, suggesting its involvement in animal motility within the marsupium.     

Effect of host nutritive value on egg production by Ceratitis capitata (Diptera,Tephritidae)

In a previous study we demonstrated that Ceratitis capitata larvae show a superior performance when they feed on the apical portion of papaya compared to larvae that feed on the basal portion, probably due to the higher levels of sugars in the apical portion. In the present study we carried out experiments to determine if larval feeding on the basal or apical portion of the fruit influences ovarian development and egg production by adult C. capitata females. The results indicate that females that feed on the apical portion during the larval stage have more developed ovaries and produce more eggs than females who feed on the basal portion during the larval stage. We also compared artificial diets of quantitatively similar chemical composition to the apical and basal portions of the papaya fruit. In this last experiment, no differences were detected in any of the parameters studied, i.e. percent emergence, time to pupation, adult size, larval preference, ovary size, egg production and estimated ingestion.     

Cycles embryonnaires et sécrétion de la cuticule chez l'embryon de blatte, Blabera craniifer

Cuticle deposition has been studied with the electron microscope in cockroach embryos (Blabera craniifer) during normal incubation in situ and in culture in vitro, in the absence or presence of inokosterone (a phytoecdysone).Two cuticles are deposited successively during embryonic life, respectively between stages 11 and 17, and stage 21 and 24 hr after hatching. The occurrence of two embryonic cycles is thus demonstrated, the first ending at stage 17 without exuviation since there is no old cuticle to be shed, the second one at hatching.In embryos explanted at stage 17 and cultured in vitro, the formation of cuticle 2 occurred at the same rate as in situ. The addition of inokosterone (50 μg/ml) to the medium resulted in the early onset of cuticle deposition (in 3 days as compared with 15 days in situ) in legs previously cut at the base of the tarsus. Cuticle 2 was completed within 9 days after explantation (as compared with about 20 days elapsing in the normal embryo between stage 17 and the completion of cuticle 2). Unsectioned appendages were insensitive to the hormone.Regeneration of sectioned legs, which occurred normally n vitro in non-treated embryos, was completely inhibited in the presence of inokosterone, presumably because the hormone caused early immobilization of cells through accelerated cuticle formation.Results suggest that embryonic cycles are controlled by the same hormonal mechanism as larval cycles.     

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.