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.     

Fine structure and function of the digestive tract of Cyathura carinata (Krøyer) (Crustacea,Isopoda)

Summary The entire gut of Cyathura carinata is lined by a cuticle indicating its completely ectodermal origin. By flattening of the epithelial folds and possibly also of reserve-folds of the plasma membrane the intestine is highly dilatable, an adaptation towards a rapid uptake of the food which is sucked in by means of specialized mouthparts, which pierce the body wall of its main prey, the polychaete Nereis diversicolor. Bundles of microtubules within the intestinal cells presumably represent cytoskeletal structures providing protection against mechanical stress. Spirally arranged muscle fibres, which form peculiar contact areas with the gut, can easily follow any dilatation. A few indications of the metabolic functions of the anterior gut epithelium have been found: Basally and apically located labyrinthine structures of the plasma membrane, apically located clear vesicles, positive reactions for lysosomal, mitochondrial and membraneous enzymes, a strikingly thin and loosely arranged cuticle through which food substances of low molecular weight may diffuse. The cells of the gut and also of the digestive caeca are interconnected by desmosomes, extensive pleated septate junctions, and gap junctions. In the pleon the gut is less dilatable and devoid of plasma membrane specializations. In this area tendon cells, particularly rich in microtubules, serve as attachment sites for the dilating muscles of the rectum. The digestive caeca synthetize and secrete digestive enzymes, mix food and enzymes in their lumen, resorb food molecules, store lipids and glycogen. In the glandular epithelium small cells, rich in rough ER, and a majority of large cells, rich in lipid droplets, occur which, however, are interconnected by a series of morphologically intermediate cells. All cells bear an apical brush border, form a basal labyrinth and contain high to medium activities of acid phosphatase, nonspecific esterases, ATPase, and succinic dehydrogenase. The ER-rich cells are far less frequent than in the omnivorous or herbivorous isopods (Sphaeroma, Idothea, Asellidae, Oniscoidea).     

The fine structure of the buccal tentacles of Holothuria forskali (Echinodermata,Holothuroidea)

Summary Ultrastructural study of the buccal tentacles of Holothuria forskali revealed that each tentacle bears numerous apical papillae. Each papilla consists of several differentiated sensory buds.The epidermis of the buds is composed of three cell types, i.e. mucus cells, ciliated cells, and glandular vesicular cells (GV cells). The GV cells have apical microvilli; they contain bundles of cross striated fibrillae associated with microtubules. Ciliated cells have a short non-motile cilium. Bud epidermal cells intimately contact an epineural nervous plate which is located slightly above the basement membrane of the epidermis. The epineural plate of each bud connects with the hyponeural nerve plexus of the tentacle. This nerve plexus consists of an axonic meshwork surrounded in places by sheath cells. The buccal tentacles have well-developed mesothelial muscles. Direct innervation of these muscles by the hyponeural nerve plexus was not seen.It is suggested that the buccal tentacles of H. forskali are sensory organs. They would recognize the organically richest areas of the sediment surface through the chemosensitive abilities of their apical buds. Tentacles presumably trap particles by wedging them between their buds and papillae.     

The association of microtubules with the plasmalemma in epidermal tendon cells of the river crab

The mode of association of microtubules (MTs) with the plasmalemma in epidermal tendon cells of the river crab, Potamon dehaani was studied by thin-section electron microscopy. In the leg muscle, the tendon cells connect striated muscle cells with the cuticle, forming specialized junctions at both ends. At the muscle-tendon cell junction, the apposed plasmalemmas are interdigitated in a zig-zag pattern separated by a uniform space of about 50 nm, where the basal lamina is shared by two cells. At the tendon cell-cuticle junction, the plasmalemma of the tendon cell forms many conical invaginations, into which dense fibrous material extends from the cuticle. Inside the tendon cell, numerous microtubules run parallel to the direction of tension transmission and are arranged into parallel bundles of various sizes. Within such bundles, fine filamentous structures cross-link adjacent MTs. MTs span the entire length of the cell and attach at their both ends to the junctional domains of the plasmalemma. The junctional plasmalemma is characterized by formation of an electron-dense undercoat, through which MTs are connected with the plasmalemma proper. The ultrastructural features of MT association with the plasmalemma are basically the same at both junctions. At the junctions, MTs usually terminate with free ends and are linked laterally to the plasmalemmal undercoat with fine filamentous structures. These observations emphasize the role of the plasmalemmal undercoat as a device of the attachment of MTs to the plasmalemma.     

Keratinization of fish skin with special reference to the catfish Bagarius bagarius

Summary Histochemical reactions indicating keratinization have previously been demonstrated in parts of the epidermis of Bagarius bagarius. Fluorescence histochemistry and electron microscopy have now confirmed these results. Elevated areas of the epidermis are capped by a layer of dead cells with altered contents. On the outer aspect of these cells a dense layer, 18 nm thick, beneath the plasma membrane corresponds to the resistant envelope found in keratinized cells in tetrapod vertebrates. In Bagarius this layer does not extend to all faces of the keratinized cells, but a similar envelope has been detected in two other sites of piscine keratinized epidermis investigated, namely in the breeding tubercles of Phoxinus phoxinus and in the teeth of Lampetra fluviatilis. In the elevated areas of Bagarius-epidermis, the epithelial cells undergo progressive changes in cytoplasmic organization as they become more superficial. The second tier from the surface is sealed by tight junctions and is separated from the overlying keratinized cells by a sub-corneal space resembling that found in keratinized amphibian epidermis. Histochemical evidence of a high lipid content in the outer layers of the epidermis correlates with the presence of lipid inclusions and lamellated membranous profiles in the material studied by electron microscopy. Histochemical results show that the fin skin of Blennius pholis is not keratinized, but secretes a cuticle, histochemically reactive for both proteins and glycoproteins.     

A putative new hydrostatic skeletal function for the epidermis in Monhysterids (Nematoda)

The ultrastructure of the epidermis of two Monhysterid nematodes (Geomonhystera disjuncta and Diplolaimella dievengatensis) is studied in detail. The epidermis is composed of discrete uninucleated cells. The cytoplasmic layer of the epidermis between the cuticle and the somatic muscles is very thin and contains bundles of filaments that attach the muscles to the cuticle. The epidermal chords are voluminous and contain the nuclei and most of the cell organelles. In the chords many large electron-transparent vacuoles are found. It is hypothesized that these vacuoles fulfill a function as a compartmentalised hydrostatic skeleton.     

Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.     

Spindle membranes and calcium sequestration during meiosis ofDysdercus intermedius (Heteroptera)

The fate of intracellular membranes stained by the osmium ferricyanide (OsFeCN) procedure was followed from premeiotic interphase to interkinesis inDysdercus intermedius. During diakinesis the centrioles forming primary cilia attach temporarily with their proximal ends to the nuclear envelope which is stretched from pole to pole. Breakdown of the nuclear envelope is preceded by deep indentations with microtubules from growing asters. Vesicles of smooth endoplasmic reticulum which accumulate gradually in the course of prophase contribute to the ensheathment of the chromosomes with membranes. When the nuclear envelope breaks down, the polar parts of the formerly perinuclear membranes follow the ingrowth of the spindle microtubules towards the cell equator where the seven bivalents are arranged in a circle with the X₁X₂ sex chromosomes in the centre. The metaphase I spindle thus contains longitudinally oriented membranes between the poles, membranous envelopes around all chromosomes and radial connections from the autosomes to the sex chromosomes in the centre. At anaphase the homologues leave their common sheath and a microtubular stembody surrounded by membranes appears between the receding dyads. In the interkinetic nucleus the gonosomes are separated from the autosomes by a common membranous sheath which may be instrumental in their joint assignment to only one pole in the second meiotic division. Calcium sequestering sites visualized by oxalate precipitation are the Golgi lamellae and vesicles derived from them that surround the whole spindle body.     

Ultrastructure of the peritrophic membrane-secreting cells in the cardia of the blowfly, Lucilia cuprina

Scanning and transmission electron microscopy are used to reveal the internal anatomy and ultrastructure of the cardia which is the source of the triple layered peritrophic membrane in the blowfly Lucilia cuprina. Within the cardia, rings of secretory cells (formation zones) and non-secretory tissue (valvula cardiaca) interlock to secrete and mould the layers of membrane. Formation zone cells have abundant rough endoplasmic reticulum, Golgi and secretory vesicles. A portion of midgut just posterior to the formation zone is covered by close-packed microvilli connected by septate-like junctions. The cuticle-lined valvula cardiaca is rich in smooth endoplasmic reticulum, glycogen and microtubules. The oesophageal cuticle is unusual in containing tubular structures. The ultrastructural features of the separate components of the cardia are discussed in terms of their secretory and non-secretory roles; modified midgut cells secrete chitin and protein whereas modified foregut tissue (valvula cardiaca) appears to be adapted to provide structural integrity (extensive junctions, microtubules), movement (muscles, possibly microtubules), a store of energy (glycogen deposits) and possibly a lipidic secretion (from smooth endoplasmic reticulum) to lubricate the passage of the membranes.     

The fine structure of wound repair in an insect (Rhodnius prolixus)

The cells surrounding a wound in the integument of Rhodnius adults show an increase in RNA content, cytochrome oxidase and esterase activity. An excision in the integument is filled by blood which coagulates and is tanned into an insoluble membrane. The basement membrane of the adjoining epidermis acts as a self-sealing membrane and contracts to cover the excision. The epidermis is attached to the cuticle by the subcuticular layer which it resorbs and by pore canal filaments which are left behind as it migrates. The epidermis migrates as a sheet in contact with the cuticle then with the coagulated blood and basement membrane which cover the excision. Blood cells migrate individually into an excision and do not adhere to a surface in the process. Microtubules cannot be identified with movement. Both epidermal and blood cells remove the cells killed by wounding as evidenced by the appearance of coated vesicles and phagocytic bodies in both cell types. The reconstituted integument consists of a surface membrane in which the layers of the epicuticle are not distinguishable, a nonlamellate cuticle secreted by an epidermis which also appears to secrete the new basement membrane.     

Ultrastructure and Cortical Morphogenesis in the Euplotine Hypotrich Certesia quadrinucleata Fabre-Domergue, 1885 (Ciliophora,Protozoa)1

The cortical development during cell division and the interphase ultrastructure of the marine interstitial hypotrich Certesia quadrinucleata is described using light microscopy and both scanning and transmission electron microscopy. Membranelles are paramembranelles; postciliary microtubules from rightmost membranellar kinetosomes line the buccal cavity and separate parallel arrays of pharyngeal discs that border the cytopharynx. A large paroral membrane is present; an endoral membrane is absent. Alveolar plates lie within alveolar membranes except in regions where organelles and organellar complexes (cirri, the condylopallium, dorsal bristles, membranelles, and the paroral membrane) emerge from the cortex. Muciferous-like bodies attach to the plasma membrane in these regions. Dorsal bristles possess transverse and postciliary microtubules as well as kinetodesmal fiber like those of other hypotrichs. Lasiosomes are present. A unique bulbous structure—the condylopallium—protrudes from the anterior right of the cell. The morphogenetic pattern is euplotine in that cortical development begins in one latitudinal zone, and the oral primordium of the opisthe develops within a subsurface pouch apart from the frontal primordia. Microtubular bundles appear beside (later attached to) developing frontal anlagen; they disappear after cirri are in final interphase locations. Although possessing unique characters, Certesia shares a close phylogenetic relationship with Euplotes.     

The fine structure of the somatic muscles and their attachment to the cuticle in two species of Pycnogonida

The fine structure of the somatic muscles and their attachment to the cuticle in the pyenogonids Nymphon (Chaetonymphon) macronyx G. O. Sars and Boreonymphon cf. abyssorum (Norman) is described. The muscles possess characteristics which are typical of arthropod slow muscle fibers: relatively long sarcomeres, a mean A-band length of about 6 μm and a ratio of thin to thick contractile filaments of 4:1. The sarcotubular system consists of distinct t-tubules, an irregular SR part and randomly distributed dyads and triads, the muscles are attached to the cuticle by specialized epidermal cells containing microtubules extending from the cuticular to the muscular side. The myoepidermal and epidermal-cuticular junctions are described.     

Integrins hold Drosophila together

The Drosophila position-specific (PS) integrins are members of the integrin family of cell surface receptors and are thought to be receptors for extracellular matrix components. Each PS integrin consists of an α subunit, α_PS1 or α_PS2, and a β_PS subunit. Mutations in the β_PS subunit and the α_PS2 subunit have been characterised and reveal that the PS integrins have an essential role in the adhesion of different cell layers to each other. The PS integrins are especially required for the function of the cell-matrix-cell junctions, where the muscles attach to the epidermis and where one surface of the developing wing adheres to the other. These junctions are similar to vertebrate focal adhesions and hemidesmosomes, which also contain integrins. Integrin-mediated cell to cell adhesion via the extracellular matrix provides a way for tissues to adhere to each other without intermingling of their cells.     

The Dufour gland closing apparatus in Formica sanguinea Latreille (Hymenoptera,Formicidae)

Summary A closing apparatus of the Dufour gland is described in the formicine ant Formica sanguinea Latreille 1798. Four sets of muscles are involved, two of which directly attach to the slit-like duct. The latter shows a considerable cuticular thickening of its intima at this level. Ultrastructural observations reveal that the muscles are attached to a cuticula by means of intracellular microtubules in the duct cells. These microtubules run parallel to the myofilaments. Together with the increased contact area for muscular attachments they are believed to ensure the accurate muscular mechanism assuring a well-controlled spraying activity of this gland. Opening of the duct is probably achieved by active muscular contractions, while its closure may be achieved by a passive return to the rest position of the thickened cuticular intima.Research assistant of the Belgian National Fund for Scientific Research     

The harvestman tarsus and tarsal flexor system with notes on appendicular sensory structures in laniatores

The tarsal flexor system, a novel system of retinacular structures, is described for the first time based on morphological and ultrastructural examinations of several Neotropical harvestmen (Opiliones: Laniatores). The tarsal flexor system is made up of many individual pulleys that function to maintain close apposition between the tendon and internal ventral surface of the cuticle in the tarsus. Pulley cells are specialized tendinous cells that form the semi‐circular, retinacular pulley system in the tarsus; these cells contain parallel arrays of microtubules that attach to cuticular fibers extending from deep within the cuticle (i.e., tonofibrillae). The tarsal flexor system is hypothesized to provide mechanical advantage for tarsal flexion and other movements of the tarsus. This system is discussed with regards to other lineages of Opiliones, especially those that exhibit prehensility of the tarsus (i.e., Eupnoi). Comparing tarsal morphology of laniatorid harvestmen to other well‐studied arachnids, we review some literature that may indicate the presence of similar tarsal structures in several arachnid orders. The general internal organization of the tarsus is described, and ultrastructural data are presented for a number of tarsal structures, including sensilla chaetica and the tarsal perforated organ. Sensilla chaetica possess an internal lumen with dendritic processes in the center and exhibit micropores at the distal tip. With respect to the tarsal perforated organ, we found no ultrastructural evidence for a sensory or secretory function, and we argue that this structure is the result of a large pulley attachment site on the internal surface of the cuticle. A small, previously undocumented muscle located in the basitarsus is also reported. J. Morphol. 274:1216–1229, 2013. © 2013 Wiley Periodicals, Inc.     

Ultrastructure of the colon of Locusta migratoria

The ultrastructure of the colon of Locusta migratoria is described. The colon is lined by a thick cuticle that, for the most part, adheres to the underlying epithelium. The cuboid epithelial cells are characterized by moderate invaginations of the apical and, to a lesser extent, basal plasma membranes; the lateral plasma membranes are relatively flat. The bulk of the mitochondria are located in the apical region of the cell and are not particularly associated with any of the plasma membranes. The basal region of the cells contains much rough endoplasmic reticulum, glycogenlike granules, and a predominance of spherical, electron-dense bodies of various sizes. Where muscle fibers make contact with the epithelium, the cells are much reduced; the cytoplasm is usually less electron-dense, and, typically, the nucleus has a thick layer of granular material associated with the inner nuclear membrane. The apical and basal plasma membranes of the reduced epithelial cells contain numerous hemidesmosomes. The apical hemidesmosomes occur in pairs around an extracellular space that contains electron-opaque material. The latter forms tonofibrillae that extend into the endocuticle. Bundles of microtubules are associated with the hemidesmosomes. The tubules traverse the cell from the apical to the basal region. The possible significance of these findings is discussed.     

Organization and differentiation of the body-wall musculature in Macrostomum (Turbellaria,Macrostomidae)

We studied the body-wall musculature, its ECM (extracellular matrix), and the junctional complexes between muscle cells and between muscle cells and ECM in Macrostomum hystricinum marinum Rieger, 1977, using Nomarski-contrast and electron microscopy. Differentiation of these body-wall components was followed by monitoring embryonic stages at 52%, 64%, and 82% of the time between egg-laying and hatching and with study of the hatchling and adult stages. For comparison, the body-wall musculature of other macrostomidans has been examined in conventional light-histological sections.Muscles form a grid of longitudinally, diagonally, and circularly oriented fibers beneath the epidermis in M. hystricinum marinum and this orientation of cells can be found already in embryos at 64% development. Younger embryos at 52% development show no muscle differentiation. The ECM forms a net-like arrangement that apparently envelops the individual muscle cells. Characteristic knob-like thickenings of the ECM occur at the base of the epidermis. Muscle cells attach to each other, to the epidermis, and to other cell types through hemidesmosome-like junctions at thickenings of the ECM in the adult and hatchling stages; no true desmosomes exist between muscle cells. Gap junctions occur commonly between longitudinal muscles of adult specimens and between perikarya of muscle cells in embryos at 64% and 82% development.More comparative studies are needed to determine the systematic value of presence or absence of the diagonal muscle fibers in the body wall of turbellarians.     

The nature of cnidarian desmocytes

The electron microscope reveals that the cnidarian desmocyte is an ectodermal cell which forms acidophil protein tonofibrillae intracellularly. One end of the cell is bound to mesogleal fibrils; the other becomes embedded in the thickening cuticle. The bundle of tonofibrillae later becomes rivetshaped and the cell dies, but still the mesoglea remains bound to the cuticle by means of the rivet. The histochemistry and formation of the rivet as well as the comparative cytology of cnidarian desmocytes are discussed.     

The fine structure of epidermal papillae of Travisia forbesii (Annelida)

The structure of the epidermis of Travisia forbesii was described using light and electron microscopy. The epidermis is a highly modified variant of the normal one-layer polychaete epithelium. It consists of basal epidermal cells and an external layer of closely sited papillae consisting of glandular and supportive epidermal cells, and extensive electron-transparent intercellular spaces. The papillae are embedded in the thick cuticle. Each papilla has a peduncle, which is formed by one cell that penetrates the inner cuticle layer to the basal epidermal cells. A fold of basement membrane forms the core of the peduncle and ends in the base of a papilla. All epidermal cells are connected to each other with apical cell junctions and to the basement membrane with hemidesmosomes, so the epithelium is continuous and uninterrupted. The epidermis has an intra-epidermal neuron plexus. The structure of the papillae is compared with papillae and tubercles of other polychaetes, and the possible functional significance and phylogenetic implications of these structures are discussed.     

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