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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.
Cuticle, chitin, microtubules, anchoring junctions, extracellular matrix, embryo
The arthropod exoskeleton performs diverse functions, including mechanical support, sensing, prevention of desiccation and protection against pathogens and predators. Locomotion of these animals is based on extensive connections between exoskeleton and muscular system. The exoskeleton consists of a complex chitin-protein matrix, secreted by a single-layered epithelium. The chitin-protein matrix is either non-calcified or calcified, as in insects and crustaceans, respectively. Specialized epithelial cells, named tendon cells, are the sites of firm mechanical connections between exoskeleton and underlying tissues (
The ultrastructure, molecular composition and differentiation of specialized anchoring complexes between exoskeleton, tendon cells and muscle cells were extensively studied in an insect Drosophila melanogaster, with emphasis on myotendinous junction characterization (
Here we report new data on the ultrastructural architecture of anchoring complexes comprising exoskeleton, tendon cells and muscles in adult premolt isopod crustaceans, in premolt marsupial mancas and in prehatching embryos. Our study is focused primarily to connections between the complex matrix of the exoskeleton and tendon cells, modified epithelial cells at the sites of muscles attachment. To the best of our knowledge, the exoskeleton anchoring to underlying tissues in embryos and marsupial mancas of crustaceans has not been characterized before and its ultrastructural organization is presented here. Comparative evaluation of the results with respect to the other arthropods, particularly to insect model organism Drosophila melanogaster, is presented. The involvement of these anchoring connections in molting and in intramarsupial motility is discussed.
A scheme showing the general architecture of the muscle attachment to the epidermis in arthropods (adapted from
Specimens of Ligia italica Fabricius, 1798 (Crustacea: Isopoda) were collected at the Piran Bay coast in Slovenia. Animals were inspected for ventral sternal deposits and premolt adult specimens were anaesthetized. The dorsal parts of pereonites were isolated, fixed in 2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.3) and postfixed by 1% OsO4. After washing and dehydration in a graded series of ethanol, samples were embedded in an epoxy resin mixture. Semithin sections were stained with Azure II. – Methylene blue. Ultrathin sections were either imaged non-contrasted or contrasted with uranyl acetate and lead citrate.
Specimens of Porcellio scaber Latreille, 1804 (Crustacea: Isopoda) prehatching embryos and marsupial mancas were isolated from brood pouches of females maintained in laboratory culture. Determination of intramarsupial developmental stages was performed as described in
Light microscopy was performed by AxioImager Z.1 microscope (Zeiss) equiped with an AxioCam HRc camera and Axiovision software. Ultrastructural imaging was performed by CM 100 transmission electron microscope (Phillips) equiped with a BioScan 792 digital camera (Gatan) and Digital Micrograph software.
Results Adult premolt specimensThe ultrastructural architecture of the exoskeleton – muscles attachment regions was analysed in the dorsal parts of pereonites in adult premolt Ligia italica. The pre-ecdysial cuticle was tightly connected to the underlying tendon cells and muscles already in the early premolt (Fig. 2). Extensive connections were established between the chitin-protein matrix of the newly forming cuticle and the apical parts of the tendon cells in the early phase of cuticle elaboration. These matrix – cell linkages consisted of numerous fibrous structures. The most intriguing result was that the fibers traversed the entire new cuticle, spanned the whole ecdysal space and protruded deep into the old cuticle (Figs 2B, C). They extended from the tendon cell apex up to the exocuticular layer in the old cuticle. The continuity of these fibers was clearly followed in some sections, revealing a direct mechanical connection of the detached exoskeleton to epithelium in these regions. Fibrous structures were arranged in parallel to one another and in the same direction as microtubules arrays in the tendon cells and myofilaments arrays in the muscle cells. This direction is roughly perpendicular to the body surface. Fibrous connections followed approximately straight lines and did not display any branching or prominent curvatures. General architecture of the preecdysal cuticle at muscle attachment sites was similar to that in the other regions, displaying epicuticular and exocuticular layers and characteristic pattern of chitin-protein fibers arrangement.
Extensive parallel arrays of microtubules inside the tendon cells were aligned in the apical to basal direction (Fig. 2D). A concourse of microtubules towards the cytoplasmic densities at the apical membrane (hemidesmosome-like structures) of tendon cells was evident. On the basal side the microtubules were positioned close to the electron dense plaques along the basal membrane, engaged in myotendinous junction.
Prominent anchoring junctions were evident between muscle cells and tendon cells (Fig. 3). The entire basal membrane of the tendon cell was intensely folded in a zigzag pattern, exactly matching the folding of the muscle cell sarcolemma beneath. Both cell surfaces contributing to this junction were closely apposed and the intervening layer of the extracellular matrix material was not conspicous. The complex connection between these two cells in premolt specimens corresponds to characteristic design in animals with one complete cuticle (Supplementary figure), comprising electron dense plaques beneath both cell membranes and extracellular material in the narrow intercellular space.
Exoskeleton – muscle attachment in the dorsal parts of pereonites in adult premolt Ligia italica. Overview of the muscle attachment in a specimen with a detached and a newly forming cuticle (2A a semithin section). The new cuticle is extensively connected to the underlying tendon cells already in the early premolt. Fibrous connections running from the apical region of tendon cells through the new pre-ecdysal cuticle and ecdysal space up to the exocuticle of the detached exoskeleton are evident (2B, C). Parallel arrays of microtubules and apical electron dense plaques are characteristic for tendon cells (2D). DC detached cuticle NC new cuticle ES ecdysal space MC muscle cell TC tendon cell Mt microtubules; arrowheads – fibrous connections.
Myotendinous junction in the dorsal parts of pereonites of premolt adult Ligia italica is an extensive anchoring junction. The entire zigzag folded basal and apical membranes of tendon and muscle cells, respectively, are engaged in this outstanding intercellular mechanical connection (3A). Prominent electron dense cytoplasmic plaques are evident along both cell membranes, separated by a thin layer of extracellular matrix (3B). MC muscle cell TC tendon cell.
Development of Porcellio scaber embryos and marsupial mancas involves renewal of the exoskeleton. Prehatching embryo and several marsupial mancas displaying morphological attributes of premolt were analysed in this study. The detachment of the old cuticle and disintegration of the basal parts of the detached cuticle were identified in the integument of these specimens. In addition, the substantial new cuticle formation was evident in marsupial mancas. The exoskeleton in the prehatching embryo and in the premolt marsupial mancas was extensively connected to tendon cells and muscles underneath and extensive intercellular junctions between muscle cells and tendon cells were established (Fig. 4).
Numerous fibrous connections between the detached cuticle and the apical membrane of tendon cells were evident in all premolt intramarsupial animals examined (Fig. 5). The newly forming cuticle in marsupial mancas, consisting of epicuticle and a few layers of the pre-ecdysal procuticle, was mechanically connected to tendon cells.
Tendon cells ultrastructurally resembled the adult arthropod tendon cells and were characterized by apicobasal arrays of microtubules. Apically, the microtubules were found close to oblong electron dense regions (hemidesmosome-like structures), aligned in the same direction (Figs 5D–F and Fig. 6). In longitudinal and in oblique sections the profiles of microtubules were evidenced in close proximity to the electron dense plaques of junctions along the tendon cell basal membrane in both, prehatching embryos (Figs 7A, B) and marsupial mancas (Figs 7C, D).
Myotendinous junctions displayed a characteristic zigzag outline, occupying the entire tendon – muscle interface (Fig. 7). From the structural point of view, the myotendinous junctions in marsupial mancas closely resembled these junctions in adult arthropods, comprising a thin layer of extracellular material between cell membranes and dense cytoplasmic plaques of approximately equal densities and thicknesses below the both cell membranes (Figs 7C, D). On the other hand, in the prehatching embryo, the intracellular membrane-associated layer of dense cytoplasmic material contributing to the anchoring junction in a tendon cell was more lucent and thinner than that contributing to the junction in a muscle cell (Figs 7A, B).
Overview of exoskeleton anchoring to tendon cells and muscles in intramarsupial developmental stages of Porcellio scaber. In prehatching embryos (4A, B) and in premolt marsupial mancas (4C a semithin section and 4D) the connections between the exoskeleton, tendon cells and muscle cells were already established. EX exoskeleton ES ecdysal space NC new cuticle TC tendon cell MC muscle cell.
Anchoring junctions between the detached cuticle and the apical part of tendon cells in the prehatching embryo (5A, D, E) and marsupial mancas (5B, C, F) of Porcellio scaber (ultrathin cross-sections). The newly forming cuticle in marsupial mancas, consisting of the epicuticle and pre-ecdysal procuticle, was mechanically connected to tendon cells. Numerous bundles of fibers (arrows) running from tendon cells through the new cuticle and ecdysal space into the detached cuticle are evident. Microtubules were found in close proximity to electron dense plaques at the apical surface of tendon cells. DC detached cuticle NC new cuticle EC epicuticle ES ecdysal space PP pre-ecdysal procuticle TC tendon cell Mt microtubules.
Electron dense plaques (hemidesmosome-like structures) at the apical surface of the tendon cells in the marsupial manca of Porcellio scaber. Electron dense plaques (arrows) are associated with microtubules (Mt) in the cytoplasm of the tendon cell and with the bundles of fibers (F) running through the ecdysal space (ES) on the opposite side. DC detached cuticle NC new cuticle.
Myotendinous junction in the Porcellio scaber prehatching embryo (7A, B) and marsupial mancas (7C, D). The cytoplasmic plaque of the anchoring junction at the basal membrane of tendon cell is more lucent and thinner than the accompanying plaque at the apical membrane of the muscle cell in the prehatching embryo (7A, B). Microtubules of the tendon cells are in close proximity to the basal dense plaques. TC tendon cell MC muscle cell Mt microtubules Myf myofilaments.
Specialized anchoring complexes between exoskeleton, tendon cells and force-generating muscle cells are essential features of the musculoskeletal system in arthropods (
Our results show that the old exoskeleton is still mechanically attached to the underlying epithelium in the regions of muscle attachment sites for a certain period of the premolt phase in isopod crustaceans. We have observed massive arrays of fibers running from tendon cells through the new cuticle and ecdysal space up to the distal layers of the detached cuticle in adult premolt Ligia italica and in premolt intramarsupial specimens of Porcellio scaber. The continuity of these fibers was clearly evidenced in both species. The fibers extending from the tendon cells deep into the cuticular matrix in non-molting specimens are known from previous studies of arthropods (
Apicobasally oriented microtubule arrays are formed in several types of polarized epithelial cells.
The myotendinous junction in the dorsal parts of pereonites in molting Ligia italica is a zigzag patterned junction of the tendon cell basal membrane and muscle cell sarcolemma, with an inconspicious layer of extracellular matrix inbetween. The entire basal surface and apical surface of tendon and muscle cell, respectively, contribute to this heterotypic adherens junction. Both interacting cell membranes are extensively folded, which increases the surface area of contact and contributes to enhanced mechanical resistance. The myotendinous connection in molting Ligia italica structurally resembles that described in non-molting specimens and in Drosophila. The myotendinous junction in Drosophila is considered to be composed of two sets of hemiadherens junctions with an intervening layer of extracellular matrix material that has a substantial thickness in certain situations (
The ultrastructural arhitecture of myotendinous junctions in the prehatching embryos and marsupial mancas of Porcellio scaber analysed in this study is similar to the general structural outline of adult arthropod muscle attachments. In marsupial mancas it appears to be structurally fully elaborated, while in the prehatching embryo it may not be completely formed. The cytoplasmic plaques engaged in anchoring junctions at the basal membrane of the tendon cell in the prehatching embryo are markedly electron lucent and thinner as compared to the opposing cytoplasmic plaques in the muscle cells, while in adult arthropods the myotendinous anchoring junctions comprise cytoplasmic plaques of similar thicknesses and densities in both cells. A similar situation was observed in Drosophila in vitro culture of primary embryonic cells by
Cell to cell and cell to matrix anchoring junctions, together with their associated cytoskeletal elements, are engaged in providing tissue structural scaffold and integrity, but more than that, they are increasingly discussed from the perspective of tissue and cell dynamics (
Exoskeleton-muscle attachment in the adult Ligia italica with the entire cuticle of the usual thickness. (doi: 10.3897/zookeys.176.2445.app) File format: Portable (Public) Network Graphic (png).