Anatomy of the ovaries of the starfish Asterias rubens (Echinodermata)

Summary The ovaries of the starfish Asterias rubens were studied histologically and ultrastructurally. The reproductive system in female specimens consists of ten separate ovaries, two in each ray. Each ovary is made up of a rachis with lateral primary and secondary folds: the acini maiores and acini minores. The ovarian wall is composed of an outer and an inner part, separated by the genital coelomic sinus. The ovarian lumen contains oocytes in various phases of oogenesis, follicle cells, nurse cells, phagocytosing cells and steroid-synthesizing cells.Oogenesis is divided into four phases: (i) multiplication phase of oogonia, (ii) initial growth phase of oocytes I, (iii) growth phase proper of oocytes I, and (iv) post-growth phase of oocytes I. The granular endoplasmic reticulum and the Golgi complex of the oocytes appear to be involved in yolk formation, while the haemal system, haemal fluid and nurse cells may also be important for vitellogenesis. The haemal system is discussed as most likely being involved in synchronizing the development of the ovaries during the annual reproductive cycle and in inducing, stimulating and regulating the function of the ovaries.Steroid-synthesizing cells are present during vitellogenesis; a correlation between the presence of these cells and vitellogenesis is discussed.     

Morphology of spines and spine joint in the crown-of-thorns starfish Acanthaster planci (Echinodermata,Asteroida)

Summary Morphology and movement of the spines of Acanthaster planci were studied. All surfaces of the animal are covered with spines. The spines on the aboral surface are cylindrical with sharp tips. The spines on the oral surface are flat; they bend over to cover the mouth and the ambulacral grooves when these soft parts are stimulated. Those on the side of the animal make a barrier of crossed spines. Thus the structure and movement of the spines are well-adapted for defense.The junction between the primary aboral spine and its pedicel makes a movable joint. The ultrastructure of the connective tissue at the joint was studied. The connective tissue is mainly composed of collagen fibers. Presumed neurosecretory cells with processes which are filled with electron-dense granules of 0.2 m diameter were found between collagen fibers. Muscle fibers are mainly found in the connective tissue at the central holes. These observations support the view that the joint connective tissue has catch properties.     

Reactive oxygen species (ROS) production by amoebocytes of Asterias rubens (Echinodermata)

An adapted peroxidase, luminol-enhanced chemiluminescence method in an EDTA-free, Ca++-containing medium is described and used to characterise reactive oxygen species (ROS) production by starfish immunocytes using a standard microplate reader luminometer. ROS production was stimulated by direct interaction of immunocytes with bacteria or bacterial wall components, but not by the soluble stimulant PMA nor the lectin concanavalin A. Produced ROS detected by this method are apparently superoxide anions, hydrogen peroxide and peroxynitrite. Comparison with other chemiluminescence methods indicates that the described method is the only one to detect the stimulation of starfish immunocytes by the Gram-positive bacteria, Micrococcus luteus, a fact that questions previous reports indicating a lack of stimulation by pathogens. The adapted method provides a rapid determination of the overall ROS production, which is suitable for both disease control and immunotoxicological studies in echinoderms.     

Secretory Radial Glia in the Ectoneural System of the Sea Star Asterias rubens (Echinodermata)

Previous studies of epithelial nervous systems have focused on the neuronal elements, but generally neglected the origin of neuro–glial interactions. In this study, we use a polyclonal antiserum directed against Reissner's substance to label non-neuronal bipolar cells in the ectoneural part of the radial nerve cord in the sea star Asterias rubens. Ultrastructural results show secretory activity in these bipolar cells. Immunolabelled material is released into the extracellular matrix in the hyaline layer as well as in the region of the basal end-feet. As a first step towards characterising the antigen, a specific protein band of 36 kD was demonstrated with immunoprecipitation. Cells of this type: (1) traverse the epithelium to full extent from the outer surface to the basal lamina; (2) carry a single apical cilium; (3) contain conspicuous bundles of intermediate filament; (4) produce a secretion which is, at least in part, homologous to the Reissner's substance which is produced by a primitive radial glia cell type in chordates. It is concluded that the bipolar cells in the ectoneural part of the surface epithelium of the sea star Asterias rubens are secretory radial glia, which evidently have a common origin to the radial glia which secretes Reissner's substance in chordates.     

Differentiation of immune cells challenged by bacteria in the common European starfish,Asterias rubens (Echinodermata)

Amoebocytes are the main effector cells of the echinoderm immune system. In starfishes, a taxon in which bacterial diseases have been rarely reported, amoebocytes are considered to be the only circulating and immune cell type. The present paper addresses the question of amoebocyte differentiation in the starfish Asterias rubens when challenged by bacteria. Starfishes were injected with FITC-coupled bacteria (Micrococcus luteus). Amoebocytes were collected at regular time intervals for 24 h. The cytometric characteristics and the phagocytic activity were studied by flow cytometry. Three amoebocyte groups of different size were identified. The cell concentrations of the two largest and more numerous of these groups (G2 and G3) were modulated by immune stimulation while the group of smallest, less numerous, cells (G1) was unaffected. All of these cell groups were phagocytic but their kinetics of cell activation and bacteria ingestion differed. G1 cells showed the lowest phagocytic activity while G3 cells had the highest and fastest phagocytic activity. Starfish amoebocytes appear to be segregated in three groups, two of them (G2 and G3) being immunomodulated and one of them presenting a very fast reaction to bacteria. It is suggested that the high efficiency of the immune system in starfishes is related to this fast reaction.     

Skeletal growth in the echinoderm Asterias rubens L. (Asteroidea,Echinodermata) estimated by 45Ca-labelling

The skeletal dry weight of the 4.4 ± 0.2 cm size class of Asterias rubens L. from Kiel Bay in the western Baltic is 0.34 ± 0.08 g. The sum of calcium and magnesium carbonates in the skeleton amounts to 94.0 ± 1.3% while the individual concentrations are 86.9 ± 1.3% CaCO₃ and 7.1 ± 0.7% MgCO₃. The MgCO₃ is 4% lower than expected for a magnesium calcite precipitated under Baltic temperature conditions (8°C). Strontium was not determined but strontium carbonate is known to be in the region of 0.4%. The remainder is organic matter and this gives rise to 2.9 ± 1.3% organic carbon.Animals studied were at the ‘waiting stage’ and their actual growth was minimal. Uptake of ⁴⁵Ca in the skeleton consists of a fast step followed by a slow step. The fast step is attributed to saturation of exchangeable skeletal pools while the slow step is due to net deposition of CaCO₃. Skeletal growth at the waiting stage calculated from the second rate constant was found to be 0.76 μg CaCO₃j-mg skeleton^?1 · day^?1 or 0.09% · day^?1 compared with 9.3 μg CaCO₃ · mg skeleton^t1̄ · day^?1 or 1.1% · day^?1 at log phase. The isotope method is considered superior to size-frequency analysis in that it is capable of detecting differences in growth rate in individuals of the same size class and thus provides an insight into asteroid population structure.     

Histochemical observations on the pyloric caeca of Asterias rubens (Echinodermata,asteroidea) in relation to the reproductive cycle

The pyloric caeca of the starfish Asterias rubens were investigated histochemically during the reproductive cycle. The median duct and the side lobes reacted differently. The median duct reacted positively for acid phosphatases and glucose-6-phosphate dehydrogenase, whereas the side lobes reacted positively for alkaline phosphatases, neutral lipids, and fatty acids. In the transition zone between the median duct and the side lobes, the reaction for alkaline phosphatases and neutral lipids increased toward the side lobes. The function of the enzymes and the histochemical results are discussed in relation to the function of the pyloric caeca and to the reproductive cycle.     

The aboral haemal system of the sea star,Asterias rubens (Echinodermata,Asteroidea): An ultrastructural and histochemical study

Summary The aboral parts of the haemal system of the sea star Asterias rubens are described, based on light and electron microscopy. These parts are (1) the mesenteric strands along the pyloric caeca and the pyloric stomach, (2) the gastric haemal tufts, and (3) the aboral haemal ring. The mesenteric haemal strands are very limited in size and distribution and, therefore, do not seem to have a major function in nutrient translocation. The myoepithelial cells of the gastric haemal tufts have the typical features of choanocytes; their ultrastructural characteristics corroborate the possible absorptive role of the gastric haemal tufts. The myoepithelial cells of the aboral haemal ring often show distinct apical bulbs of cytoplasm suggesting apocrine secretion of PAS-positive materials which are found in the surrounding aboral coelomic ring. These cells contain large stores of particulate glycogen and typical 1–2 m electrondense globules.The ground substance of the haemal tissues, which contains collagen fibers, reticular fibrils, and numerous amoeboid phagocytes, has been analyzed histochemically. Sulfated glycosaminoglycans are almost completely absent; the predominant components are polysaccharides, proteins, and/or glycoproteins. Lipids have not been demonstrated.The possible functions of the haemal tissues and associated coelomic channels are discussed.     

Patterns of bromodeoxyuridine incorporation and neuropeptide immunoreactivity during arm regeneration in the starfish Asterias rubens

Regeneration of the arm of the starfish, Asterias rubens (L.) (Echinodermata: Asteroidea) was examined using two preparations. The first involved regeneration of the entire arm tip and its associated sensory structures and the second examined regeneration of a small section of radial nerve cord in the mid-arm region. Cell cycle activity was investigated by incorporation of the thymidine analogue, bromodeoxyuridine (BrdU). Details of neuroanatomy were obtained by immunocytochemistry (ICC) using an antiserum to the recently isolated starfish neuropeptide, GFNSALMFamide (S1). BrdU labelling indicated that initial events occur by morphallaxis, with cell cycle activity first apparent after formation of a wound epidermis. As regeneration proceeded, BrdU immunoreactive (IR) nuclei revealed cell cycle activity in cells at the distal ends of the radial nerve cord epidermis, in the coelomic epithelium, the perihaemal and water vascular canal epithelia, and in the forming tube feet of both preparations. By varying the time between BrdU pulses and tissue fixation, the possible migration or differentiation of labelled cells was investigated. Neuropeptide ICC indicated the extension of S1-IR nerve fibres into the regenerating area, soon after initial wound healing processes were complete. These fibres were varicose and disorganized in appearance, when compared to the normal pattern of S1-IR in the radial nerve. S1-IR was also observed in cell bodies, which reappeared in the reforming optic cushion and radial nerve at later stages of regeneration. Double labelling studies with anti-BrdU and anti-S1 showed no co-localization in these cell bodies, in all the stages examined. It appeared that S1-IR cells were not undergoing, and had not recently undergone, cell cycle activity. It cannot be confirmed whether S1-IR neurons were derived from proliferating cells of epithelial origin, or from transdifferentiation of epithelial cells, although the former mechanism is suggested. Differentiation of the regenerating structures to replace cells such as S1-containing neurons, is thought to involve cell cycle activity and differentiation of epithelial cells in the epidermal tissue, possibly in association with certain types of coelomocytes which move into the regenerating area.     

Occurrence of a microcanalicular system within the ossicles and dermal tissue of Asterias rubens and Marthasterias glacialis (Echinodermata: Asteroidea)

Summary A microcanalicular network is demonstrated within the ossicle stroma and the dermal tissue of two asteroid species. Microcanaliculi are presumed to be mesodermal structures. They consist of convoluted tubular ducts lined by epithelial cells associated with scattered basiepithelial nervous processes. Such a microcanalicular system has not been reported previously from any echinoderm species. Its discovery in asteroids entails some conceptual changes, especially considering the physiology of the body wall.Research assistants of the National Fund for Scientific Research (NFSR, Belgium)     

Structure and function of clypeasteroid miliary spines (Echinodermata,Echinoides)

Summary Histological and ultrastructural techniques have been used to describe the functional morphology of clypeasteroid miliary spines, with special reference to their supposed mucus-secreting role. Mucus cells were not found in the miliary spines of any members of the Arachnoididae, Fibulariidae, Laganidae, Echinarachniidae, Dendrasteridae, Astriclypeidae, or Mellitidae examined in this study. Only members of the Clypeasteridae have mucus-secreting cells in these spines. Characteristics of the skeleton, ultrastructure of the nervous system, and histology of the musculature and epithelia of the base, shaft and tip are also discussed. Miliary spines have two bands of cilia running along the entire length of opposite sides of the shaft. The geometric packing of cilium-bearing cells in these bands is described for the first time, as is the remarkable form of the sacs found at the tips of dendrasterid, astriclypeid, and mellitid miliary spines. These sacs are definitely not mucous sacs, as previously described, but are balloons of single-celled epithelium internally tethered to the skeletal tip by copious quantities of collagenous connective tissue. Miliary spines prevent obstruction of aboral nutritive and ventilatory ciliary currents caused by substrate particles falling to the test surface during burrowing. They do this in two ways: (1) they help generate ciliary currents that sweep finer material off the test, and (2) they contribute to the formation of a spine canopy that mechanically blocks larger particles from falling between the spines. Members of the Clypeasteridae secrete an interspine mucous tent that traps potentially clogging material. The miliary spine sacs of sand dollars are deformable space-fillers that plug holes between primary spines in the aboral canopy, even as the spines rock on their tubercles to push sand backwards over the test. Allometry of spines from Echinarachnius parma suggests that aboral military spines and club-shaped spines exhibit co-ordinated growth that maintains the aboral canopy throughout post-metamorphic ontogeny, and that aboral spins have an overall lower growth rate than spines on the oral surface.     

The analysis of cellular elements in coelomic fluid during early regeneration of of the starfish Asterias rubens L

Three main cell types were found in the coelomic fluid (CF) of intact starfishes: agranulocytes (55-80%) varying in size and form (spherical and ovoid) and with occasional pseudopodia, granulocytes (15-45%), and small cells (up to 2 %) with a high nuclear-cytoplasmic ratio. The starfish response to injury depends on the degree of coelomic fluid loss. After a slight wounding, when only insignificant portion of CF is lost, the cellular composition of circulating fluid changed only slightly. Unlike, a significant injury resulted in rising the share of small cells, regarded presumably as young cells. Besides, after injury the functional characteristics of SF also changed: the proportion of cells with decondensed chromatin and stained nucleoli increased, and coelomocytes acquired ability to form nets at adhesion. Moreover, some new cell types can be found (fusiform cells), with granulocyte proportion in nets increasing. We suppose that after slight wounding circulating coelomocytes may restore from the existing store of differentiated cells beyond the circulation, whereas after significant injury young undifferentiated coelomocytes are involved in the process of restoration.     

EGFR signaling regulates cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis

Similarly to development, the process of regeneration requires that cells accurately sense and respond to their external environment. Thus, intrinsic cues must be integrated with signals from the surrounding environment to ensure appropriate temporal and spatial regulation of tissue regeneration. Identifying the signaling pathways that control these events will not only provide insights into a fascinating biological phenomenon but may also yield new molecular targets for use in regenerative medicine. Among classical models to study regeneration, freshwater planarians represent an attractive system in which to investigate the signals that regulate cell proliferation and differentiation, as well as the proper patterning of the structures being regenerated. Recent studies in planarians have begun to define the role of conserved signaling pathways during regeneration. Here, we extend these analyses to the epidermal growth factor (EGF) receptor pathway. We report the characterization of three epidermal growth factor (EGF) receptors in the planarian Schmidtea mediterranea. Silencing of these genes by RNA interference (RNAi) yielded multiple defects in intact and regenerating planarians. Smed-egfr-1(RNAi) resulted in decreased differentiation of eye pigment cells, abnormal pharynx regeneration and maintenance, and the development of dorsal outgrowths. In contrast, Smed-egfr-3(RNAi) animals produced smaller blastemas associated with abnormal differentiation of certain cell types. Our results suggest important roles for the EGFR signaling in controlling cell proliferation, differentiation and morphogenesis during planarian regeneration and homeostasis.     

Wound healing and arm regeneration in Ophioderma longicaudum and Amphiura filiformis (Ophiuroidea, Echinodermata): comparative morphogenesis and histogenesis

All species of the Ophiuroidea have exceptional regenerative capabilities; in particular, they can replace arms lost following traumatic or self-induced amputation. In order to reconstruct this complex phenomenon, we studied arm regeneration in two different ophiuroids, Ophioderma longicaudum (Retzius, 1805) and Amphiura filiformis O. F. Müller, 1776, which are quite distantly related. These species present contrasting regeneration and differentiation rates and differ in several ecological traits. The aim of this paper is to interpret the primary sequence of morphogenetic and histogenetic events leading to the complete reconstruction of a new arm, comparing the arm regenerative processes of these two ophiuroid species with those described in crinoids. Arm regeneration in ophiuroids is considered an epimorphic process in which new structures develop from a typical blastema formed from an accumulation of presumptive undifferentiated cells. Our results showed that although very different in some respects such as, for instance, the regeneration rate (0.17 mm/week for O. longicaudum and 0.99 mm/week for A. filiformis), morphogenetic and histogenetic aspects are surprisingly similar in both species. The regenerative process presents similar characteristics and follows a developmental scheme which can be subdivided into four phases: a repair phase, an early regenerative phase, an intermediate regenerative phase and an advanced regenerative phase. In terms of histogenesis, the regenerative events involve the development of new structures from migratory pluripotent cells, which proliferate actively, in addition in both cases there is a significant contribution from dedifferentiated cells, in particular dedifferentiating myocytes, although to varying extents. This evidence confirms the plasticity of the regenerative phenomenon in echinoderms, which can apparently follow different pathways in terms of growth and morphogenesis, but nevertheless involve both epimorphic and morphallactic contributions at the cellular level.     

Progesterone metabolism by the echinoderms Asterias rubens and Marthasterias glacialis (Short Communication)

The echinoderms Asterias rubens and Marthasterias glacialis metabolize injected [4-(14)C]progesterone to give labelled 3beta-hydroxy-5alpha-pregnan-20-one and 3beta,6alpha-dihydroxy-5alpha-pregnan-20-one. These radioactive products are converted by the animals into conjugated forms that are soluble in aqueous methanol, and which have mobilities on t.l.c. similar to the asterosaponins.     

Evolutionary history of regeneration in crinoids (Echinodermata)

The fossil record indicates that crinoids have exhibited remarkable regenerative abilities since their origin in the Ordovician, abilities that they likely inherited from stem-group echinoderms. Regeneration in extant and fossil crinoids is recognized by abrupt differences in the size of abutting plates, aberrant branching patterns, and discontinuities in carbon isotopes. While recovery is common, not all lost body parts can be regenerated; filling plates and overgrowths are evidence of non-regenerative healing. Considering them as a whole, Paleozoic crinoids exhibit the same range of regenerative and non-regenerative healing as Recent crinoids. For example, Paleozoic and extant crinoids show evidence of crown regeneration and stalk regrowth, which can occur only if the entoneural nerve center (chambered organ) remains intact. One group of Paleozoic crinoids, the camerates, may be an exception in that they probably could not regenerate their complex calyx-plating arrangements, including arm facets, but their calyxes could be healed with reparative plates. With that exception, and despite evidence for increases in predation pressure, there is no compelling evidence that crinoids have changed though time in their ability to recover from wounds. Finally, although crinoid appendages may be lost as a consequence of severe abiotic stress and through ontogenetic development, spatiotemporal changes in the intensity and frequency of biotic interactions, especially direct attacks, are the most likely explanation for observed patterns of regeneration and autotomy in crinoids.