Dumbbell-Shaped Ossicles Discovered in Pedicellaria of Flower Sea Urchins.

Sea urchins have a globiferous pedicellaria that stands from a test with a stalk on which lies a head made of three movable jaws with venom-injecting teeth. The globiferous pedicellariae of the flower sea urchin Toxopneustes pileolus, one of the most developed among sea urchins, are unique in that the jaws are provided with a jaw membrane that gives the pedicellaria an appearance of a flower when the jaws are open. We observed this membrane in an ionic liquid that does not require processes, such as fixation, dehydration, or coating with conductive materials, for observation with a scanning electron microscope. Using this technique, we discovered dumbbell-shaped ossicles, which consist of two spheres of similar size connected by a cylinder. The diameter of the sphere is 4-8 µm, and the total length of the ossicle is 10-20 µm. The jaw membrane is trimmed with an edge zone. The ossicles were found sparsely in the connective tissue of general part of the membrane, but in the edge zone their density was so high that neighboring ossicles were in close contact with each other. Some neighboring ossicles crossed their cylinders and some inserted one of their spheres to snugly fit in the gap between the spheres of neighboring ossicles. Their structural role is very likely in strengthening the jaw membrane, probably serving as fillers in the general part of the membrane; in the edge zone the interlocking of adjacent ossicles forms a loose network providing a firm frame for the head of the globiferous pedicellaria. When opened, the stiff frame prevents the membrane from sagging. When clasped, it works as a closed door, firmly keeping prey trapped.

Ultrastructural Changes Associated with Reversible Stiffening in Catch Connective Tissue of Sea Cucumbers.

The dermis of sea cucumbers is a catch connective tissue or a mutable collagenous tissue that shows rapid, large and reversible stiffness changes in response to stimulation. The main component of the dermis is the extracellular material composed of collagen fibrils embedded in a hydrogel of proteoglycans. The stiffness of the extracellular material determines that of the dermis. The dermis has three mechanical states: soft (Sa), standard (Sb) and stiff (Sc). We studied the ultrastructural changes associated with the stiffness changes. Transverse sections of collagen fibrils in the dermis showed irregular perimeters with electron-dense protrusions or arms that cross-bridged between fibrils. The number of cross-bridges increased in stiffer dermis. The distance between the fibrils was shorter in Sc than that in other states, which was in accord with the previous report that water exuded from the tissue in the transition Sb→Sc. The ultrastructure of collagen fibrils that had been isolated from the dermis was also studied. Fibrils aggregated by tensilin, which causes the transition Sa→Sb possibly through an increase in cohesive forces between fibrils, had larger diameter than those dispersed by softenin, which antagonizes the effect of tensilin. No cross-bridges were found in isolated collagen fibrils. From the present ultrastructural study we propose that three different mechanisms work together to increase the dermal stiffness. 1.Tensilin makes collagen fibrils stronger and stiffer in Sa→Sb through an increase in cohesive forces between subfibrils that constituted fibrils; 2. Cross-bridging by arms caused the fibrils to be a continuous network of bundles both in Sa→Sb and in Sb→Sc; 3. The matrix embedding the fibril network became stiffer in Sb→Sc, which was produced by bonding associated with water exudation.

Coordination between catch connective tissue and muscles through nerves in the spine joint of the sea urchin Diadema setosum.

Echinoderms have catch connective tissues that change their stiffness as a result of nervous control. The coordination between catch connective tissue and muscles was studied in the spine joint of the sea urchin Diadema setosum. Spine joints are equipped with two kinds of effector: spine muscles and a kind of catch connective tissue, which is called the catch apparatus (CA). The former is responsible for spine movements and the latter for maintenance of spine posture. Diadema show a shadow reaction in which they wave spines when a shadow falls on them, which is a reflex involving the radial nerves. Dynamic mechanical tests were performed on the CA in a joint at which the muscles were severed so as not to interfere with the mechanical measurements. The joint was on a piece of the test that contained other spines and a radial nerve. Darkening of the preparation invoked softening of the CA and spine waving (the shadow reaction). Electrical stimulation of the radial nerve invoked a similar response. These responses were abolished after the nerve pathways from the radial nerve to spines had been cut. A touch applied to the CA stiffened it and the adjacent spines inclined toward the touched CA. A touch to the base of the adjacent spine softened the CA and the spines around the touched spine inclined towards it. The softening of the CA can be interpreted as a response that reduces the resistance of the ligaments to spine movements. Our results clearly show coordination between catch connective tissue and muscles through nerves.

Softenin, a novel protein that softens the connective tissue of sea cucumbers through inhibiting interaction between collagen fibrils.

The dermis in the holothurian body wall is a typical catch connective tissue or mutable collagenous tissue that shows rapid changes in stiffness. Some chemical factors that change the stiffness of the tissue were found in previous studies, but the molecular mechanisms of the changes are not yet fully understood. Detection of factors that change the stiffness by working directly on the extracellular matrix was vital to clarify the mechanisms of the change. We isolated from the body wall of the sea cucumber Stichopus chloronotus a novel protein, softenin, that softened the body-wall dermis. The apparent molecular mass was 20 kDa. The N-terminal sequence of 17 amino acids had low homology to that of known proteins. We performed sequential chemical and physical dissections of the dermis and tested the effects of softenin on each dissection stage by dynamic mechanical tests. Softenin softened Triton-treated dermis whose cells had been disrupted by detergent. The Triton-treated dermis was subjected to repetitive freeze-and-thawing to make Triton-Freeze-Thaw (TFT) dermis that was softer than the Triton-treated dermis, implying that some force-bearing structure had been disrupted by this treatment. TFT dermis was stiffened by tensilin, a stiffening protein of sea cucumbers. Softenin softened the tensilin-stiffened TFT dermis while it had no effect on the TFT dermis without tensilin treatment. We isolated collagen from the dermis. When tensilin was applied to the suspending solution of collagen fibrils, they made a large compact aggregate that was dissolved by the application of softenin or by repetitive freeze-and-thawing. These results strongly suggested that softenin decreased dermal stiffness through inhibiting cross-bridge formation between collagen fibrils; the formation was augmented by tensilin and the bridges were broken by the freeze-thaw treatment. Softenin is thus the first softener of catch connective tissue shown to work on the cross-bridges between extracellular materials.

Energy expenditure associated with softening and stiffening of echinoderm connective tissue.

Catch connective tissue of echinoderms at rest (in the standard state) either stiffens or softens in response to different kinds of stimulation. The energy consumption associated with the changes was estimated by measurement of the oxygen consumption rate (VO(2)) in three types of connective tissues-echinoid catch apparatus (CA), holothuroid body-wall dermis (HD), and asteroid body-wall dermis (AD). Mechanical stimulation by repetitive compression (10%-15% strain), which increased viscosity measured by creep tests, was employed for inducing the stiff state. Noradrenaline (10(-3) mol l(-1)), which decreased viscosity of CA, and static 80% compressive strain, which decreased viscosity of HD, were used to induce the soft state in the respective tissues. The VO(2) (in µl/g/h) values of the standard state were 2.91 (CA), 1.41 (HD), and 0.56 (AD), which were less than 1/4 of the VO(2) of the resting body-wall muscle of the starfish. The VO(2) of the stiff state was about 1.5 times greater than that of the standard state in all types of connective tissues. The VO(2) of the soft state was 3.4 (CA)-9.1 (HD) times greater than that of the standard state. The economical nature of catch connective tissue in posture maintenance is discussed.

Mechanical mutability in connective tissue of starfish body wall.

Stiffness changes in response to mechanical and chemical stimulation were studied in muscle-free dermal samples from the body wall of the starfish Linckia laevigata. The ultrastructural study showed that the dermis was packed with collagen fibrils between which only a small number of cells were observed. Muscles were found only in the walls of coelomic extensions leading to papulae. Stress-strain tests were performed on isolated dermis containing no muscles. The tangent modulus was 27.5 MPa at 0.04% strain rate in the stress-strain tests. It was increased to 40.7 MPa by mechanical stimulation, which also increased the tensile strength and breaking-strain energy density. Dynamic mechanical tests showed that the increase in stiffness in response to mechanical stimulation was transient. Acetylcholine (10(-6)-10(-3) mol l(-1)) and artificial seawater with an elevated potassium concentration (KASW) stiffened the dermis. Mechanical stimulation caused a 12% mass loss. KASW also caused mass loss, which was inhibited by anesthesia. These results clearly showed that the stiffness changes in the starfish dermis were based on a non-muscular mechanism that was similar to that of other echinoderm connective tissues with mechanical mutability.

A novel stiffening factor inducing the stiffest state of holothurian catch connective tissue.

The dermis of sea cucumbers is a catch connective tissue or mutable collagenous tissue that shows large changes in stiffness. Extensive studies on the dermis revealed that it can adopt three different states having different mechanical properties that can be reversibly converted. These are the stiff, standard and soft states. The standard state is readily produced when a dermal piece is immersed in the sea water containing Ca²+, whereas the soft state can be produced by removal of Ca²+. A stiffening protein, tensilin, has been isolated from some sea cucumbers (Cucumaria frondosa and Holothuria leucospilota). Although tensilin converts the state of the dermis from soft to standard, it cannot convert from standard to stiff. In this study, we isolated and partially purified a novel stiffening factor from the dermis of Holothuria leucospilota. The factor stiffened the dermis in normal artificial sea water (ASW) but did not stiffen the soft dermis in Ca²+-free ASW. It also stiffened the dermis that had been converted to the standard state in Ca²+-free ASW by the action of tensilin. These results suggest that the factor produces the stiff dermis from the standard state but cannot work as a stiffener on the soft dermis. Its addition to longitudinal muscles of the sea cucumber produced no effects, suggesting that its effect is specific to the catch connective tissue. Its stiffening activity was susceptible to trypsin, meaning that it is a polypeptide, and its molecular mass estimated from gel filtration chromatography was 2.4 kDa.

Evidence that water exudes when holothurian connective tissue stiffens.

The dermis of the body wall of sea cucumbers is composed mainly of extracellular materials such as collagens, proteoglycans and water; the water content is as high as 80%. Yet it shows rapid changes in stiffness under neural control. The dermis has been proposed to assume three mechanical states, soft, standard, and stiff. We investigated the relationship between the stiffness and the dermal mass and volume. Both the mass and volume decreased by 15% when the dermis stiffened from the standard state to the stiff state by mechanical stimulation and by chemical stimulation with potassium-rich seawater. The effect of the latter was abolished by anesthesia. The mass decrease was caused largely by water exudation. Tensilin, a holothurian protein that stiffens the soft dermis to form the standard state, did not cause any changes in mass. These results suggested that the stiffening mechanisms responsible for the transition from the soft state to the standard state, and that from the standard to the stiff state, are different. The removal of water from the dermis in the standard state, by soaking in hypertonic solution, caused only slight stiffening, which suggested that water exudation was not the direct cause of the stiffening. A change of pH of the surrounding medium, either more acidic or basic, was not associated with mass changes, although it caused a large increase in stiffness. The implications of the present results for the molecular mechanisms of the stiffness changes are discussed.

Low oxygen consumption and high body content of catch connective tissue contribute to low metabolic rate of sea cucumbers.

The energy consumption of echinoderms is low in comparison with that of other invertebrates. We demonstrated this by measuring the oxygen consumption rate per unit of body weight (VO2) of the sea cucumber Actinopyga mauritiana: VO2 was 1/8 that of the "standard" invertebrates. Low energy consumption in echinoderms has been attributed to their high skeletal content and to catch connective tissues (CCTs) that maintain body posture by altering their mechanical properties with little energy expenditure. The former is not applicable to holothurians, and the latter has not been proven experimentally. We postulated that the large content of dermal connective tissue, which maintaines posture economically, contributes to the low energy consumption in holothurians. Body-wall dermis occupied 53.5% of wet body weight, whereas body-wall muscles, including those of tube feet, occupied 5.1%. VO2 of the dermis in the stiff state (2.45 microl x g(-1) x h(-1)) was 1/10 that of the longitudinal body-wall muscle in contraction. the mechanical tests revealed that the stress at an imposed strain of 2% strain was 7 times greater in CCT than in muscles. These results showed that CCT could maintain posture more economically than muscles could. We concluded that the high content of connective tissue with energy-saving posture-maintenance activities contributed to the low energy consumption of holothurians.

Stichopin-containing nerves and secretory cells specific to connective tissues of the sea cucumber.

Stichopin, a 17-amino acid peptide isolated from a sea cucumber, affects the stiffness change of the body-wall catch connective tissues and the contraction of the body-wall muscles. The localization of stichopin in sea cucumbers was studied by indirect immunohistochemistry using antiserum against stichopin. Double staining was performed with both stichopin antiserum and 1E11, the monoclonal antibody specific to echinoderm nerves. A stichopin-like immunoreactivity (stichopin-LI) was exclusively found in the connective tissues of various organs. Many fibres and cells with processes were stained by both the anti-stichopin antibody and 1E11. They were found in the body-wall dermis and the connective tissue layer of the cloacae and were suggested to be connective tissue-specific nerves. Oval cells with stichopin-LI (OCS) without processes were found in the body-wall dermis, the connective tissue sheath of the longitudinal body-wall muscles, the connective tissue layer of the tube feet and tentacles, and the connective tissue in the radial nerves separating the ectoneural part from the hyponeural part. Electron microscopic observations of the OCSs in the radial nerves showed that they were secretory cells. The OCSs were located either near the well-defined neural structures or near the water-filled cavities, such as the epineural sinus and the canals of the tube feet. The location near the water-filled cavities might suggest that stichopin was secreted into these cavities to function as a hormone.

Possible self-fertilization in the brooding coral Acropora (Isopora) brueggemanni.

We examined gametogenesis and the periodicity of planula release in the brooding coral Acropora (Isopora) brueggemanni (Brook, 1893) on Akajima Island, Okinawa, Japan. We captured the moment when A. brueggemanni would be in the process of self-fertilization. Whole colonies of this species were cultured separately or together with other colonies in plastic containers. We observed no apparent periodicity of planula release in the collected colonies. A few planulae were released intermittently during the observation period, regardless of whether the colonies were cultured individually or with other colonies. Serial paraffin sections of A. brueggemanni showed follicle-like cells surrounding the oocyte during vitellogenesis. In September and October, some spermaries looked half-broken and some ova were surrounded by sperm or spermaries instead of follicle-like cells. Such ova were heart-shaped and may have been cells at the first cleavage stage. These observations suggest that the migration of spermaries and/or oocytes resulted in the close proximity of oocytes and sperm, which would allow self-fertilization. This possibility, together with the production of planula larvae by isolated colonies, suggests that this brooding coral engages in self-fertilization.

Embryogenesis in the reef-building coral Acropora spp.

Embryogenesis in the reef building corals Acropora intermedia, A. solitaryensis, A. hyacinthus, A. digitifera, and A. tenuis was studied in detail at the morphological level, and the relationships among the animal pole, blastopore, and mouth were investigated for the first time in corals. These species showed essentially the same sequence of development. The embryo undergoes spiral-like holoblastic cleavage despite the presence of a dense isolecithal yolk. After the morula stage, the embryo enters the "prawn-chip" stage, which consists of an irregularly shaped cellular bilayer. The embryo begins to roll inward to form the bowl stage; the round shape observed during this stage suggests that it may be the beginning of gastrulation. However, the blastopore closes and the stomodeum (mouth and pharynx) is formed via invagination at a site near the closed blastopore. During the planula stage, a concavity forms in the aboral region in conjunction with numerous spirocysts, suggesting that spirocysts are used to attach to the substrate before the onset of metamorphosis.

NGIWYamide-induced contraction of tube feet and distribution of NGIWYamide-like immunoreactivity in nerves of the starfish Asterina pectinifera.

NGIWYamide, a neuropeptide recently isolated from sea cucumbers, was tested on tube feet of the starfish Asterina pectinifera. NGIWYamide (10(-6)-10(-4) M) caused contraction of isolated tube feet. NGIWYamide-like immunoreactivity (NGIWYa-LI) was investigated with an antiserum against NGIWYamide. NGIWYa-LI was found in the radial nerve cord (RNC), the marginal nerve, and the tube feet. Both ectoneural and hyponeural parts of the RNC showed NGIWYa-LI. Immunoreactive cell bodies were found in both parts of RNC. Extensive labeling in the basal region of the ectoneural part suggests that a substantial proportion of axons in this part contains NGIWYamide or a similar substance. In tube feet, NGIWYa-LI was found in the sub-epithelial nerve plexus and in the basal nerve ring. Double labeling along with 1E11, a neuron-specific monoclonal antibody developed from A. pectinifera, indicated that the structures with NGIWYa-LI are neurons. These results suggest that NGIWYamide or an NGIWYamide-like peptide exists in starfish and functions as a neurotransmitter or a neuromodulator.

Tensilin-like stiffening protein from Holothuria leucospilota does not induce the stiffest state of catch connective tissue.

The dermis of sea cucumbers is a catch connective tissue or mutable connective tissue that exhibits large changes in mechanical properties. A stiffening protein, tensilin, has been isolated from the sea cucumber Cucumaria frondosa. We purified a similar protein, H-tensilin, from Holothuria leucospilota, which belongs to a different family to C. frondosa. H-tensilin appeared as a single band with an apparent molecular mass of 34 kDa on SDS-PAGE. No sugar chain was detected. Tryptic fragments of the protein had homology to known tensilin. H-tensilin aggregated isolated collagen fibrils in vitro in a buffer containing 0.5 mol l(-1) NaCl with or without 10 mmol l(-1) Ca(2+). The activity of H-tensilin was quantitatively studied by dynamic mechanical tests on the isolated dermis. H-tensilin increased stiffness of the dermis in the soft state, induced by Ca(2+)-free artificial seawater, to a level comparable to that of the standard state, which was the state found in the dermis rested in artificial seawater with normal ionic condition. H-tensilin decreased the energy dissipation ratio of the soft dermis to a level comparable to that of the standard state. When H-tensilin was applied on the dermis in the standard state, it did not alter stiffness nor dissipation ratio. The subsequent application of artificial seawater in which the potassium concentration was raised to 100 mmol l(-1) increased stiffness by one order of magnitude. These findings suggest that H-tensilin is involved in the changes from the soft state to the standard state and that some stiffening factors other than tensilin are necessary for the changes from the standard to the stiff state.

Experimental allometry: effect of size manipulation on metabolic rate of colonial ascidians.

The allometric scaling of metabolic rate of organisms, the three-quarters power rule, has led to a questioning of the basis for the relation. We attacked this problem experimentally for the first time by employing the modular organism, the ascidian that forms a single layered flat colony, as a model system. The metabolic rate and colony size followed the three-quarters power relation, which held even after the colony size was experimentally manipulated. Our results established that the three-quarters power relation is a real continuous function, not an imaginary statistical regression. The fact that all the hypotheses failed to explain why the two-dimensional organism adhered to the three-quarters power relation led us to propose a new hypothesis, in which the allometric relation derives from the self-organized criticality based on local interaction between modulus-comprising organisms.

Mechanical properties of the isolated catch apparatus of the sea urchin spine joint: muscle fibers do not contribute to passive stiffness changes.

The catch apparatus (CA) is the collagenous ligament at the spinal joint of sea urchins. It maintains spine posture by stiffening and allows spine movement by softening. A CA preparation, which was isolated from ossicles, was used to test the hypothesis that frictional forces between collagen fibers and ossicles are the source of stiffness changes. Isolated preparations of the CA changed in stiffness, thus falsifying the hypothesis. Another hypothesis proposes that muscle fibers, which represent a relatively small component of the CA, cause stiffening of the CA by contraction. Chemicals that evoked contraction in spine muscles did not always stiffen the CA: the CA of Heterocentrotus mammillatus softened in response to artificial seawater with potassium concentration elevated to 100 mM. This provided evidence against the muscle-based hypothesis. The present results suggest that the stiffness changes of the CA are based on changes in the mechanical properties of the extracellular components of the connective tissue and are therefore related to the connective tissue catch that is widespread in other echinoderms.

Contraction and stiffness changes in collagenous arm ligaments of the stalked crinoid Metacrinus rotundus (Echinodermata).

Shortening and stiffness were measured simultaneously in the aboral ligament of arms of sea lilies. Arm pieces were used from which oral tissues (including muscles) were removed, leaving only collagenous ligaments connecting arm ossicles. Chemical stimulation by means of artificial seawater with an elevated concentration of potassium caused both a bending movement and stiffness changes (either softening or stiffening). The movement lasted for 1.5-10 min, and bent posture was maintained. The observation that contraction was not necessarily associated with softening provided evidence against the hypothesis that the shortening of the aboral ligaments was driven by the elastic components that had been charged by the oral muscles and released their strain energy at the softening of the aboral ligaments. The speed of ligamental shortening was slower by at least one order of magnitude than that of muscles. Acetylcholine (10(-5)-10(-3) M) caused both contraction and softening. We conclude that the aboral ligament shows two mechanical activities based on different mechanisms: one is active contraction and the other is connective tissue catch in which passive mechanical properties show mutability. We suggest that there is neural coordination between the two mechanisms.

Dynamic mechanical properties of body-wall dermis in various mechanical states and their implications for the behavior of sea cucumbers.

The dermis of the sea cucumber body wall is a typical catch connective tissue that rapidly changes its mechanical properties in response to various stimuli. Dynamic mechanical properties were measured in stiff, standard, and soft states of the sea cucumber Actinopyga mauritiana. Sinusoidal deformations were applied, either at a constant frequency of 0.1 Hz with varying maximum strain of 2%-20% or at a fixed maximum strain of 1.8% with varying frequency of 0.0005-50 Hz. The dermis showed viscoelasticity with both strain and strain-rate dependence. The dermis in the standard state showed a J-shaped stress-strain curve with a stiffness of 1 MPa and a dissipation ratio of 60%; the curve of the stiff dermis was linear with high stiffness (3 MPa) and a low dissipation ratio (30%). Soft dermis showed a J-shaped curve with low stiffness (0.3 MPa) and a high dissipation ratio (80%). The strain-induced softening was observed in the soft state. Stiff samples had a higher storage modulus and a lower tangent delta than soft ones, implying a larger contribution of the elastic component in the stiff state. A simple molecular model was proposed that accounted for the mechanical behavior of the dermis. The model suggested that stiffening stimulation increased inter-molecular bonds, whereas softening stimulation affected intra-molecular bonds. The adaptive significance of each mechanical state in the behavior of sea cucumbers is discussed.

Switching of metabolic-rate scaling between allometry and isometry in colonial ascidians.

The metabolic rate and its scaling relationship to colony size were studied in the colonial ascidian Botrylloides simodensis. The colonial metabolic rate, measured by the oxygen consumption rate (V(O2) in millilitres of O(2) per hour) and the colony mass (wet weight M(w) in grams) showed the allometric relationship (V(O2) = 0.0412 M(w)(0.799). The power coefficient was statistically not different from 0.75, the value for unitary organisms. The size of the zooids and the tunic volume fraction in a colony were kept constant irrespective of the colonial size. These results, together with the two-dimensional colonial shape, excluded shape factors and colonial composition as possible causes of allometry. Botryllid ascidians show a takeover state in which all the zooids of the parent generation in a colony degenerate and zooids of a new generation develop in unison. The media for connection between zooids such as a common drainage system and connecting vessels to the common vascular system experienced reconstruction. The metabolic rate during the takeover state was halved and was directly proportional to the colonial mass. The scaling thus changed from being allometric to isometric. The alteration in the scaling that was associated with the loss of the connection between the zooids strongly support the hypothesis that the allometry was derived from mutual interaction among the zooids. The applicability of this hypothesis to unitary organisms is discussed.

Innervation of holothurian body wall muscle: inhibitory effects and localization of 5-HT.

We investigated innervation to body wall muscles as well as distribution of 5-HT (serotonin) and its effects on longitudinal muscles of body wall (LMBW) of the sea cucumber Apostichopus japonicus. With serial sections we found neural branches and fibers extending from hyponeural part of radial nerve towards LMBW and circular muscles of body wall. With the aqueous aldehyde (Faglu) method yellow fluorescence indicating indolamines was observed in LMBW and in the mesentery connecting LMBW to the body wall. With indirect immunohistochemistry 5-HT-like immunoreactivity was observed in LMBW and in mesentery. These results strongly suggested that both LMBW and mesentery contained 5-HT. The effects of monoamine neurotransmitters were studied in LMBW. Putative neurotransmitters tested were 5-HT, adrenaline, noradrenaline, dopamine, and DOPA at the concentration of 10(-6) M. The application of 5-HT caused no contraction or relaxation, but it inhibited the contraction induced by 10(-6)-10(-5) M acetylcholine (ACh). Catecholamines were ineffective by themselves and had no effects on the contraction induced by ACh. The present histological, histochemical, and pharmacological studies strongly suggested that holothurian LMBW was innervated by inhibitory serotonergic neurons of the hyponeural nervous system.