Tatsuo Motokawa

Embryogenesis in the Reef-Building Coral Acropora spp.

Abstract 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.

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 δ 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.

LOCALIZATION OF THE NEUROPEPTIDE NGIWYAMIDE IN THE HOLOTHURIAN NERVOUS SYSTEM AND ITS EFFECTS ON MUSCULAR CONTRACTION

NGIWYamide is a peptide recently isolated from the sea cucumber Apostichopus japonicus. It stiffens the connective tissue of the holothurian body wall. Localization of NGIWYamide was investigated by immunohistochemical staining with antiserum raised against NGIWYamide. In holothurian nervous systems NGIWYamide–like immunoreactivity (NGIWYa–LI) was observed in the hyponeural and ectoneural regions of the radial nerve cord, as well as in the circumoral nerve ring, podial nerves, tentacular nerves, the basiepithelial nerve plexus of the intestine and in cellular processes running through the body wall dermis. Labelled nerve fibres from the hyponeural part of the radial nerve running towards the circular muscle and from the podial nerve into the body wall dermis suggest that NGIWYamide controls both muscle and connective tissue. We examined the effect on muscle activity of the sea cucumber. NGIWYamide (10–7 to 10–4 M) caused contraction of the longitudinal body wall muscle. Tentacles showed contraction only at a higher dose (10–4 M). NGIWYamide (10–4 M) inhibited spontaneous contraction of the intestine.

Contractile Connective Tissue in Crinoids

Active movements in animals are usually attributed to cellular protein engines, e.g., the actin-myosin system of muscle cells. Here we report the first evidence of an extracellular contractile connective tissue, which we have found in sea lilies and feather stars (Echinodermata, Crinoida). These marine animals have arm muscles that are antagonized, not by other muscles, but by ligaments consisting of extracellular fibrils interspersed with neuron-like cell processes. Contractile cells are lacking, yet these arm ligaments actively contracted upon stimulation. The ligaments stayed in a contracted condition even after the stimulus had stopped. The stresses generated were lower than those of typical skeletal muscles. Additional data from crinoid cirri, which lack muscles entirely, corroborate the hypothesis that the connective tissue of the ligaments is contractile.

Effects of ionic environment on viscosity of Triton-extracted catch connective tissue of a sea cucumber body wall

Abstract Mechanical properties of catch connective tissue are greatly affected by its ionic environment. In order to understand the role of ions, a preparation was developed in which cellular activities were suppressed by treatment with 1% Triton X-100. The material used was body-wall dermis of the sea cucumber Holothuria leucospilota Brandt. The effects of the main cations in seawater (H + , Na + , K + , Ca 2+ , Mg 2+ ) on the creep viscosity of the Triton model were compared with those of intact dermis. The comparison distinguished the site of action of ions. K + had its main effect on cells that control the catch mechanism, whereas Ca 2+ worked directly on extracellular materials. H + , Na + and Mg 2+ had both effects.

Morphological basis and mechanics of arm movement in the stalked crinoid Metacrinus rotundus (Echinodermata, Crinoida)

We obtained live specimens of the stalked crinoid Metacrinus rotundus from Suruga Bay, Japan, in 1992 and managed to keep them for several months in aquaria. Video observations of crawling individuals showed that both the aboral bending of the arm and the oral bending can be power strokes. When climbing up a lattice, the crinoids bend their arms aborally to pull the body upward. The oral side of the arm contains muscles, but the aboral side has ligaments without muscles. We found that ligaments on the aboral side are divided into two parts, the aboral ligament and the newly found fossa ligament. Ultrastructurally, the aboral ligament resembles typical echinoderm catch connective tissue. Its microfibrils exhibit a variable banding pattern after special staining and might be an elastic material. The fossa ligament contains only collagen fibrils which are often closely connected. Biomechanically the frozen and rethawed aboral ligament behaves like a spring. It shows between 2.4 and 29.1% stress relaxation, and it is so stiff that the oral muscles would not be able to stretch it. We suggest that the connective tissue of the ligament is a catch connective tissue and softens when the arm moves. During long-term filtration posture the ligament would stiffen so as to maintain its posture for a long time without muscle contraction and thus without using much energy.

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 (Vo2 in millilitres of O2 per hour) and the colony mass (wet weight Mw in grams) showed the allometric relationship (Vo2 = 0.0412 Mw0.799. 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.

Penetration and storage of sponge spicules in tissues and coelom of spongivorous echinoids

When echinoids feed on sponges, silicate spicules of the sponge were found to enter their body either by penetration through the wall of the food canal into coelomic cavities or by penetration into skeletal plates and spines. The spicules, which have penetrated into the coelom, obviously evoke a kind of protective answer. They were found to be entangled by clusters of cell remnants, the so-called brown bodies. The brown bodies contain melanin and gather at special sites of the echinoid body; these are the Stewart Organs, the gills and the inner side of the ambulacral plates. Sometimes the silicate spicule becomes surrounded by a calcareous sheath. The length of the sponge spicules makes their removal impossible, so that they are stored. The spicules penetrating into the plates are partly incorporated into the stereom. The four species examined in this study were Asthenosoma ijimai, Araeosoma owstoni, Diadema setosum (collected in Sagami Bay, Japan in 1991) and Hapalosoma gemmiferum (collected in Suruga Bay, Japan in 1991).

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

SUMMARY 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 Ca2+. 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 Ca2+-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.

Cirri of the stalked crinoid Metacrinus rotundus: neural elements and the effect of cholinergic agonists on mechanical properties

Sea lilies are enigmatic animals due to their scarcity and their biology is comparatively neglected. Cirri, arranged in whorls of five along the sea lily stalk, anchor and support the animal. They consist of ossicles interconnected by collagenous ligaments and by a central canal. Cirri have a welldeveloped nervous system but lack muscular cells. A light and electron microscopic study was performed to clarify the morphology of the nervous system of the cirri. Two cellular networks were found, one of neuron‐like cells and one of cells filled with bullet‐shaped organelles. Both networks ramify throughout the cirral ossicles up to the interossicle ligaments. Mechanical tests were performed to analyse the influence of cholinergic agonists on the mechanical properties of these ligaments. In the tests, the cirral ligaments softened after the application of acetylcholine, muscarinic agonists and nicotinic agonists. The reaction time to muscarinic agonists was much slower than to acetylcholine and nicotinic agonists. At low concentrations, muscarinic agonists caused active development of force. No reaction to stimuli was observed in anaesthetized cirri. The data clearly establish the existence of catch connective tissue which can change its mechanical properties under nervous control mediated via nerves with cholinergic receptors. The possible sources of the observed force production are discussed and it is concluded that active contraction of collagenous ligaments causes movement of cirri.