Nanoscopic X-ray tomography for correlative microscopy of a small meiofaunal sea-cucumber.

In the field of correlative microscopy, light and electron microscopy form a powerful combination for morphological analyses in zoology. Due to sample thickness limitations, these imaging techniques often require sectioning to investigate small animals and thereby suffer from various artefacts. A recently introduced nanoscopic X-ray computed tomography (NanoCT) setup has been used to image several biological objects, none that were, however, embedded into resin, which is prerequisite for a multitude of correlative applications. In this study, we assess the value of this NanoCT for correlative microscopy. For this purpose, we imaged a resin-embedded, meiofaunal sea cucumber with an approximate length of 1 mm, where microCT would yield only little information about the internal anatomy. The resulting NanoCT data exhibits isotropic 3D resolution, offers deeper insights into the 3D microstructure, and thereby allows for a complete morphological characterization. For comparative purposes, the specimen was sectioned subsequently to evaluate the NanoCT data versus serial sectioning light microscopy (ss-LM). To correct for mechanical instabilities and drift artefacts, we applied an alternative alignment procedure for CT reconstruction. We thereby achieve a level of detail on the subcellular scale comparable to ss-LM images in the sectioning plane.

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.

On some sea cucumbers (Echinodermata: Holothuroidea) from off the south and west coasts of South Africa collected by the South African Environmental and Observation Network (SAEON).

Twenty four specimens of holothuroids recently received from the South African Environmental and Observation Network (SAEON), collected from off the south and west coasts of South Africa, are herein recorded and/or described. The specimens comprise eight nominal and one indeterminate species and represent both shallow-water and deep-sea forms, distributed from Plettenberg Bay to just north of Lambert's Bay in the Western Cape Province. There are no new species but two new records for the South African region and extensions of horizontal and bathymetric distributions of the other species. Additions to the South African fauna are Zygothuria lactea (Théel, 1886) and Synallactes cf. challengeri (Théel, 1886). The paper also contains the first definite record of Thyone venusta Selenka, 1868, originally described from the Red Sea. Distribution ranges of the following species have been altered Synallactes viridilimus Cherbonnier, 1952; S. mollis Cherbonnier, 1952 and Psuedostichopus langeae Thandar, 2009.

Posterior regeneration following fission in the holothurian Cladolabes schmeltzii (Dendrochirotida: Holothuroidea).

The regeneration of the posterior portion of the body after fission was studied in the holothurian Cladolabes schmeltzii using electron microscopy methods. Following fission, the aquapharyngeal complex, gonad and anterior portion of the first descending part of the intestine remain in the anterior fragment of the body. The entire regeneration process is divided into five stages. In the first three stages, the digestive system and damaged ends of the longitudinal muscle bands regenerate. The intestine is formed through the rearrangement and growth of the remaining portion of the first descending part of the intestine. The gut anlage grows down the mesentery and joins the regenerating cloaca. The cloaca is formed from two sources: its posterior portion appears as a result of immersion of the epidermis, while the anterior portion develops from the terminal segment of the growing intestine. Regeneration of muscles progresses in the typical manner for echinoderms: through immersion and myogenic transformation of the coelomic epithelium. Respiratory trees appear in animals when the growth of the external part of the body has begun (fourth stage). They are formed as an outgrowth of the dorsal wall of the anterior portion of the cloaca. It was concluded that regeneration of the posterior portion of the body in the holothurian C. schmeltzii following fission is realized through morphallactic rearrangements of the remaining parts of organs. The main mechanism through which the digestive, respiratory, and contractile systems are formed is epithelial morphogenesis.

Benthodytes violeta, a new species of a deep-sea holothuroid (Elasipodida: Psychropotidae) from Mar del Plata Canyon (south-western Atlantic Ocean).

A new species of elasipodid holothuroid, Benthodytes violeta sp. nov., is described from the Mar del Plata Canyon off Buenos Aires Province (around 38ºS-54ºW). It was taken at four locations at depths ranging from 1500 to 1950 m. This new species has a violet gelatinous body of up to 200 mm in length, with eight pairs of dorsal appendages, lateral festooned edges and four rows of tube feet ventrally. Body wall ossicles comprise rods and crosses with three or four arms and a central bipartite apophysis borne on the primary cross; tentacles and gonad deposits comprise rods and crosses with three and four arms. This is the first report of a holothuroid from the Mar del Plata Canyon area.

The caudinid sea cucumbers of New Zealand (Echinodermata: Holothuroidea: Molpadida: Caudinidae).

Five species of Caudinidae occur in New Zealandwaters. Two new species are described: Paracaudina alta sp. nov.; Paracaudina reductia sp. nov. Two species reported previously are discussed: Paracaudina chilensis (Müller) and Paracaudina coriacea (Hutton). A lectotype has been established for P. coriacea (Hutton). Hedingia albicans var. glabra (Théel) is raised out of synonymy with Hedingia albicans (Théel), and the variety elevated to species status as Hedingia glabra (Théel). A key is provided for the New Zealand species of Caudinidae.

Spermatogenesis and chromatin condensation in male germ cells of sea cucumber Holothuria leucospilota (Clark, 1920).

Male germ cells in the testis of Holothuria leucospilota can be divided into 12 stages based on ultrastructure and patterns of chromatin condensation. The spermatogonium (Sg) is a spherical-shaped cell with a diameter of about 6.5-7microm. Its nucleus mostly contains euchromatin and small blocks of heterochromatin scattered throughout the nucleus. The nucleolus is prominent. Primary spermatocytes are divided into six stages, i.e., leptotene (LSc), zygotene (ZSc), pachytene (PSc), diplotene (DSc), diakinesis (DiSc) and metaphase (MSc). The early cells are round while in DiSc and in MSc cells are oval in shape. From LSc to MSc, the sizes of cells range from 3.5 to 4microm. LSc contains large blocks of heterochromatin as a result of increasingly condensed 17nm fibers. In ZSc, the nucleus contains prominent synaptonemal complexes but a nucleolus is absent. In PSc, heterochromatin blocks are tightly packed together by 26nm fibers and appeared as large patches in DSc. Heterochromatin patches were enlarged to form chromosomes in DiSc and MSc and then the chromosome are moved to be aligned along equatorial region. The secondary spermatocyte (SSc) is an oval cell about 4.5-5.5microm. Their nuclei contain large clumps of heterochromatin along the nuclear envelope and in the center nuclear region. Spermatids are divided into two stages, i.e., early spermatid (ESt) and late spermatid (LSt). The nuclei decrease in size by a half and become spherical; thus the chromatin fibers condensed into 20nm and are closely packed together leaving only small spaces in LSt. The spermatozoa (Sz), with chromatin tightly packed in the spherical nucleus with a diameter of 2microm and a small acrosome situated at the anterior of the nucleus. The tail consists of a pair of centrioles lying perpendicular to each other and surrounded by a mitochondrial ring, and an axonemal complex, surrounded by a plasma membrane.

Polysaccharides from Astragalus membranaceus promote phagocytosis and superoxide anion (O2-) production by coelomocytes from sea cucumber Apostichopus japonicus in vitro.

The potential immunostimulatory effects of Astralagus membranaceus polysaccharides (APS) on sea cucumber, Apostichopus japonicus (Selenka), were investigated in vitro. Phagocytosis and superoxide anion (O(2)(-)) production by phagocytic amoebocytes (PA) from A. japonicus coelomic fluid were measured during incubation at 18 degrees C, 22 degrees C, or 25 degrees C with APS at 0, 10, 20, or 40 microg mL(-1) (n=3). Phagocytic activity against yeast cells was quantified by direct visualization, and O(2)(-) production by nitroblue tetrazolium (NBT) reduction assay. Compared with controls, including APS at 20 microg mL(-1) significantly increased (P<0.05) the percentage of phagocytic capacity (PC) and phagocytic index (PI) at 18 degrees C and 22 degrees C, but no significant enhancement was observed at 25 degrees C. In contrast, the coelmocytes of A. japonicus can have an obvious generation of O(2)(-) after the stimulation. The concentration of 20 microg mL(-1) APS resulted in a significant increase in nitroblue tetrazolium (NBT) positive cells (P<0.05) at different temperature and even 10 microg mL(-1) APS could increase O(2)(-) generation significantly at 18 degrees C and 22 degrees C. Both phagocytosing and O(2)(-) production increased with the increase of APS concentration from 0 to 20 microg mL(-1) at different temperature, and when APS at 40 microg mL(-1), they were decreased. It suggested that immunocytes activity in A. japonicus decreased with the temperature increasing from 18 degrees C to 25 degrees C, and APS could be an effective immunostimulant to enhance phagocytic activity and O(2)(-) production.

Developmental origin of the adult nervous system in a holothurian: an attempt to unravel the enigma of neurogenesis in echinoderms.

In adult echinoderms, the nervous system includes the ectoneural and hyponeural subsystems. The former has been believed to develop from the ectoderm, whereas the latter is considered to be mesodermal in origin. However, this view has not been substantially supported by embryological examinations. Our study deals with the developmental origin of the nervous system in the direct-developing sea cucumber Eupentacta fraudatrix. The rudiment of the adult nervous system develops from ectodermally derived cells, which ingress into the primary body cavity from the floor of the vestibule. At the earliest stages, only the rudiment of the ectoneural nerve ring is laid down. The radial nerve cords and tentacular nerves grow out from this subcutaneous rudiment. The ectoneural cords do not develop simultaneously but make their appearance in the following order: unpaired mid-ventral cord, paired dorsal lateral cords, and ventral lateral cords. These transitional developmental stages probably recapitulate the evolution of the echinoderm body plan. The holothurian hyponeural subsystem, as other regions of the metazoan nervous system, has an ectodermal origin. It originally appears as a narrow band of tissue, which bulges out of the basal region of the ectoneural neuroepithelium. Our data combined with those of other workers strongly suggest that the adult nervous tissue in echinoderms develops separately from the superficial larval system of ciliary nerves. Therefore, our data are neither in strict accordance with Garstang's hypothesis nor do they allow to refuse it. Nevertheless, in addition to ciliary bands, other areas of neurogenetic epidermis must be taken into account.

Acute peristome edema disease in juvenile and adult sea cucumbers Apostichopus japonicus (Selenka) reared in North China.

Acute peristome edema disease (APED) is a new disease that broke out in cultured sea cucumber along the Shangdong and Liaoning province coasts in China, PR, and has caused a great deal of death in Apostichopus japonicus (Selenka) since 2004. Here we report virus-like particles found in intestine epithelium of sea cucumbers reared in North China. It is the first time that sea cucumbers are reported to be infected by virus. Histological examinations showed that the viral inclusion bodies existed in intestine epithelium cells. Electron microscopic examinations show that the virions were spherical, 80-100nm in diameter, and composed of a helical nucleocapsid within an envelope with surface projections. Detailed studies on the morphogenesis of these viruses found many characteristics previously described for coronaviruses. Virus particles always congregated, and formed a virus vesicle with an encircling membrane. The most obvious cellular pathologic feature is large granular areas of cytoplasm, relatively devoid of organelles. Tubular structures within virus-containing vesicles, nucleocapsid inclusions, and double-membrane vesicles are also found in the cytopathic cells. No rickettsia, chlamydia, bacteria, or other parasitic organisms were found.

Morphology, structure of dimorphic sperm, and reproduction in the hermaphroditic commensal bivalve Pseudopythina tsurumaru (Galeommatoidea: Kellidae).

In Japan Pseudopythina tsurumaru is an up to 10.8 mm-long commensal of the burrowing sea cucumber Protankyra bidentata, whereas in Hong Kong the same species is smaller and associated with the crab Hexapus anfractus, itself a commensal of P. bidentata. Japanese P. tsurumaru is a hermaphrodite tending towards protogyny maturing to a female when > or = 7 mm, and entering the hermaphroditic condition when > or = 9 mm long. In addition to normal euspermatozoa, the species produces 30-32 microm long and 7 x 8 microm broad spindle-shaped paraspermatozoa provided with a conical acrosome, a nucleus, and a bundle of approximately 15-16 flagella issuing from the head region. Paired pouch-formed seminal receptacles normally occur in bivalves > or = 6 mm. Bulk sperm transfer presumably takes place by way of spermatozeugmata formed by the two types of sperm cells. Exogenous euspermatozoa attach to particular nonepithelial cells that occupy the interior of the receptacles. These cells, together with their associated sperm, are probably released as syncytial sperm-carrying bodies into the suprabranchial chamber, where the ova are fertilized.

Visceral regeneration in holothurians.

Holothurians, or sea cucumbers, exhibit two processes that have intrigued biologists for decades: autotomy and regeneration. Autotomy includes the loss of body parts by evisceration or fission, and regeneration is the extraordinary process by which the lost organs are replaced. In this article, we review the literature on evisceration, transection, and visceral regeneration in holothurians and compare these processes in different orders and lower taxa. Focusing mainly on the digestive tube, we analyze regeneration from a cellular perspective, considering especially the origin, migration, and proliferation of the cellular components of the regenerated organ. The data highlight the most interesting aspects of holothurian regeneration and indicate those critical problems requiring new information and new approaches.

Muscle regeneration in holothurians.

The muscle system of holothurians includes visceral (coelomic epithelium) and somatic (longitudinal muscle bands, retractors of aquapharyngeal complex) musculature. Visceral musculature regeneration is achieved by the transformation of myoepithelial cells via their dedifferentiation, migration, proliferation, and redifferentiation. During somatic muscle regeneration the new muscle bundles are formed due to dedifferentiation, migration, and immersion of the coelomic epithelial cells into the connective tissue. While submerging, the epithelial cells transform into myocytes and begin to produce myofibrils in their cytoplasm. Concomitantly, a basal lamina is formed around the group of myogenic cells, separating them from the surrounding extracellular matrix. The myohistogenesis is accompanied by a conspicuous DNA-synthetic activity. Proliferation is insignificant and seems to be of no essential importance for muscle regeneration. The synthesis of DNA followed by no cytokinesis results in an increase in the amount of DNA of myocyte nuclei.

Maintaining the line of defense: regeneration of Cuvierian tubules in the sea cucumber Holothuria forskali (Echinodermata, Holothuroidea).

When irritated, individuals of the sea cucumber Holothuria forskali expel a few Cuvierian tubules which lengthen, instantly become sticky, and rapidly immobilize most organisms with which they come into contact. After expulsion, the lost tubules are readily regenerated. When only a few tubules have been expelled, there is often a latent period before the regeneration starts. In contrast, when many tubules have been expelled, the regenerative process starts immediately but proceeds in successive waves of 10 to 30 tubules that begin to regenerate at 10-day intervals. However, in all cases, the complete regeneration of a given tubule takes about 5 weeks and may be divided into three successive phases: an initial repair phase including the overall 48-h post-autotomy period, a true regenerative phase taking about 4 weeks to complete, and a growth phase of about one more week. Initial regeneration events occur by epimorphosis, cell proliferation being essential to the regenerative process, whereas late events occur mainly by morphallaxis, with migration of the newly differentiated cells. The mesothelium is the tissue layer in which cell proliferation is the most precocious and the most important, involving both peritoneocytes and undifferentiated cells (which seem to be dedifferentiated peritoneocytes). As regeneration proceeds, the percentage of undifferentiated cells regularly decreases in parallel with the differentiation of granular (adhesive-secreting) cells and myocytes. The myocytes then separate off from the mesothelium and migrate within the connective tissue layer. Three types of pseudopodial cells follow one another in the tubule connective tissue during regeneration. Type 1 cells have all the characteristics of echinoderm phagocytes and may have a fibroblastic function, cleaning the connective tissue compartment before new collagen synthesis starts. Type 2 cells are rather undifferentiated and divide actively. The presence of type 3 cells is closely associated with the appearance of collagen fibers, and it is suggested that they have a fibroblastic function. In the inner epithelium, cells also divide actively, but only those in which spherules have not yet differentiated in the basal intraconnective processes. It appears, therefore, that in the three tissue layers of the tubules, regeneration proceeds by cell dedifferentiation, then proliferation, and finally by differentiation. Cuvierian tubules thus constitute a very efficient defensive mechanism: their large number, sparing use, and particular regeneration dynamics make them an almost inexhaustible line of defense maintained at limited energy cost.

Partial biochemical and immunologic characterization of fibrillin microfibrils from sea cucumber dermis.

The dermis of the sea cucumber Cucumaria frondosa is a mutable collagenous tissue composed of collagen fibrils, microfibrils, proteoglycans, and other soluble and insoluble components. A major constituent of the dermis is a network of 10-14 nm microfibrils which surrounds and penetrates bundles of collagen fibrils. These microfibrils, which are morphologically very similar to the fibrillin microfibrils of vertebrates, were found to be insoluble in protein denaturants, including chaotropic agents and ionic and nonionic detergents, regardless of the reduction of disulfide bonds. The microfibrils are covalently crosslinked by epsilon-(gamma-glutamyl)lysine at a concentration of 3.725 nmol/mg dry weight of purified insoluble material. The network is susceptible to proteolysis by trypsin, chymotrypsin, and pancreatic elastase, but not by bacterial collagenase. Amino acid compositional analysis of the network shows it to be composed of 25% ASX and GLX residues. Comparison with the proteins in the SwissProt database gives the network protein a high probability of being related to the mammalian protein fibrillin. The network is glycosylated: approximately 7% of the mass is constituted by neutral and amino sugars. The intact microfibrillar network cross-reacted with a well-characterized antiserum to mammalian fibrillin.

Glycosaminoglycan involved in the cation-induced change of body wall structure of sea cucumber Stichopus japonicus.

The body wall of sea cucumber Stichopus japonicus was treated with various concentrations of several cations, and examined for changes in toughness, taking punch force as parameter. Toughness of the body wall tended to decrease with increasing concentration of each cation, but in different modes depending mainly upon the valency of cation: e.g., the body wall completely lost toughness in 0.3 M Na+ or 0.4 M K+, whereas it retained more than half the initial toughness even in 0.4 M Ca2+ or Mg2+. Glycosaminoglycan (GAG) from the body wall was dissolved in water, and examined for viscosity changes as caused by those cations. Specific viscosity (eta sp) decreased from 0.71 (without cation) to 0.47-0.57 in the presence of 0.1 M monovalent and divalent cations. At 0.4 M, monovalent cations reduced eta sp to 0.38-0.46, but divalent cations increased eta sp to 0.56-0.63. Electron microscopy demonstrated that GAG matrix was clearly observed in the absence of cation, but disappeared in 0.4 M NaCl, forming wide free spaces in the body wall. These results all suggested that GAG is closely involved in the change of toughness of sea cucumber body wall.

The diameter of chromatin fibres depends on linker length.

We have studied the diameter of chromatin fibres embedded in epoxy resins for three different materials: mouse thymus, chicken erythrocytes and sea cucumber spermatozoa. We confirm that the diameter of chromatin fibres increases with linker length, both values being influenced by the protein composition of chromatin.

[Peptidergic and monoaminergic innervation of the gonads in the holothurian Cucumaria japonica]. / Peptidergicheskaia i monoaminergicheskaia innervatsiia goloturii Cucumaria japonica.

In the holothurian gonad structure of the peptidergic and monoaminergic systems has been described. Axons of their cells form tissue and hemal terminals. Epithelial cells and smooth myocytes of the gonadal wall get direct innervation, having contacts with the axonal terminals that are separated by the cleft 50-75 nm in the diameter. It is possible that neuropeptides and biogenic monoamines are transported to the germ and follicular cells of the germinative gonadal zone via hemolymph of the hemal sinus.

Close apposition of muscle cells in the longitudinal bands of the body wall of a holothurian, Isostichopus badionotus.

Electron microscopy reveals that sarcolemmata of adjacent muscle cells form pentalaminar junctions by fusion of apposed trilaminar double leaflet membranes. These junctions appear to be candidates for low resistance pathways between muscle fibers. The muscles depolarize slowly when bathed in solutions containing elevated concentrations of KCl, and the sucrose gap method can then be used to measure the potential difference between polarized and depolarized regions. Thus the junctions which we have observed may provide the structural basis for electrical transmission through the sucrose gap.

The subunit structure of chromatin fibres.

Optimal conditions for studying the ultrastructure of chromatin fibers of histone-containing spermatozoa in thin sections have been determined. Better results for preservation in sperm of the sea cucumber Holothuria tubulosa, have been found than in different frog species studied. The fine structure of chromatin fibers after different treatments was studied by computer materials. A clear superbead structure was found under all conditions which preserve the chromatin fibres. These have a diameter of 30 nm, with superbeads about 33 nm long. In the best preserved cases an additional periodicity of 11 nm along the fibres was found. There is no clear relationship of this periodicity with an eventual solenoidal structure of the chromatin fibers.