Ultrastructure of Male Sexual Apparatus of Scutellonema brachyurum

Electron micrographs of serial sections show that the male sexual apparatus of Scutellonema brachyurum includes two morphologically identical spicules. Each is composed of a swollen tubular head, crescentic shaft, and leaf-like blade with membranous velum expanded from the central trunk. The spicules are concave and grooved on the ventral side and convex on the dorsal side near the trunk. The trunk is continuous with the shaft and head. Nerve tissue occupies the core of the spicule and includes a dendritic process which gains access to the exterior via a small pore on the lateral side of the spicule tip. Three protractor and two retractor muscles are associated with each spicule. A sensory accessory piece connects with the tip of the gubernaculum and protrudes from the lower side of the opening of the spicular pouch; it protracts and retracts with the muscularized gubernaculum. The gubernaculum varies from bow-shaped in the distal part to boat-shaped in the mid region. A sac exits beneath the accessory piece as a buffer for its movement. A cuticular guiding bar originating from the dorsal wall of the spicular pouch has a tongue. The ventral surface of the tongue is sclerotized to separate the two spicules. It is mobile by muscles of the protractor gubernaculi, retractor gubernaculi, and seductor gubernaculi.     

Morphological Comparison and Taxonomic Utility of Copulatory Structures of Selected Nematode Species

Spicules of 9 Meloidogyne, 2 Heterodera, 3 Globodera, and 12 other plant-parasitic, insect-parasitic, and free-living nematodes were excised and examined using scanning electron microscopy (SEM). Gubernacula of some of the species were also excised, and their structure was determined. The two spicules of all species examined were symmetrically identical in morphology. The spicule typically consisted of three parts: head, shaft, and blade with dorsal and ventral vela. The spicular nerve entered through the cytoplasmic core opening on the lateral outer surface of the spicule head and generally communicated with the exterior through one or two pores at the spicule tip. Spicules of Xiphinema sp. and Aporcelaimellus sp. were not composed of three typical parts, were less sclerotized, and lacked a cytoplasmic core opening and distal pores. Spicules of Aphelenchoides spp. had heads expanded into apex and rostrum and had very arcuate blades with thick dorsal and ventral edges (limbs). Gubernaculum shapes were stable within a species, but differed among species examined. The accessory structures of Hoplolaimus galeatus consisted of a tongue-shaped gubernaculum with two titillae at its distal end and a plate-like capitulum terminating distally in two flat, wing-like structures. A comparison of spicules of several species of Meloidogyne by SEM and light microscopy revealed no striking morphological differences.     

Effects of Germanium (Ge) on the silica spicules of the marine sponge Suberites domuncula: Transformation of spicule type

Germanium (Ge), in the form of germanic acid, at a Ge/Si molar ratio of 1.0 inhibits gemmule development and silica deposition in the marine demosponge Suberites domuncula. Lower Ge/Si ratios inhibit the growth in length of the silica spicules (tylostyles) producing short structures, but with relatively normal morphology and close to normal width; spherical protuberances occasionally occur on these spicules. A few of the short spicules possess completely round rather than pointed tips. Many of the latter develop when Ge is added (pulsed) to growing animals, thus inducing a change in spicule type. These results indicate that the growth in length of the axial filament is more sensitive to Ge inhibition than is silica deposition and that pointed spicule tips normally develop because the growth of the axial filament at the spicule tip is more rapid than silica deposition. Newly formed spicules initiate silica deposition at the spicule head but the absence of Ge-induced bulbs as in freshwater spicules (oxeas) leaves open the question of whether there is a silicification center(s) present in Suberites tylostyles. The morphogenesis of freshwater oxeas and of marine tyolstyles appears fundamentally different-bidirectional growth in the former and unidirectional growth in the latter. X-ray analysis demonstrate relatively uniform Ge incorporation into the silica spicules with considerable variation from spicule to spicule in the incorporated level. Increased silicic acid concentration induces the formation of siliceous spheres, suggesting that the axial filament becomes prematurely encased in silica.     

Loss of phospholipid asymmetry in dilauroylphosphatidylcholine induced plasma membrane vesicles from human platelets

Incubation of human platelets with unilamellar vesicles composed of dilauroylphosphatidylcholine (DLPC) induces shedding of small vesicular structures from the platelet plasma membrane. No significant cell lysis is observed during the process of shedding. Isolated spicules contain the major membrane glycoproteins, Ib, IIb, and IIIa, which are used to define the sidedness of the spicule membrane. These glycoproteins are completely susceptible to chymotrypsin treatment, whereas cytoskeletal proteins are inaccessible towards this enzyme. This demonstrates that the spicule membranes have a right-side-out orientation in as far as membrane proteins are concerned. Isolated spicules were 30-fold more active than platelets in stimulating prothrombin conversion to thrombin by the prothrombinase complex (factors Xa, Va and Ca2+). The increased prothrombinase activity reflects an increased amount of phosphatidylserine in the outer leaflet of the spicule membrane. Protein analysis of platelet spicules and native platelets reveals a number of differences, the most conspicuous of which is the virtual absence of myosin in the spicule preparations. It is proposed that a lack of myosin produces a different cytoskeletal organization in the spicules. This enables phosphatidylserine to become exposed at the outer surface of the spicule membrane.     

Spicule Formation-Inducing Substance in Sea Urchin Embryo

Isolated micromeres of sea urchin produced spicules in sea water containing blastocoelic fluid (BCF) taken from embryos, or in a medium in which embryos had previously been dissociated (dissociated solution, DS). When isolated micromeres were cultured in vitro , their descendants initiated spicule formation only when BCF was added to the culture medium by the time when, in normal development, primary mesenchyme cells form two aggregates in the vegetal region. After the initiation of spicule formation, growth of spicules occurred under the continuous influence of DS. Spicule formation-inducing (SFI) activity in DS was first detected at the mesenchme blastula stage. The activity in BCF was heat-labile and was inactivated by trypsin.     

Ultrastructural Aspects of Spicule Formation in the Solitary Ascidian Herdmania momus (Urochordata,Ascidiacea)

The solitary stolidobranch ascidian Herdmania momus contains numerous calcium carbonate spicules in its tunic and body tissues. The slender body spicules form inside complex sheaths in the body wall and branchial basket, where they remain for the life of the animal. The much smaller tunic spicules form inside the tunic blood vessels and then migrate to the tunic surface, where they become anchored by their spiny base. This paper is an ultrastructural investigation of the formation of the body spicules; the tunic spicules, which apparently form quite differently, will be the focus of a future study. The body spicules are composed of rows of closely packed acicular spines which form completely extracellularly. The spine tips are covered by flattened, highly pseudopodial sclerocytes bound together by tightly interdigitating cell processes. The basal regions of contiguous spines are covered by very thin sclerocyte cell processes. An organic matrix is present within the spines; its exact nature is not clear. A very dense extracellular inter-spine matrix is located between the spine tips and the contiguous basal regions. Presclerocytes within the sheaths between the spicules are probably responsible for formation of the extracellular structures of the sheaths. The presclerocytes appear to aggregate and transform into sclerocytes at the apical end of the spicule. New spines are added at the apical end of the spicule as well as between larger spines. Comparisons are made between body spicule formation in H. momus and skeletogenesis in echinoids.     

CRYSTAL PROPERTY OF THE LARVAL SEA URCHIN SPICULE*

A pair of pluteus skeletal spicules arises from a pair of calcareous granules via the triradiate form. In polarized light, each spicule behaves as though carved out of a single crystal of magnesian calcite. The optic axis lies perpendicular to the plane of the triradiate and parallel to the body rod of the pluteus. However, in the scanning electron microscope, the spicule surface appeared smooth or somewhat spongy and manifested no crystal faces. Neither etching nor fracturing revealed underlying crystalline texture. Nevertheless, rhombohedral calcite crystals could be grown epitaxially onto isolated spicules immersed in a medium containing CaCl₂ and NaHCO₃. The optic axes of all crystals coincided with the optic axis of the spicule on which they were grown. Corresponding faces of the crystals were all aligned parallel to each other despite the complex shape of each spicule. Where the left and right spicules joined, two mutually tilted sets of crystals were observed but not crystals of intermediate orientation. Thus, the sea urchin larval spicule is built from a stack of molecularly contiguous microcrystals but its overall shape is generated by the mesenchyme cells independent of the magnesian calcite crystal habit.     

The biology of the marine sponge Microciona prolifera (Ellis and Solander). III. Spicule secretion and the effect of temperature on spicule size

The secretion of siliceous spicules in the marine demosponge Microciona prolifera (Ellis and Solander) is by three different means. Styles are secreted by sclerocytes with archeocyte characteristics (nucleolate nucleus, phagosomes). chelas are formed by small sclerocytes with anucleolate nuclei, and toxas are apparently formed extracellularly within membranous material. Genetically and physiologically equivalent explants of this sponge were grown at 15, 20, and 25 C for four weeks. Analyses of spicule dimensions show little correlation of temperature with spicule length, except in the case of toxas. but a clear inverse relationship of spicule width with temperature. It is suggested that thicker spicules are formed at lower temperatures due to the more efficient entrapment of silicon rather than to effects upon silicon transport. Chela dimensions are very uniform implying an all or none process in their secretion. Differences in spicule dimensions between individual sponges grown at these temperatures may be due to the highly complex pathways of silicon transport and/or to genetic differences.     

Asymmetric inhibition of spicule formation in sea urchin embryos with low concentrations of gadolinium ion

As gastrulation proceeds during sea urchin embryogenesis, primary mesenchyme cells (PMCs) fuse to form syncytial cables, within which calcium is deposited as CaCO₃, and a pair of spicules is formed. Earlier studies suggested that calcium, previously sequestered by primary mesenchyme cells, is secreted and incorporated into growing spicules. We examined the effects of gadolinium ion (Gd³⁺), a Ca²⁺ channel blocker, on spicule formation. Gd³⁺ did not lead to a retardation of embryogenesis prior to the initiation of gastrulation and did not inhibit the ingression of PMCs from the blastula wall or their migration along the inner blastocoel surface. However, when embryos were raised in seawater containing submicromolar to a few micromolar Gd³⁺, of which levels are considered to be insufficient to block Ca²⁺ channels, a pair of triradiate spicules was formed asymmetrically. At 1–3 μmol/L Gd³⁺, many embryos formed only one spicule on either the left or right side, or embryos formed a very small second spicule. Induction of the spicule abnormality required the presence of Gd³⁺ specifically during late blastula stage prior to spicule formation. An accumulation or adsorption of Gd³⁺ was not detected anywhere in the embryos by X‐ray microanalysis, which suggests that Ca²⁺ channels were not inhibited. These results suggest that Gd³⁺ exerts an inhibitory effect on spicule formation through a mechanism that does not involve inhibition of Ca²⁺ channels.     

The isotopic effects of D2O in developing sea urchin eggs

When developing sea urchin eggs were treated with sea water containing 40% D2O (D2O-SW) at the 8-cell stage, the micromere formation was delayed and micromeres were larger than those seen in the control. But eggs returned to normal artificial sea water (NASW) at the 16- to 32-cell stage did not form abnormal spicules in larvae of Pseudocentrotus depressus. Little effect on the spicule formation of Hemicentrotus pulcherrimus was also noted. If the culture period in D2O-SW was extended until the hatching stage, the number of plutei with abnormal spicules increased. Primary mesenchyme cells of Pseudocentrotus depressus larvae failed to make two aggregated spicule rudiments on the ventral side of the larva and developed a ring-like spicule. This ring-like spicule, however, only occasionally occurred in the larvae of Hemicentrotus pulcherrimus. The cell cycle was longer in the presence of D2O. However, blastomeres managed to divide throughout the development. Larvae reared in 20% D2O-SW after the hatching stage developed into quasi-normal plutei but smaller than control. We found no exogastrulation in these larvae. Exogastrulation was found only in larvae continuously cultured in 40% D2O-SW from the early development. These results are inconsistent with previous reports made by other authors.     

Biomineralization of the spicules of sea urchin embryos

The formation of calcareous skeletal elements by various echinoderms, especially sea urchins, offers a splendid opportunity to learn more about some processes involved in the formation of biominerals. The spicules of larvae of euechinoids have been the focus of considerable work, including their developmental origins. The spicules are composed of a single optical crystal of high magnesium calcite and variable amounts of amorphous calcium carbonate. Occluded within the spicule is a proteinaceous matrix, most of which is soluble; this matrix constitutes about 0.1% of the mass. The spicules are also enclosed by an extracellular matrix and are almost completely surrounded by cytoplasmic cords. The spicules are deposited by primary mesenchyme cells (PMCs), which accumulate calcium and secrete calcium carbonate. A number of proteins specific, or highly enriched, in PMCs, have been cloned and studied. Recent work supports the hypothesis that proteins found in the extracellular matrix of the spicule are important for biomineralization.     

Véligère planctotrophe du doridien Aegires punctilucens (D'Orbigny) (Mollusca: Nudibranchia: Notodorididae): Description et métamorphose

Different larval planktonic stages of the nudibranch Aegires punctilucens (D'Orbigny) are described. The youngest has a shell of the protoconch type 1 (Thompson) and a bilobed velum. After loss of the shell, the next stage is characterized by a large velum with broad and thick lobes. The mantel covers the body and has tubercles which grow progressively. Spicules appear in the mantle and in the foot; they are simple, triradiate or cross-shaped. A zone of hyaline denticles are present in the stomach lumen.Metamorphosis has been obtained under laboratory rearing. After the gradual resorption of the velum, the animal looks like a small dorid and is grey with white spots. The foot is slender and there are 14 tubercles always arranged in the same way and bristled with spicules.After discussing the species identification, the veliger is compared with other nudibranch larvae. The development of Aegires is very unusual with a two-stage metamorphosis, the first at the time of loss of the shell, and the second at loss of the velum cast. The intermediate stage between those two stages is planktonic.     

Dynamics of spicule production in the marine sponge <Emphasis Type="Italic">Hymeniacidon perlevis</Emphasis> during in vitro cell culture and seasonal development in the field

To characterize the formation of silica spicules, the dynamics of spiculogenesis of an intertidal marine sponge Hymeniacidon perlevis (Montagu 1818) (Porifera: Demospongiae) were investigated by measuring the gene expression of silicatein (the enzyme responsible for spicule silicification) and the dimensional changes of spicules during the developmental process of individual sponges and in cell cultures of primmorphs of archaeocyte-dominant cell populations. The different developmental stages of spicules were documented by time-lapse microscopy and observed by transmission electron microscopy during a 1-month culture period. During its annual life cycle, H. perlevis has four different developmental stages: dormancy, resuscitation, bloom, and decline. Field-grown individual sponge samples at different stages were collected over 7 months (March to September 2005). The dimensions of the silica spicules from these samples were microscopically measured and statistically analyzed. This analysis and the material properties of the spicules allowed them to be classified into four groups representing the different developmental stages of spiculogenesis. Silicatein expression in the bloom stage was more than 100 times higher than that in the other stages and was correlated with the spicule developmental stage. The trend of spicule formation in field-grown sponges was consistent with the trend in cell culture. A new parameter, the maturation degree (MD) of spicules (defined as the ratio of actual to theoretical silica deposition of mature spicules), was introduced to quantify spicule development. Silica spiculogenesis during H. perlevis development was delineated by comparing MD and silicatein expression.     

Spicule Formation in the Invertebrates with Special Reference to the Gorgonian Leptogorgia virgulata

Many of the invertebrates possess calcium carbonate spicules.This paper is a review of the formation of these structuresin the Porifera, Coelenterata, Platyhelminthes, Mollusca, Echinodermataand Ascidiacea. Mature spicules appear to be extracellular structures.Sponge spicules initiate intercellularly then become extracellular.Alcyonarian, turbellarian, echinoid and ascidian spicule depositionbegins intracellularly and then becomes extracellular. The continuationof growth in the extracellular environment has not been documentedexcept for the echinoids. Placophoran spicules initiate andremain as extracellular structures. Early spicule growth seemsto occur from or within a single cell. However, cell aggregationand/or neighboring cells appear to be important to the processof spicule formation. The spicule forming cells, in general,are found in a collagenous medium which may be associated withspicule growth. The organic matrix from the spicules of the gorgonian Leptogorgiavirgulata is a glycoprotein. Autoradiography reveals that thismatrix is apparently synthesized in the rough endoplasmic reticulumand Golgi complexes and then transported to the spicule formingvacuole via Golgi vesicles. To gain information about the entryand transport of calcium ions, the effects of ouabain and vanadateon calcium uptake were examined. Ouabain had no effect on calciumuptake. Vanadate treatment increased the uptake of calcium inscleroblasts and epithelial tissue and decreased its uptakein spicules. This may suggest that vanadate sensitive ATPasesare involved in the pumping of calcium out of scleroblasts,out of epithelial cells into the mesoglea, and into scleroblastorganelles. Autoradiography using ⁴⁵Ca indicates that the majorityof these ions initially accumulate in the branch axis. The labelmoves through the axial epithelium to the mesoglea and reachesthe spiculeforming vacuoles in the scleroblasts via dense bodies     

Characterization of proteins from the matrix of spicules from the alcyonarian, Lobophytum crassum

The organic matrix of spicules of the alcyonarian coral, Lobophytum crassum, was studied to investigate its molecular characteristics and functional properties. The shape of the spicules was identified using scanning electron microscopy. The soluble organic matrix comprised 0.03% of the spicule weight. The SDS-PAGE analysis of the preparation showed four protein bands with apparent molecular weights of 37, 48, 67 and 102 kDa. The 67- and 102-kDa proteins appeared to be calcium binding proteins, detected as radioactive bands by ⁴⁵Ca autoradiography. The 67-kDa protein appears to be glycosylated. The N-terminal amino acid sequence of the 67 kDa was determined; 7 of 20 residues were acidic. A database search for homologous proteins did not give a clear indication of the function of the 67-kDa protein. The isolated organic matrix possesses carbonic anhydrase activity which functions in calcium carbonate crystal formation, indicating that organic matrix is not only structural protein but also a catalyst. An interpretation of these results is that the spicule of alcyonarian corals has a proteinaceous organic matrix related to the calcification process.     

Socket cells mediate spicule morphogenesis in Caenorhabditis elegans males.

Caenorhabditis elegans male spicule morphogenesis requires the coordinated cellular behaviors of several types of cells. We found that the spicule neurons and sheath cells, although important for spicule function, are dispensable for spicule morphology. In contrast, the spicule socket cells are essential for both spicule elongation and formation of spicule cuticle. The socket cells are not only necessary but also sufficient to produce spicule cuticle. This functional aspect of socket cells is genetically separable from their function in mediating spicule elongation: elongated spicules with defective spicule cuticle can be formed. During spicule morphogenesis, the expression of an egl-17::GFP reporter gene is found in the spicule socket cells and its expression appears to be regulated in the socket cells. Mutants defective in TGF-beta signaling display a crumpled spicules phenotype as a result of failure of socket cell movement during spicule morphogenesis. These observations suggest that both the FGF and the TGF-beta signaling pathways might be involved in spicule elongation.     

Can the morphology of the integumentary spicules be used to distinguish genera and species of phyllidiid nudibranchs (Porostomata: Phyllidiidae)?

Thirty-eight specimens belonging to four genera and 15 species of the nudibranch family Phyllidiidae were examined to investigate whether the morphology of their integumentary calcareous spicules and/or the occurrence of the spicules within the regions of the body could be used to distinguish genera and species. The spicules were studied separately from five regions of the body of each specimen—the foot, gills, mantle, dorsal pustules (or ridges in Reticulidia) and rhinophores. The mantle itself plus its pustules were found to possess the full complement of spicules in every individual. Four types of spicules were recorded overall—smooth diactines, centro-polytylote diactines, triactines and tetractines. Different regions of the body were found to possess different spicule types: (a) only smooth diactines in the gills, (b) both smooth diactines and triactines in the foot and (c) all of smooth diactines, centro-polytylote diactines and triactines in the mantle, dorsal pustules and the rhinophores. Among the genera, three types of spicules (smooth diactine, triactine, and tetractine) are present in Phyllidia, Phyllidiopsis and Reticulidia, but the form of the spicules is not diagnostic between these genera or between the constituent species. The fourth type of spicule (centro-polytylote diactine) is present exclusively in Phyllidiella, and is diagnostic for that genus. However, we failed to find any difference in spicule form, or composition, or location in the body between the three (closely related) species of Phyllidiella we investigated. Therefore, our key conclusion is that spicule morphology is an extremely important character to tell the genus Phyllidiella apart from all the other genera of the family, but it is not taxonomically informative at the level of species.     

Isolation,culture, and differentiation of echinoid primary mesenchyme cells

Summary Methods are described for isolation and culture of primary mesenchyme cells from echinoid embryos. Ninety-five percentpure primary mesenchyme cells were isolated from early gastrulae ofStrongylocentrotus purpuratus, exploiting the biological segregation of these cells within the blastocoel. When cultured, more than 90% of the isolated cells reached the differentiated state, spicule formation, in synchrony with in vivo controls. Isolated primary mesenchyme cells were cultured with and without various cellular and acellular components of normal embryos in order to study the potential involvement of these components in the morphogenesis of the primary mesenchyme. Our data indicate that: 1. primary mesenchyme cells lack the ability to form the annular pattern of the primary mesenchymal ring autonomously; 2. they autonomously produce spicules of a characteristic morphology that differs from that of embryonic spicules; 3. morphogenesis of the primary mesenchyme is not affected by association with embryonic basal lamina, blastocoel matrix, or loosely aggregated epithelial cells, or by close confinement of each set of primary mesenchyme cells within the blastocoelar space; and 4. reaggregated, tightly associated epithelial cells can promote normal primary mesenchyme ring formation, and modify the primary mesenchyme-intrinsic spicule pattern to produce more normal spicule forms.