Phylogenetic comparison of spicule networks in cryptobranchiate dorid nudibranchs (Gastropoda, Euthyneura, Nudibranchia, Doridina)

Many dorid nudibranchs possess large numbers of calcareous spicules in their mantle, gill, rhinophores and foot. However, the arrangements of these structures and their differences among taxa are poorly known. Spicule networks were stained with Alizarin red and compared among 12 species of cryptobranchiate dorid nudibranchs and four outgroups. Three general types of networks were found: a cobweb-like, unbraced framework of one or few spicules per side; a ramifying system of thick, spiculated tracts; and a lattice-like arrangement of distinct radial and circumferential tracts. The Discodorididae species investigated shared a cobweb-like network and papillae supported by a ring of spicules, while the Porostomata showed consistent characters leading to a lattice-like network with larger spicules in the central notum. The Dorididae studied were not cohesive, but each species shared characters with the aforementioned groups. Therefore, spicule network form may provide new characters to help resolve the phylogeny of Doridina.     

Chemical and physical defenses against predators in Cystodytes (Ascidiacea)

Ascidians utilize both physical (spicules, tunic toughness) and chemical defenses (secondary metabolites, acidity) and suffer relatively little predation by generalist predators. The genus Cystodytes (Polycitoridae) is distributed widely in both tropical and temperate waters. Secondary metabolite composition, calcareous spicules and tunic acidity (pH < 1) may act as redundant defense mechanisms against predation in this genus. To assess the relative importance of chemical and physical defenses against predation in ascidians, we studied purple and blue morphs of Cystodytes from the western Mediterranean (formerly assigned to Cystodytes dellechiajei, but recently shown to belong to two different species), and a purple morph from Guam (USA), identified as Cystodytes violatinctus. Crude extracts, spicules, ascididemin (the major alkaloid of the blue morph) and acidity were used in feeding trials to evaluate chemical and physical defense mechanisms in Cystodytes spp. We performed feeding experiments in the field with a guild of generalist fish (mostly damselfish), and in the laboratory with a sea urchin and a puffer fish. Our results showed that all crude extracts and ascididemin significantly deterred fish predation, but not sea urchin predation. However, neither acidity alone nor spicules at natural concentrations deterred feeding. These results and other studies on sponges and gorgonians suggest that secondary metabolites are the primary means of defense against fish predators. Spicules and tunic acidity may perform other ecological roles and/or target certain specialist predators.     

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     

Extracellular Formation of Body and Tunic Spicules in the New Zealand Solitary Ascidian Pyura pachydermatina (Urochordata, Ascidiacea)

The New Zealand ascidian Pyura pachydermatina has a 7–10 cm long body at the end of a stalk up to 1 m long and 1–2 cm in diameter. Two different spicule types are present: dumbbell-shaped spicules of calcite in the fibrous tunic that covers the body and stalk, and antler-shaped spicules of amorphous calcium carbonate in the soft body tissues. Both types form extracellularly within a closed compartment surrounded by an epithelium of sclerocytes. In adults the tunic spicules form in 2–3 weeks in the lumen of the tunic blood vessels, as determined by calcein uptake studies. They add mineral only while surrounded by the sclerocyte epithelium, which is anchored to the vessel wall. Ultimately the sclerocytes rupture at one or more leading points on the spicule. The blood vessel epithelium also becomes very thin at these points and either ruptures or the cells separate. allowing the spicules to migrate out into the tunic. The sclerocytes degenerate and the blood vessel closes behind the migrating spicule, thus maintaining the vessel's integrity. Tunic spicules accumulate in the subcuticular region of the stalk, but the outermost layer of tunic covering the body is periodically sloughed off along with some spicules. This gives the "neck" between body and stalk a flexibility that allows it to orient to currents, and prevents an accumulation of epizoic organisms on the body. The antler spicules form within blood sinuses of the body tissues. The mineral and organic material are arranged in concentric layers. In the branchial sac, oral tentacles, gut and endostyle, where antler spicules occur most densely, the branches interlock, providing support to the soft tissues. They are of many sizes and apparently remain where they form, increasing in number and size throughout the animal's lifespan.     

Thyroxine and vitamin D in the gorgonian Leptogorgia virgulata.

The gorgonian coral Leptogorgia virgulata contains thyroxine, or a thyroxine-like substance, referred to here as G-T(4). The G-T(4) levels were significantly higher in colonies collected in the summer vs. winter months. Using immunocytochemical techniques, G-T(4) was localized in the axis, polyp epithelium, and within the electron dense bodies of scleroblasts (spicule-forming cells), as well as on the periphery of spicules. G-T(4) was also localized in the mesoglea between closely adjacent scleroblasts. The effects of exogenous T(4) on the uptake of Ca(45) was determined in spicule, tissue and axis fractions of L. virgulata. The uptake of Ca(45) increased in T(4) treated spicules but decreased in the tissue fraction for all time periods tested. The uptake of Ca(45) into axes was not affected by exogenous T(4) until day 10 of the study. These data suggest that G-T(4) may function in the process of spicule formation. 1,25-dihydroxyvitamin D apparently is synthesized via ultraviolet radiation. Colonies deprived of ultraviolet radiation had significantly more 'irregular' spicules than colonies maintained in ultraviolet radiation. Exposure to sunlight therefore may be associated with the process of normal spicule formation.     

Differential distribution of spicule matrix proteins in the sea urchin embryo skeleton

Spicule matrix proteins are the products of primary mesenchyme cells, and are present in calcite spicules of the sea urchin embryo. To study their possible roles in skeletal morphogenesis, monoclonal antibodies against SM50, SM30 and another spicule matrix protein (29 kDa) were obtained. The distribution of these proteins in the embryo skeleton was observed by immunofluorescent staining. In addition, their distribution inside the spicules was examined by a 'spicule blot' procedure, direct immunoblotting of proteins embedded in crystallized spicules. Our observations showed that SM50 and 29 kDa proteins were enriched both outside and inside the triradiate spicules of the gastrulae, and also existed in the corresponding portions of growing spicules in later embryos and micromere cultures. The straight extensions of the triradiate spicules and thickened portions of body rods in pluteus spicules were also rich in these proteins. The SM30 protein was only faintly detected along the surface of spicules. By examination using the spicule blot procedure, however, SM30 was clearly detectable inside the body rods and postoral rods. These results indicate that SM50 and 29 kDa proteins are concentrated in radially growing portions of the spicules (normal to the c-axis of calcite), while SM30 protein is in the longitudinally growing portions (parallel to the c-axis). Such differential distribution suggests the involvement of these proteins in calcite growth during the formation of three-dimensionally branched spicules.     

Testing for defensive synergy in Caribbean sponges: Bad taste or glass spicules?

Chemical and physical defenses of sessile organisms against consumers are well described for both terrestrial and marine systems. However, previous studies have focused on chemical or physical defenses in isolation, and have not considered their interaction. Marine sponges provide a model system for testing this interaction. Some sponge species produce secondary metabolites that deter predation; they may also contain siliceous spicules, but previous studies have provided little evidence that spicules in isolation offer any defense against generalist fish predators. To determine whether the two components have an additive, antagonistic, or synergistic interaction, crude organic extracts and spicules from individuals of 8 Caribbean sponge species were isolated and tested in laboratory feeding assays. These included one chemically defended reef sponge (Agelas clathrodes) and seven known to be intermediately deterrent: six from reef habitats (Cinachyrella alloclada, Clathria virgultosa, Cribrochalina infundibulum, Niphates digitalis, Svenzea zeai, and Xestospongia muta) and one from mangrove habitats (Tedania ignis). Extracts and spicules were assayed at various concentrations, both individually and in combination, in laboratory feeding assays with the bluehead wrasse, Thalassoma bifasciatum. A SAS based GENMOD procedure based on an isobolographic analysis model was used for statistical comparisons. Four sponges (A. clathrodes, C. alloclada, C. virgultosa, and one of three individuals of X. muta) showed evidence of synergisms. Of these, synergy in C. alloclada, C. virgultosa, and X. muta was caused by approximately natural concentrations of extracts and spicules. The extract of A. clathrodes was deterrent, but combination assays required nearly a 3-fold reduction in extract concentration and an 8-fold increase in spicule concentration to show the synergistic effect. Contrary to previous findings, spicules from C. infundibulum and two of three individuals of X. muta were deterrent at natural concentrations. Sponge spicules may be defensive in isolation, or may enhance chemical defenses against consumers, but the lack of synergisms for individuals in 4 of 7 species with intermediate levels of chemical defense suggests that defensive synergy is not the general rule and, when present, may be an example of an exaptation.     

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.     

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.     

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.     

Thyroxine and vitamin D in the gorgonian Leptogorgia virgulata

The gorgonian coral Leptogorgia virgulata contains thyroxine, or a thyroxine-like substance, referred to here as G-T₄. The G-T₄ levels were significantly higher in colonies collected in the summer vs. winter months. Using immunocytochemical techniques, G-T₄ was localized in the axis, polyp epithelium, and within the electron dense bodies of scleroblasts (spicule-forming cells), as well as on the periphery of spicules. G-T₄ was also localized in the mesoglea between closely adjacent scleroblasts. The effects of exogenous T₄ on the uptake of Ca⁴⁵ was determined in spicule, tissue and axis fractions of L. virgulata. The uptake of Ca⁴⁵ increased in T₄ treated spicules but decreased in the tissue fraction for all time periods tested. The uptake of Ca⁴⁵ into axes was not affected by exogenous T₄ until day 10 of the study. These data suggest that G-T₄ may function in the process of spicule formation. 1,25-dihydroxyvitamin D apparently is synthesized via ultraviolet radiation. Colonies deprived of ultraviolet radiation had significantly more ‘irregular’ spicules than colonies maintained in ultraviolet radiation. Exposure to sunlight therefore may be associated with the process of normal spicule formation.     

A reconsideration of the relationship between polyaxonid and monaxonid spicules in Demospongiae: new data from the genera Crambe and Discorhabdella (Porifera)

The relationships between different spicule lineages of Demospongiae are revised through the ontogenetic study of the main spicules of the genera Crambe and Discorhabdella. The presence of terminal orifices in the basal spines of the asterose acanthostyles of Discorhabdella, and the actines of the desmas of Crambe have been shown by examining young spicules under high magnification. Thus, the polyaxonid origin of both spicule types is hereby supported by the ontogenetic information, and their homology is also supported by their equivalent arrangement in the skeleton. The current differences in shape between both spicule types are considered the result of a divergent morphological evolution from an ancestral polyactinal corpuscle, by the atrophy/hypertrophy of a different number of actines. Arguments are also presented to support the homology of these two spicule types with the sphaeroclons of Vetulina, and other fossil genera. Moreover, the presence of axial canals inside the tubercles of the tuberose tylostyles of Discorhabdella and Crambe tuberosa indicates that the tubercles are actually atrophied actines as in the case of the hadromerid genus Terpios. According to the ontogeny, the tuberose morphology of these spicules may correspond to the retention of an ancestral characteristic in the Poecilosclerida and Hadromerida; in this case, a monophyletic origin, is suggested between both taxa. From the overall results here presented, the tetraxonid spicule, presently considered by most authors as the primitive morphotype, as well as some monaxons, could be considered as evolving from a polyaxial form.     

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.     

External Morphology of the Spicules of some Trichodoridae

External morphology of spicules in several species of Trichodoridae was studied by scanning electron and light microscopy. The bristles on the spicules observed in the light microscope in several species were revealed as small scales forming a sheath which covers most of the spicule body. Some species have smooth spicules, whereas other species exhibited complicated structures formed by projections. In all of the species studied, either a ventro-terminal or terminal opening of the spicules was observed.     

Morphological plasticity in the tropical sponge Anthosigmella varians: responses to predators and wave energy

The goal of the research presented here was to examine phenotypic plasticity exhibited by three morphotypes of the common Caribbean sponge Anthosigmella varians (Duchassaing & Michelotti). We were interested in examining the biotic (and, to a lesser extent, abiotic) factors responsible for branch production in this species. We also tested the hypothesis that the skeleton may serve an antipredator function in this sponge, focusing on vertebrate fish predators (i.e., angelfish) in this work. In transplant and caging experiments, unprotected forma varians replicates were immediately consumed by angelfish, while caged replicates persisted on the reef for several months. These findings support the hypothesis that predators (and not wave energy) restrict forma varians to lagoonal habitats. Branch production was not observed in A. varians forma incrustans when sponges were protected from predators or placed in predator-free, low-wave-energy environments. It is not clear from our work whether forma incrustans is capable of producing branches (i.e., whether branch production is a plastic trait in this morph). Additional field experiments demonstrated that A. varians forma varians increased spicule concentrations, compared to uninjured sponges, in response to artificial predation events, and A. varians forma rigida reduced spicule concentrations, compared to uncaged controls, when protected from predators. These findings indicate that spicule concentration is a plastic morphological trait that can be induced by damage, and that A. varians may be able to reduce spicule concentrations when environmental conditions change (e.g., in the absence of predators). The potential significance of inducible defenses and structural anti-predator defenses in sponges is discussed in relation to recent work on sponge chemical defenses.     

Growth patterns of Lower Palaeozoic sponges

Detailed studies of the growth patterns of modern siliceous sponges are restricted to demosponges and theoretical models. It is generally assumed that sponge growth is essentially incremental, with completion of one arbitrary unit being followed by external addition. All recent species are thick-walled, but Lower Palaeozoic sponges are dominated by thin-walled hexactinellids, with most Cambrian taxa consisting of a single spicule layer. Large populations of a primitive dictyospongiid have allowed the reconstruction of the growth patterns of their spicules and body morphology. The results indicate that growth occurred through continuous expansion of the globose body, accompanied by continuous enlargement of existing spicules, with a spicule size limit being reached only during the lifetime of a few individuals. It is noted that this skeletal growth pattern is otherwise restricted to deuterostomes. Consecutive appearance of successive spicule size orders appears to have maintained a maximum inhalant pore area. Comparisons with more limited data from two acanthose hexactinellids and a hazeliid demosponge indicate that an identical growth pattern operated in these species. The subsequent evolution of growth patterns is discussed, with various mechanisms producing the later thick-walled morphologies of hexactinellids and demosponges. The implications of these observations are discussed with reference to identification and systematics, since spicule size and arrangement are shown to vary during growth.     

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.     

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.     

Micro-computed tomography of spicule networks in three genera of dorid sea-slugs (Gastropoda: Nudipleura: Doridina) shows patterns of phylogenetic significance

Dorid nudibranchs (Gastropoda: Nudipleura) are a key taxon for studying the evolution and interaction of chemical defence, colour patterns and feeding specialization, but we lack a robust phylogeny for hypothesis testing. To provide new morphological characters, we investigated the extensive interior spicule networks of several dorid taxa. We compared traditional staining methods versus micro-computed tomography (μCT) of 31 specimens representing 10 species of Aldisa, Cadlina and Onchidoris. We found that μCT offered a nondestructive view of sufficient resolution to study the gross morphology of networks, although images of fine structures were too grainy to allow robust comparisons. Network form did not vary within species and was consistent within genera. The three genera varied in several obvious characters, such as the presence of a pleural sinus channel, large dorsal spicules and multispicular tracts, as well as in relative size, shape and orientation of spicules. These characters, combined with those from the literature, supported recent molecular phylogenies that group Cadlina with Aldisa, and that question the monophyly of the Cryptobranchia. This suggests that these network forms will prove a fruitful source of phylogenetic characters with at least genus-level resolution.