Skeleton construction upon local regression of the sponge body

We previously revealed that the mechanism of demosponge skeleton construction is self-organization by multiple rounds of sequential mechanical reactions of player cells. In these reactions, “transport cells” dynamically carry fine skeletal elements (spicules) on epithelia surrounding the inner body space of sponges (basal epithelium (basopinacoderm) and the endodermal epithelium (ENCM)). Once spicules pierce ENCM and apical pinacoderm, subsequently they are cemented to the substratum under the sponge body, or connected to other skeleton-constructing spicules. Thus, the “pierce” step is the key to holding up spicules in the temporary periphery of growing sponges’ bodies. Since sponges can regress as well as grow, here we asked how skeleton construction occurs during local regression of the body. We found that prior to local basopinacoderm retraction (and thus body regression), the body became thinner. Some spicules that were originally carried outward stagnated for a while, and were then carried inwards either on ENCM or basopinacoderm. Spicules that were carried inwards on ENCM pierced epithelia after a short transport, and thus became held up at relatively inward positions compared to spicules carried on outwardly extending basopinacoderm. The switch of epithelia on which transport cells migrate efficiently occurred in thinner body spaces where basopinacoderm and ENCM became close to each other. Thus, the mechanisms underlying this phenomenon are rather mechanical: the combination of sequential reactions of skeleton construction and the narrowed body space upon local retraction of basopinacoderm cause spicules to be held up at more-inward positions, which might strengthen the basopinacoderm's attachment to substratum.     

Skeletal development in <Emphasis Type="Italic">Acropora palmata</Emphasis> (Lamarck 1816): a scanning electron microscope (SEM) comparison demonstrating similar mechanisms of skeletal extension in axial versus encrusting growth

Many Acropora palmata colonies consist of an encrusting basal portion and erect branches. Linear growth of the skeleton results in extension along the substrate (encrusting growth), lengthening of branches (axial growth) and thickening of branches and crust (radial growth). Scanning Electron Microscopy is used to compare the mechanisms of skeletal extension between encrusting growth and axial growth. In encrusting growth, the distal margin of the skeleton lacks corallites (which develop about 1 mm from the edge); in contrast, in axial growth, axial corallites along the branch tip form the distal portion of the skeleton. In both locations, the distal margin of the skeleton consists of a lattice-like structure composed of rods that extend from the body of the skeleton and bars that connect these rods. An actively extending skeleton is characterized by sharply pointed rods and partially developed bars. Distal growth of rods (and formation of bars) is effected by the formation of new sclerodermites. Each sclerodermite begins with the deposition of fusiform crystals (that range in length from 1 to 5 μm). These provide a surface for nucleation and growth of spherulitic tufts, clusters of short (<1 μm long) aragonite needles. The needles that are oriented perpendicular to the axis of the skeletal element (rod or bar), and perpendicular to the overlying calicoblastic epithelium, continue extension to appear on the surface of the skeleton as 10–15 μm wide bundles (of needle tips) called fasciculi. However, some crusts that abut competitors for space have a different morphology of skeletal elements (rods and bars). The distal edge of these crusts terminates in blunt coalescing rods, and bars that are fully formed. Absence of fusiform crystals, lack of sharply pointed rods and bars, and full development of sclerodermites characterize a skeletal region that has ceased, perhaps only temporarily, skeletal extension.     

Ultrastructure and cytochemistry of the early calcification site and of its mineralization organic matrix in Paracentrotus lividus (Echinodermata: Echinoidea)

 The ultrastructure and cytochemistry of skeleton formation sites prior to mineralization are described for the first time in echinoderms. These early sites are intracellular vacuoles located in syncytial pseudopodia of skeleteton-forming cells. They contain a mineralization organic matrix, which shows a calcium-binding ability and is framed in a tridimensional structure made of concentric layers bridged by radial threads. This organic matrix presents repetitive structures which could be implicated in mineralization control. Both the tridimensional organization of the organic matrix and its framing, before mineralization starts, question the current theories which suggest that the echinoderm organic matrix is soluble at the onset of mineralization and adsorbs on the forming crystal. Accepted: 19 March 1998     

Soluble proteins control growth of skeleton crystals in three coralline demosponges

Summary Calcified demosponges (coralline sponges, sclero-sponges), the first metazoa producing a carbonate skeleton, used to be important reef building organisms in the past. The relatives of this group investigated here,Spirastrella (Acanthochaetetes) wellsi, Astrosclera willeyana andVaceletia cf.crypta, are restricted to cryptic niches of modern Pacific coral reefs and may be considered as “living fossils’. They are characterized by a basic biologically controlled metazoan biomineralization process. Each of the investigated taxa forms its calcareous basal skeleton in a highly specialized way. Moreover, each taxon secretes distinct Ca²⁺-binding macromolecules which were entrapped within the calcium carbonate crystals during skeleton formation. Therefore these Ca²⁺-binding macromolecules were also described as intracrystalline macromolecules. When isolated and separated by SDS polyacrylamide gel electrophoresis, the organic skeleton matrix of the three species revealed to be composed of a respective distinct array of EDTA-soluble proteins. A single protein of 41 kDa was detected inS. wellsi, two proteins of 38 and 120 kDa inA. willeyana, and four proteins of 18 kDa, 30 kDa, 33 kDa, and 37 kDa inVaceletia sp. When run on IEF gel, the Ca²⁺-binding proteins gave staining bands at pH values between 5.25 and 5.65. As proved by anin vitro mineralization assay, the extracted proteins effectively inhibit CaCO₃ and SrCO₃ precipitation, respectively, in a saturated solution. Biochemical properties and behavior of the extracted proteins strongly suggest that they are involved in crystal nucleation and skeleton carbonate formation within the calcified sponges studied here.     

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state ¹³C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40–100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state ¹³C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The ¹³C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of α-chitin than β-chitin.     

In vitro evaluation of natural marine sponge collagen as a scaffold for bone tissue engineering

The selection of a suitable scaffold matrix is critical for cell-based bone tissue engineering. This study aimed to identify and characterize natural marine sponges as potential bioscaffolds for osteogenesis. Callyspongiidae marine sponge samples were collected from the Fremantle coast of Western Australia. The sponge structure was assessed using scanning electron microscopy (SEM) and Hematoxylin and eosin. Mouse primary osteoblasts were seeded onto the sponge scaffold and immunostained with F-actin to assess cell attachment and aggregation. Alkaline phosphatase expression, von Kossa staining and real-time PCR were performed to examine the osteogenic potential of sponge samples. SEM revealed that the sponge skeleton possessed a collagenous fibrous network consisting of interconnecting channels and a porous structure that support cellular adhesion, aggregation and growth. The average pore size of the sponge skeleton was measured 100 to 300 μm in diameter. F-actin staining demonstrated that osteoblasts were able to anchor onto the surface of collagen fibres. Alkaline phosphatase expression, a marker of early osteoblast differentiation, was evident at 7 days although expression decreased steadily with long term culture. Using von Kossa staining, mineralisation nodules were evident after 21 days. Gene expression of osteoblast markers, osteocalcin and osteopontin, was also observed at 7, 14 and 21 days of culture. Together, these results suggest that the natural marine sponge is promising as a new scaffold for use in bone tissue engineering.     

Skeletal structures and habitats of Recent and fossil Acanthochaetetes (subclass Tetractinomorpha,Demospongiae, Porifera)

The investigation of the habitats, the spicular skeletons, and the structure and chemistry of the nonspicular high-Mg calcite skeletons of a fossil Acanthochaetetes from the Late Albian (Cretaceous) of Northern Spain and the extant Acanthochaetetes wellsi from Pacific reefs demonstrates an astonishing correspondence. The skeletons of both species are hemispherical or pyriform with the lower part containing an epitheca. They are built up of single calicles which are subdivided by tabulae. Spines protrude from the walls into the calicles. Scanning electron microscopy and thin sections reveal that the high-Mg calcite skeleton consists of two different microstructures: a irregular ssensu Wendt 1979 or microlamellar (sensu Cuif et al. 1979) and a completely irregular structure. AAS and EDAX analysis of the calcite skeletons produce roughly the same Mg and Sr contents. Tylostyle megascleres and aster-like microscleres are observed in the spicular skeletons of both species. The only difference between the two species is the greater variability of the microscleres in the extant species. Moreover, the fossil species incorporates the scleres in the non-spicular skeleton, while the extant species does not. Both species live/lived in the same niches of tropical reefs: the cryptic habitats of submarine caves in the reef core and the dimly lighted habitats of the deeper fore-reef.     

Calcium content of liming material and its effect on sulphur release in a coniferous forest soil

Soil columns with O + A (Experiment I) or Ohorizons (Experiment II) from a Haplic Podsol wereincubated at 15 °C for 368 and 29 + 106 days,respectively. Three types of liming material differingin Ca²⁺ content, i.e. calcium carbonate(CaCO₃), dolomite (CaMg(CO₃)₂) andmagnesium carbonate (MgCO₃), were mixed into theO horizons in equimolar amounts corresponding to 6000kg of CaCO₃ per ha. In the limed treatments ofExperiment I, the leaching of dissolved organic carbon(DOC) and the net sulphur mineralization (estimated asaccumulated SO ₄ ^2– leaching corrected forchanges in the soil pools of adsorbed and waterextractable SO ₄ ^2– ) increased with decreasingCa²⁺ content of the lime and increasing degree oflime dissolution. In relation to the controltreatment, only the MgCO₃ treatment resulted ina significantly higher net sulphur mineralization. InExperiment I the net sulphur mineralization was 4.06,1.68, 0.57, and 2.14 mg S in the MgCO₃,CaMg(CO₃)₂, CaCO₃ and control treatment,respectively. The accumulated SO ₄ ^2– leachingin Experiment II during the first 29 days was 1.70,0.74 and 0.48 mg S in the MgCO₃,CaMg(CO₃)₂ and control treatment,respectively. In the two experiments there wereconsistently significant positive correlations betweenleached amounts of SO ₄ ^2– and DOC. It wasconcluded that net sulphur mineralization was stronglyconnected to the solubilization of the organic matter(DOC formation) and that pH and/or Ca²⁺ ionsaffected the net sulphur mineralization through theireffects on organic matter solubility.     

An ultrastructural investigation of muscle attachment in the opercular filament of a polychaete annelid

The ultrastructure of peduncle muscle attachment to the cuticular flange in the opercular filament of the serpulid Pomatoceros lamarckii Quatrefages is described. The cuticular flange is composed of layers of orthogonally arranged fibres. Specialized epithelial cells (tendon cells) and a collagenous matrix intervene between the peduncle muscles and the cuticular flange. The tendon cells are characterized by hemidesmosomes at both apical and basal ends, connected by thick bundles of tonofilaments. Apically long specialized microvilli from the tendon cells penetrate the cavities in the orthogonally arranged layers of fibres of the cuticular flange. The basal surfaces of the tendon cells and the terminal ends of the peduncle muscles anchor independently of one another in the collagenous matrix. The peduncle muscles appear to be smooth muscles which contain thin filaments, 5 nm in diameter, and thick filaments, 40-100 nm in diameter, with a faint axial periodicity 12-14 nm. The method of peduncle muscle attachment in the opercular filament is compared with those of other invertebrates.     

Non-rigid cryptic sponges in oyster patch reefs (Lower Kimmeridgian, Langenberg/Oker, Germany)

Summary Two patch reefs which predominately consist of the oysterNanogyra nana (Sowerby 1822) are exposed in Lower Kimmeridigian strata of the Langenberg hillrange, central Germany. Left oyster valves making up the frame-work of the reefs formed small abundant cavities that were inhabited by a unique sponge community. The excellent preservation of non-rigid sponges was related to early organomineralization within the decaying sponge tissue. As a process of sponge taphonomy, different types of microbially induced carbonates precipitated preserving spicule aggregates. Organomineralization within sponge soft tissues is especially favored with the Langenberg patch reefs due to the closed or semi-closed system conditions with the cavities. The δ¹³C values ofin situ formed microbialities reveal that carbonate precipitation was in equilibrium with Jurassic seawater. The carbon of the microbialites does not derive from the bacterial remineralization of organic matter, but is of a marine source. Likewise, organomineralization is probably related to bacterial EPS or decaying sponge tissues providing an organic matrix for initial carbonate precipitation. Biomarker analyses revealed, that the patch reef microbialites contain terminally branched fatty acids (iso-andanteiso-pentadecanoic acid) in significant concentrations. These fatty acids, like hopanoid hydrocarbons, are most likely of a bacterial source. This is in agreement with sulfate-reducing bacteria remineralizing the decaying sponges as further indicated by the occurrence of framboidal pyrite in sponge microbialites.     

Functional morphology of Tethya species (Porifera): 2. Three-dimensional morphometrics on spicules and skeleton superstructures of T. minuta

The biomechanics of body contraction in Porifera is almost unknown, although sponge contraction has been observed already in ancient times. Some members of the genus Tethya represent the most contractile poriferan species. All of them show a highly ordered skeleton layout. Based on three main spicule types, functional units are assembled, termed skeleton superstructures here. Using synchrotron radiation based x-ray microtomography and quantitative image analysis with specially developed particle and structure recognition algorithms allowed us to perform spatial allocation and 3D-morphometric characterizations of single spicules and skeleton superstructures in T. minuta. We found and analyzed three skeleton superstructures in the investigated specimen: (1) 85 megasclere bundles, (2) a megaster sphere, composed by 16,646 oxyasters and (3) a pinacoderm–tylaster layer composed by micrasters. All three skeleton superstructures represent composite materials of siliceous spicules and extracellular matrix. From structure recognition we developed an abstracted mathematical model of the bundles and the sphere. In addition, we analyzed the megaster network interrelation topology and found a baso-apical linear symmetry axis for the megaster density inside the sphere. Based on our results, we propose a hypothetical biomechanical contraction model for T. minuta and T. wilhelma, in which the skeleton superstructures restrain physical stress generated by contraction in the tissue. While skeletal structures within the genus Tethya have been explained using R. Buckminster Fullers principle of tensegrity by other authors, we prefer material science based biomechanical approaches, to understand skeletal superstructures by referring to their composite material properties.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Sobralispongia densespiculata, a new coralline sponge from the Upper Jurassic of Portugal

From the Kimmeridgian (Upper Jurassic) of Portugal, the coralline sponge Sobralispongia densespiculata nov. gen. and nov. sp. is described. Main characteristics are a crustose habit, a primary spicule skeleton of very densely packed styles and subtylostyles arranged in a plumose architecture, microscleres of possibly aster-type, and a microgranular to fibrous secondary calcareous skeleton. The primary mineralogy of the calcareous skeleton was probably high-Mg calcitic. An assignment to the demosponge Order Axinellida is proposed.     

Dynamic structure of the mesohyl in the sponge Chondrosia reniformis (Porifera, Demospongiae)

The common demosponge Chondrosia reniformis possesses the capacity to undergo an unusual creep process which results in the formation of long outgrowths from the parent body. These shape changes, which have been interpreted as adaptive strategies related to environmental factors, asexual reproduction or localised locomotor phenomena, are due mainly to the structural and mechanical adaptability of the collagenous mesohyl. This contribution describes the morphological correlates of mesohyl plasticisation in C. reniformis. The microscopic anatomy of the mesohyl was examined when it was in different physiological conditions: (1) standard ”resting” condition, (2) ”stiffened” condition and (3) dynamic ”creep” condition. In this last case four representative regions of the sponge body were analysed: the parent region, the elongation region, the transition region and the propagule region. The results show that the histological modification of the sponge mesohyl during plasticisation is limited and localised. The most significant structural changes involve mainly cytological features of specific cellular components characterised by granule inclusions (i.e. the spherulous cells) and the arrangement and density of the collagenous extracellular framework, though the integrity of the collagen fibrils themselves is not affected. Morphological and functional aspects of mesohyl plasticisation invite comparison with the mutable collagenous tissue of echinoderms. Possible functional analogies between these two tissues are hypothesised. Accepted: 29 June 2001     

Microstructure, growth banding and age determination of a primnoid gorgonian skeleton (Octocorallia) from the late Younger Dryas to earliest Holocene of the Bay of Biscay

A fossil primnoid gorgonian skeleton (Octocorallia) was recovered on the eastern Galician Massif in the Bay of Biscay (NE Atlantic) from 720 m water depth. The skeleton shows a growth banding of alternating Mg–calcitic and organic (gorgonin) increments in the inner part, surrounded by a ring of massive fibrous calcite. Three calcite-dominated cycles, bounded by thick organic layers, consist of five light-dark couplets of calcite and gorgonin. Two AMS-¹⁴C datings of the fossil skeleton give ages of 10,880 and 10,820 ± 45 ¹⁴C years before present (BP). We arrive at a calibrated age range of 11,829–10,072 cal. years BP (two σ), which comprises the late Younger Dryas to the earliest part of the Holocene. The cyclic calcitic–organic growth banding may be controlled by a constant rate of calcite secretion with a fluctuating rate of gorgonin production, possibly related to productivity cycles. The skeletal fabric change of alternating calcitic–organic increments to massive fibrous calcite may be the result of hydrographic changes during the deglaciation as reflected by preliminary stable isotope data. If this hypothesis proves to be correct, primnoid gorgonians are able to match with varying hydrodynamic conditions by changing their biomineralisation mode.     

Biomineralization of calcium carbonate in the cell wall of Lithothamnion crispatum (Hapalidiales,Rhodophyta): correlation between the organic matrix and the mineral phase

Over the past few decades, progress has been made toward understanding the mechanisms of coralline algae mineralization. However, the relationship between the mineral phase and the organic matrix in coralline algae has not yet been thoroughly examined. The aim of this study was to describe the cell wall ultrastructure of Lithothamnion crispatum, a cosmopolitan rhodolith‐forming coralline algal species collected near Salvador (Brazil), and examine the relationship between the organic matrix and the nucleation and growth/shape modulation of calcium carbonate crystals. A nanostructured pattern was observed in L. crispatum along the cell walls. At the nanoscale, the crystals from L. crispatum consisted of several single crystallites assembled and associated with organic material. The crystallites in the bulk of the cell wall had a high level of spatial organization. However, the crystals displayed cleavages in the (104) faces after ultrathin sectioning with a microtome. This organism is an important model for biomineralization studies as the crystallographic data do not fit in any of the general biomineralization processes described for other organisms. Biomineralization in L. crispatum is dependent on both the soluble and the insoluble organic matrix, which are involved in the control of mineral formation and organizational patterns through an organic matrix‐mediated process. This knowledge concerning the mineral composition and organizational patterns of crystals within the cell walls should be taken into account in future studies of changing ocean conditions as they represent important factors influencing the physico‐chemical interactions between rhodoliths and the environment in coralline reefs.     

The two-step mode of growth in the scleractinian coral skeletons from the micrometre to the overall scale

It has been known since the 19th century that coral skeletons are built of aragonite crystals with taxonomy-linked arrangements, but the way by which each coral species controls this crystallization process remains an unsolved question. The problem became still more intriguing when it was shown that isotopic compositions of coral aragonite were subject to taxonomy-linked influences (the "vital effect"). On the other hand, presence of an organic component in coral skeletons is also long known, but localization of these compounds is admittedly restricted to particular structures called "centres of calcifications." Fibres, the largely predominant part of the coral skeletons, are usually considered as purely mineral units. In this paper, it is shown that in both "centres of calcification" and fibres, organic compounds are associated with the mineral material at a deep structural level. A series of variously scaled observations and localized measurements allow recognition of the presence of an organic component at the nanometre scale. Far from being a freely operating process, crystallization of coral fibres is thus permanently controlled by the polyp basal ectoderm through a cyclic two-step process acting at the micrometre-scale. The biomineralization cycle begins by secretion of a proteoglycan matrix. As the composition of these sugars-proteins assemblages has been shown taxonomy dependent, the hypothesis can be made that multiple and long recognized specificities of coral skeletons are linked to this biochemically driven crystallization process. Additionally, this new concept of the biomineralization process in coral skeletons provides us with an access to the long term evolution of the Scleractinia. Remarkably, results of a skeleton-based approach using microstructural criteria (i.e., the spatial relationships of "centres of calcification" and the three-dimensional arrangements of fibres), are consistent with a molecular phylogenetic analysis carried out on the same species. Clearly, at the overall ontogenic level, the two-step growth mode of coral skeletons is also a valuable tool to reconstruct the evolutionary history of Scleractinia.     

Effects of nutrient enrichment on C and N stable isotope ratios of invertebrates,fish and their food resources in boreal streams

Carbon and nitrogen stable isotopes are frequently used to study energy sources and food web structure in ecosystems, and more recently, to study the effects of anthropogenic stress on aquatic ecosystems. We investigated the effect of nutrient enrichment on δ¹³C and δ¹⁵N in fine (FPOM), coarse (CPOM) particulate organic matter, periphyton, invertebrates and fish in nine boreal streams in south-central Sweden. In addition, we analysed the diet of benthic consumers using stable isotope data. Increases in δ¹⁵N of periphyton (R ² = 0.88), CPOM (0.78), invertebrates (0.92) and fish (0.89) were related to nutrient enrichment. In contrast, δ¹³C signatures did not change along the nutrient gradient. Our results show that δ¹⁵N has potential as a sensitive indicator of nutrient enrichment in boreal streams. Carbon and nitrogen isotopes failed to elucidate putative diets of selected aquatic consumers. Indeed, comparison of low- and high-impact sites showed that δ¹³C of many consumers were found outside the ranges of basal resource δ¹³C. Moreover, ranges of basal resource δ¹³C and δ¹⁵N overlapped at both low and high sites, making discrimination between the importance of allochthonous and autochthonous production difficult. Our findings show that a fractionation rate of 3.4‰ is not always be appropriate to assess trophic interactions, suggesting that more studies are needed on fractionation rates along gradients of impairment. Handling editor: M. Power     

The reef cave dwelling ultraconservative coralline demospongeAstrosclera willeyana Lister 1900 from the Indo-Pacific

Summary Astrosclera willeyana Lister 1900 is a pyriform-half spherical, predominantly bright orange colored, coralline demosponge with a mean size of about 20 mm in height and maximum head diameter. The habitat ofAstrosclera is generally restricted to cryptic and light reduced environments of the Indo-Pacific, found mainly in reef caves, but sometimes also in the dim-light areas of cave entrances and overhangs, where it is green colored at the side towards the light. Caves of Indo-Pacific coral reefs were divided into four major facies zones, named 1 to 4 with decreasing light intensities.Astrosclera occurs in reef caves on a carbonate basement in Zone 2, 3, and 4, reaching maximum abundance in Zone 3 and the proximal part of Zone 4, but rare in the distal, very dark areas of Zone 4. Other abundant coralline sponges in reef caves areSpirastrella (Acanthochaetetes) wellsi andVaceletia crypta. Astrosclera is the most common coralline sponge throughout the studied sites of the Indo-Pacific. The living tissue ofAstrosclera penetrates the basal skeleton to a maximum of 50% depending on specimen size. The soft tissue shows a stromatoporoid grade of organization and can be divided into three major zones. The ectosome is the area directly beneath the exopinacoderm reaching a thickness of about 100–300 μm. The ectosome is characterized by the absence of choanocyte chambers, an enrichment of storage and supporting cells (SSC's), and the archaeocyte-like large vesicle cells (LVC's), which are responsible for the initial formation of aragonitic spherulites. Megasclerocytes were only rarely observed, but show remarkably numerous cytoplasmatic digitations. The choanosome, contiguous with the ectosome but with a more-or-less sharp transition, comprises the major part of the living tissue inAstrosclera, characterized by small choanocyte chambers (±15 μm diameter) and a high density of bacterial symbionts. Other cellular components of the choanosomal mesohyle are archaeocytes, typical pluripotent, phagocytotic demosponges cells, SSC's, and rare fiber cells. Bacteriocytes, which inhabit up to 30 bacteria in a large vacuole, are described for the first time from coralline sponges, as well as a new cell type, the ‘waste cell’, characterized by a large vacuole which is filled up with a fluffy substance, probably non-digested remains of bacteria (membranes, fibrilar EPS). These cells probably derive from archaeocytes or bacteriocytes and are often observed close to excurrent canals. Most of the ground substance of the choanosome lacks collagen and spongin, but has abundant polysaccharide mucus (EPS) produced by the symbiotic bacteria. The zone of epitaxial backfill (ZEB) is considered as a subzone of the choanosome due to its important role for thesyn vivo cementation of the lowest parts of the basal skeleton. It is characterized by a reduced number (or absence) of choanocytes and bacteria and sometimes an enrichment of SSC's. Astrosclera has a viviparous mode of reproduction. The bacterial symbionts are transferred from the parent sponge in the parenchymella larvae. A large proportion of the population inhabit the choanosome, where the bacteria can reach up to 70% of the total biomass in some areas. The ectosome is nearly free of bacteria. Four major bacterial morphotypes are recognized. Bacteria act either as a direct food source for the sponge (archaeocytes), or the sponge benefits from certain metabolic products of the bacteria (bacteriocytes). Bacteria are at least in part facultative anaerobic, eliminating metabolic waste products of the sponge through fermentative processes during times of reduced or ceasated pumping, or at any time. The spicular skeleton ofAstrosclera consists of megascleres; microscleres are absent. The basic spicule type is a sub-verticillate—verticillate acanthostyle, of theAgelas type, with a mean length of 80 μm. The spicule morphology and size is highly variable, depending on the geographic origin of the specimen. At the broader proximal end of the spicule (showing a thickening in nearly all populations) the spicule is embedded in spongin microfibrils. As the sponge grows, the spicules get entrapped in the basal skeleton, initially at the proximal end. Based on similar spicule morphology, six ‘groups with similar spicule morphology’ (GSSM's) are differentiated. These GSSM's probably represent geographic sub-species. Restriction fragment length analysis of the ribosomal DNA (ITS1, 5.8S, ITS2, and small parts of the 18S and 28S regions) was analyzed to test the genetic variation of twenty speciments from five geographical distinct areas (representing three of the GSSM groups based on the spicule morphology). However, the restriction fragment length analysis did not detect any genetic differences that would indicate that there is more than one species ofAstrosclera amongst the specimens examined. The aragonitic calcareous basal skeleton ofAstrosclera is composed of 20–60 μm-sized aragonitic spherulites, produced by a combination of three processes. First, the spherulites are formed in LVC's in the ectosome. In a second process, the basopinacocytes transport the spherulites to the tips of the skeletal pillars, where they fuse together by epitaxial growth; and in a third process, during upward growth, the soft tissue is slowly rejected from the lowermost-parts of the skeletal cavities and the remaining spaces are subsequently filled by epitaxially-growing aragonite fibers. It is hypothesized thatAstrosclera is able to control the rate of calcification by the regulation of its bacterial population. The mean growth rate ofAstrosclera was measured at 230 μm per year. Aminoacid and monosaccharide composition of the insoluble intracrystalline matrix (IOM) of the largestAstrosclera ever found (with a maximum age of 550 years) is very stable in all portions of the skeleton. No strong diagenetic effect on the IOM is visible due to the stable composition in all parts of the skeleton, in contrast to the SOM which shows strong diagenetic effects with increasing age of the skeleton, both in aminoacid and monosaccharide composition and quantity. The IOM is dominated by proteins and is represented by the intravacuolar fibers and sheets forming the containers for the seed crystals in the first intracellular process of biocalcification. The nature of the IOM during the second and third process remains unknown without further investigation. Collagen was not detected as a major part of the IOM. The character of the SOM is very typical for Ca²⁺ binding mucus substances. Two proteins present in the SOM were detected by HPLC gel filtration chromatography, visible at 280 nm wave length detection: a large molecule with a molecular weight of 130 kD, and a smaller one with about 40 kD. Both macromolecules are attributed to have a different function in the biocalcification processes. Stable isotope time series (46 samples) of δ¹⁸O and δ¹³C were measured in successive growth layers of the largeAstrosclera from Ribbon Reef#10 (GBR).Astrosclera forms its skeletal aragonite in equilibrium with the ambient seawater, and represents, therefore, a high precision recorder of the isotopic history of the ambient seawater. δ¹³C of surface water dissolved inorganic carbon in the northern Great Barrier Reef has apparently decreased continuously since the mid-16^th century. The total decrease is 0.7‰. The major decline of 0.5‰ occurred during the industrial period of the 19th and 20th century, likely to be due to the increased release of CO₂ by deforestation and burning of fossil fuel during the period of industrialization after 1850 (increased input of lighter carbon isotopes). The oxygen isotope history shows a slightly colder (and/or dryer) phase before 1850, which correlates with the Little Ice Age. A considerable shift to lighter values occurred during the 20th century (warming of SST). This may be due to an anthropogenic greenhouse effect. Most of the major climatic changes caused by ENSO/El Ni?o events as well as by large volcano eruptions in the last four and a half centuries were recorded in the oxygen isotope record ofAstrosclera. A large number of sponges with spherulitic microstructures were observed in the Triassic deposits of St. Cassian (Carnian, Dolomites) and of the Alacir Clay Valley near Antalya (lower Norian, Turkey). At least some of the sponge skeletal morphologies, certainly belonging to different taxa thanAstrosclera, appear to have been formed by the same processes as observed in extantAstrosclera. It was found that morphological characters of the ‘spherulitic skeleton’, even though they show signs of same formation processes, have no higher taxonomic value. The spherulitic skeleton must have had appeared independently in different lineages, and thus can be regarded as a convergent character. Although the spherulitic basal skeleton was determined to have no higher taxonomic value, it was nevertheless used to identify sponges with affinities to the extantAstrosclera, since the Triassic ‘astrosclerid’ sponges lack spicules. Only one type of sponge belonging to the taxonAstrosclera was found in the Triassic deposits of Antalya. This sponge shows a gross morphology similar to extantA. willeyana, and all the features of the three distinct biocalcification processes leading to the formation of the basal skeleton are present. Spicules, thick, short subacanthostyles/styles, were found in this specimen. A new species,Astrosclera cuifi n. sp. is described in this present work, and it is shown that the ultraconservative taxonAstrosclera has persisted at least since the late Triassic.

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Micromorphology, Ultrastructure, Biocalcification, Isotope Record, Taxonomy, Biogeography, Phylogeny

     

Plant and soil natural abundance <Emphasis Type="Italic">δ</Emphasis><Superscript>15</Superscript>N: indicators of relative rates of nitrogen cycling in temperate forest ecosystems

Watersheds within the Catskill Mountains, New York, receive among the highest rates of nitrogen (N) deposition in the northeastern United States and are beginning to show signs of N saturation. Despite similar amounts of N deposition across watersheds within the Catskill Mountains, rates of soil N cycling and N retention vary significantly among stands of different tree species. We examined the potential use of δ ¹⁵N of plants and soils as an indicator of relative forest soil N cycling rates. We analyzed the δ ¹⁵N of foliage, litterfall, bole wood, surface litter layer, fine roots and organic soil from single-species stands of American beech (Fagus grandifolia), eastern hemlock (Tsuga canadensis), red oak (Quercus rubra), and sugar maple (Acer saccharum). Fine root and organic soil δ ¹⁵N values were highest within sugar maple stands, which correlated significantly with higher rates of net mineralization and nitrification. Results from this study suggest that fine root and organic soil δ ¹⁵N can be used as an indicator of relative rates of soil N cycling. Although not statistically significant, δ ¹⁵N was highest within foliage, wood and litterfall of beech stands, a tree species associated with intermediate levels of soil N cycling rates and forest N retention. Our results show that belowground δ ¹⁵N values are a better indicator of relative rates of soil N cycling than are aboveground δ ¹⁵N values.     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999