Live tissue imaging shows reef corals elevate pH under their calcifying tissue relative to seawater

The threat posed to coral reefs by changes in seawater pH and carbonate chemistry (ocean acidification) raises the need for a better mechanistic understanding of physiological processes linked to coral calcification. Current models of coral calcification argue that corals elevate extracellular pH under their calcifying tissue relative to seawater to promote skeleton formation, but pH measurements taken from the calcifying tissue of living, intact corals have not been achieved to date. We performed live tissue imaging of the reef coral Stylophora pistillata to determine extracellular pH under the calcifying tissue and intracellular pH in calicoblastic cells. We worked with actively calcifying corals under flowing seawater and show that extracellular pH (pHe) under the calicoblastic epithelium is elevated by ～0.5 and ～0.2 pH units relative to the surrounding seawater in light and dark conditions respectively. By contrast, the intracellular pH (pHi) of the calicoblastic epithelium remains stable in the light and dark. Estimates of aragonite saturation states derived from our data indicate the elevation in subcalicoblastic pHe favour calcification and may thus be a critical step in the calcification process. However, the observed close association of the calicoblastic epithelium with the underlying crystals suggests that the calicoblastic cells influence the growth of the coral skeleton by other processes in addition to pHe modification. The procedure used in the current study provides a novel, tangible approach for future investigations into these processes and the impact of environmental change on the cellular mechanisms underpinning coral calcification.     

Calcification process dynamics in coral primary polyps as observed using a calcein incubation method

Calcification processes are largely unknown in scleractinian corals. In this study, live confocal imaging was used to elucidate the spatiotemporal dynamics of the calcification process in aposymbiotic primary polyps of the coral species Acropora digitifera. The fluorophore calcein was used as a calcium deposition marker and a visible indicator of extracellular fluid distribution at the tissue-skeleton interface (subcalicoblastic medium, SCM) in primary polyp tissues. Under continuous incubation in calcein-containing seawater, initial crystallization and skeletal growth were visualized among the calicoblastic cells in live primary polyp tissues. Additionally, the distribution of calcein-stained SCM and contraction movements of the pockets of SCM were captured at intervals of a few minutes. Our experimental system provided several new insights into coral calcification, particularly as a first step in monitoring the relationship between cellular dynamics and calcification in vivo. Our study suggests that coral calcification initiates at intercellular spaces, a finding that may contribute to the general understanding of coral calcification processes.     

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.     

Calcite Formation in Soft Coral Sclerites Is Determined by a Single Reactive Extracellular Protein

Calcium carbonate exists in two main forms, calcite and aragonite, in the skeletons of marine organisms. The primary mineralogy of marine carbonates has changed over the history of the earth depending on the magnesium/calcium ratio in seawater during the periods of the so-called “calcite and aragonite seas.” Organisms that prefer certain mineralogy appear to flourish when their preferred mineralogy is favored by seawater chemistry. However, this rule is not without exceptions. For example, some octocorals produce calcite despite living in an aragonite sea. Here, we address the unresolved question of how organisms such as soft corals are able to form calcitic skeletal elements in an aragonite sea. We show that an extracellular protein called ECMP-67 isolated from soft coral sclerites induces calcite formation in vitro even when the composition of the calcifying solution favors aragonite precipitation. Structural details of both the surface and the interior of single crystals generated upon interaction with ECMP-67 were analyzed with an apertureless-type near-field IR microscope with high spatial resolution. The results show that this protein is the main determining factor for driving the production of calcite instead of aragonite in the biocalcification process and that –OH, secondary structures (e.g. α-helices and amides), and other necessary chemical groups are distributed over the center of the calcite crystals. Using an atomic force microscope, we also explored how this extracellular protein significantly affects the molecular-scale kinetics of crystal formation. We anticipate that a more thorough investigation of the proteinaceous skeleton content of different calcite-producing marine organisms will reveal similar components that determine the mineralogy of the organisms. These findings have significant implications for future models of the crystal structure of calcite in nature.     

The skeletal organic matrix from Mediterranean coral Balanophyllia europaea influences calcium carbonate precipitation

Scleractinian coral skeletons are made mainly of calcium carbonate in the form of aragonite. The mineral deposition occurs in a biological confined environment, but it is still a theme of discussion to what extent the calcification occurs under biological or environmental control. Hence, the shape, size and organization of skeletal crystals from the cellular level through the colony architecture, were attributed to factors as diverse as mineral supersaturation levels and organic mediation of crystal growth. The skeleton contains an intra-skeletal organic matrix (OM) of which only the water soluble component was chemically and physically characterized. In this work that OM from the skeleton of the Balanophyllia europaea, a solitary scleractinian coral endemic to the Mediterranean Sea, is studied in vitro with the aim of understanding its role in the mineralization of calcium carbonate. Mineralization of calcium carbonate was conducted by overgrowth experiments on coral skeleton and in calcium chloride solutions containing different ratios of water soluble and/or insoluble OM and of magnesium ions. The precipitates were characterized by diffractometric, spectroscopic and microscopic techniques. The results showed that both soluble and insoluble OM components influence calcium carbonate precipitation and that the effect is enhanced by their co-presence. The role of magnesium ions is also affected by the presence of the OM components. Thus, in vitro, OM influences calcium carbonate crystal morphology, aggregation and polymorphism as a function of its composition and of the content of magnesium ions in the precipitation media. This research, although does not resolve the controversy between environmental or biological control on the deposition of calcium carbonate in corals, sheds a light on the role of OM, which appears mediated by the presence of magnesium ions.     

Modern carbonate microbialites from an asbestos open pit pond, Yukon, Canada

Microbialites were discovered in an open pit pond at an abandoned asbestos mine near Clinton Creek, Yukon, Canada. These microbialites are extremely young and presumably began forming soon after the mine closed in 1978. Detailed characterization of the periphyton and microbialites using light and scanning electron microscopy was coupled with mineralogical and isotopic analyses to investigate the mechanisms by which these microbialites formed. The microbialites are columnar in form (cm scale), have an internal spherulitic fabric (mm scale), and are mostly made of aragonite, which is supersaturated in the subsaline pond water. Initial precipitation is seen as acicular aragonite crystals nucleating onto microbial biomass and detrital particles. Continued precipitation entombs benthic diatoms (e.g. Brachysira vitrea), filamentous algae (e.g. Oedogonium sp.), dinoflagellates, and cyanobacteria. The presence of phototrophs at spherulite centers strongly suggests that these microbes play an important initial role in aragonite precipitation. Substantial growth of individual spherulites occurs abiotically through periodic precipitation of aragonite that forms concentric laminations around spherulite centers while pauses in spherulite growth allow for colonization by microbes. Aragonite associated with biomass (δ(13)C = -4.6‰ VPDB) showed a (13)C-enrichment of 0.8‰ relative to aragonite exhibiting no biomass (δ(13)C = -5.4‰ VPDB), which suggests a modest removal of isotopically light dissolved inorganic carbon by phototrophs. The combination of a low sedimentation rate, high calcification rate, and low microbial growth rate appears to result in the formation of these microbialites. The formation of microbialites at an historic mine site demonstrates that an anthropogenically constructed environment can foster microbial carbonate formation.     

Effects of variations in carbonate chemistry on the calcification rates of Madracis auretenra (= Madracis mirabilis sensu Wells, 1973): bicarbonate concentrations best predict calcification rates

Physiological data and models of coral calcification indicate that corals utilize a combination of seawater bicarbonate and (mainly) respiratory CO₂ for calcification, not seawater carbonate. However, a number of investigators are attributing observed negative effects of experimental seawater acidification by CO₂ or hydrochloric acid additions to a reduction in seawater carbonate ion concentration and thus aragonite saturation state. Thus, there is a discrepancy between the physiological and geochemical views of coral biomineralization. Furthermore, not all calcifying organisms respond negatively to decreased pH or saturation state. Together, these discrepancies suggest that other physiological mechanisms, such as a direct effect of reduced pH on calcium or bicarbonate ion transport and/or variable ability to regulate internal pH, are responsible for the variability in reported experimental effects of acidification on calcification. To distinguish the effects of pH, carbonate concentration and bicarbonate concentration on coral calcification, incubations were performed with the coral Madracis auretenra (= Madracis mirabilis sensu Wells, 1973) in modified seawater chemistries. Carbonate parameters were manipulated to isolate the effects of each parameter more effectively than in previous studies, with a total of six different chemistries. Among treatment differences were highly significant. The corals responded strongly to variation in bicarbonate concentration, but not consistently to carbonate concentration, aragonite saturation state or pH. Corals calcified at normal or elevated rates under low pH (7.6–7.8) when the seawater bicarbonate concentrations were above 1800 μm . Conversely, corals incubated at normal pH had low calcification rates if the bicarbonate concentration was lowered. These results demonstrate that coral responses to ocean acidification are more diverse than currently thought, and question the reliability of using carbonate concentration or aragonite saturation state as the sole predictor of the effects of ocean acidification on coral calcification.     

Short term viability of soft tissue detached from the skeleton of reef-building corals

We have induced soft tissue detachment from the skeleton of two colonial hard corals of the Pocilloporid family, both in vivo and in vitro. A parallel was made between polyp “bail-out”, i.e. field and laboratory-observed detachment of tissue fragments alone from the skeleton, and the dissociation method used for initiation of coral primary cell cultures. The in vitro approach provided insights into the active cellular re-arrangement mechanisms underlying coral tissue detachment. Functional polyps were not regenerated. Viability of tissue isolates detached from coral skeleton was probed for their use as a model for short-term biological assays. Cell viability dropped from 70% to 30% within the first week maintenance in vitro. Short-term isolate cultures limited to 3 days are a compromise allowing attachment of coral cells, yet preserving viability at about 70% of the total coral cell population.     

Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification

The mechanisms behind the transfer of molecules from the surrounding sea water to the site of coral calcification are not well understood, but are critical for understanding how coral reefs are formed. We conducted experiments with the fluorescent dye calcein, which binds to calcium and is incorporated into growing calcium carbonate crystals, to determine the permeability properties of coral cells and tissues to this molecule, and to determine how it is incorporated into the coral skeleton. We also compared rates of calcein incorporation with rates of calcification measured by the alkalinity anomaly technique. Finally, by an electrophysiological approach, we investigated the electrical resistance of coral tissues in order to better understand the role of tissues in ionic permeability. Our results show that (i) calcein passes through coral tissues by a paracellular pathway, (ii) intercellular junctions control and restrict the diffusion of molecules, (iii) intercellular junctions should have pores of a size higher than 13 Å and lower than 20 nm, and (iv) the resistance of the tissues owing to paracellular junctions has a value of 477 ± 21 Ohm cm². We discuss the implication of our results for the transport of calcium involved in the calcification process.     

Intracellular crystal-bearing vesicles in the epidermis of scleractinian corals, Astrangia danae (Agassiz) and Porites porites (Pallas).

Orthorhombic aragonitic crystals, embedded with a granular lipo-protein matrix and surrounded by a trilaminar membrane, are localized in the apical cytoplasm of epidermal cells of Scleractinian corals. Adult specimens of Astrangia danae (Agassiz) and settled planulae of Porites porites (Pallas) contain crystals averaging 0.7 mu by 0.1 mu by 0.3 mu within Golgi-derived vesicles. Short-term labelling with 45Ca reveals distribution of radioactivity amont a basic tissue fraction (92%) an acid tissue fraction (5%) and a skeletal fraction (3%). Identification of the primordial crystal population within membrane-bound visicles provides overwhelming evidence for the intracellular mode of calcification in Scleractinia. Moreover, it permits development of a novel concept of cellular regulation over these dynamic events. The membrane-bound vesicel is a miniature crystal fabrication station and a vehicle responsible for transportation of seed crystals and an organic matrix material to sites of discharge from the cell. The vesicle membrane becomes a probable locus of active transport and enzymatic activity as well as a physical barrier to be penetrated for release of vesicle contents into the extracellular milieu. Contact between the vesicle membrane and the plasmalemma would result in exocytosis and the onset of skeletogenesis. Principles governing crystal growth would prevail from then on. The released crystal becomes a nucleation catalyst and the organic matrix, a supply of ionic calcium for self-limiting crystallization. Crystals are produced by the organism spontaneously and continuously from shortly after larval attachment throughout the life of the polyp. Therefore, these membrane-bound vesicles signal the dynamic process by which initiation, differentiation, growth and limitation of the coral skeleton is regulated.     

In vivo light-microscopic documentation for primary calcification processes in the hermatypic coral Stylophora pistillata

Skeletogenesis in the hermatypic coral Stylophora pistillata was studied by using the lateral skeleton preparative (LSP) assay, viz., a coral nubbin attached to a glass coverslip glued to the bottom of a Petri dish. Observations on tissue and skeletal growth were made by polarized microscopy and by using vital staining. The horizontal distal tissue edges developed thin transparent extensions of ectodermal and calicoblastic layers only. Four stages (I-IV) of skeletogenesis were observed at these edges, underneath the newly developed tissue. In stage I, a thin clear layer of coral tissue advanced 3–40 μm beyond the existing LSP peripheral zone, revealing no sign of spiculae deposition. At stage II, primary fusiform crystals (1 μm each) were deposited, forming a primary discontinuous skeletal front 5–30 μm away from the previously deposited skeleton. During stage III, needle-like crystals appeared, covering the primary fusiform crystals. Stage IV involved further lengthening of the needle-like crystals, a process that resulted in occlusion of the spaces between adjacent crystals. Calcification stages I-III developed within hours, whereas stage IV was completed in several days to weeks. Two basic skeletal structures, “scattered” and “laminar” skeletons, were formed, integrating the growth patterns of the needle-like crystals. High variation was recorded in the expression of the four calcification stages, either between different locations along a single LSP or between different preparations observed at the same diurnal time. All four skeletogenesis stages took place during both day and night periods, indicating that an intrinsic process controls S. pistillata calcification. This study was supported by the Israel Science Foundation (206/01 to J.E.), by the BARD, US-Israel Bi-National Agricultural Research and Development, by INCO-DEV project (REEFRES), and by CORALZOO, EC Collective Research project.     

Carbonic anhydrase in the scleractinian coral Stylophora pistillata: characterization, localization, and role in biomineralization

Carbonic anhydrases (CA) play an important role in biomineralization from invertebrates to vertebrates. Previous experiments have investigated the role of CA in coral calcification, mainly by pharmacological approaches. This study reports the molecular cloning, sequencing, and immunolocalization of a CA isolated from the scleractinian coral Stylophora pistillata, named STPCA. Results show that STPCA is a secreted form of alpha-CA, which possesses a CA catalytic function, similar to the secreted human CAVI. We localized this enzyme at the calicoblastic ectoderm level, which is responsible for the precipitation of the skeleton. This localization supports the role of STPCA in the calcification process. In symbiotic scleractinian corals, calcification is stimulated by light, a phenomenon called "light-enhanced calcification" (LEC). The mechanism by which symbiont photosynthesis stimulates calcification is still enigmatic. We tested the hypothesis that coral genes are differentially expressed under light and dark conditions. By real-time PCR, we investigated the differential expression of STPCA to determine its role in the LEC phenomenon. Results show that the STPCA gene is expressed 2-fold more during the dark than the light. We suggest that in the dark, up-regulation of the STPCA gene represents a mechanism to cope with night acidosis.     

Oxygen and Heterotrophy Affect Calcification of the Scleractinian Coral Galaxea fascicularis

Heterotrophy is known to stimulate calcification of scleractinian corals, possibly through enhanced organic matrix synthesis and photosynthesis, and increased supply of metabolic DIC. In contrast to the positive long-term effects of heterotrophy, inhibition of calcification has been observed during feeding, which may be explained by a temporal oxygen limitation in coral tissue. To test this hypothesis, we measured the short-term effects of zooplankton feeding on light and dark calcification rates of the scleractinian coral Galaxea fascicularis (n = 4) at oxygen saturation levels ranging from 13 to 280%. Significant main and interactive effects of oxygen, heterotrophy and light on calcification rates were found (three-way factorial repeated measures ANOVA, p<0.05). Light and dark calcification rates of unfed corals were severely affected by hypoxia and hyperoxia, with optimal rates at 110% saturation. Light calcification rates of fed corals exhibited a similar trend, with highest rates at 150% saturation. In contrast, dark calcification rates of fed corals were close to zero under all oxygen saturations. We conclude that oxygen exerts a strong control over light and dark calcification rates of corals, and propose that in situ calcification rates are highly dynamic. Nevertheless, the inhibitory effect of heterotrophy on dark calcification appears to be oxygen-independent. We hypothesize that dark calcification is impaired during zooplankton feeding by a temporal decrease of the pH and aragonite saturation state of the calcifying medium, caused by increased respiration rates. This may invoke a transient reallocation of metabolic energy to soft tissue growth and organic matrix synthesis. These insights enhance our understanding of how oxygen and heterotrophy affect coral calcification, both in situ as well as in aquaculture.     

珊瑚礁生态系统中珊瑚藻的生态作用研究进展

珊瑚藻是海洋红藻中的大型钙化藻类,全球分布623种,中国现有记录共77种。随着生态科学研究的广泛展开,人们越来越认识到,珊瑚藻在海洋生态系统中,尤其在维持珊瑚礁生态系统的生物多样性及生态功能中发挥着重要作用。目前,科研人员对有关珊瑚藻的初级生产力、钙化作用以及在诱导底栖无脊椎动物幼虫的附着与变态等方面已有多方面的研究和探索。然而,有关珊瑚藻生态功能的深层次机理问题有待进一步深入研究。文章着重围绕目前珊瑚藻研究中的一些热点问题,从近年来珊瑚藻在珊瑚礁生态系统中的生态功能方面的研究概况进行综述,以期加深人们对珊瑚藻的认识,并促进对珊瑚藻生态功能的进一步深入研究。     

Hierarchically structured scleractinian coral biocrystals

Microscopic (AFM and FESEM) observations show that scleractinian coral biomineral fibers in extant Desmophyllum and Favia, and fossil Jurassic Isastrea are composed of nanocrystalline grains of about 30-100 nm in size. In contrast to these findings, SR diffraction data on the same coral materials exhibit narrow Bragg peaks suggesting much larger crystallite size. These seemingly contradicting results of microscopic and diffraction studies are reconciled within a new, minute-scale model of scleractinian biomineral fibers. In this model, nanocrystalline aragonite units are interconnected by mineral bridges and form aggregates usually larger than 200 nm. Most likely, the size of the aggregates is resulting from physiological biomineralization cycles that control cellular secretion of ions and biopolymeric species. Intercalation of biopolymers into crystal lattice may influence consistently several structural parameters of the scleractinian coral bio-aragonite in all studied samples: (i) the lattice parameters and internal strains of the bio-aragonite are larger than in mineral aragonite, (ii) lattice parameter elongations and internal strains reveal directional anisotropy with respect to crystallographic axes.     

Skeletal microstructure of Galaxea fascicularis exsert septa: a high-resolution SEM study

The deposition of four crystal types at the growth surface of the septa of several color morphs of the coral Galaxea fascicularis was investigated over a 24-h period. Results suggest that nanocrystals, on denticles at the apices of exsert septa, may be the surface manifestation of centers of calcification. These crystals were also found on the septa of the axial corallite of Acropora formosa. The deposition of nanocrystals appears to be independent of diurnal rhythms. Internally and proximal to the septal apices, distinct clusters of polycrystalline fibers originate from centers of calcification and form fanlike fascicles. Upon these fascicles, acicular crystals grow and extend to form the visible fasciculi at the skeletal surface. Deposition of aragonitic fusiform crystals in both G. fascicularis and A. formosa occurs without diurnal rhythm. Nucleation of fusiform crystals appears to be independent of centers of calcification and may occur by secondary nucleation. Formation of semi-solid masses by fusiform crystals suggests that the crystals may play a structural role in septal extension. Lamellar crystals, which have not been reported as a component of scleractinian coral skeletons before, possess distinct layers of polyhedral plates, although these layers also do not appear to be associated with daily growth increments. The relationship of lamellar crystals to other components of the scleractinian coral skeleton and their involvement in skeletal growth is unknown.     

Cellular microenvironment influences the ability of mammary epithelia to undergo cell cycle

The use of cell culture models is a principal and fundamental technology used in understanding how mammalian cells work. However, for some cell types such as mammary epithelia, the lines selected for extended culture are often transformed or have chromosomal abnormalities, while primary cultures have such a curtailed lifespan that their use is restricted. For example, mammary luminal epithelial cells (MECs) are used to study mechanisms of breast cancer, but the proliferation of primary cell cultures is highly limited. Here we describe the establishment of a new culture system to allow extended analysis of cultures of primary mouse MECs. In 2D monolayer culture, primary MECs showed a burst of proliferation 2-3 days post isolation, after which cell cycle decreased substantially. Addition of mammary epithelial growth factors, such as Epidermal Growth Factor, Fibroblast Growth Factor-2, Hepatocyte Growth Factor, and Receptor Activator for Nuclear Factor κB Ligand, or extracellular matrix proteins did not maintain their proliferation potential, neither did replating the cells to increase the mitogenic response. However, culturing MECs directly after tissue extraction in a 3D microenvironment consisting of basement membrane proteins, extended the time in culture in which the cells could proliferate. Our data reveal that the cellular microenvironment has profound effects on the proliferative properties of the mammary epithelia and is dominant over growth factors. Moreover, manipulating the cellular environment using this novel method can maintain the proliferative potential of primary MECs, thus enabling cell cycle to be studied as an endpoint after gene transfer or gene deletion experiments.     

High density micromass cultures of embryonic limb bud mesenchymal cells: An in vitro model of endochondral skeletal development

Summary To study the mechanisms regulating endochondral skeletal development, we examined the characteristics of long-term, high density micromass cultures of embryonic chicken limb bud mesenchymal cells. By culture Day 3, these cells underwent distinct chondrogenesis, evidenced by cellular condensation to form large nodules exhibiting cartilage-like morphology and extracellular matrix. By Day 14, extensive cellular hypertrophy was seen in the core of the nodules, accompanied by increased alkaline phosphatase activity, and the limitation of cellular proliferation to the periphery of the nodules and to internodular areas. By Day 14, matrix calcification was detected by alizarin red staining, and calcium incorporation increased as a function of culture time up to 2 to 3 wk and then decreased. X-ray probe elemental analysis detected the presence of hydroxyapatite. Analogous to growth cartilage developing in vivo, these cultures also exhibited time-dependent apoptosis, on the basis of DNA fragmentation detected in situ by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL), ultrastructural nuclear morphology, and the appearance of internucleosomal DNA degradation. These findings showed that cellular differentiation, maturation, hypertrophy, calcification, and apoptosis occurred sequentially in the embryonic limb mesenchyme micromass cultures and indicate their utility as a convenient in vitro model to investigate the regulatory mechanisms of endochondral ossification.     

Skeletal development in Acropora cervicornis: I. Patterns of calcium carbonate accretion in the axial corallite

Summary Scanning electron microscopy and serial petrographic thin sections were used to investigate skeletal elongation and mineralization in the perforate coral, Acropora cervicornis. The axial corallite extends by the formation of randomly oriented fusiform crystals which are deposited on its distal edge. Aragonitic needle-like crystals grow in random directions from the surface of these fusiform crystals. Only those needle-like crystals growing toward the calicoblastic epithelium (i.e. crystals whose growth axis is perpendicular to the plane of the calicoblastic cell membrane) continue to elongate. Groups of these growing crystals join to form well-defined fasciculi which make up the primary skeletal elements comprising the septotheca. The resulting skeleton is highly porous with all surfaces covered by the continuous calicoblastic epithelium. This cell layer is separated by thin mesoglea from the flagellated gastrodermis which lines the highly ramified coelenteron. Porosity and permeability of the skeleton decrease with distance from the tip. Density correspondingly increases due to the addition of aragonite to the fasciculi whose boundaries become less distinct as channels fill with calcium carbonate.     

Processes Forming Daily Lamination in a Microbe-Rich Travertine Under Low Flow Condition at the Nagano-yu Hot Spring,Southwestern Japan

A daily rhythm of microbial processes, in terms of sub-mm order lamination, was identified for a microbe-rich aragonite travertine formed at a low-flow site of the Nagano-yu Hot Spring in Southwestern Japan. Continuous observation and sampling clearly showed that the lamination consisted of diurnal microbe-rich layers (M-layers) and nocturnal crystalline layers (C-layers). The M-layers originated from biofilm formed by growth and upward migration of filamentous cyanobacteria related to Microcoleus sp., which can rapidly glide and secrete extracellular polymeric substances (EPS). During the daytime, cyanobacterial biofilm development inhibited aragonite precipitation on the travertine surface due to the calcium-binding ability of EPS. After sunset, aragonite precipitation started on the surface where aerobic heterotrophic bacteria decomposed EPS, which induced precipitation of micritic crystals. This early stage of C-layer formation was followed by abiotic precipitation of fan-shaped aragonite aggregates. Despite their major role in lamina formation, the cyanobacteria were readily degraded within 6–10 days after embedding, and the remaining open spaces in the M-layers were sparsely filled with crystal clots. These lamina-forming processes were different from those observed in a high-flow site where the travertine has a dense texture of aragonite crystals. The microbial travertine at Nagano-yu is similar to some Precambrian stromatolites in terms of in situ mineral precipitation, regular sub-mm order lamination, and arrangement of filamentous microbes; therefore, the lamination of these stromatolites possibly occur with a daily rhythm. The microbial processes demonstrated in this study may revise the interpretation of ancient stromatolite formation.