Evolution of calcium-carbonate skeletons in the Hydractiniidae

Biomineralization has mostly been studied in the class Anthozoa (Phylum Cnidaria), but very little is known about the evolution of the calcified skeleton in the class Hydrozoa or about the processes leading to its formation. The evolution of the calcified skeleton is here investigated in the hydrozoan family Hydractiniidae. A phylogenetic analysis of ribosomal, mitochondrial, and nuclear-protein-coding DNA sequences supported two independent origins of the calcified skeleton within the Hydractiniidae and indicates a case of parallel evolution, as suspected in the Anthozoa. Neither of the two origins of skeleton in the Hydractiniidae has led to either speciose or numerically abundant species, in contrast with other skeletonized hydrozoan families. Finally, we show that the origin of calcified skeletons in the Hydractiniidae is significantly correlated with the distribution of species with calcium carbonate granules within a polyp's gastrodermal cells. This suggests that the presence of these granules precedes the origin of a full skeleton.     

Cambrian anemones with preserved soft tissue from the Chengjiang biota, China

The group Cnidaria includes 'jellyfish', soft-bodied anemone and anemone-like forms and calcified corals. These diploblastic organisms have a fossil record extending back to the earliest metazoans of the Neoproterozoic; however certain cnidarians of the subclass Zoantharia, characterized by soft-bodied anemone-like forms, are absent or poorly represented in the fossil record. Despite the paucity of fossils, it is thought that calcification by soft anemone-like animals was responsible for producing the skeleton that allowed the preservation of the first corals. We report discovery of an abundant assemblage of in situ soft-bodied polyps with tissues. They are preserved in exquisite detail and come from the well-known Lower Cambrian Chengjiang biota of Yunnan, China. The soft-bodied polyps display a simple anatomy that is comparable to some extant anemones of the order Actinaria. The new fossils are assigned to Archisaccophyllia kunmingensis n. gen. et n. sp. Their simple and conservative form suggests that these fossils may represent some kind of ancestral rootstock. The preserved life assemblage provides a unique snapshot of Lower Cambrian anemone life and provides clues for relationships with extant actiniarians as well as calcified corals.     

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.     

Dead-end evolution of the Cnidaria as deduced from 5S ribosomal RNA sequences

Using nucleotide sequences of 5S ribosomal RNAs from 2 hydrozoan jellyfishes, 3 scyphozoan jellyfishes and 2 sea anemones, a phylogenetic tree of Cnidaria has been constructed to elucidate the evolutionary relationships of radial and bilateral symmetries. The 3 classes of Cnidaria examined herein belong to one branch, which does not include other metazoan phyla such as the Platyhelminthes. The Hydrozoa (having radial symmetry without septa) and the Scyphozoa (having radial symmetry with septa) are more closely related to each other than to the Anthozoa (having bilateral symmetry with septa). In classical taxonomy, multicellular animals are considered to have evolved through organisms with radial symmetry (e.g., Cnidaria) to bilateral symmetry. Our results, however, indicate that the emergence of the Bilateria was earlier than that of the Radiata, suggesting (in opposition to Haeckel's view) that the radial symmetry of Cnidaria is an evolutionary dead end.     

Evolutionary origin of rhopalia: insights from cellular‐level analyses of Otx and POU expression patterns in the developing rhopalial nervous system

SUMMARY In Cnidaria, the medusae of Scyphozoa and its sister‐group Cubozoa uniquely possess rhopalia at their bell margin. These sensory centers coordinate behavior and development. We used fluorescent in situ hybridization and confocal microscopy to examine mRNA expression patterns in Aurelia sp.1 (Cnidaria, Scyphozoa) during early medusa formation, while simultaneously visualizing the developing nervous system by immunofluorescence. The genes investigated include AurOtx1, and the POU genes, AurPit1, and AurBrn3, homologs of genes known to function in cephalar neural organization and sensory cell differentiation across Bilateria. Our results show that AurOtx1 expression defines the major part of the oral neuroectodermal domain of the rhopalium, within which distinct populations of AurBrn3‐ and AurPit1‐expressing sensory cells develop. Thus, despite the unique attributes of rhopalial evolution, we suggest that the rhopalial nervous system of scyphozoan medusae involves similar patterns of differential expression of genes that function in bilaterian cephalic structure and neuroendocrine system development. We propose that rhopalia evolved from preexisting sensory structures that developed distinct populations of sensory cells differentially expressing POU genes within Otx oral‐neuroectodermal domains. This implies some commonality of developmental genetic functions involving these genes in the still poorly constrained common ancestor of bilaterians and cnidarians.     

The impact of seawater temperature on coral growth parameters of the colonial coral Cladocora caespitosa (Anthozoa, Scleractinia) in the eastern Adriatic Sea

The scleractinian coral Cladocora caespitosa deserves a special place among the major carbonate bioconstructors of the Mediterranean Sea. Annual coral skeleton growth, coral calcification, and skeleton density of the colonial coral C. caespitosa taken from 25 locations in the eastern Adriatic Sea were analyzed and compared with annual sea surface temperatures (SST). The growth rates of the coral C. caespitosa from the 25 stations in the Adriatic Sea ranged from 1.92 to 4.19?mm per year, with higher growth rates of the investigated corallites in the southern part of the Adriatic Sea. These growth rates are similar to those measured in other areas of the Mediterranean Sea. The correlation between coral growth and sea temperatures in the Adriatic Sea is seen as follows: An X-radiograph analysis of coral growth in C. caespitosa colonies that are over 60?years old showed that higher growth rates of this coral coincided with a warmer period in the Mediterranean Sea. A positive significant correlation exists between corallite growth rates and SST and coral calcification and SST. A negative correlation exists between coral density and SST. Coral growth rates also showed a correlation with higher eutrophication caused by nearby fish farms, along with a greater depth of the investigated colonies and high bottom currents.     

Chlamydia pneumoniae in foci of "early" calcification of the tunica media in arteriosclerotic arteries: an incidental presence?

Only a few previous works investigated the involvement of Chlamydia pneumoniae (Chlamydophila pneumoniae) in arterial calcification. The present study investigated a possible association between C. pneumoniae and medial calcification. Carotid artery segments obtained by endarterectomy from 60 patients were examined by PCR and immunohistochemistry to identify the presence of C. pneumoniae. Arterial specimens showing double-positive (n = 17), double-negative (n = 22), and single-positive results (n = 21) were further analyzed by a combination of histology, immunohistochemistry, and electron microscopy. Medial calcification occurred in 10 of 17 (58.8%) C. pneumoniae double-positive arterial specimens, but no medial calcification was observed in any of 22 C. pneumoniae double-negative arterial specimens. Electron microscopy indicated C. pneumoniae in smooth muscle cells (SMCs) in foci of medial calcification. Medial SMCs showing damage to the cytoplasm and basement membrane contained the structures with the appearance of elementary, reticulate, and aberrant bodies of C. pneumoniae. The presence of C. pneumoniae in SMCs was confirmed by electron-microscopic immunocytochemistry. In the extracellular matrix, calcification was observed in C. pneumoniae aberrant bodies that exited the SMCs. The findings offer a new hypothesis of arterial calcification: they suggest that C. pneumoniae infection of medial SMCs may be associated with the pathophysiological events of arteriosclerotic calcification of the tunica media.     

Low temperature FESEM of the calcifying interface of a scleractinian coral

The ultrastructural nature of the calcifying interface in the scleractinian coral Galaxea fascicularis has been investigated using high-resolution, low temperature field emission scanning electron microscopy (FESEM). This technique permitted structural analyses of soft tissue and skeleton in G. fascicularis in a frozen-hydrated state, without the need for chemical fixation or decalcification. Structural comparisons are made between frozen-hydrated polyps and polyps that have undergone conventional fixation and decalcification. Vesicles expelled by the calicoblastic ectodermal cells into sub-skeletal spaces and previously suggested to play a role in calcification were commonly observed in fixed samples but were distinctly absent in frozen-hydrated preparations. We propose that these vesicles are fixation artefacts. Two distinct types of vesicles (380 and 70 nm in diameter, respectively), were predominant throughout the calicoblastic ectodermal cells of frozen-hydrated preparations, but these were never seen to be entering, or to be contained within, sub-skeletal spaces, nor did they contain any crystalline material. In frozen-hydrated preparations, membranous sheets were seen to surround and isolate portions of aboral mesogloea and to form junctional complexes with calicoblastic cells. The calicoblastic ectoderm was closely associated with the underlying skeleton, with sub-skeletal spaces significantly smaller (P<0.0001) in frozen-hydrated polyps compared to fixed polyps. A network of organic filaments (26 nm in diameter) extended from the apical membranes of calicoblastic cells into these small sub-skeletal cavities. A thin sheath was also frequently observed adjacent to the apical membrane of calicoblastic cells.     

Embryonic development and metamorphosis of the scyphozoan <Emphasis Type="Italic">Aurelia</Emphasis>

We investigated the development of Aurelia (Cnidaria, Scyphozoa) during embryogenesis and metamorphosis into a polyp, using antibody markers combined with confocal and transmission electron microscopy. Early embryos form actively proliferating coeloblastulae. Invagination is observed during gastrulation. In the planula, (1) the ectoderm is pseudostratified with densely packed nuclei arranged in a superficial and a deep stratum, (2) the aboral pole consists of elongated ectodermal cells with basally located nuclei forming an apical organ, which is previously only known from anthozoan planulae, (3) endodermal cells are large and highly vacuolated, and (4) FMRFamide-immunoreactive nerve cells are found exclusively in the ectoderm of the aboral region. During metamorphosis into a polyp, cells in the planula endoderm, but not in the ectoderm, become strongly caspase 3 immunoreactive, suggesting that the planula endoderm, in part or in its entirety, undergoes apoptosis during metamorphosis. The polyp endoderm seems to be derived from the planula ectoderm in Aurelia, implicating the occurrence of “secondary” gastrulation during early metamorphosis.     

Fauna of hydromedusae (Cnidaria: Hydrozoa) of the northwestern Sea of Japan

A brief overview of studies of hydromedusae (Cnidaria: Hydrozoa) of the northwestern Sea of Japan is provided. Based on literature data and plankton sampling by the authors, an annotated checklist of hydromedusae of the northwestern Sea of Japan was compiled for the first time. It includes 25 species belonging to 20 families and 5 orders.     

Features of development of fetal bone organ culture in space flight

Fetal mouse metatarsals cultured for 4 days onboard international space laboratory IML-1 (STS-42) were investigated using light microscopy and electron microscopy combined with X-ray microanalysis. Bones cultured in microgravity were equal in length to both ground and inflight (1 g) controls. Three zones: epiphyseal, proliferative, and hypertrophic chondrocytes were distinguished and measured in metatarsals isolated from 16-day-old fetuses. In bone cultures exposed to microgravity, hypertrophic zone tended to decrease and epiphyseal area was increased compared to controls. Proliferative zone has equal length both in bones cultured under microgravity and in controls. The same tendency was observed in bone cultures from 17-day-old fetuses. Metatarsals cultured in microgravity have less spreading calcification zone of diaphysis in comparison with both controls. The results suggest that maturation of chondrocytes and calcification of cartilage, but not cell proliferation, are microgravity sensitive processes in developing bones isolated from the organism.     

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.     

Microsatellite abundance across the Anthozoa and Hydrozoa in the phylum Cnidaria

Background

Microsatellite loci have high mutation rates and thus are indicative of mutational processes within the genome. By concentrating on the symbiotic and aposymbiotic cnidarians, we investigated if microsatellite abundances follow a phylogenetic or ecological pattern. Individuals from eight species were shotgun sequenced using 454 GS-FLX Titanium technology. Sequences from the three available cnidarian genomes (Nematostella vectensis, Hydra magnipapillata and Acropora digitifera) were added to the analysis for a total of eleven species representing two classes, three subclasses and eight orders within the phylum Cnidaria.

Results

Trinucleotide and tetranucleotide repeats were the most abundant motifs, followed by hexa- and dinucleotides. Pentanucleotides were the least abundant motif in the data set. Hierarchical clustering and log likelihood ratio tests revealed a weak relationship between phylogeny and microsatellite content. Further, comparisons between cnidaria harboring intracellular dinoflagellates and those that do not, show microsatellite coverage is higher in the latter group.

Conclusions

Our results support previous studies that found tri- and tetranucleotides to be the most abundant motifs in invertebrates. Differences in microsatellite coverage and composition between symbiotic and non-symbiotic cnidaria suggest the presence/absence of dinoflagellates might place restrictions on the host genome.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-939) contains supplementary material, which is available to authorized users.     

Gender-related differences in the apparent timing of skeletal density bands in the reef-building coral Siderastrea siderea

Density banding in skeletons of reef-building corals is a valuable source of proxy environmental data. However, skeletal growth strategy has a significant impact on the apparent timing of density-band formation. Some corals employ a strategy where the tissue occupies previously formed skeleton during as the new band forms, which leads to differences between the actual and apparent band timing. To investigate this effect, we collected cores from female and male colonies of Siderastrea siderea and report tissue thicknesses and density-related growth parameters over a 17-yr interval. Correlating these results with monthly sea surface temperature (SST) shows that maximum skeletal density in the female coincides with low winter SSTs, whereas in the male, it coincides with high summer SSTs. Furthermore, maximum skeletal densities in the female coincide with peak Sr/Ca values, whereas in the male, they coincide with low Sr/Ca values. Both results indicate a 6-month difference in the apparent timing of density-band formation between genders. Examination of skeletal extension rates also show that the male has thicker tissue and extends faster, whereas the female has thinner tissue and a denser skeleton—but both calcify at the same rate. The correlation between extension and calcification, combined with the fact that density banding arises from thickening of the skeleton throughout the depth reached by the tissue layer, implies that S. siderea has the same growth strategy as massive Porites, investing its calcification resources into linear extension. In addition, differences in tissue thicknesses suggest that females offset the greater energy requirements of gamete production by generating less tissue, resulting in differences in the apparent timing of density-band formation. Such gender-related offsets may be common in other corals and require that environmental reconstructions be made from sexed colonies and that, in fossil corals where sex cannot be determined, reconstructions must be duplicated in different colonies.     

NCX3 is a major functional isoform of the sodium-calcium exchanger in osteoblasts

The calcium phosphate-based skeleton of vertebrates serves as the major reservoir for metabolically available calcium ions. The skeleton is formed by osteoblasts which first secrete a proteinaceous matrix and then provide Ca++ for the calcification process. The two calcium efflux ports found in most cells are the plasma membrane Ca-ATPase (PMCA) and the sodium-calcium exchanger (NCX). In osteoblasts, PMCA and NCX are located on opposing sides of the cell with NCX facing the mineralizing bone surface. Two isoforms of NCX have been identified in osteoblasts NCX1, and NCX3. The purpose of this study was to determine the extent to which each of the two NCX isoforms support delivery of Ca++ into sites of calcification and to discern if one could compensate for the other. SiRNA technology was used to knockdown each isoform separately in MC3T3-E1 osteoblasts. Osteoblasts in which either NCX1 or NCX3 was impaired were tested for Ca++ efflux using the Ca++ specific fluorophore, fluo-4, in a sodium-dependent calcium uptake assay adapted for image analysis. NCX3 was found to serve as a major contributor of Ca++ translocation out of osteoblasts into calcifying bone matrix. NCX1 had little to no involvement.     

Intracellular precipitation of hydroxyapatite mineral and implications for pathologic calcification

In contrast to physiologic biomineralization occurring in bones, teeth and otoconia in vertebrates, calcification of soft tissues occurs in many pathologic conditions. Although similarities have been noted between the two processes, and despite the important clinical consequences of ectopic calcification, the molecular mechanisms regulating ectopic calcification are poorly understood. Although calcification is mainly extracellular, intracellular calcification has been reported and might indeed contribute to pathologic calcification of soft tissues. To better understand the process of intracellular calcification as a potential origin for pathologic calcification, and to examine the role of proteoglycans in this process, we investigated a pattern of intracellular nucleation and growth of hydroxyapatite in Madin–Darby Canine Kidney (MDCK) epithelial cells using electron microscopy, secondary ion mass spectroscopy (NanoSIMS), cytochemical staining, immunolabeling and biochemical analysis. We report here that under mineralizing cell culture conditions where β-glycerophosphate (βGP) was added as an exogenous organic source of phosphate, βGP-cleaving alkaline phosphatase activity increased and hydroxyapatite crystals subsequently nucleated within intracellular, membrane-bounded compartments. The small, leucine-rich proteoglycan decorin was also upregulated and associated with mineral in these cultures. Such information provides insight into the mechanisms leading to pathologic calcification and describes a process whereby hydroxyapatite deposition in cells might lead to ectopic calcification.     

Symmetry and the tentacular apparatus in Cnidaria

Comparative analysis provides evidence that bilateral symmetry is a primary character of Cnidaria. All anthozoan taxa are characterized by bilateral symmetry. The anthozoan pharyngeal plane is a plane of bilateral symmetry of mesenteries and, at the same time, it is a plane of bilateral symmetry of regulatory gene expression in anthozoan morphogenesis. In Medusozoa, the bilateral symmetry is replaced by radial symmetry, but some hydrozoans (for example, Corymorphidae) demonstrate bilateral symmetry. The bilateral symmetry of Cnidaria is thought to be inherited from the common ancestors of both cnidarians and triploblastic bilaterians. The secondary radial symmetry of Cnidaria evidently is a result of the adaptation to the sessile mode of life. The presence of both the marginal and labial rings of tentacles is supposed to be a plesiomorphic character of Cnidaria. In some groups of cnidarians, one of the tentacle rings may be reduced.     

Inferred calcification rate of a temperate azooxanthellate caryophylliid coral along a wide latitudinal gradient

Correlations between environmental parameters (depth temperature and solar radiation) and growth parameters (bulk skeletal density, linear extension rate and net calcification rate) of the solitary azooxanthellate coral, Caryophyllia inornata, were investigated along an 8° latitudinal gradient on the western Italian coasts. Net calcification rate correlated positively with both bulk skeletal density and linear extension rate, showing that C. inornata allocates calcification resources evenly to thickening the skeleton and increasing linear growth. Overall, the three growth parameters did not follow gradients in the two environmental parameters, showing a different trend compared to most studies on zooxanthellate corals. However, the results are in agreement with the only previous analysis of an azooxanthellate coral, Leptopsammia pruvoti, studied along the same latitudinal gradient. In a comparison of the response to temperature of all Mediterranean species whose growth has been investigated to date, azooxanthellate corals were more tolerant to temperature increases than zooxanthellate corals.     

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.