Comprehensive characterization of skeletal tissue growth anomalies of the finger coral Porites compressa

The scleractinian finger coral Porites compressa has been documented to develop raised growth anomalies of unknown origin, commonly referred to as “tumors”. These skeletal tissue anomalies (STAs) are circumscribed nodule-like areas of enlarged skeleton and tissue with fewer polyps and zooxanthellae than adjacent tissue. A field survey of the STA prevalence in Oahu, Kaneohe Bay, Hawaii, was complemented by laboratory analysis to reveal biochemical, histological and skeletal differences between anomalous and reference tissue. MutY, Hsp90a1, GRP75 and metallothionein, proteins known to be up-regulated in hyperplastic tissues, were over expressed in the STAs compared to adjacent normal-appearing and reference tissues. Histological analysis was further accompanied by elemental and micro-structural analyses of skeleton. Anomalous skeleton was of similar aragonite composition to adjacent skeleton but more porous as evidenced by an increased rate of vertical extension without thickening. Polyp structure was retained throughout the lesion, but abnormal polyps were hypertrophied, with increased mass of aboral tissue lining the skeleton, and thickened areas of skeletogenic calicoblastic epithelium along the basal floor. The latter were highly metabolically active and infiltrated with chromophore cells. These observations qualify the STAs as hyperplasia and are the first report in poritid corals of chromophore infiltration processes in active calicoblastic epithelium areas.     

Skeletal growth, ultrastructure and composition of the azooxanthellate scleractinian coral Balanophyllia regia

The biomineralization process and skeletal growth dynamics of azooxanthellate corals are poorly known. Here, the growth rate of the shallow-water dendrophyllid scleractinian coral Balanophyllia regia was evaluated with calcein-labeling experiments that showed higher lateral than vertical extension. The structure, mineralogy and trace element composition of the skeleton were characterized at high spatial resolution. The epitheca and basal floor had the same ultrastructural organization as septa, indicating a common biological control over their formation. In all of these aragonitic skeletal structures, two main ultrastructural components were present: “centers of calcification” (COC) also called rapid accretion deposits (RAD) and “fibers” (thickening deposits, TD). Heterogeneity in the trace element composition, i.e., the Sr/Ca and Mg/Ca ratios, was correlated with the ultrastructural organization: magnesium was enriched by a factor three in the rapid accretion deposits compared with the thickening deposits. At the interface with the skeleton, the skeletogenic tissue (calicoblastic epithelium) was characterized by heterogeneity of cell types, with chromophile cells distributed in clusters regularly spaced between calicoblasts. Cytoplasmic extensions at the apical surface of the calicoblastic epithelium created a three-dimensional organization that could be related to the skeletal surface microarchitecture. Combined measurements of growth rate and skeletal ultrastructural increments suggest that azooxanthellate shallow-water corals produce well-defined daily growth steps.     

Confocal laser scanning light microscopy of the extra-thecal epithelia of undecalcified scleractinian corals

The extra-thecal epithelia of cryofixed undecalcified, freeze-substituted polyps of the scleractinian corals Galaxea fascicularis and Tubastrea faulkneri and axial and basal polyps of Acropora formosa have been examined, in anhydrously prepared thick slices, by confocal laser scanning light microscopy. The avoidance of chemical fixation and decalcification makes it possible to determine whether previously seen structures are real or artefactual products of swelling, shrinkage and distortion. All of the epithelia of all the corals examined are characterised by well defined intercellular spaces. Mucocytes are present in all cell layers in Galaxea and Tubastrea but are not present in any cell layers in the axial polyp of Acropora although they are abundant in the oral ectoderm of the basal polyps in this coral. Zooxanthellae are absent in Tubastrea, the epithelia of the exert septa of Galaxea and the axial polyp of Acropora. The calicoblastic ectoderm is generally composed of thin squamous cells with large intercellular spaces. At rapidly calcifying regions such as the tips of the exert septa of Galaxea, the calicoblastic cells are elongated with extensive arborisation of the basal regions of the cells. They are separated by large intercellular spaces and contain numerous fluorescent granules. The apical regions of these cells appear to be closely applied to the surface of the skeleton. There is no evidence of a space between the apical region of the calicoblastic cells and the skeleton.     

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.     

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.     

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic–related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in–vivo imaging with cryo-electron microscopy and cryo–elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.     

The nature and construction of skeletal spines inPocillopora damicornis (Linnaeus)

Extreme variability in the size, shape and spacing of skeletal spines ofPocillopora damicornis has been demonstrated both within single colonies and also between colonies from different environments. Preliminary studies indicated that the majority of spines from branch tips at the apex of the colony display a ‘fasciculate’ growth surface in contrast to partly fasciculate or ‘smooth’ growth surfaces exhibited by spines from branch tips at the base of the colony. No significant differences in the height and width of costal spines from apical and basal branch tips within a single colony were observed, although spines from colonies exposed to strong wave action tended to be significantly shorter and narrower than those from more sheltered environments. Both costal and coenosteal spines from wave-exposed colonies displayed branching and divided extremities while those from sheltered environments consisted of simple cones. Spines develop as an outgrowing of the calicoblastic ectoderm which secretes the skeleton. Growing costal and coenosteal spines are enveloped by a layer of calicoblastic ectoderm which penetrates through mesogloea, aboral gastroderm, coelenteron, oral gastroderm, mesogloea and finally oral ectoderm. Spines within the corallite are surrounded by calicoblastic ectoderm, mesogloea and aboral gastroderm only. A scheme for the growth of the spines is discussed.     

Morphological studies of the soft tissues involved in skeletal dissolution in the coralFungia fungites

Light and transmission electron microscopy were used to study mechanisms involved in the separation of the disc from the stalk in juvenileFungia fungites (Scleractinia, Fungiidae). Separation occurs because the skeleton is weakened by dissolution across a distinct plane at the junction of the stalk and disc. The tissue layer adjacent to the skeleton in the stalk was composed of typical, squamose, calicoblastic cells. In contrast, calicoblastic cells in the region of skeletal dissolution were tall and columnar. They contained many microvilli, abundant mitochondria and several different types of vesicles. It is assumed that these calicoblastic cells are actively involved in skeletal dissolution.     

Diurnal patterns of skeleton formation in Pocillopora damicornis (Linnaeus)

Scanning, transmission and X-ray microanalytical electron microscopy were used to investigate the skeleton, organic matrix and calicoblastic ectoderm of the reef coral Pocillopora damicornis over a diurnal cycle. All skeletal surfaces, both during day and hight, are fasciculate except for skeletal spines on the branch tip apex at night where small (0.5 m) fusiform crystals are deposited. X-ray microanalysis shows that the fusiform crystals and needle-shaped crystals that compose the fasciculi are distinct forms of calcium carbonate. Demineralization of the skeleton reveals an organic matrix with two components which are related to the formation of fusiform crystals and fasciculi. During the day the calicoblastic ectoderm overlying all skeletal surfaces is 1–3 m thick. At night ultrastructural evidence suggests that skeletal deposition occurs only on those skeletal spines at the branch tip apex which are growing parallel with the branch growth axis. The calicoblastic ectoderm overlying apical skeletal spines at night shows a greater degree of cellular activity, and is thicker, than calicoblastic ectoderm overlying both other skeletal surfaces at night (<8 m cf. >6 m) and all skeletal surfaces during the day (<8 m cf. >3 m). The deposition of fusiform crystals on skeletal spines at the branch tip apex is proposed to promote deposition of fasciculi during the day, relative to other skeletal surfaces, providing a mechanism determining apical growth of branch tips. The results are discussed with respect to previous concepts of skeletal deposition in scleractinian corals.     

New type of organic matrix in cirals formed at the decalcified site: Structure and composition

This is the first demonstration that the organic matrix appears at the decalcified site of the skeleton in a juvenile coral of Fungia fungites (Linnaeus). This matrix was secreted by the calicoblastic layer and is composed of fibrous matters as seen in mesoglea. If formed a thick sheet at the advanced stage. The amino acid composition of this matrix was similar to that in the skeleton rather than mesogleal protein. Morphologic and functional features suggest that the organic sheet must be similar to the sheet which is secreted extracellularly by planula larva at the time of settlement. The sheet in F. fungites may have a role of protecting calicoblastic epithelium from exposure as a result of skeletal dissolution.     

Calcium associated with a fibrillar organic matrix in the scleractinian coral Galaxea fascicularis

Summary.  Field emission scanning electron microscopy of frozen-hydrated preparations of the scleractinian coral Galaxea fascicularis revealed organic fibrils which have a diameter of 26 nm and are located between calicoblastic ectodermal cells and the underlying CaCO₃ skeleton. Small (37 nm in diameter) nodular structures observed upon this fibrillar organic material possibly correspond to localised Ca-rich regions detected throughout the calcifying interfacial region of freeze-substituted preparations by X-ray microanalysis. We propose that these Ca-rich regions associated with the organic material are nascent crystals of CaCO₃. Significant amounts of S were also detected throughout the calcifying interfacial region, further verifying the likely presence of organic material. However, the bulk of this S is unlikely to be derived from mucocytes within the calicoblastic ectoderm. It is suggested that in the scleractinian coral G. fascicularis, nodular crystals of CaCO₃ establish upon a fibrillar, S-containing, organic matrix within small but distinct extracellular pockets formed between calicoblastic ectodermal cells and skeleton. This arrangement conforms with the criteria necessary for biomineralisation and with the long-held theory that organic matrices may act as templates for crystal formation and growth in biological mineralising systems. Received April 30, 2002; accepted September 11, 2002; published online March 11, 2003     

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.     

Fine-scale mapping of physicochemical and microbial landscapes of the coral skeleton

The coral skeleton harbours a diverse community of bacteria and microeukaryotes exposed to light, O₂ and pH gradients, but how such physicochemical gradients affect the coral skeleton microbiome remains unclear. In this study, we employed chemical imaging of O₂ and pH, hyperspectral reflectance imaging and spatially resolved taxonomic and inferred functional microbiome characterization to explore links between the skeleton microenvironment and microbiome in the reef-building corals Porites lutea and Paragoniastrea benhami. The physicochemical environment was more stable in the deep skeleton, and the diversity and evenness of the bacterial community increased with skeletal depth, suggesting that the microbiome was stratified along the physicochemical gradients. The bulk of the coral skeleton was in a low O₂ habitat, whereas pH varied from pH 6–9 with depth. Physicochemical gradients of O₂ and pH of the coral skeleton explained the β-diversity of the bacterial communities, and skeletal layers that showed O₂ peaks had a higher relative abundance of endolithic algae, reflecting a link between the abiotic environment and the microbiome composition. Our study links the physicochemical, microbial and functional landscapes of the coral skeleton and provides new insights into the involvement of skeletal microbes in the coral holobiont metabolism.     

Stable isotopic records of bleaching and endolithic algae blooms in the skeleton of the boulder forming coral Montastraea faveolata

Within boulder forming corals, fixation of dissolved inorganic carbon is performed by symbiotic dinoflagellates within the coral tissue and, to a lesser extent, endolithic algae within the coral skeleton. Endolithic algae produce distinctive green bands in the coral skeleton, and their origin may be related to periods of coral bleaching due to complete loss of dinoflagellate symbionts or “paling” in which symbiont populations are patchily reduced in coral tissue. Stable carbon isotopes were analyzed in coral skeletons across a known bleaching event and 12 blooms of endolithic algae to determine whether either of these types of changes in photosynthesis had a clear isotopic signature. Stable carbon isotopes tended to be enriched in the coral skeleton during the initiation of endolith blooms, consistent with enhanced photosynthesis by endoliths. In contrast, there were no consistent δ¹³C patterns directly associated with bleaching, suggesting that there is no unique isotopic signature of bleaching. On the other hand, isotopic values after bleaching were lighter 92% of the time when compared to the bleaching interval. This marked drop in skeletal δ¹³C may reflect increased kinetic fractionation and slow symbiont recolonization for several years after bleaching.     

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.     

Seasonal growth bands in Famennian rugose coral Scruttonia kunthi and their environmental significance

Large colonies of rugose coral Scruttonia kunthi occurring in the upper Famennian of Sudetes (southern Poland) reveal distinct growth banding in their skeletons. They were investigated for internal structural characteristics and stable isotopic composition. The skeletal tissue consists of alternating light and dark bands which differ in thickness, density and morphology of structural elements, and in occurrence of corallite contraction and rejuvenescense. Darker parts with densely arranged thick skeletal elements are thin in comparison to lighter parts. In addition, they include frequently offsets and contraction of corallites. A couplet of dense and less dense bands is interpreted to represent most probably an annual cycle. The calculated growth rate for Scruttonia kunthi varied from 6 mm/yr to 12 mm/yr. Growth-band formation was influenced environmentally. Oxygen isotopic data provide an evidence that high-density bands were formed in the season of higher environmental stress, with relatively warmer temperatures and higher sedimentation rates. Carbon isotopic signatures are very uniform, and thus enigmatic. They indicate that at least growth rate of the skeleton and seawater temperature had no influence on the coral δ¹³C.     

Wound repair in Montipora capitata

We documented the microscopic morphology of tissue healing in Montipora capitata. Fragments from two healthy coral colonies were traumatized by scraping tissue and skeleton and monitored in flow-through seawater tables every 2-4 days for 40 days for gross and cellular changes. Grossly, corals appeared healed and repigmented by Day 40. Histologically, traumatized issues were undistinguishable from intact untraumatized tissues by Day 12. We suspect that the calicoblastic epidermis of basal body wall is pluripotential and can develop into surface epidermis when needed.     

Baseline shifts in coral skeletal oxygen isotopic composition: a signature of symbiont shuffling?

Decades-long records of the stable isotopic composition of coral skeletal cores were analyzed from four sites on the Mesoamerican Reef. Two of the sites exhibited baseline shifts in oxygen isotopic composition after known coral bleaching events. Changes in pH at the calcification site caused by a change in the associated symbiont community are invoked to explain the observed shift in the isotopic composition. To test the hypothesis that changes in symbiont clade could affect skeletal chemistry, additional coral samples were collected from Belize for paired Symbiodinium identification and skeletal stable isotopic analysis. We found some evidence that skeletal stable isotopic composition may be affected by symbiont clade and suggest this is an important topic for future investigation. If different Symbiodinium clades leave consistent signatures in skeletal geochemical composition, the signature will provide a method to quantify past symbiont shuffling events, important for understanding how corals are likely to respond to climate change.     

Desmocytes in the calicoblastic epithelium of the stony coral Mycetophyllia reesi and their attachment to the skeleton

Desmocytes scattered over the surface of the corallum of the scleractinian Mycetophyllia reesi attach the calicoblastic tissue to the skeleton. The structure of the desmocyte is generally consistent with that of other scleractinians except for their more rectangular profiles and greater size. However, the extent of attachment is distinctive, and the mode of attachment to mineral is described for the first time. The skeleton contains dual rows of interconnected pits between the septa, within and among which desmocytes form virtually uninterrupted sheets. Desmocytes terminate with hemidesmosomes that attach the epithelium to a fibrillar basal lamina. Fibrils extend from the basal lamina into the skeletal matrix anchoring tissue firmly to the skeleton. In addition, the basal lamina itself appears to be incorporated within the organic matrix during growth, partitioning the skeleton into compartments. Because the skeletal organic matrix is physicochemically labile during demineralization, these intraskeletal details cannot be observed unless polycationic dyes such as Ruthenium red or other glycan precipitating agents are employed in the fixative sequence.     

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.