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 biology of coral metamorphosis: molecular responses of larvae to inducers of settlement and metamorphosis

Like many other cnidarians, corals undergo metamorphosis from a motile planula larva to a sedentary polyp. In some sea anemones such as Nematostella this process is a smooth transition requiring no extrinsic stimuli, but in many corals it is more complex and is cue-driven. To better understand the molecular events underlying coral metamorphosis, competent larvae were treated with either a natural inducer of settlement (crustose coralline algae chips/extract) or LWamide, which bypasses the settlement phase and drives larvae directly into metamorphosis. Microarrays featuring > 8000 Acropora unigenes were used to follow gene expression changes during the 12 h period after these treatments, and the expression patterns of specific genes, selected on the basis of the array experiments, were investigated by in situ hybridization. Three patterns of expression were common—an aboral pattern restricted to the searching/settlement phase, a second phase of aboral expression corresponding to the beginning of the development of the calicoblastic ectoderm and continuing after metamorphosis, and a later orally-restricted pattern.     

Metamorphosis and acquisition of symbiotic algae in planula larvae and primary polyps of Acropora spp.

Coral planulae settle, then metamorphose and form polyps. This study examined the morphological process of metamorphosis from planulae into primary polyps in the scleractinian corals Acropora nobilis and Acropora microphthalma, using the cnidarian neuropeptide Hym-248. These two species release eggs that do not contain Symbiodinium. The mode of acquisition of freshly isolated Symbiodinium (zooxanthellae) (FIZ) by the non-symbiotic polyp was also examined. Non-Hym-248 treated swimming Acropora planulae did not develop blastopore, mesenteries or coelenteron until the induction of metamorphosis 16 days after fertilization. The oral pore was formed by invagination of the epidermal layer after formation of the coelenteron in metamorphosing polyps. At 3 days after settlement and metamorphosis, primary polyps exposed to FIZ established symbioses with the Symbiodinium. Two–four days after exposure to FIZ, the distribution of Symbiodinium was limited to the gastrodermis of the pharynx and basal part of the polyps. Eight–ten days after exposure to FIZ, Symbiodinium were present in gastrodermal cells throughout the polyps.     

Microstructural characteristics of the stony coral genus Acropora useful to coral reef paleoecology and modern conservation

Identification of fossil corals is often limited due to poor preservation of external skeleton morphology, especially in the genus Acropora which is widespread across the Indo‐Pacific. Based on skeleton characteristics from thin section, we here develop a link between the internal skeleton structure and external morphology. Ten characteristics were summarized to distinguish Acropora and five related genera, including the type and differentiation of corallites, the skeleton nature of corallites (septa, columellae, dissepiments, wall), and calcification centers within septa. Acropora is distinctive for its dimorphic corallites: axial and radial. Isopora is similar to Acropora but possess more than a single axial corallites. Montipora and Astreopora (family Acroporidae) have monomorphic corallites and a synapticular ring wall, with clustered calcification center in the former and medial lines in the latter. Pocillopora and Porties are classified by distinctive dissepiments, columellae and septa. These microstructural skeleton characteristics were effective in the genus identification of fossil corals from drilled cores in the South China Sea. Eighteen detailed characteristics (ten of axial corallites, four of radial corallites, and four of coenosteum) were used in the Acropora species classification. The axial corallites size and structure (including corallite diameter, synapticular rings, and septa), the septa of radial corallites, and the arrangement of coenosteum were critical indicators for species identification. This identification guide can help paleoenvironmental and paleoecological analyses and modern coral reef conservation and restoration.     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

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.     

Sexual reproduction of Acropora reef corals at Moorea, French Polynesia

Little information is available on reproductive processes among corals in isolated central Pacific reef regions, including French Polynesia. This study examined the timing and mode of sexual reproduction for Acropora reef corals at Moorea. Spawning was observed and/or inferred in 110 Acropora colonies, representing 12 species, following full moon periods in September through November 2002. Gamete release was observed and inferred in four species of Acropora between 9 and 13 nights after the full moon (nAFM) in September 2002. Twelve Acropora spp. spawned gametes between 5 and 10 nAFM in October 2002, with six species spawning 7 nAFM and four species spawning 9 nAFM. In November 2002, spawning of egg and sperm bundles was observed and inferred in 27 colonies of Acropora austera, 6 nAFM. These are the first detailed records of spawning by Acropora corals in French Polynesia.     

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.     

Bud formation in the scyphozoan Cassiopea andromeda: epithelial dynamics and fate map

Cassiopea andromeda scyphistomae reproduce asexually by forming spindle shaped buds which, after detachment, metamorphose into polyps. Parent polyps appear to contribute to the buds' ecto- and endodermal epithelial cells, septal muscle cells, nematocytes and some zooxanthellae. Herein we describe bud morphogenesis, define 5 bud stages, and investigate the recruitment of bud ectoderm. India ink vital marking experiments reveal permanent apicobasal displacement of ectoderm. Labelled polyp cells are displaced towards and incorporated into the emerging bud. Ectoderm is recruited from all angular positions and cells labelled at increasing distances from the bud center are traced at increasingly more proximal positions on the buds. Unlike in Hydra attenuata, the recruitment area appears to be asymmetric since the zone contributing ectoderm from below is smaller than the zones above and lateral to the buds.     

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     

Signaling pathways of heat- and hypersalinity-induced polyp bailout in Pocillopora acuta

Polyp bailout is a drastic response to acute stress where coral coloniality breaks down and polyps detach. We induced polyp bailout in Pocillopora acuta with heat stress and tested for differential gene expression using RNAseq and a qPCR assay. Furthermore, we induced polyp bailout with hypersalinity and compared the results to identify stressor-independent signals and pathways active during polyp bailout. Both stressors led to the onset of polyp bailout and the detachment of vital polyps. We observed activation of microbe-associated molecular pattern receptors and downstream signaling pathways of the innate immune system. Further, we detected growth factors and genes active during Wnt-signaling potentially contributing to wound healing, regeneration, and proliferation. Upregulation of several genes encoding for matrix metalloproteinases and the fibroblast growth factor signaling pathway are the most likely involved in the remodeling of the extracellular matrix, as well as in the detachment of polyps from the calcareous skeleton during polyp bailout. Expression of genes of interest in our qPCR assay of vital polyps from our heat-stress experiment, showed a trend for a normalization of gene expression after polyp bailout. Our results provide new insights into the signaling cascades leading to the observed physiological responses during polyp bailout. Comparison between the two stressors showed that certain signaling pathways are independent of the stressor and suggested that polyp bailout is a general response of corals to acute stress. Furthermore, immune system responses during polyp bailout indicate that microbe-associated partners of corals may lead to the polyp bailout response.

     

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.     

Polyp oriented modelling of coral growth

The morphogenesis of colonial stony corals is the result of the collective behaviour of many coral polyps depositing coral skeleton on top of the old skeleton on which they live. Yet, models of coral growth often consider the polyps as a single continuous surface. In the present work, the polyps are modelled individually. Each polyp takes up resources, deposits skeleton, buds off new polyps and dies. In this polyp oriented model, spontaneous branching occurs. We argue that branching is caused by a so called “polyp fanning effect” by which polyps on a convex surface have a competitive advantage relative to polyps on a flat or concave surface. The fanning effect generates a more potent branching mechanism than the Laplacian growth mechanism that we have studied previously (J. Theor. Biol. 224 (2003) 153). We discuss the application of the polyp oriented model to the study of environmentally driven morphological plasticity in stony corals. In a few examples we show how the properties of the individual polyps influence the whole colony morphology. In our model, the spacing of polyps influences the thickness of coral branches and the overall compactness of the colony. Density variations in the coral skeleton may also be important for the whole colony morphology, which we address by studying two variants of the model. Finally, we discuss the importance of small scale resource translocation in the coral colony and its effects on the morphology of the colony.     

Observations of a new source of coral mortality along the Kenyan coast

In early 2002 coral mortality occurred along 600 km of coastline from Tanzania to Kenya. Astreopora, Echinopora , and Montipora species were severely affected, with Montipora being nearly eliminated from Kenyan reefs. Acropora , Platygyra , Goniopora , and massive Porites were also affected; however, Porites and Goniopora rarely died and often recovered, whereas death for most other species occurred within 2 weeks. In Echinopora and Montipora , a dull ashy tissue color and brittle skeletons characterized the early stages of this event with a mucus layer on the tissue surface in intermediate stages. Mucus and embedded debris then disappeared and surfaces were left covered in a white calcareous dust that sometimes capped a black layer. Astreopora tissues became dull and pale, and seldom produced mucus; eventually the skeleton became bare and white. Either a colorless translucent or brownish thin margin of tissue was visible between living tissue and bare skeleton, depending on species. Scanning electron micrographs of affected corals revealed the presence of fungi. Histology and staining showed that the fungi were mostly in the three genera that died from the syndrome and it may be that fungi invaded and killed corals weakened by another unidentified pathogen.     

An early Cambrian polyp reveals a potential anemone-like ancestor for medusozoan cnidarians

Cnidarians form a disparate phylum of animals and their diploblastic body plan represents a key step in animal evolution. Cnidarians are split into two main classes; anthozoans (sea anemones, corals) are benthic polyps, while medusozoans (hydroids, jellyfishes) generally have alternating life cycle stages of polyps and medusae. A sessile polyp is present in both groups and is widely regarded as the ancestral form of their last common ancestor. However, the nature and anatomy of the ancestral polyp, particularly of medusozoans, is controversial, owing to the divergent body plans of the extant lineages and the scarcity of medusozoan soft tissues in the fossil record. Here, we redescribe Conicula striata Luo & Hu from the early Cambrian Chengjiang biota, south China, which has previously been interpreted as a polyp, lophophorate or deuterostome. Through re-examination of the holotype and 51 exceptionally preserved specimens, we show that C. striata possessed features of both anthozoans and medusozoan polyps. A conical, annulated organic skeleton (periderm) fully encasing a polyp is found in fossil and living medusozoans, while a tubular pharynx extending from the mouth into a gut partitioned by c. 28 mesenteries, resembling the actinopharynx of anthozoans. Our phylogenetic analyses recover C. striata as a stem-group medusozoan, implying that the wealth of medusozoan diversity derived, ultimately, from an anemone-like ancestor.     

Grazing by a small fish affects the early stages of a post-settlement stony coral

Short-term experiments were used to isolate the detrimental effects of grazer disturbance on young corals, and determine the stage of development at which recruits are no longer susceptible to this disturbance. Artificial substrata containing an algal matrix and coral recruits of different life stages were exposed to grazing by epilithic algal matrix (EAM) feeding combtoothed blennies, Salarias fasciatus. Single polyp recruits were vulnerable to grazer disturbance, while multi-polyp recruits (ca. 6–8 polyps) survived with evidence of minor damage in the form of tissue and polyp loss. The result indicates that blennies, although small and possessing weak dentition, can negatively influence the survival of young coral recruits. The protruding structure of micro-nubbins, representing juvenile corals were not damaged, suggesting that coral achieving that size and form can escape such damage. Communicated by Ecology Editor Prof. Peter Mumby     

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.     

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.     

Diversity and dynamics of bacterial communities in early life stages of the Caribbean coral Porites astreoides

In this study, we examine microbial communities of early developmental stages of the coral Porites astreoides by sequence analysis of cloned 16S rRNA genes, terminal restriction fragment length polymorphism (TRFLP), and fluorescence in situ hybridization (FISH) imaging. Bacteria are associated with the ectoderm layer in newly released planula larvae, in 4-day-old planulae, and on the newly forming mesenteries surrounding developing septa in juvenile polyps after settlement. Roseobacter clade-associated (RCA) bacteria and Marinobacter sp. are consistently detected in specimens of P. astreoides spanning three early developmental stages, two locations in the Caribbean and 3 years of collection. Multi-response permutation procedures analysis on the TRFLP results do not support significant variation in the bacterial communities associated with P. astreoides larvae across collection location, collection year or developmental stage. The results are the first evidence of vertical transmission (from parent to offspring) of bacteria in corals. The results also show that at least two groups of bacterial taxa, the RCA bacteria and Marinobacter, are consistently associated with juvenile P. astreoides against a complex background of microbial associations, indicating that some components of the microbial community are long-term associates of the corals and may impact host health and survival.