Nutritional resources as positional information for morphogenesis in the stony coral Stylophora pistillata

We are interested in deciphering the mechanisms for morphogenesis in the Red Sea scleractinian coral Stylophora pistillata with the help of mathematical models. Previous mathematical models for coral morphogenesis assume that skeletal growth is proportional to the amount of locally available energetic resources like diffusible nutrients and photosynthetic products. We introduce a new model which includes factors like dissolved nutrients and photosynthates, but these resources do not serve as building blocks for growth but rather provide some kind of positional information for coral morphogenesis. Depending on this positional information side branches are generated, splittings of branches take place and branch growth direction is determined. The model results are supported by quantitative comparisons with experimental data obtained from young coral colonies.     

Molecular analyses of protein components of the organic matrix in the exoskeleton of two scleractinian coral species

Biochemical and histological studies on the exoskeleton of scleractinian corals had demonstrated presence of the organic matrix containing proteins, lipids and chitin. Examination at the electron microscopic level had shown that the initial phase of calcification occurred in close association with organic substances secreted by calicoblastic cells. The possibility was thereby proposed that certain organic substances induce formation of calcium carbonate crystals, presumably functioning as templates for nucleation. In search for such a molecule, biochemical and molecular analyses were initiated on protein components of the organic matrix extracted from the calcified exoskeleton of the hermatypic coral, Galaxea fascicularis and the ahermatype, Tubastrea aurea. In SDS-PAGE analyses of the extracts, one major protein and a few other minor bands were detected in each of the two species. A cDNA encoding the major protein (named galaxin) in G. fascicularis was cloned and its primary structure was deduced. It consisted mostly of tandem repeats of a unit sequence of about 30 residues, and its sequence did not exhibit significant similarity to known proteins. Preliminary characterization of the T. aurea proteins showed that two proteins bound Ca²⁺, and suggested that the major protein of 46 kDa was not homologous to galaxin.     

Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization

Soluble organic matrix (SOM) synthesis and secretion were investigated in two scleractinian corals using antibodies raised against this organic matrix. Results demonstrate that even if other cell types, including zooxanthellae, can supply precursors for SOM synthesis, only calicoblastic cells facing the skeleton are directly responsible for the synthesis and secretion of the SOM components. Results also indicate that, as is the case for other biominerals, skeleton formation is biologically controlled and not chemically dominated as originally believed. In addition to advancing the understanding of mechanisms of coral biomineralization, these antibodies could have numerous applications: for example as markers of skeletogenesis, as tools for cell culture, and in comparative studies among calcifying organisms.     

Effect of nutrient enrichment on growth and photosynthesis of the zooxanthellate coral Stylophora pistillata

The effect of prolonged (9 week) nutrient enrichment on the growth and photosynthetic rates of the zooxanthellate coral Stylophora pistillata was investigated. The main questions were: (1) what is the exposure time needed to induce measurable change in growth rate? (2) which are the concentrations of nitrogen and phosphorus required to cause changes in these rates? (3) what is the recovery potential of the corals after the nutrient stress? For this purpose, three tanks (N, P, NP) were enriched with ammonium (N), phosphorus (P) or both nutrients (NP), respectively. A fourth tank (C) served as a control. The growth of 40 nubbins (10 in each tank) was monitored during four periods: period 1 (nutrient-poor conditions), period 2 (10?μm NH₄ and/or 2?μm PO₄ enrichment), period 3 (20?μm NH₄ and/or 2?μm PO₄) and period 4 (nutrient-poor conditions). Period 4 was performed to study the recovery potential of corals after a nutrient stress. During period 1, growth rates remained constant in all tanks. In the P tank, growth rates declined during the two enrichment periods, with a total decrease of 60% by the end of period 3. In the N tank, growth rates remained nearly constant during period 2 but decreased in period 3 (60% decrease). In the NP tank, 50% and 25% decreases were observed during periods 2 and 3. At the end of the recovery period, a regain in growth rate was observed in the N and NP tanks (35 and 30% increase, respectively, compared with the rates measured at the end of period 3) and growth rates returned to 60% of the initial rates. By contrast, in the P tank, there was no regain in growth and a further decrease of 5% was observed. Rates of photosynthesis were often higher during the enriched than the nutrient-poor period (up to 150% increase). Corals with the highest percent increases in maximal gross photosynthetic rate (P ^g _max ) had the smallest decreases in growth rate due to nutrient enrichment. In conclusion, high ammonium (20?μm) and relatively low phosphorus concentrations (2?μm) are required to induce a significant decrease in coral growth rate. The largest reduction was observed with both ammonium and phosphorus enrichment. The decrease in growth rate was rapid following nutrient enrichment, since a 10% decrease or more could be observed after the first week of treatment.     

Identification of low molecular weight molecules as new components of the nacre organic matrix

Nacre of Pinctada margaritifera displays a number of interesting biological activities on bone, mainly concentrated in a water-soluble organic matrix representing 0.24% of the nacre weight. Dialysis of that matrix through 8 kDa and 1 kDa cut-off membranes showed that 60% of it is made of small molecules of molecular masses below 1 kDa. Reversed-phase high-performance liquid chromatography of the small molecule fractions and subsequent electrospray ionization mass spectrometric analysis of 19 fractions thereof indicated the presence of at least 110 different molecules, in the range 100 Da–700 Da. Evidence for aggregate-forming properties of the small molecules was given. Amino acid analysis revealed that most of the small molecules were not peptides and tandem mass spectrometric gas-phase fragmentations clearly indicated a structural relationship between several molecules. Intriguingly, differences of a single Dalton between mono-charged ions peaks were observed. Further, approximately 40 analytes could be arranged in a ladder-like manner with mass spaces of 57 Da. Some of the water-soluble peptide sequences obtained after MS/MS fragmentation revealed that the 57 Da shift corresponds to the repetition of glycine residues. Furthermore, the exchange of glycine against alanine explains the 14 Da shift observed between some peptides. These data show for the first time that small molecules, especially peptides, are prevalent components of nacre. The molecular species described in this report might have a functional role in nacre.     

Bioaccumulation of zinc in the scleractinian coral Stylophora pistillata

The uptake kinetics of zinc (Zn), an essential nutrient for both photosynthesis and calcification, in the tissue of S. pistillata showed that the transport of Zn is composed of a linear component (diffusion) at high concentrations and an active carrier-mediated component at low concentrations. The carrier affinity (K _m=28 pmol l⁻¹) was very low, indicating a good adaptation of the corals to low levels of Zn in seawater. Zn accumulation in the skeleton was linear; its level was dependent on the length of the incubation as well as on the external concentration of dissolved Zn. There was also a light-stimulation of Zn uptake, suggesting that zooxanthellae, through photosynthesis, are involved in this process. An enrichment of the incubation medium with 10 nM Zn significantly increased the photosynthetic efficiency of S. pistillata. This result suggests that corals living in oligotrophic waters might be limited in essential metals, such as zinc.     

Soluble organic matrix of two Scleractinian corals: partial and comparative analysis

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.     

Evidence of density-independent mortality in a settling coral reef damselfish, Chromis viridis

To know if the variation in the number of settling fish larvae can be dampened by density-dependent postsettlement mortality, we investigated the relationship between settler density and predator-induced mortality of a coral reef damselfish, Chromis viridis. Totals of 2, 3, 5, 8, 10, 12, 14, 16, 18, and 20 fish of 10 or 20 mm total length were released in experimental cages enclosing a coral head of Porites rus (to provide settlement habitat) and five predators. The results showed that the mortality rate of both 10- and 20-mm fish was density independent.     

Gene expression associated with white syndromes in a reef building coral,Acropora hyacinthus

Background

Corals are capable of launching diverse immune defenses at the site of direct contact with pathogens, but the molecular mechanisms of this activity and the colony-wide effects of such stressors remain poorly understood. Here we compared gene expression profiles in eight healthy Acropora hyacinthus colonies against eight colonies exhibiting tissue loss commonly associated with white syndromes, all collected from a natural reef environment near Palau. Two types of tissues were sampled from diseased corals: visibly affected and apparently healthy.

Results

Tag-based RNA-Seq followed by weighted gene co-expression network analysis identified groups of co-regulated differentially expressed genes between all health states (disease lesion, apparently healthy tissues of diseased colonies, and fully healthy). Differences between healthy and diseased tissues indicate activation of several innate immunity and tissue repair pathways accompanied by reduced calcification and the switch towards metabolic reliance on stored lipids. Unaffected parts of diseased colonies, although displaying a trend towards these changes, were not significantly different from fully healthy samples. Still, network analysis identified a group of genes, suggestive of altered immunity state, that were specifically up-regulated in unaffected parts of diseased colonies.

Conclusions

Similarity of fully healthy samples to apparently healthy parts of diseased colonies indicates that systemic effects of white syndromes on A. hyacinthus are weak, which implies that the coral colony is largely able to sustain its physiological performance despite disease. The genes specifically up-regulated in unaffected parts of diseased colonies, instead of being the consequence of disease, might be related to the originally higher susceptibility of these colonies to naturally occurring white syndromes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1540-2) contains supplementary material, which is available to authorized users.     

Carbonic anhydrase inhibitors. Inhibition studies with anions and sulfonamides of a new cytosolic enzyme from the scleractinian coral Stylophora pistillata

The catalytic activity and the inhibition of a new coral carbonic anhydrase (CA, EC 4.2.1.1), from the scleractinian coral Stylophora pistillata, STPCA-2, has been investigated. STPCA-2 has high catalytic activity for the physiological reaction being less sensitive to anion and sulfonamide inhibitors compared to STPCA, a coral enzyme previously described. The best STPCA-2 anion inhibitors were sulfamide, sulfamic acid, phenylboronic acid, and phenylarsonic acid (K_Is of 5.7-67.2 μM) whereas the best sulfonamide inhibitors were acetazolamide and dichlorophenamide (K_Is of 74-79 nM). Because this discriminatory effect between these two coral CAs, sulfonamides may be useful to better understand the physiological role of STPCA and STPCA-2 in corals and biomineralization processes.     

Molecular cloning and characterization of first organic matrix protein from sclerites of red coral, Corallium rubrum

We report here for the first time the isolation and characterization of a protein from the organic matrix (OM) of the sclerites of the alcyonarian, Corallium rubrum. This protein named scleritin is one of the predominant proteins extracted from the EDTA-soluble fraction of the OM. The entire open reading frame (ORF) was obtained by comparing amino acid sequences from de novo mass spectrometry and Edman degradation with an expressed sequence tag library dataset of C. rubrum. Scleritin is a secreted basic phosphorylated protein which exhibits a short amino acid sequence of 135 amino acids and a signal peptide of 20 amino acids. From specific antibodies raised against peptide sequences of scleritin, we obtained immunolabeling of scleroblasts and OM of the sclerites which provides information on the biomineralization pathway in C. rubrum.     

Habitat choice,recruitment and the response of coral reef fishes to coral degradation

The global degradation of coral reefs is having profound effects on the structure and species richness of associated reef fish assemblages. Historically, variation in the composition of fish communities has largely been attributed to factors affecting settlement of reef fish larvae. However, the mechanisms that determine how fish settlers respond to different stages of coral stress and the extent of coral loss on fish settlement are poorly understood. Here, we examined the effects of habitat degradation on fish settlement using a two-stage experimental approach. First, we employed laboratory choice experiments to test how settlers responded to early and terminal stages of coral degradation. We then quantified the settlement response of the whole reef fish assemblage in a field perturbation experiment. The laboratory choice experiments tested how juveniles from nine common Indo-Pacific fishes chose among live colonies, partially degraded colonies, and dead colonies with recent algal growth. Many species did not distinguish between live and partially degraded colonies, suggesting settlement patterns are resilient to the early stages of declining coral health. Several species preferred live or degraded corals, and none preferred to associate with dead, algal-covered colonies. In the field experiment, fish recruitment to coral colonies was monitored before and after the introduction of a coral predator (the crown-of-thorns starfish) and compared with undisturbed control colonies. Starfish reduced live coral cover by 95–100%, causing persistent negative effects on the recruitment of coral-associated fishes. Rapid reductions in new recruit abundance, greater numbers of unoccupied colonies and a shift in the recruit community structure from one dominated by coral-associated fishes before degradation to one predominantly composed of algal-associated fish species were observed. Our results suggest that while resistant to coral stress, coral death alters the process of replenishment of coral reef fish communities.     

Long-term persistence of low coral cover and abundance on a disturbed coral reef flat in the northern Red Sea

Levels of coral cover and abundance on a coral reef flat in Eilat (Israeli Red Sea) were estimated in 2001 by surveying nineteen 10-m transects, and compared to the levels reported in the same area between 1966 and 1973. Lower values compared to 1966 levels are evident, and there has been only a modest recovery following a catastrophic low tide that killed a large proportion of the corals in 1970. Percent cover of soft and stony corals (16.1%) was less than half of that reported for 1969 (35%), when a sharp decrease in coral abundance had already been observed. The total number of soft and stony coral colonies observed was 300, compared to 541 in 1966. In contrast to 1966, when half of the transects surveyed contained more than 30 coral colonies, no transects with this number of corals were observed. The cover of seven of the most common stony coral species was 841 cm, which is twice the coral cover of that in 1973, but only 22% of the 1969 level. Millepora dichotoma, an abundant species before 1970, has almost disappeared, and the soft coral Litophyton, abundant in 1972, was not observed. Anthropogenic nutrient enrichment is apparently among the causes for the lack of coral recovery in the studied reef flat. Reefs located further away from sources of pollution have recovered quickly after natural and anthropogenic disturbances and have retained their coral abundance and diversity.     

Aspartic acid concentrations in coral skeletons as recorders of past disturbances of metabolic rates

The composition of total hydrolysable amino acids (THAAs) in a skeleton of the coral Porites australiensis, collected from Ishigaki Island, Japan, was examined in order to determine whether amino acids (AA) can be used as biomarkers of past changes in coral physiology (metabolism). Micro-samples, corresponding to a time resolution of 1 month, were collected along the growth axis of the coral. Of the 20 AAs analyzed, aspartic acid (Asp) was the most abundant, and its mole concentration relative to the sum of all other AAs (mole%Asp) showed a clear seasonal pattern of low content during winters and high during summers. A growth disturbance in the coral skeleton during 1988–1990, shown by X-ray scans and oxygen and carbon stable isotope data, was marked by a high mole%Asp ratio. Variability in carbon isotope data has often been attributed to metabolic effects, or changes in the isotopic composition of seawater, or both. The changes in mole%Asp shown here suggest that metabolic effects are mainly responsible for sharp changes in carbon isotope profiles during periods of growth disturbance.     

A simple technique for measuring buoyant weight increment of entire, transplanted coral colonies in the field

Estimating the impacts of global and local threats on coral reefs requires monitoring reef health and measuring coral growth and calcification rates at different time scales. This has traditionally been mostly performed in short-term experimental studies in which coral fragments were grown in the laboratory or in the field but measured ex situ. Practical techniques in which growth and measurements are performed over the long term in situ are rare. Apart from photographic approaches, weight increment measurements have also been applied. Past buoyant weight measurements under water involved a complicated and little-used apparatus. We introduce a new method that combines previous field and laboratory techniques to measure the buoyant weight of entire, transplanted corals under water. This method uses an electronic balance fitted into an acrylic glass underwater housing and placed atop of an acrylic glass cube. Within this cube, corals transplanted onto artificial bases can be attached to the balance and weighed at predetermined intervals while they continue growth in the field. We also provide a set of simple equations for the volume and weight determinations required to calculate net growth rates. The new technique is highly accurate: low error of weight determinations due to variation of coral density (< 0.08%) and low standard error (< 0.01%) for repeated measurements of the same corals. We outline a transplantation technique for properly preparing corals for such long-term in situ experiments and measurements.     

Electrophoretic subfractionation of low and high molecular weight humic acids fractions

Summary Soil humic acid was fractionated on a molecular weight basis either using Sephadex gel filtration or electrophoresis on a discontinuous polyacrylamide gel. Low and high molecular weight fractions obtained by these two methods were choosen for subsequent subfractionation using electrophoretic methods. The high and low molecular weight fractions yielded several subfractions after separation by isotachophoresis or isoelectric focusing. Components of the high molecular weight fractions occupied the upper portion of the mobility train; components of the low molecular weight fractions lead the mobility train. Adsorption by Sephadex was avoided by using 4M urea as an eluent. The elution of the humic substances adsorbed to the polyacrylamide gel matrix was achieved by using a 0.1M Tris –0.025M EDTA solution.     

Electron microscopical and laser diffraction studies of the nucleation and growth of crystals in the organic matrix of dentine

Summary The growing ends of rat incisors were freeze-dried and embedded in methacrylate without contact with any other solution. Dentine from alcohol and formalin fixed human teeth was also embedded in methacrylate. Ultrathin sections were prepared and electron micrographs taken at original magnifications of X20 000 and X40 000. The best focussed pictures from through focus series were selected for photographic enlargement to a total of X200 000. 507 measurements of the distance between dot-like nuclei in the calcium phosphate needles and chains and 536 measurements of the distance between the neighbouring parallel chains and needles were made using a measuring microscope. In addition, the most commonly occurring separation distances between the dot-like nuclei — within individual rows or between neighbouring rows—were measured by laser diffraction of the x20 000 EM negatives.The most commonly occurring range for the distance between the dot-like nuclei and the lateral distance between the rows as determined morphologically was 3.7–6.3 nm. The corresponding value as determined by laser diffraction for the recently formed rat incisor dentine lay in the region 3.0–5.2 nm, whereas the same value reached to 6.5 nm in the case of mature human dentine. The distances between the dot-like nuclei are regarded as representing the distances between active nucleus-inducing centres on a chain-like matrix. From a study of the morphology of the nuclei it is concluded that the plate-like crystallites usually arise through fusion of needle-like rows of dot-like nuclei when these lie close and parallel to one another.We thank Frau G. Neinhardt, Frau R. Höhling and Mr. P. S. Reynolds for their valuable technical assistance. We thank the Deutsche Forschungsgemeinschaft and the Medical Research Council for their financial support.We thank the Landesamt für Forschung des Landes N.R.W. for the laser equipment.     

Direct measurements of carbon and carbonate export from a coral reef ecosystem (Moorea Island , French Polynesia)

The export of carbon and carbonate from coral reefs was investigated through a multidisciplinary investigation of the hydrological, geochemical, sedimentological and biological features of Tiahura reef on the northwestern coast of Moorea Island (French Polynesia). The hydrology of the fore-reef is characterised by prevailing longshore western currents and a strong thermocline. As revealed by turbidity structures (benthic and intermediate nepheloid layers) and by the amount of particles collected by near-bottom sediment traps, horizontal and downslope advections of particles dominate over offshore vertical transport. The exported material is rich in carbonate (ca. 80%) and poor in organic matter (ca. 4%). Sedimentation rates at 430 m depth, i.e. definitive export, reached 209.6 mg m^-2 d^-1 (dry weight). Estimates of carbon and carbonates export for Tiahura reef also reported here represent respectively 47% and 21% of the organic and inorganic carbon produced within the reef.