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.     

Response of a scleractinian coral, Stylophora pistillata, to iron and nitrate enrichment

The purpose of this study was to determine whether the addition of iron alone or in combination with nitrate affects growth and photosynthesis of the scleractinian coral, Stylophora pistillata, and its symbiotic dinoflagellates. For this purpose, we used three series of two tanks for a 3-week enrichment with iron (Fe), nitrate (N) and nitrate+iron (NFe). Two other tanks were kept as a control (C). Stock solutions of FeCl(3) and NaNO(3) were diluted to final concentrations of 6 nM Fe and 2 &mgr;M N and continuously pumped from batch tanks into the experimental tanks with a peristaltic pump. Results obtained showed that iron addition induced a significant increase in the areal density of zooxanthellae (ANOVA, p=0.0013; change from 6.3+/-0.7x10(5) in the control to 8.5+/-0.6x10(5) with iron). Maximal gross photosynthetic rates normalized per surface area also significantly increased following iron enrichment (ANOVA, p=0.02; change from 1.23+/-0.08 for the control colonies to 1.81+/-0.24 &mgr;mol O(2) cm(-2) h(-1) for the iron-enriched colonies). There was, however, no significant difference in the photosynthesis normalized on a per cell basis. Nitrate enrichment alone (2 &mgr;M) did not significantly change the zooxanthellae density or the rates of photosynthesis. Nutrient addition (both iron and nitrogen) increased the cell-specific density of the algae (CSD) compared to the control (G-test, p=0.3x10(-9)), with an increase in the number of doublets and triplets. CSD was equal to 1.70+/-0.04 in the Fe-enriched colonies, 1.54+/-0.12 in the N- and NFe-enriched colonies and 1.37+/-0.02 in the control. Growth rates measured after 3 weeks in colonies enriched with Fe, N and NFe were 23%, 34% and 40% lower than those obtained in control colonies (ANOVA, p=0.011).     

Calcification and associated physiological parameters during a stress event in the scleractinian coral Stylophora pistillata

High calcification rates observed in reef coral organisms are due to the symbiotic relationship established between scleractinian corals and their photosynthetic dinoflagellates, commonly called zooxanthellae. Zooxanthellae are known to enhance calcification in the light, a process referred as "light-enhanced calcification". The disruption of the relationship between corals and their zooxanthellae leads to bleaching. Bleaching is one of the major causes of the present decline of coral reefs related to climate change and anthropogenic activities. In our aquaria, corals experienced a chemical pollution leading to bleaching and ending with the death of corals. During the time course of this bleaching event, we measured multiple parameters and could evidence four major consecutive steps: 1) at month 1 (January 2005), the stress affected primarily the photosystem II machinery of zooxanthellae resulting in an immediate decrease of photosystem II efficiency, 2) at month 2, the stress affected the photosynthetic production of O2 by zooxanthellae and the rate of light calcification, 3) at month 3, there was a decrease in both light and dark calcification rates, the appearance of the first oxidative damage in the zooxanthellae, the disruption of symbiosis, 4) and finally the death of corals at month 6.     

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.     

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.     

Photo-acclimation of the hermatypic coral Stylophora pistillata while subjected to either starvation or food provisioning

This study investigated the photo-acclimation capacity of the coral Stylophora pistillata (Esper). Outer branches of coral colonies, taken from 2 m, were subjected to 90, 20, or 3% of incident surface photosynthetic active radiation (PAR(0)), or kept in total darkness. The corals were maintained either in filtered seawater (i.e., under starvation), or in seawater that had daily additions of zooplankton (rotifers). The experiments were maintained for 31 days. Zooxanthellae population densities and chlorophyll concentrations increased in S. pistillata fragments subjected to 20 and 3% PAR(0). The zooxanthellae densities decreased after 6 days in corals kept in total darkness, although chlorophyll concentrations remained higher. Corals that were fed and subjected to 90% PAR(0) showed lower degrading zooxanthellae frequencies, higher photosynthetic and respiration rates, and higher chlorophyll concentrations than corals in the same light regime under starvation. Complete acclimation to dim (20% PAR(0)) and low (3% PAR(0)) light was only apparent for corals fed with zooplankton. Changes in zooxanthellae population densities occurred through differential rates of zooxanthellae division and degradation.     

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.     

Copper enrichment reduces thermal tolerance of the highly resistant Red Sea coral Stylophora pistillata

Coral Reefs - Corals in the Gulf of Aqaba (GoA) in the northern Red Sea show high thermal tolerance. The GoA has therefore been suggested as a coral reef refuge from climate change. However, as a...     

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.     

Maternal-larval population genetic traits in Stylophora pistillata, a hermaphroditic brooding coral species

Aspects of maternal-planula larval genetics in the monoecious scleractinian coral Stylophora pistillata (Red Sea, Eilat) were studied by amplified fragment length polymorphism (AFLP) methodology in two successive reproductive seasons. In total, 293 planulae and 10 adult colonies were analyzed. In June 2006, 147 planulae were collected from 10 shallow water colonies. In March, April and June 2007, 146 additional planulae were sampled from five of the ten 2006 sampled colonies. All AFLP products showed unalike band profiles indicating a fully sexual production pattern. We used 181 and 210 putative AFLP loci, of which the overall level of polymorphism in 2006 was 92 and 99 % in 2007 (respectively). Differences were also observed between 2006 and 2007 reproductive seasons in terms of total average gene diversity (0.191 vs. 0.247, respectively), suggesting fast turnover of sperm donor genotypes. In addition, increased numbers of potential sperm donor colonies in the vicinity of gravid females showed no impact on genetic differentiation levels in released larvae. UPGMA tree revealed clustering of maternal genotypes and their offspring, suggesting, as expected, high relatedness between planulae and their mothers. In addition, the average heterozygosity of each group of siblings was persistently lower than heterozygosity calculated for the respective maternal colony, suggesting the possibility of partial inbreeding. This trend of reduced genetic heterogeneity in Stylophora pistillata is an alarming sign for populations residing in the northern Red Sea coral reefs.     

A profile of an endosymbiont-enriched fraction of the coral Stylophora pistillata reveals proteins relevant to microbial-host interactions

This study examines the response of Symbiodinium sp. endosymbionts from the coral Stylophora pistillata to moderate levels of thermal "bleaching" stress, with and without trace metal limitation. Using quantitative high throughput proteomics, we identified 8098 MS/MS events relating to individual peptides from the endosymbiont-enriched fraction, including 109 peptides meeting stringent criteria for quantification, of which only 26 showed significant change in our experimental treatments; 12 of 26 increased expression in response to thermal stress with little difference affected by iron limitation. Surprisingly, there were no significant increases in antioxidant or heat stress proteins; those induced to higher expression were generally involved in protein biosynthesis. An outstanding exception was a massive 114-fold increase of a viral replication protein indicating that thermal stress may substantially increase viral load and thereby contribute to the etiology of coral bleaching and disease. In the absence of a sequenced genome for Symbiodinium or other photosymbiotic dinoflagellate, this proteome reveals a plethora of proteins potentially involved in microbial-host interactions. This includes photosystem proteins, DNA repair enzymes, antioxidant enzymes, metabolic redox enzymes, heat shock proteins, globin hemoproteins, proteins of nitrogen metabolism, and a wide range of viral proteins associated with these endosymbiont-enriched samples. Also present were 21 unusual peptide/protein toxins thought to originate from either microbial consorts or from contamination by coral nematocysts. Of particular interest are the proteins of apoptosis, vesicular transport, and endo/exocytosis, which are discussed in context of the cellular processes of coral bleaching. Notably, the protein complement provides evidence that, rather than being expelled by the host, stressed endosymbionts may mediate their own departure.     

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.     

Molecular cloning and localization of a PMCA P-type calcium ATPase from the coral Stylophora pistillata

Plasma-membrane calcium pumps (PMCAs) are responsible for the expulsion of Ca(2+) from the cytosol of all eukaryotic cells and are one of the major transport systems involved in long-term regulation of resting intracellular Ca(2+) concentration. An important feature of stony corals, one of the major groups of calcifying animals, is the continuous export of large quantities of Ca(2+) for skeletogenesis. Here, we report the cloning and functional expression of the stpPMCA gene from the coral Stylophora pistillata, and whose features resemble those of the plasma-membrane Ca(2+)-ATPase family of mammalian cells. This is the first known example of a Ca(2+)-ATPase from the phylum Cnidaria, and thus, the most phylogenetically distant PMCA sequence in the animal kingdom described to date. We demonstrate that the localization of stpPMCA within calicoblastic cells is fully coherent with its role in calcification. We also show that the coral Ca(2+) pump is more closely related to vertebrate PMCAs than to Caenorhabditis elegans PMCAs. The cloning of evolutionarily conserved genes from cnidarian species repeatedly shows that these genes encode similar functional domains. Moreover, this high level of gene conservation further validates the use of cnidarian model systems for studying processes shared by Eumetazoans.     

Cloning of a calcium channel alpha1 subunit from the reef-building coral, Stylophora pistillata

While the mechanisms of cellular Ca2+ entry associated with cell activation are well characterized, the pathway of continuous uptake of the large amount of Ca2+ needed in the biomineralization process remains largely unknown. Scleractinian corals are one of the major calcifying groups of organisms. Recent studies have suggested that a voltage-dependent Ca2+ channel is involved in the transepithelial transport of Ca2+ used for coral calcification. We report here the cloning and sequencing of a cDNA coding a coral alpha1 subunit Ca2+ channel. This channel is closely related to the L-type family found in vertebrates and invertebrates. Immunohistochemical analysis shows that this channel is present within the calicoblastic ectoderm, the site involved in calcium carbonate precipitation. These data and previous results provide molecular evidence that voltage-dependent Ca2+ channels are involved in calcification. Cnidarians are the most primitive organisms in which a Ca2+ channel has been cloned up to now; evolutionary perspectives on Ca2+ channel diversity are discussed.     

Acclimation of the Hermatypic Coral Stylophora pistillata to Bright Light

Photoacclimation dynamics to bright light was studied in the symbiotic reef-building coral Stylophora pistillata. Coral colonies were collected from shallow shaded sites (2 m, 40–20% PAR_s) from a fringing reef at Sesoko Island, Okinawa, Japan. Outer branches were broken off from the colonies and placed in an outdoor aquarium until the start of the experiments. After maintenance of the branches in an aquarium under a light intensity of 30% PAR_s for 30 days (experiment 1) or for 90 days (experiment 2), the samples were exposed to 95% PAR_s for 120 days in the same aquarium. The population density of zooxanthellae, chlorophyll concentration, locations of zooxanthellae, proliferating zooxanthellae frequency (PZF), and degrading zooxanthellae frequency (DZF) were examined. It was shown that after acclimation of coral branches to bright light, the population density of zooxanthellae, chlorophyll concentration calculated per 1000 polyps, and chlorophyll concentration in zooxanthellae decreased. The size of zooxanthellae significantly decreased. A decrease in the population density of zooxanthellae was detected by the eighth day of acclimation, and stabilization in the density of the symbionts occurred in the period from the 40th to the 60th day of the experiment. The chlorophyll concentration in zooxanthellae significantly decreased by the second day of exposure to bright light and stabilized by the fourth day. The PZF level sharply dropped on the second day, while the DZF level sharply increased and was higher than the PZF level for 40 days of exposure to bright light. We conclude, therefore, that the population density of zooxanthellae is regulated by the rates of two processes: cell division and the cell degradation.     

The reef building coral Stylophora pistillata uses stored carbohydrates to maintain ATP levels under thermal stress

Coral Reefs - Coral reefs are on the brink of collapse from global warming and associated coral bleaching. Coral bleaching is the loss of algal symbionts from the coral tissue. The reduction in...     

Effects of increased pCO2 on zinc uptake and calcification in the tropical coral Stylophora pistillata

Zinc (Zn) is an essential element for corals. We investigated the effects of ocean acidification on zinc incorporation, photosynthesis, and gross calcification in the scleractinian coral Stylophora pistillata. Colonies were maintained at normal pH_T (8.1) and at two low-pH conditions (7.8 and 7.5) for 5 weeks. Corals were exposed to ⁶⁵Zn dissolved in seawater to assess uptake rates. After 5 weeks, corals raised at pH_T (8.1) exhibited higher ⁶⁵Zn activity in the coral tissue and skeleton, compared with corals raised at a lower pH. Photosynthesis, photosynthetic efficiency, and gross calcification, measured by ⁴⁵Ca incorporation, were however unchanged even at the lowest pH.     

Evidence of low molecular weight components in the organic matrix of the reef building coral, Stylophora pistillata

Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization.     

Gene expression profiles during short‐term heat stress in the red sea coral Stylophora pistillata

During the past several decades, corals worldwide have been affected by severe bleaching events leading to wide‐spread coral mortality triggered by global warming. The symbiotic Red Sea coral Stylophora pistillata from the Gulf of Eilat is considered an opportunistic ‘r’ strategist. It can thrive in relatively unstable environments and is considered a stress‐tolerant species. Here, we used a S. pistillata custom microarray to examine gene expression patterns and cellular pathways during short‐term (13‐day) heat stress. The results allowed us to identify a two‐step reaction to heat stress, which intensified significantly as the temperature was raised to a 32 °C threshold, beyond which, coping strategies failed at 34 °C. We identified potential ‘early warning genes’ and ‘severe heat‐related genes’. Our findings suggest that during short‐term heat stress, S. pistillata may divert cellular energy into mechanisms such as the ER‐unfolded protein response (UPR) and ER‐associated degradation (ERAD) at the expense of growth and biomineralization processes in an effort to survive and subsequently recover from the stress. We suggest a mechanistic theory for the heat stress responses that may explain the success of some species which can thrive under a wider range of temperatures relative to others.