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Cnidarians living in symbiosis with photosynthetic dinoflagellates (commonly named zooxanthellae) are exposed to high concentrations of reactive oxygen species (ROS) upon illumination. To quench ROS production, both the cnidarian host and zooxanthellae express a full suite of antioxidant enzymes. Studying antioxidative balance is therefore crucial to understanding how symbiotic cnidarians cope with ROS production. We characterized glutathione peroxidases (GPx) in the symbiotic cnidarian Anemonia viridis by analysis of their isoform diversity, their activity distribution in the three cellular compartments (ectoderm, endoderm and zooxanthellae) and their involvement in the response to thermal stress. We identified a GPx repertoire through a phylogenetic analysis showing 7 GPx transcripts belonging to the A. viridis host and 4 GPx transcripts strongly related to Symbiodinium sp. The biochemical approach, used for the first time with a cnidarian species, allowed the identification of GPx activity in the three cellular compartments and in the animal mitochondrial fraction, and revealed a high GPx electrophoretic diversity. The symbiotic lifestyle of zooxanthellae requires more GPx activity and diversity than that of free-living species. Heat stress induced no modification of GPx activities. We highlight a high GPx diversity in A. viridis tissues by genomic and biochemical approaches. GPx activities represent an overall constitutive enzymatic pattern inherent to symbiotic lifestyle adaptation. This work allows the characterization of the GPx family in a symbiotic cnidarian and establishes a foundation for future studies of GPx in symbiotic cnidarians., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

The temperate symbiotic sea anemone Anemonia viridis, a member of the Cnidaria phylum, is a relevant experimental model to investigate the molecular and cellular events involved in the preservation or in the rupture of the symbiosis between the animal cells and their symbiotic microalgae, commonly named zooxanthellae. In order to increase research tools for this model, we developed a primary culture from A. viridis animal cells. By adapting enzymatic dissociation protocols, we isolated animal host cells from a whole tentacle in regeneration state. Each plating resulted in a heterogeneous primary culture consisted of free zooxanthellae and many regular, small rounded and adherent cells (of 3-5 μm diameter). Molecular analyses conducted on primary cultures, maintained for 2 weeks, confirmed a specific signature of A. viridis cells. Further serial dilutions and micromanipulation allowed us to obtain homogenous primary cultures of the small rounded cells, corresponding to A. viridis "epithelial-like cells". The maintenance and the propagation over a 4 weeks period of primary cells provide, for in vitro cnidarian studies, a preliminary step for further investigations on cnidarian cellular pathways notably in regard to symbiosis interactions.

The only symbiotic Mediterranean gorgonian, Eunicella singularis, has faced several mortality events connected to abnormal high temperatures. Since thermotolerance data remain scarce, heat-induced necrosis was monitored in aquarium by morphometric analysis. Gorgonian tips were sampled at two sites: Medes (Spain) and Riou (France) Islands, and at two depths: -15 m and-35 m. Although coming from contrasting thermal regimes, seawater above 28 °C led to rapid and complete tissue necrosis for all four populations. However, at 27 °C, the time length leading to 50% tissue necrosis allowed us to classify samples within three classes of thermal sensitivity. Irrespectively of the depth, Medes specimens were either very sensitive or resistant, while Riou fragments presented a medium sensitivity. Microsatellite analysis revealed that host and symbiont were genetically differentiated between sites, but not between depths. Finally, these genetic differentiations were not directly correlated to a specific thermal sensitivity whose molecular bases remain to be discovered., (Copyright © 2013 Académie des sciences. Published by Elsevier SAS. All rights reserved.)

Over the last few decades, sessile benthic organisms from the Mediterranean Sea have suffered from the global warming of the world's oceans, and several mass mortality events were observed during warm summers. It has been hypothesized that mortality could have been due to a nutrient (food) shortage following the stratification of the water column. However, the symbiotic gorgonian Eunicella singularis has also presented a locally exceptional mortality, despite its autotrophic capacities through the photosynthesis of its dinoflagellate symbionts. Thus, this study has experimentally investigated the response of E. singularis to a thermal stress (temperature increase from 18 to 26°C), with colonies maintained more than 2 months under four nutritional diets: autotrophy only (AO), autotrophy and inorganic nitrogen addition (AN), autotrophy and heterotrophy (AH), heterotrophy only (HO). At 18°C, and contrary to many other anthozoans, supplementation of autotrophy with either inorganic nitrogen or food (heterotrophy) had no effect on the rates of respiration, photosynthesis, as well as in the chlorophyll, lipid and protein content. In the dark, heterotrophy maintained the gorgonian's metabolism, except a bleaching (loss of pigments), which did not affect the rates of photosynthesis. At 24°C, rates of respiration, and photosynthesis significantly decreased in all treatments. At 26°C, in addition to a decrease in the lipid content of all treatments, a bleaching was observed after 1 week in the AO treatment, while the AH and AN treatments resisted three weeks before bleaching. These last results suggest that, temperatures above 24°C impair the energetic reserves of this species and might explain the mortality events in the Mediterranean.

Increase in seawater temperature is one of the major effects of global climate change that affects marine organisms, including Cnidaria. Among them, gorgonians from the NW Mediterranean Sea, such as the species Eunicella singularis, have suffered spectacular and extensive damage. We thus investigated in a controlled laboratory experiment the response of E. singularis to a long-term increase in temperature and we took a special interest in its photosynthetic and calcification response to the stress. Two populations collected at 15 and 35 m depths were studied in order to determine whether there was a difference in sensitivity to thermal stress between living depths. Our results show: (a) that calcification and photosynthesis were impacted only when gorgonians were maintained for more than two weeks at 26 degrees C, and (b) that colonies of E. singularis living in shallow waters were less tolerant than those living in deep waters. Because E. singularis is a symbiotic species, we have also discussed the potential role of symbiosis in the thermotolerance response.

Symbiotic cnidarians are marine invertebrates harboring photosynthesizing microalgae (named zooxanthellae), which produce great amounts of oxygen and free radicals upon illumination. Studying antioxidative balance is then crucial to understanding how symbiotic cnidarians cope with ROS production. In particular, it is suspected that oxidative stress triggers cnidarian bleaching, i.e., the expulsion of zooxanthellae from the animal host, responsible for symbiotic cnidarian mass mortality worldwide. This study therefore investigates catalase antioxidant enzymes and their role in bleaching of the temperate symbiotic sea anemone Anemonia viridis. Using specific separation of animal tissues (ectoderm and endoderm) from the symbionts (zooxanthellae), spectrophotometric assays and native PAGE revealed both tissue-specific and activity pattern distribution of two catalase electrophoretypes, E1 and E2. E1, expressed in all three tissues, presents high sensitivity to the catalase inhibitor aminotriazole (ATZ) and elevated temperatures. The ectodermal E1 form is responsible for 67% of total catalase activity. The E2 form, expressed only within zooxanthellae and their host endodermal cells, displays low sensitivity to ATZ and relative thermostability. We further cloned an ectodermal catalase, which shares 68% identity with mammalian monofunctional catalases. Last, 6 days of exposure of whole sea anemones to ATZ (0.5 mM) led to effective catalase inhibition and initiated symbiont expulsion. This demonstrates the crucial role of this enzyme in cnidarian bleaching, a phenomenon responsible for worldwide climate-change-induced mass mortalities, with catastrophic consequences for marine biodiversity.

The symbiotic life style involves mutual ecological, physiological, structural, and molecular adaptations between the partners. In the symbiotic association between anthozoans and photosynthetic dinoflagellates (Symbiodinium spp., also called zooxanthellae), the presence of the endosymbiont in the animal cells has constrained the host in several ways. It adopts behaviors that optimize photosynthesis of the zooxanthellae. The animal partner has had to evolve the ability to absorb and concentrate dissolved inorganic carbon from seawater in order to supply the symbiont's photosynthesis. Exposing itself to sunlight to illuminate its symbionts sufficiently also subjects the host to damaging solar ultraviolet radiation. Protection against this is provided by biochemical sunscreens, including mycosporine-like amino acids, themselves produced by the symbiont and translocated to the host. Moreover, to protect itself against oxygen produced during algal photosynthesis, the cnidarian host has developed certain antioxidant defenses that are unique among animals. Finally, living in nutrient-poor waters, the animal partner has developed several mechanisms for nitrogen assimilation and conservation such as the ability to absorb inorganic nitrogen, highly unusual for a metazoan. These facts suggest a parallel evolution of symbiotic cnidarians and plants, in which the animal host has adopted characteristics usually associated with phototrophic organisms.

Cnidarians in symbiosis with photosynthetic protists must withstand daily hyperoxic/anoxic transitions within their host cells. Comparative studies between symbiotic (Anemonia viridis) and non-symbiotic (Actinia schmidti) sea anemones show striking differences in their response to oxidative stress. First, the basal expression of SOD is very different. Symbiotic animal cells have a higher isoform diversity (number and classes) and a higher activity than the non-symbiotic cells. Second, the symbiotic animal cells of A. viridis also maintain unaltered basal values for cellular damage when exposed to experimental hyperoxia (100% O(2)) or to experimental thermal stress (elevated temperature +7 degrees C above ambient). Under such conditions, A. schmidti modifies its SOD activity significantly. Electrophoretic patterns diversify, global activities diminish and cell damage biomarkers increase. These data suggest symbiotic cells adapt to stress while non-symbiotic cells remain acutely sensitive. In addition to being toxic, high O(2) partial pressure (P(O(2))) may also constitute a preconditioning step for symbiotic animal cells, leading to an adaptation to the hyperoxic condition and, thus, to oxidative stress. Furthermore, in aposymbiotic animal cells of A. viridis, repression of some animal SOD isoforms is observed. Meanwhile, in cultured symbionts, new activity bands are induced, suggesting that the host might protect its zooxanthellae in hospite. Similar results have been observed in other symbiotic organisms, such as the sea anemone Aiptasia pulchella and the scleractinian coral Stylophora pistillata. Molecular or physical interactions between the two symbiotic partners may explain such variations in SOD activity and might confer oxidative stress tolerance to the animal host.

Cnidarians living in symbiosis with photosynthetic cells--called zooxanthellae--are submitted to high oxygen levels generated by photosynthesis. To cope with this hyperoxic state, symbiotic cnidarians present a high diversity of superoxide dismutases (SOD) isoforms. To understand better the mechanism of resistance of cnidarian hosts to hyperoxia, we studied copper- and zinc-containing SOD (CuZnSOD) from Anemonia viridis, a temperate symbiotic sea anemone. We cloned two CuZnSOD genes that we call AvCuZnSODa and AvCuZnSODb. Their molecular analysis suggests that the AvCuZnSODa transcript encodes an extracellular form of CuZnSOD, whereas the AvCuZnSODb transcript encodes an intracellular form. Using in situ hybridization, we showed that both AvCuZnSODa and AvCuZnSODb transcripts are expressed in the endodermal and ectodermal cells of the sea anemone, but not in the zooxanthellae. The genomic flanking sequences of AvCuZnSODa and AvCuZnSODb revealed different putative binding sites for transcription factors, suggesting different modes of regulation for the two genes. This study represents a first step in the understanding of the molecular mechanisms of host animal resistance to permanent hyperoxia status resulting from the photosynthetic symbiosis. Moreover, AvCuZnSODa and AvCuZnSODb are the first SODs cloned from a diploblastic animal, contributing to the evolutionary understanding of SODs.

The soluble organic matrix (OM) of various biominerals (red coral skeleton, oyster shell, sea urchin test, turbot otolith, chicken eggshell) was extracted after demineralization with acetic acid. The protein content of the OM varies strongly from 0.02 to 1.6 microg/mg biomineral whereas proteoglycans present less variations (from 0.7 to 1.4 microg/mg biomineral). Electrophoresis of biominerals OM shows differences in their protein pattern although several bands are present in all matrices. OM of all biominerals shows carbonic anhydrase activity but no activity was detectable in the endolymph. OM of all biominerals also displays an anticalcifying activity. After separation of the OM extracts by chloroform-methanol, 80% of the anticalcifying activity was found in the methanol phase except in the urchin test. After OM precipitation with trichloracetic acid, 70% of the activities was found in the supernatants. Partial biochemical characterization suggests that the anticalcifying factor is a polyanionic and water-soluble molecule, which could be proteoglycans. The endolymph surrounding the otolith also displays an anticalcifying activity although its inhibitous activity was 50 times lower than that of the otolith OM. However, the anticalcifying activity of the endolymph is assumed by a proteic structure (80% activity precipitated with TCA treatment). Our results suggest that both carbonic anhydrase and anticalcifying activities are widespread and play a significant role in the regulation of biomineral formation. Results are discussed in relation to the calcification process that takes place at the fluid-mineral interface.

Many cnidarians, such as sea anemones, contain photosynthetic symbiotic dinoflagellates called zooxanthellae. During a light/dark cycle, the intratentacular O(2) state changes in minutes from hypoxia to hyperoxia (3-fold normoxia). To understand the origin of the high tolerance to these unusual oxic conditions, we have characterized superoxide dismutases (SODs) from the three cellular compartments (ectoderm, endoderm and zooxanthellae) of the Mediterranean sea anemone Anemonia viridis. The lowest SOD activity was found in ectodermal cells while endodermal cells and zooxanthellae showed a higher SOD activity. Two, seven and six SOD activity bands were identified on native PAGE in ectoderm, endoderm and zooxanthellae, respectively. A CuZnSOD was identified in both ectodermal and endodermal tissues. MnSODs were detected in all compartments with two different subcellular localizations. One band displays a classical mitochondrial localization, the three others being extramitochondrial. FeSODs present in zooxanthellae also appeared in endodermal host tissue. The isoelectric points of all SODs were distributed between 4 and 5. For comparative study, a similar analysis was performed on the whole homogenate of a scleractinian coral Stylophora pistillata. These results are discussed in the context of tolerance to hyperoxia and to the transition from anoxia to hyperoxia.

Fibroblast growth factor-2 (FGF-2), administered to the isolated rat heart by perfusion and under constant pressure, is protective against ischemia-reperfusion (I-R). Here we have investigated whether FGF-2 cardioprotection: (a) is dependent on flow modulation; (b) is linked to effects on contractility; (c) is mediated by protein kinase C (PKC); and (d) is linked to PKC and/or mitogen activated protein kinase (MAPK) associated with the sarcolemma. The isolated rat heart was used as a model. Under conditions of constant flow FGF-2 induced significant improvement in recovery of contractile function during I-R. Under constant perfusion pressure, FGF-2 induced a negative inotropic effect (15% decrease in developed pressure). Chelerythrine, a specific PKC inhibitor, prevented both the FGF-2-induced negative inotropic effect before ischemia, and cardioprotection during I-R. FGF-2 induced a chelerythrine-preventable, five-fold increase in sarcolemmal calcium-independent PKC activity. It also increased the association of PKC subtypes -epsilon and -delta with sarcolemmal membranes, detected by Western blotting, as well as, for PKC delta, by immunolocalization. FGF-2 increased the association of PKC epsilon with the membrane fraction of adult cardiomyocyte in culture, confirming that it can affect PKC signaling in cardiomyocytes directly and in a manner similar to its effects in situ. Finally, FGF-2 induced increased active MAPK at sarcolemmal as well as cytosolic sites. Active sarcolemmal MAPK remained elevated when the FGF-2-induced protection was prevented by chelerythrine. In conclusion, we have provided evidence that cardioprotection by FGF-2 is independent of flow modulation. PKC activation mediates both the FGF-2-induced negative inotropic effect before ischemia and the cardioprotective effect assessed during reperfusion, suggesting a cause and effect relationship. Furthermore, FGF-2 cardioprotection is linked to targeting of sarcolemmal sites by calcium-independent PKC.

Basic fibroblast growth factor (bFGF) has been implicated in the changes in gene expression that, under pathological conditions such as ischemia or volume overload, lead to adult cardiomyocyte hypertrophy. In many tissues, one of the first events following cell activation by growth factors is an enhancement of the intracellular free calcium concentration, generated by fluxes from internal storage compartments and through channels of the plasma membrane. The present study was undertaken to determine whether cardiac myocytes isolated from adult rat ventricles express Ca2+-permeable channels activated by bFGF. Using the cell-attached mode of the patch-clamp technique, we observed that bFGF (from 0.1-10 nM) induced an increase of fast burst openings, mediated by Ca2+-permeable channels with low conductance (15 pS) and voltage-independence. Inside-out patch-clamp experiments revealed that inositol 1,4,5-trisphosphate (5 microM) enhanced the opening of Ca2+-permeable channels with similar properties as the bFGF-induced channels, indicating that IP3 may be a second messenger of this process. Confocal fluorescence imaging of intracellular free calcium provided direct evidence that bFGF induced an increase of cytoplasmic and nucleoplasmic free Ca2+ concentrations which were generated, in part, by Ca2+ influx through the plasma membrane. In conclusion, this study supports the presence, in the plasma membrane of adult cardiac myocytes, of messenger-activated calcium channels which could play key roles in the calcium-dependent pathways that are activated in response to growth factors., (Copyright 1997 Academic Press Limited.)

Basic fibroblast growth factor (bFGF) is a potent mitogen for many cell lineages including fetal cardiomyocytes. Furthermore, bFGF has been shown to modify gene expression, in vitro, in adult nonproliferative ventricular myocytes. This effect is suspected to be partly responsible for the genetic modifications that occur in vivo under pathophysiological conditions such as ischemia or pressure overload and that lead to myocardial hypertrophy. However, little is known about the first steps of the molecular mechanisms that take place soon after cell activation by bFGF. In this study, using biochemical and electrophysiological approaches, we have established, on cardiomyocytes cultured from neonatal rat ventricles, that (i) differentiated beating cells express at least two classes of bFGF-receptors having high and low affinity (Kd = 10 +/- 2 pM and 1 +/- 0.5 nM); (ii) the stimulation of these bFGF receptors promotes an increase in the beating frequencies of cultured cardiomyocytes (40 +/- 10%); (iii) bFGF provokes the activation of poorly specific and voltage-independent calcium channels (12pS); (iv) inositol 1,4,5-trisphosphate enhances similar bFGF-induced Ca2+ currents and is therefore suspected to be a second messenger triggering this activation. These results support the presence, in cultured cardiomyocytes, of new calcium channels whose activation after bFGF binding may be partly responsible for the cell response to this growth factor.