Cécile Sabourault

Oxidative stress and apoptotic events during thermal stress in the symbiotic sea anemone, Anemonia viridis

Symbiosis between cnidarian and photosynthetic protists is widely distributed over temperate and tropical seas. These symbioses can periodically breakdown, a phenomenon known as cnidarian bleaching. This event can be irreversible for some associations subjected to acute and/or prolonged environmental disturbances, and leads to the death of the animal host. During bleaching, oxidative stress has been described previously as acting at molecular level and apoptosis is suggested to be one of the mechanisms involved. We focused our study on the role of apoptosis in bleaching via oxidative stress in the association between the sea anemone Anemonia viridis and the dinoflagellates Symbiodinium species. Characterization of caspase‐like enzymes were conducted at the biochemical and molecular level to confirm the presence of a caspase‐dependent apoptotic phenomenon in the cnidarian host. We provide evidence of oxidative stress followed by induction of caspase‐like activity in animal host cells after an elevated temperature stress, suggesting the concomitant action of these components in bleaching.

Adaptations to endosymbiosis in a cnidarian-dinoflagellate association: differential gene expression and specific gene duplications.

Trophic endosymbiosis between anthozoans and photosynthetic dinoflagellates forms the key foundation of reef ecosystems. Dysfunction and collapse of symbiosis lead to bleaching (symbiont expulsion), which is responsible for the severe worldwide decline of coral reefs. Molecular signals are central to the stability of this partnership and are therefore closely related to coral health. To decipher inter-partner signaling, we developed genomic resources (cDNA library and microarrays) from the symbiotic sea anemone Anemonia viridis. Here we describe differential expression between symbiotic (also called zooxanthellate anemones) or aposymbiotic (also called bleached) A. viridis specimens, using microarray hybridizations and qPCR experiments. We mapped, for the first time, transcript abundance separately in the epidermal cell layer and the gastrodermal cells that host photosynthetic symbionts. Transcriptomic profiles showed large inter-individual variability, indicating that aposymbiosis could be induced by different pathways. We defined a restricted subset of 39 common genes that are characteristic of the symbiotic or aposymbiotic states. We demonstrated that transcription of many genes belonging to this set is specifically enhanced in the symbiotic cells (gastroderm). A model is proposed where the aposymbiotic and therefore heterotrophic state triggers vesicular trafficking, whereas the symbiotic and therefore autotrophic state favors metabolic exchanges between host and symbiont. Several genetic pathways were investigated in more detail: i) a key vitamin K–dependant process involved in the dinoflagellate-cnidarian recognition; ii) two cnidarian tissue-specific carbonic anhydrases involved in the carbon transfer from the environment to the intracellular symbionts; iii) host collagen synthesis, mostly supported by the symbiotic tissue. Further, we identified specific gene duplications and showed that the cnidarian-specific isoform was also up-regulated both in the symbiotic state and in the gastroderm. Our results thus offer new insight into the inter-partner signaling required for the physiological mechanisms of the symbiosis that is crucial for coral health.

The transcriptomic response to thermal stress is immediate, transient and potentiated by ultraviolet radiation in the sea anemone Anemonia viridis.

Among the environmental threats to coral reef health, temperature and ultraviolet increases have been proposed as major agents, although the relative contribution of each in the cnidarian/zooxanthellae symbiosis breakdown has been poorly addressed. We have investigated the transcriptomic response to thermal stress, with and without ultraviolet radiation (UVR), in the symbiotic sea anemone Anemonia viridis. Using the Oligo2K A. viridis microarray, dedicated to genes potentially involved in the symbiosis interaction, we monitored the gene expression profiles after 1, 2 and 5 days of stresses that further lead to massive losses of zooxanthellae. Each stress showed a specific gene expression profile with very little overlap. We showed that the major response to thermal stress is immediate (24 h) but returns to the baseline gene expression profile after 2 days. UVR alone has little effect but potentiates thermal stress, as a second response at 5 days was observed when the two stresses were coupled. Several pathways were highlighted, such as mesoglea loosening, cell death and calcium homeostasis and described in more details. Finally, we showed that the dermatopontin gene family, potentially involved in collagen fibrillogenesis, issued from actinarian‐specific duplication events, with one member preferentially expressed in the gastroderm and specifically responding to stress.

Tissue-specific induction and inactivation of cytochrome P450 catalysing lauric acid hydroxylation in the sea bass, Dicentrarchus labrax.

Microsomal cytochrome P450-dependent lauric acid hydroxylase activities were characterized in liver, kidney, and intestinal mucosa of the sea bass (Dicentrarchus labrax). Microsomes from these organs generated (omega-1)-hydroxylauric acid and a mixture of positional isomers including (omega)-, (omega-2)-, (omega-3)- and (omega-4)-hydroxylauric acids, which were identified by RP-HPLC and GC-MS analysis. Peroxisome proliferators, such as clofibrate and especially di(2-ethylhexyl) phthalate, increased kidney microsomal lauric acid hydroxylase activities. The synthesis of 11-hydroxylauric acid was enhanced 5.3-fold in kidney microsomes. Liver microsomal lauric acid hydroxylase activities were weakly affected and no significant induction was found in small intestine microsomes from clofibrate or di(2-ethylhexyl) phthalate-treated fish. The differences in lauric acid metabolisation and the tissue-specific induction by peroxisome proliferators suggest the involvement of several P450s in this reaction. Incubations of liver and kidney microsomes with lauric acid analogues (11- or 10-dodecynoic acids) resulted in a time- and concentration-dependent loss of lauric acid hydroxylase activities. The induction of these activities in fish by phthalates, which are widely-distributed environmental pollutants, may be taken into consideration for the development of new biomarkers.

Cnidarian–Dinoflagellate Symbiosis-Mediated Adaptation to Environmental Perturbations

The symbiotic relationships between corals and their phototroph dinoflagellate, Symbiodinium sp., are at the basis of the ecology of coral reefs flourishing in tropical waters. While coral reefs present a very high biological and economic value, they are presently under stress due to climate change (seawater temperature increase and acidification). In order to live together, animal hosts and their symbionts have evolved and acquired specific adaptations ranging from trophic exchange to acquisition of new mechanisms of CO2- and N-compound uptake, to hyperoxia tolerance and more. We think that these mutualistic relationships have also produced tolerance towards environmental stress.

ISOLATION OF CYTOCHROME P450 CDNAS (CYP1A1 AND CYP4T2) FROM THE SEA BASS (DICENTRARCHUS LABRAX) : TOOLS FOR BIOMONITORING SEA POLLUTION.

Abstract Cytochrome P450 are key detoxification enzymes which are potentially induced by xenobiotics such as environmental contaminants. Thus, induction features of some cytochrome P450 are used as biomarkers. Many studies use enzyme activities to detect this induction, but this can also be monitored by protein or mRNA quantitation. There are about 80 different cytochrome P450 genes in vertebrates, but only few sequences are available in fish. Family 1, 2 and 3 are now well studied, but mostly in trout. We isolated CYP1A and CYP4T2 sequences in the sea bass Dicentrarchus labrax, which is considered as an excellent sentinel species in the Mediterranean sea. CYP1A isoform is induced by PAHs, PCBs, and dioxins, and some CYP4 isoforms are known to be induced by phthalate esters plasticizers and chlorinated aryl phenoxy herbicides. We characterize the induction of the genes by these environmental contaminants in several organs. This study gives some knowledge about cytochrome P450 gene regulation in the sea bass, which is essential for further use of these genes as biomarkers. Quantitation of mRNA could be used as additional tools to report CYP1A and CYP4T2 induction, and could lead to the development of efficient biomarkers for these class of marine pollutants.