Paola Furla

Symbiosis-induced adaptation to oxidative stress.

SUMMARY 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% O2) or to experimental thermal stress (elevated temperature +7°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 O2 partial pressure (PO2) 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.

The Symbiotic Anthozoan: A Physiological Chimera between Alga and Animal.

Abstract 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 symbionts 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.

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.

Characterization of superoxide dismutases in anoxia- and hyperoxia-tolerant symbiotic cnidarians

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.

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.

Functional polarity of the tentacle of the sea anemone Anemonia viridis: role in inorganic carbon acquisition

The oral epithelial layers of anthozoans have a polarized morphology: photosynthetic endosymbionts live within endodermal cells facing the coelenteric cavity and are separated from the external seawater by the ectodermal layer and the mesoglea. To study if this morphology plays a role in the supply of inorganic carbon for symbiont photosynthesis, we measured the change in pH and the rate of OH- (H+) fluxes induced by each cell layer on a tentacle of the sea anemone Anemonia viridis. Light-induced pH increase of the medium bathing the endodermal layers led to the generation of a transepithelial pH gradient of approximately 0.8 pH units across the tentacle, whereas darkness induced acidification of this medium. The light-induced pH change was associated with an increase of total alkalinity. Only the endodermal layer was able to induce a net OH- secretion (H+ absorption). The light-induced OH- secretion by the endodermal cell layer was dependent on the presence of HCO3- in the compartment facing the ectoderm and was sensitive to several inhibitors of ion transport. [14C] HCO3- incorporation into photosynthates confirmed the ectodermal supply, the extent of which varied from 25 to > 90%, according to HCO3- availability. Our results suggest that the light-induced OH- secretion by the endodermal cell layer followed the polarized transport of HCO3- and its subsequent decarboxylation within the endodermal cell layer. This polarity may play a significant role both in inorganic carbon absorption and in the control of light-enhanced calcification in scleractinian corals.The oral epithelial layers of anthozoans have a polarized morphology: photosynthetic endosymbionts live within endodermal cells facing the coelenteric cavity and are separated from the external seawater by the ectodermal layer and the mesoglea. To study if this morphology plays a role in the supply of inorganic carbon for symbiont photosynthesis, we measured the change in pH and the rate of OH-(H+) fluxes induced by each cell layer on a tentacle of the sea anemone Anemonia viridis. Light-induced pH increase of the medium bathing the endodermal layers led to the generation of a transepithelial pH gradient of ∼0.8 pH units across the tentacle, whereas darkness induced acidification of this medium. The light-induced pH change was associated with an increase of total alkalinity. Only the endodermal layer was able to induce a net OH- secretion (H+ absorption). The light-induced OH- secretion by the endodermal cell layer was dependent on the presence of[Formula: see text] in the compartment facing the ectoderm and was sensitive to several inhibitors of ion transport. [14C][Formula: see text]incorporation into photosynthates confirmed the ectodermal supply, the extent of which varied from 25 to >90%, according to[Formula: see text] availability. Our results suggest that the light-induced OH-secretion by the endodermal cell layer followed the polarized transport of [Formula: see text] and its subsequent decarboxylation within the endodermal cell layer. This polarity may play a significant role both in inorganic carbon absorption and in the control of light-enhanced calcification in scleractinian corals.

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.

Symbiodinium isolation by NaOH treatment

SUMMARY The presence of photosynthetic zooxanthellae (dinoflagellates) in the tissue of many cnidarians is the main reason for their ecological success (i.e. coral reefs). It could also be the main cause of their demise, as the worldwide bleaching of reef-building coral is nothing less than the breakdown of this symbiotic association. The stability of this relationship is the principal marker for the biomonitoring of cnidarian health. We have therefore developed a new, simple method to isolate zooxanthellae in a few steps using NaOH solution. The protocol was validated in three symbiotic cnidarian species: a sea anemone, a gorgonian and a coral. Our method allows the isolation of intact and viable zooxanthellae with better yields than classic methods, especially for species with a calcareous skeleton. Moreover, the isolated zooxanthellae were free of host nucleic contaminants, facilitating subsequent specific molecular analyses.

Physiological Adaptation to Symbiosis in Cnidarians

Up to the nineteenth century, cnidarians, among other organisms such as echinoderms and sponges, were classified as zoophytes, or animal – plant. This term, initially used by Wotton in 1552 and later by Linne and Cuvier (Daudin 1926), was only referring at this time to the external shape of the organisms, fixed branched one. This term was abandoned in the twentieth century; it is however curious to note that biological and physiological reasons might support this term. Indeed, Brandt at the end of the nineteenth century showed the presence of photosynthetic algae inside the tissues of these animals. He suggested that these algae were symbiotic and called them zooxanthellae (Brandt 1881; see Perru 2003, for a review). Zooxanthellae belong to the dinoflagellate phylum and were initially considered as a single species, called Symbiodinium microadriaticum (Freudenthal 1962). It was presently shown to be highly diverse and subdivided in Symbiodinium clades (Pochon et al. 2006). Associations of these different clades with their host did not evolve randomly and members of the same cnidarian species generally harbor the same Symbiodinium clade(s) (see review by Coffroth and Santos 2005, Stambler 2010 this book).

General Ecological Aspects of Anthozoan-Symbiodinium Interactions in the Mediterranean Sea

The aim of this chapter is to provide a general overview of the main ecological aspects of Anthozoan-Symbiodinium mutualisms in the Mediterranean Sea. There are reports of at least twelve species of symbiotic anthozans in the basin. These anthozoans establish symbiotic relations with Symbiodinium Temperate A and B2 (Symbiodinium psygmophilum), corresponding to the only two species of Symbiodinium described in the region. A synthesis of the trophic and biochemical aspects of the interaction between Symbiodinum and their cnidarian hosts is given to contribute to the understanding of the mechanisms that maintain this special association. Finally, current knowledge about the ecological importance of this interaction in engineering species is examined. This review is framed to highlight the ecological importance of this symbiotic relationship in ecosystem construction and maintenance on an enclosed, temperate marine basin.