Symbiodinium isolation by NaOH treatment

International audienceThe 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 response of the symbiotic gorgonian Eunicella singularis to a long-term temperature increase

9 pages, 9 figures, 2 tablesIncrease 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°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.This research was supported by the Government of the Principality of Monaco and the Medchange project (www.medchange.org) funded by the Agence Nationale pour la Recherche (ANR).Peer reviewe

Conspicuous morphological differentiation without speciation in Anemonia viridis (Cnidaria, Actiniaria)

Anemonia viridis is a model species for studies of physiological and transcriptomic response to symbiosis and environmental stress (temperature, light, symbiosis breakdown). Five morphs are described in this species, based on morphology, pigment protein content, and major mode of reproduction. Up to now, the taxonomic status of these morphs remains unclear, without clear knowledge of whether the morphological variation amongst the morphs is due to phenotypic plasticity or adaptation. In the present study, we assess the species status of the three most commonly found morphs by coalescent analyses. For this purpose, five markers were designed for genes whose expression is modified under stress (ca2m, duf140, RNAbinding5, tyrK, sym32) and analysed in 34 individuals representing the morphs A. viridis var rufescens, rustica and smaragdina from eight geographic sites (two in the English Channel and six in the Mediterranean Sea). Phylogenetic analyses of individual gene trees showed no clear separation of the morphs. Furthermore, multilocus coalescent analyses using SpedeSTEM and BPP regrouped all the morphs into one species, showing very few genetic differences amongst them. In a further analysis, we checked for clonality amongst 80 individuals of A. viridis var. smaragdina from one geographic site using three microsatellite loci. This morph proved to be as clonal as var. rustica, indicating a similar potential for asexual reproduction

Horizontal acquisition of Symbiodiniaceae in the Anemonia viridis (Cnidaria, Anthozoa) species complex

International audienc

Anemonia viridis primary cell culture: a new tool for cnidarian studies

International audienceno abstrac

Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis

International audienceThe 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 mu m 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

Horizontal acquisition of Symbiodiniaceae in the Anemonia viridis (Cnidaria, Anthozoa) species complex

All metazoans are in fact holobionts, resulting from the association of several organisms, and organismal adaptation is then due to the composite response of this association to the environment. Deciphering the mechanisms of symbiont acquisition in a holobiont is therefore essential to understanding the extent of its adaptive capacities. In cnidarians, some species acquire their photosynthetic symbionts directly from their parents (vertical transmission) but may also acquire symbionts from the environment (horizontal acquisition) at the adult stage. The Mediterranean snakelocks sea anemone, Anemonia viridis (Forskål, 1775), passes down symbionts from one generation to the next by vertical transmission, but the capacity for such horizontal acquisition is still unexplored. To unravel the flexibility of the association between the different host lineages identified in A. viridis and its Symbiodiniaceae, we genotyped both the animal hosts and their symbiont communities in members of host clones in five different locations in the North Western Mediterranean Sea. The composition of within-host-symbiont populations was more dependent on the geographical origin of the hosts than their membership to a given lineage or even to a given clone. Additionally, similarities in host-symbiont communities were greater among genets (i.e. among different clones) than among ramets (i.e. among members of the same given clonal genotype). Taken together, our results demonstrate that A. viridis may form associations with a range of symbiotic dinoflagellates and suggest a capacity for horizontal acquisition. A mixed-mode transmission strategy in A. viridis, as we posit here, may help explain the large phenotypic plasticity that characterizes this anemone.publishe

Resilience to ocean acidification: decreased carbonic anhydrase activity in sea anemones under high pCO2 conditions

Non-calcifying photosynthetic anthozoans have emerged as a group that may thrive under high carbon dioxide partial pressure ( pCO2) conditions via increased productivity. However, the physiological mechanisms underlying this potential success are unclear. Here we investigated the impact of high pCO2 on the dissolved inorganic carbon (DIC) use in the temperate sea anemone Anemonia viridis. We assessed the impacts of long-term exposure to high pCO2, i.e. sampling in situ natural CO2 vents (Vulcano, Italy), and short-term exposure, i.e. during a 3 wk controlled laboratory experiment. We focused on photo-physiological parameters (net photosynthesis rates, chlorophyll a content and Symbiodinium density) and on carbonic anhydrase (CA) activity, an enzyme involved in the energy-demanding process of DIC absorption. Long-term exposure to high pCO2 had no impact on Symbiodinium density and chlorophyll a content. In contrst, short-term exposure to high pCO2 induced a significant reduction in Symbiodinium density, which together with unchanged net photosynthesis resulted in the increase of Symbiodinium productivity per cell. Finally, in both in situ long-term and laboratory short-term exposure to high pCO2, we observed a significant decrease in the CA activity of sea anemones, suggesting a change in DIC use (i.e. from an HCO3- to a CO2 user). This change could enable a shift in the energy budget that may increase the ability of non-calcifying photosynthetic anthozoans to cope with ocean acidification

Resilience to ocean acidification: decreased carbonic anhydrase activity in sea anemones under high pCO2 conditions

Non-calcifying photosynthetic anthozoans have emerged as a group that may thrive under high carbon dioxide partial pressure ( pCO2) conditions via increased productivity. However, the physiological mechanisms underlying this potential success are unclear. Here we investigated the impact of high pCO2 on the dissolved inorganic carbon (DIC) use in the temperate sea anemone Anemonia viridis. We assessed the impacts of long-term exposure to high pCO2, i.e. sampling in situ natural CO2 vents (Vulcano, Italy), and short-term exposure, i.e. during a 3 wk controlled laboratory experiment. We focused on photo-physiological parameters (net photosynthesis rates, chlorophyll a content and Symbiodinium density) and on carbonic anhydrase (CA) activity, an enzyme involved in the energy-demanding process of DIC absorption. Long-term exposure to high pCO2 had no impact on Symbiodinium density and chlorophyll a content. In contrst, short-term exposure to high pCO2 induced a significant reduction in Symbiodinium density, which together with unchanged net photosynthesis resulted in the increase of Symbiodinium productivity per cell. Finally, in both in situ long-term and laboratory short-term exposure to high pCO2, we observed a significant decrease in the CA activity of sea anemones, suggesting a change in DIC use (i.e. from an HCO3- to a CO2 user). This change could enable a shift in the energy budget that may increase the ability of non-calcifying photosynthetic anthozoans to cope with ocean acidification

Conspicuous morphological differentiation without speciation in <i>Anemonia viridis</i> (Cnidaria, Actiniaria)

<p><i>Anemonia viridis</i> is a model species for studies of physiological and transcriptomic response to symbiosis and environmental stress (temperature, light, symbiosis breakdown). Five morphs are described in this species, based on morphology, pigment protein content, and major mode of reproduction. Up to now, the taxonomic status of these morphs remains unclear, without clear knowledge of whether the morphological variation amongst the morphs is due to phenotypic plasticity or adaptation. In the present study, we assess the species status of the three most commonly found morphs by coalescent analyses. For this purpose, five markers were designed for genes whose expression is modified under stress (<i>ca2m</i>, <i>duf140</i>, <i>RNAbinding5</i>, <i>tyrK</i>, <i>sym32</i>) and analysed in 34 individuals representing the morphs <i>A. viridis</i> var <i>rufescens</i>, <i>rustica</i> and <i>smaragdina</i> from eight geographic sites (two in the English Channel and six in the Mediterranean Sea). Phylogenetic analyses of individual gene trees showed no clear separation of the morphs. Furthermore, multilocus coalescent analyses using SpedeSTEM and BPP regrouped all the morphs into one species, showing very few genetic differences amongst them. In a further analysis, we checked for clonality amongst 80 individuals of <i>A. viridis</i> var. <i>smaragdina</i> from one geographic site using three microsatellite loci. This morph proved to be as clonal as var. <i>rustica</i>, indicating a similar potential for asexual reproduction.</p