Advertisement

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

  • 578 Accesses

  • 3 Citations

Abstract

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.

This is a preview of subscription content, log in to check access.

Access options

Buy single article

Instant unlimited access to the full article PDF.

US$ 39.95

Price includes VAT for USA

Subscribe to journal

Immediate online access to all issues from 2019. Subscription will auto renew annually.

US$ 199

This is the net price. Taxes to be calculated in checkout.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

References

  1. Apte S, Khoury F, Roth W, Schlichter D (1996) Transport of amino acids into freshly isolated cells from a sea anemone. Endocytobiosis Cell Res 11:129–146

  2. Domart-Coulon IJ, Elbert DC, Scully EP, Calimlim PS, Ostrander GK (2001) Aragonite crystallization in primary cell cultures of multicellular isolates from a hard coral, Pocillopora damicornis. Proc Natl Acad Sci USA 98:11885–11890

  3. Domart-Coulon I, Tambutte S, Tambutte E, Allemand D (2004) Short term viability of soft tissue detached from the skeleton of reef-building corals. J Exp Mar Biol Ecol 309:199–217

  4. Frank U, Rinkevich B (1999) Scyphozoan jellyfish’s mesoglea supports attachment, spreading and migration of anthozoans’ cells in vitro. Cell Biol Int 23:307–311

  5. Frank U, Rabinowitz C, Rinkevich B (1994) In vitro establishment of continuous cell cultures and cell lines from ten colonial cnidarians. Mar Biol 120:491–499

  6. Furla P, Bénazet-Tambutté S, Jaubert J, Allemand D (1998) Functional polarity of the tentacle of the sea anemone Anemonia viridis: role in inorganic carbon acquisition. Am J Physiol 274:303–310

  7. Furla P, Allemand D, Orsenigo MN (2000) Involvement of H(+)-ATPase and carbonic anhydrase in inorganic carbon uptake for endosymbiont photosynthesis. Am J Physiol Regul Integr Comp Physiol 278:870–881

  8. Ganot P, Moya A, Magnone V, Allemand D, Furla P, Sabourault C (2011) Adaptations to endosymbiosis in a cnidarian-dinoflagellate association: differential gene expression and specific gene duplications. PLoS Genet 7:e1002187

  9. Gates RD, Muscatine L (1992) Three methods for isolating viable anthozoan endoderm cells with their intracellular symbiotic dinoflagellates. Coral Reefs 11:143–145

  10. Kopecky EJ, Ostander G (1999) Isolation and primary culture of viable multicellular endothelial isolates from hard corals. In Vitro Cell Dev Biol Anim 35:616–624

  11. Merle PL, Sabourault C, Richier S, Allemand D, Furla P (2007) Catalase characterization and implication in bleaching of a symbiotic sea anemone. Free Radic Biol Med 42:236–246

  12. Moya A, Ganot P, Furla P, Sabourault C (2012) The transcriptomic response to thermal stress is immediate, transient and potentiated by ultraviolet radiation in the sea anemone Anemonia viridis. Mol Ecol 21:1158–1174

  13. Richier S, Merle PL, Furla P, Pigozzi D, Sola F, Allemand D (2003) Characterization of superoxide dismutases in anoxia- and hyperoxia-tolerant symbiotic cnidarians. Biochim Biophys Acta 162:84–91

  14. Richier S, Sabourault C, Courtiade J, Zucchini N, Allemand D, Furla P (2006) Oxidative stress and apoptotic events during thermal stress in the symbiotic sea anemone, Anemonia viridis. FEBS J 273:4186–4198

  15. Rinkevich B (1999) Cell cultures from marine invertebrates: obstacles, new approaches and recent improvements. J Biotechnol 170:133–153

  16. Rinkevich B (2005) Marine invertebrate cell cultures: new millennium trends. Mar Biotechnol 7:429–439

  17. Rinkevich B (2011) Cell cultures from marine invertebrates: new insights for capturing endless stemness. Mar Biotechnol 13:345–354

  18. Roberts JM, Davies PS, Fixter LM, Preston T (1999) Primary site and initial products of ammonium assimilation in the symbiotic sea anemone Anemonia viridis. Mar Biol 135:223–236

  19. Sabourault C, Ganot P, Deleury E, Allemand D, Furla P (2009) Comprehensive EST analysis of the symbiotic sea anemone, Anemonia viridis. BMC Genomics 10:333

  20. Savage AM, Goodson MS, Visram S, Trapido-Rosenthal H, Wiedenmann J, Douglas AE (2002) Molecular diversity of symbiotic algae at the latitudinal margins of their distribution: dinoflagellates of the genus Symbiodinium in corals and sea anemones. Mar Ecol Prog Ser 244:17–26

  21. Zamoum T, Furla P (2012) Symbiodinium isolation by NaOH treatment. J Exp Biol 215:3875–3880

Download references

Acknowledgments

S.B.V. is grateful to Drs. Philippe Ganot and Cécile Sabourault for npc1 primers gift and PCR technical advice. Authors are also grateful to Brigitte Poderini, for sea anemone maintenance.

Author information

Correspondence to Stéphanie Barnay-Verdier.

Additional information

Nathalie Joli and Juliette Olivré contributed equally to this work.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Barnay-Verdier, S., Dall’Osso, D., Joli, N. et al. Establishment of primary cell culture from the temperate symbiotic cnidarian, Anemonia viridis . Cytotechnology 65, 697–704 (2013) doi:10.1007/s10616-013-9566-2

Download citation

Keywords

  • Cnidarian
  • Primary cell culture
  • Symbiosis