Organisms Diversity & Evolution

, Volume 17, Issue 1, pp 87–99 | Cite as

Examining the retention of functional kleptoplasts and digestive activity in sacoglossan sea slugs

  • Elise M. J. Laetz
  • Peter T. Rühr
  • Thomas Bartolomaeus
  • Angelika Preisfeld
  • Heike Wägele
Original Article


Solar-powered sea slugs (Sacoglossa: Gastropoda) have long captured the attention of laymen and scientists alike due to their remarkable ability to steal functional chloroplasts from their algal food, enslaving them to withstand long starvation periods. Recently, a wealth of data has shed insight into this remarkable relationship; however, the cellular mechanisms governing this process are still completely unknown. This study explores these mechanisms, providing insight into the chloroplast retention and delayed digestion, occurring within the slug’s digestive gland. We examine the relationships between functional chloroplast and lysosome abundances during starvation, in live material, for the long-term retaining species Elysia timida, the ambiguous long/short-term retaining Elysia viridis, and the short-term retaining Thuridilla hopei, to elucidate digestive differences that contribute to the development of functional kleptoplasty. Functional chloroplast and lysosome abundance are measured using chlorophyll a autofluorescence and the pH-dependent stain acridine orange. In each species, the number of chloroplasts and lysosomes is indirectly proportional, with the plastid density decreasing when starvation begins. We also present a new FIJI/Image J Plugin, the 3D—Accounting and Measuring Plugin, 3D-AMP, which enables the reliable analysis of large image sets.


Sacoglossa Kleptoplast Lysosomes Intracellular digestion Acridine orange FIJI Plugin 



We would like to thank Claudia Müller, Jörn van Döhren, Ekin Tilic, Daria Krämer, Albert Haas, Gregor Christa, Cessa Rauch, Jan de Vries, and Sven Gould and Ulf Bickmeyer for their expertise and help in this project. We also appreciate the feedback we received from our two anonymous reviewers.

This study was funded by the Deutsche Forschungsgemeinschaft project Wa 618/17, the Alexander Koenig Gesellschaft, and a personal grant to EMJL from the University of Bonn, Germany.

Author contributions

EMJL and HW devised the project concept and EMJL conducted the lab work. PTR and EMJL designed 3D-AMP and PTR wrote this software. AP and TB help troubleshoot staining and all authors contributed to the analysis and manuscript.

Compliance with ethical standards

Ethical statement

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.


  1. Baumgartner, F. A., Pavia, H., & Toth, G. B. (2015). Acquired phototrophy through retention of functional chloroplasts increases growth efficiency of the sea slug Elysia viridis. PLoS ONE, 10(4), e0120874. doi: 10.1371%2Fjournal.pone.0120874.CrossRefPubMedPubMedCentralGoogle Scholar
  2. Christa, G. (2014). Functional kleptoplasty in a limapontiodean genus: phylogeny, food preferences, and photosynthesis in costasiella with a focus on C. ocellifera (Gastropoda: Sacoglossa). Journal of Molluscan Studies.Google Scholar
  3. Christa, G., Wescott, L., Schäberle, T. F., König, G. M., & Wägele, H. (2013a). What remains after 2 months of starvation? Analysis of sequestered algae in a photosynthetic slug, Plakobranchus ocellatus (Sacoglossa, Opisthobranchia), by barcoding. Planta, 237(2), 559–572. doi: 10.1007/s00425-012-1788-6.CrossRefPubMedGoogle Scholar
  4. Christa, G., Zimorski, V., Woehle, C., Tielens, A. G. M., Wägele, H., Martin, W. F., & Gould, S. B. (2013b). Plastid-bearing sea slugs fix CO2 in the light but do not require photosynthesis to survive. Proceedings. Biological sciences / The Royal Society, 281(1774), 20132493.
  5. Christa, G., Händeler, K., Kück, P., Vleugels, M., Franken, J., Karmeinski, D., & Wägele, H. (2014a). Phylogenetic evidence for multiple independent origins of functional kleptoplasty in Sacoglossa (Heterobranchia, Gastropoda). Organisms Diversity & Evolution, 15(1), 23–36.CrossRefGoogle Scholar
  6. Christa, G., Händeler, K., Schäberle, T. F., König, G. M., & Wägele, H. (2014b). Identification of sequestered chloroplasts in photosynthetic and non-photosynthetic sacoglossan sea slugs (Mollusca, Gastropoda). Frontiers in Zoology, 11(5).Google Scholar
  7. Curtis, N. E., Massey, S. E., & Pierce, S. K. (2006). The symbiotic chloroplasts in the sacoglossan Elysia clarki are from several algal species. Invertebrate Biology, 125(4), 336–345.CrossRefGoogle Scholar
  8. Curtis, N. E., Middlebrooks, M. L., Schwartz, J. A., & Pierce, S. K. (2015). Kleptoplastic sacoglossan species have very different capacities for plastid maintenance despite utilizing the same algal donors. Symbiosis, 65(1), 23–31.CrossRefGoogle Scholar
  9. De Duve, C. (1963). The lysosome concept. In Ciba Foundation Symposium-Lysosomes (pp. 1–35). Wiley Online Library.Google Scholar
  10. de Vries, J., Christa, G., Gould, SB. (2014). Plastid survival in the cytosol of animal cells. Trends in Plant Science 19(6):347–50. doi: 10.1016/j.tplants.2014.03.010.
  11. de Vries, J., Woehle, C., Christa, G., Wägele, H., Tielens, A. G. M., Jahns, P., & Gould, S. B. (2015). Comparison of sister species identifies factors underpinning plastid compatibility in green sea slugs. Proceedings of the Royal Society of London B: Biological Sciences, 282(1802).
  12. Driessche, T. V. (1966). Circadian rhythms in Acetabularia: photosynthetic capacity and chloroplast shape. Experimental Cell Research, 42(1), 18–30.CrossRefGoogle Scholar
  13. Gallop, A., Bartrop, J., & Smith, D. C. (1980). The biology of chloroplast acquisition by Elysia viridis. Proceedings of the Royal Society of London. Series B. Biological Sciences, 207(1168), 335–349.CrossRefGoogle Scholar
  14. Greene, R. W. (1970). Symbiosis in sacoglossan opisthobranchs: functional capacity of symbiotic chloroplasts. Marine Biology, 7(2), 138–142. doi: 10.1007/BF00354917.CrossRefGoogle Scholar
  15. Händeler, K., Grzymbowski, Y. P., Krug, P. J., & Wägele, H. (2009). Functional chloroplasts in metazoan cells—a unique evolutionary strategy in animal life. Frontiers in Zoology, 6(1), 28.CrossRefPubMedPubMedCentralGoogle Scholar
  16. Johnson, M. D. (2011). The acquisition of phototrophy: adaptive strategies of hosting endosymbionts and organelles. Photosynthesis Research, 107(1), 117–132.CrossRefPubMedGoogle Scholar
  17. Kusuzaki, K., Matsubara, T., Satonaka, H., Matsumine, A., Nakamura, T., Sudo, A., et al. (2014). Intraoperative Photodynamic Surgery (iPDS) with acridine orange for musculoskeletal sarcomas. Cureus, 6(9).Google Scholar
  18. Lindholm, T., & Mork, A.-C. (1989). Symbiotic algae and plastids in planktonic ciliates. Memoranda Societatis pro Fauna et Flora Fennica, 65(1), 17–22.Google Scholar
  19. Martin, R., Walther, P., & Tomaschko, K.-H. (2013). Phagocytosis of algal chloroplasts by digestive gland cells in the photosynthesis-capable slug Elysia timida (Mollusca, Opisthobranchia, Sacoglossa). Zoomorphology, 132(3), 253–259. doi: 10.1007/s00435-012-0184-x.CrossRefGoogle Scholar
  20. McLean, N. (1976). Phagocytosis of chloroplasts in Placida dendritica (Gastropoda: Sacoglossa). Journal of Experimental Zoology, 197(3), 321–329.CrossRefGoogle Scholar
  21. Moriyama, Y., Takano, T., & Ohkuma, S. (1982). Acridine orange as a fluorescent probe for lysosomal proton pump. Journal of Biochemistry, 92(4), 1333–1336.
  22. Pelletreau, K. N., Weber, A. P. M., Weber, K. L., & Rumpho, M. E. (2014). Lipid accumulation during the establishment of kleptoplasty in Elysia chlorotica. PLoS ONE, 9(5), e97477. doi: 10.1371/journal.pone.0097477.CrossRefPubMedPubMedCentralGoogle Scholar
  23. Rauch, C., de Vries, J., Rommel, S., Rose, L. E., Woehle, C., Christa, G., et al. (2015). Why it is time to look beyond algal genes in photosynthetic slugs. Genome Biology and Evolution, 7(9), 2602–2607.CrossRefPubMedPubMedCentralGoogle Scholar
  24. Rumpho, M. E., Summer, E. J., & Manhart, J. R. (2000). Solar-powered sea slugs. Mollusc/algal chloroplast symbiosis. Plant Physiology, 123(1), 29–38.CrossRefPubMedPubMedCentralGoogle Scholar
  25. Schmitt, V. (2011). Behavioral adaptations in relation to long-term retention of endosymbiotic chloroplasts in the Sea Slug Elysia timida. Thalassas, 27(2), 225–238.Google Scholar
  26. Schmitt, V., Händeler, K., Gunkel, S., Escande, M.-L., Menzel, D., Gould, S. B., et al. (2014). Chloroplast incorporation and long-term photosynthetic performance through the life cycle in laboratory cultures of Elysia timida (Sacoglossa, Heterobranchia). Frontiers in Zoology, 11(1), 5. doi: 10.1186/1742-9994-11-5.CrossRefPubMedPubMedCentralGoogle Scholar
  27. Stoecker, D. K., Johnson, M. D., de Vargas, C., & Not, F. (2009). Acquired phototrophy in aquatic protists.Google Scholar
  28. Taylor, D. L. (1968). Chloroplasts as symbiotic organelles in the digestive gland of Elysia viridis [Gastropoda: opisthobranchia]. Journal of the Marine Biological Association of the United Kingdom, 48(01), 1–15. doi: 10.1017/S0025315400032380.CrossRefGoogle Scholar
  29. Ventura, P., Calado, G., & Jesus, B. (2013). Photosynthetic efficiency and kleptoplast pigment diversity in the sea slug Thuridilla hopei (Vérany, 1853). Journal of Experimental Marine Biology and Ecology, 441, 105–109. doi: 10.1016/j.jembe.2013.01.022.CrossRefGoogle Scholar
  30. Wägele, H., & Johnsen, G. (2001). Observations on the histology and photosynthetic performance of “solar-powered” opisthobranchs (Mollusca, Gastropoda, Opisthobranchia) containing symbiotic chloroplasts or zooxanthellae. Organisms Diversity & Evolution, 1(3), 193–210. doi: 10.1078/1439-6092-00016.CrossRefGoogle Scholar
  31. Wägele, H., & Martin, W. (2014). Endosymbioses in sacoglossan seaslugs: plastid-bearing animals that keep photosynthetic organelles without borrowing genes. In W. Löffelhardt (Ed.), Endosymbiosis SE - 14 (pp. 291–324). Vienna: Springer. doi: 10.1007/978-3-7091-1303-5_14.CrossRefGoogle Scholar
  32. Wägele, H., Deusch, O., Händeler, K., Martin, R., Schmitt, V., Christa, G., et al. (2011). Transcriptomic evidence that longevity of acquired plastids in the photosynthetic slugs Elysia timida and Plakobranchus ocellatus does not entail lateral transfer of algal nuclear genes. Molecular Biology and Evolution, 28(1), 699–706. doi: 10.1093/molbev/msq239.CrossRefPubMedGoogle Scholar

Copyright information

© Gesellschaft für Biologische Systematik 2016

Authors and Affiliations

  • Elise M. J. Laetz
    • 1
    • 2
  • Peter T. Rühr
    • 1
  • Thomas Bartolomaeus
    • 2
  • Angelika Preisfeld
    • 3
  • Heike Wägele
    • 1
  1. 1.Zoological Research Museum Alexander KoenigBonnGermany
  2. 2.Institute of Evolutionary Biology and Animal EcologyRheinische Friedrich-Wilhelms-Universität BonnBonnGermany
  3. 3.Bergische University WuppertalFaculty of Mathematics and SciencesWuppertalGermany

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