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Laboratory culturing of Elysia chlorotica reveals a shift from transient to permanent kleptoplasty

Abstract

The kleptoplastic sacoglossan Elysia chlorotica shares a requisite, intracellular symbiosis with the plastids (= chloroplasts) of the Xanthophyte alga Vaucheria litorea. Although wild specimens have been used to address a range of biological questions, no studies have thoroughly characterized animal development during the initial establishment of the symbiosis under controlled laboratory conditions. Laboratory culture conditions were modified and the time required for successful metamorphosis was reduced by 40 % relative to previous work. Plastids were not initially stable within the host; “permanent kleptoplasty” was obtained only after ≥7 days of feeding on V. litorea. Feeding for shorter time periods resulted in the loss of plastids and abnormal development; this phase was characterized as “transient kleptoplasty”. Individuals in the transient state exhibited a significantly greater decrease in length compared to animals with permanent kleptoplasts after the same starvation period. To test the effect of food availability after obtaining permanent kleptoplasty, animals were subjected to various dietary regimes followed by a recovery period of constant feeding. Thirty percent of animals survived prolonged starvation (>4 weeks) after only the initial week of feeding required to establish permanent kleptoplasty. All treatments showed rapid growth when re-exposed to Vaucheria. Thus, during initial development E. chlorotica experiences enhanced fitness when Vaucheria is available for consumption. However, the animal rapidly establishes permanent kleptoplasty, which bestows flexible food requirements and resistance to food limitation, a likely advantage for E. chlorotica in salt marsh environments where Vaucheria sp. abundance is sporadic.

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References

  1. Capo TR, Bardales AT, Gillette PR, Lara MR, Schmale MC, Serafy JE (2009) Larval growth, development, and survival of laboratory-reared Aplysia californica: effects of diet and veliger density. Comp Biochem Physiol C 149(2):215–223

    Google Scholar 

  2. Clark KB, Jensen KR, Strits HM (1990) Survey for functional kleptoplasty among west Atlantic Ascoglossa (Sacoglossa) (Mollusca: Opisthobranchia). Veliger 33:339–345

    Google Scholar 

  3. Davis GM, Mazurkiewicz M, Mandracchia M (1982) Spurwinkia—morphology, systematics, and ecology of a new genus of North American marshland Hydrobiidae (mollusca, gastropoda). Proc Acad Nat Sci Phila 134:143–177

    Google Scholar 

  4. de Negri A, de Negri G (1876) Ber Deut Chem Ges Berl 9:84

    Google Scholar 

  5. Devine SP, Pelletreau KN, Rumpho ME (2012) 16S rDNA-based metagenomic analysis of bacterial diversity associated with two populations of the kleptoplastic sea slug Elysia chlorotica and its algal prey Vaucheria litorea. Biol Bull 223(1):138–154

    PubMed  CAS  Google Scholar 

  6. Evertsen J, Burghardt I, Johnsen G, Wägele H (2007) Retention of functional chloroplasts in some sacoglossans from the Indo-pacific and Mediterranean. Mar Biol 151:2159–2166

    Article  Google Scholar 

  7. Fujita T, Matsushita M, Endo Y (2004) The lectin-complement pathway—its role in innate immunity and evolution. Immunol Rev 198:185–202

    PubMed  Article  CAS  Google Scholar 

  8. Gordon AH, Darcy Hart P, Young MR (1980) Ammonia inhibits phagosome-lysosome fusion in macrophages. Nature 286(5768):79–80

    PubMed  Article  CAS  Google Scholar 

  9. Goren MB (1977) Phagocyte lysosomes—interactions with infectious agents, phagosomes, and experimental perturbations in function. Annu Rev Microbiol 31:507–533

    PubMed  Article  CAS  Google Scholar 

  10. Green BJ, Li WY, Manhart JR, Fox TC, Summer EJ, Kennedy RA, Pierce SK, Rumpho ME (2000) Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus. Plant Physiol 124(1):331–342

    PubMed  Article  CAS  Google Scholar 

  11. Green BJ, Fox TC, Rumpho ME (2005) Stability of isolated algal chloroplasts that participate in a unique mollusc/kleptoplast association. Symbiosis 40(1):31–40

    Google Scholar 

  12. Gross J, Bhattacharya D, Pelletreau KN, Rumpho ME, Reyes-Prieto A (2012) Secondary and tertiary endosymbiosis and kleptoplasty. In: Bock R, Knoop V, (eds) Advances in photosynthesis and respiration—Genomics of chloroplasts and mitochondria. Springer Science+Business Media B.V., 35, pp 000–000, doi:10.1007/978-94-007-2920-9_2

  13. Händeler K, Grzymbowski YP, Krug PJ, Wägele H (2009) Functional chloroplasts in metazoan cells—a unique strategy in animal life. Front Zool 6:28

    PubMed  Article  Google Scholar 

  14. Harrigan JF, Alkon DL (1978) Laboratory cultivation of Haminoea solitaria (Say, 1822) and Elysia chlorotica (Gould, 1870). Veliger 21(2):299–305

    Google Scholar 

  15. Hart PDA (1979) Phagosome-lysosome fusion in macrophages: a hinge in the intracellular fate of ingested microorganisms. Front Biol 48:409–423

    Google Scholar 

  16. Hibino T, Loza-Coll M, Messier C, Majeske AJ, Cohen AH, Terwilliger DP, Buckley KM, Brockton V, Nair SV, Berney K, Fugmann SD, Anderson MK, Pancer Z, Cameron RA, Smith LC, Rast JP (2006) The immune gene repertoire encoded in the purple sea urchin genome. Dev Biol 300(1):349–365

    PubMed  Article  CAS  Google Scholar 

  17. Hohman TC, McNeil PL, Muscatine L (1982) Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis. J Cell Biol 94(1):56–63

    PubMed  Article  CAS  Google Scholar 

  18. Jones TC, Hirsch JG (1972) Interactions between Toxoplasma gondii and mammalian cells 2. Absence of lysosomal fusion with phagocytic vacuoles containing living parasites. J Exp Med 136(5):1173–1194

    PubMed  Article  CAS  Google Scholar 

  19. Karakashian SJ, Rudzinska MA (1981) Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria. Exp Cell Res 131(2):387–393

    PubMed  Article  CAS  Google Scholar 

  20. Kawaguti S, Yamasu T (1965) Electron microscopy on the symbiosis between and elysioid gastropod and chloroplasts of a green alga. Biol J Okayama Univ 11:57–65

    Google Scholar 

  21. Kodama Y, Fujishima M (2010) Secondary symbiosis between Paramecium and Chlorella cells. Int Rev Cell Mol Biol 279:33–77

    PubMed  Article  CAS  Google Scholar 

  22. Kriegstein AR, Castellucci V, Kandel ER (1974) Metamorphosis of Aplysia californica in laboratory culture. Proc Natl Acad Sci USA 71(9):3654–3658

    PubMed  Article  CAS  Google Scholar 

  23. Krug PJ (1998) Poecilogony in an estuarine opisthobranch: planktotrophy, lecithotrophy, and mixed clutches in a population of the ascoglossan Alderia modesta. Mar Biol 132(3):483–494

    Article  Google Scholar 

  24. Krug PJ (2007) Poecilogony and larval ecology in the gastropod genus Alderia. Am Malacol Bull 23(1–2):99–111

    Article  Google Scholar 

  25. McFall-Ngai M, Nyholm SV, Castillo MG (2010) The role of the immune system in the initiation and persistence of the Euprymna scolopes-Vibrio fischeri symbiosis. Semin Immunol 22(1):48–53

    PubMed  Article  CAS  Google Scholar 

  26. Messier-Solek C, Buckley KM, Rast JP (2010) Highly diversified innate receptor systems and new forms of animal immunity. Semin Immunol 22(1):39–47

    PubMed  Article  CAS  Google Scholar 

  27. Mondy WL, Pierce SK (2003) Apoptotic-like morphology is associated with annual synchronized death in kleptoplastic sea slugs (Elysia chlorotica). Invert Biol 122(2):126–137

    Article  Google Scholar 

  28. Müjer CV, Andrews DL, Manhart JR, Pierce SK, Rumpho ME (1996) Chloroplast genes are expressed during intracellular symbiotic association of Vaucheria litorea plastids with the sea slug Elysia chlorotica. Proc Natl Acad Sci USA 93(22):12333–12338

    PubMed  Article  Google Scholar 

  29. Muscatine L, McNeil PL (1989) Endosymbiosis in Hydra and the evolution of internal defense systems. Am Zool 29(2):371–386

    Google Scholar 

  30. Nyholm SV, McFall-Ngai MJ (2004) The winnowing: establishing the squid-Vibrio symbiosis. Nat Rev Microbiol 2(8):632–642

    PubMed  Article  CAS  Google Scholar 

  31. Nyholm SV, Stewart JJ, Ruby EG, McFall-Ngai MJ (2009) Recognition between symbiotic Vibrio fischeri and the haemocytes of Euprymna scolopes. Environ Microbiol 11(2):483–493

    PubMed  Article  Google Scholar 

  32. Pelletreau KN, Bhattacharya D, Price DC, Worful JM, Moustafa A, Rumpho ME (2011) Sea slug kleptoplasty and plastid maintenance in a metazoan. Plant Physiol 155(4):1561–1565

    PubMed  Article  CAS  Google Scholar 

  33. Pethe K, Swenson DL, Alonso S, Anderson J, Wang C, Russell DG (2004) Isolation of Mycobacterium tuberculosis mutants defective in the arrest of phagosome maturation. Proc Natl Acad Sci USA 101(37):13642–13647

    PubMed  Article  CAS  Google Scholar 

  34. Pierce SK, Curtis NE (2012) Cell biology of the chloroplast symbiosis in sacoglossan sea slugs. In: Jeon KW (ed) International review of cell and molecular biology, 293. Academic, Burlington, pp 123–148

    Chapter  Google Scholar 

  35. Pierce SK, Biron RW, Rumpho ME (1996) Endosymbiotic chloroplasts in molluscan cells contain proteins synthesized after plastid capture. J Exp Biol 199:2323–2330

    PubMed  CAS  Google Scholar 

  36. Pierce SK, Maugel TK, Rumpho ME, Hanten JJ, Mondy WL (1999) Annual viral expression in a sea slug population: life cycle control and symbiotic chloroplast maintenance. Biol Bull 197(1):1–6

    Article  Google Scholar 

  37. Pierce SK, Massey SE, Hanten JJ, Curtis NE (2003) Horizontal transfer of functional nuclear genes between multicellular organisms. Biol Bull 204(3):237–240

    PubMed  Article  Google Scholar 

  38. Pierce SK, Curtis NE, Hanten JJ, Boerner SL, Schwartz JA (2007) Transfer, integration and expression of functional nuclear genes between multicellular species. Symbiosis 43(2):57–64

    CAS  Google Scholar 

  39. Pierce SK, Curtis NE, Schwartz JA (2009) Chlorophyll a synthesis by an animal using transferred algal nuclear genes. Symbiosis 49(3):121–131

    Article  CAS  Google Scholar 

  40. Plaut I, Borut A, Spira ME (1995) Growth and metamorphosis of Aplysia oculifera larvae in laboratory culture. Mar Biol 122(3):425–430

    Article  Google Scholar 

  41. Pluddemann A, Mukhopadhyay S, Gordon S (2011) Innate immunity to intracellular pathogens: macrophage receptors and responses to microbial entry. Immunol Rev 240:11–24

    PubMed  Article  CAS  Google Scholar 

  42. Rast JP, Messier-Solek C (2008) Marine invertebrate genome sequences and our evolving understanding of animal immunity. Biol Bull 214(3):274–283

    PubMed  Article  CAS  Google Scholar 

  43. Rast JP, Smith LC, Loza-Coll M, Hibino T, Litman GW (2006) Review—genomic insights into the immune system of the sea urchin. Science 314(5801):952–956

    PubMed  Article  CAS  Google Scholar 

  44. Rosenstiel P, Philipp ER, Schreiber S, Bosch TG (2009) Evolution and function of innate immune receptors—insights from marine invertebrates. J Innate Immun 1(4):291–300

    PubMed  Article  CAS  Google Scholar 

  45. Rowley AF, Powell A (2007) Invertebrate immune systems-specific, quasi-specific, or nonspecific? J Immunol 179(11):7209–7214

    PubMed  CAS  Google Scholar 

  46. Rumpho ME, Summer EJ, Manhart JR (2000) Solar-powered sea slugs. Mollusc/algal chloroplast symbiosis. Plant Physiol 123(1):29–38

    PubMed  Article  CAS  Google Scholar 

  47. Rumpho ME, Summer EJ, Green BJ, Fox TC, Manhart JR (2001) Mollusc/algal chloroplast symbiosis: how can isolated chloroplasts continue to function for months in the cytosol of a sea slug in the absence of an algal nucleus? Zoology 104(3–4):303–312

    PubMed  Article  CAS  Google Scholar 

  48. Rumpho ME, Dastoor FP, Manhart JR, Lee J (2006) The kleptoplast. In: Wise RR, Hoober JK (eds) Advances in photosynthesis and respiration: the structure and function of plastids. Springer, Dordrecht, pp 451–473

    Chapter  Google Scholar 

  49. Rumpho ME, Worful JM, Lee J, Kannan K, Tyler MS, Bhattacharya D, Moustafa A, Manhart JR (2008) Horizontal gene transfer of the algal nuclear gene psbO to the photosynthetic sea slug Elysia chlorotica. Proc Natl Acad Sci USA 105(46):17867–17871

    PubMed  Article  CAS  Google Scholar 

  50. Rumpho ME, Pochareddy S, Worful JM, Summer EJ, Bhattacharya D, Pelletreau KN, Tyler MS, Lee J, Manhart JR, Soule KM (2009) Molecular characterization of the Calvin cycle enzyme phosphoribulokinase in the stramenopile alga Vaucheria litorea and the plastid hosting mollusc Elysia chlorotica. Mol Plant 2(6):1384–1396

    PubMed  Article  CAS  Google Scholar 

  51. Rumpho ME, Pelletreau KN, Moustafa A, Bhattacharya D (2011) The making of a photosynthetic animal. J Exp Biol 214:303–311

    PubMed  Article  Google Scholar 

  52. Russell DG, Mwandumba HC, Rhoades EE (2002) Mycobacterium and the coat of many lipids. J Cell Biol 158(3):421–426

    PubMed  Article  CAS  Google Scholar 

  53. Schwartz JA, Curtis NE, Pierce SK (2010) Using algal transcriptome sequences to identify transferred genes in the sea slug, Elysia chlorotica. Evol Biol 37(1):29–37

    Article  Google Scholar 

  54. Soule KM, Rumpho ME (2012) Light-regulated photosynthetic gene expression and phosphoribulokinase enzyme activity in the heterokont alga Vaucheria litorea (Xanthophyceae) and its symbiotic molluscan partner Elysia chlorotica (Gastropoda). J Phyc 48(2):373–383

    Article  CAS  Google Scholar 

  55. Switzer-Dunlap M, Hadfield MG (1977) Observations on development, larval growth and metamorphosis of four species of Aplysiidae (Gastropoda: Opisthobranchia). J Exp Mar Biol Ecol 29:245–261

    Article  Google Scholar 

  56. Trench RK (1975) Of ‘leaves that crawl’: functional chloroplasts in animal cells. In: Jennings DH (ed) Symp Soc Exp Biol, Cambridge University Press, London, pp 229–265

  57. Trench RK, Trench ME, Muscatine L (1972) Symbiotic chloroplasts—their photosynthetic products and contribution to mucus synthesis in two marine slugs. Biol Bull 142(2):335–349

    PubMed  Article  CAS  Google Scholar 

  58. Trowbridge CD (2000) The missing links: larval and post-larval development of the ascoglossan opisthobranch Elysia viridis. J Mar Biol Assoc UK 80(6):1087–1094

    Article  Google Scholar 

  59. Vergne I, Chua J, Singh SB, Deretic V (2004) Cell biology of Mycobacterium tuberculosis phagosome. Annu Rev Cell Dev Biol 20:367–394

    PubMed  Article  CAS  Google Scholar 

  60. West HH (1979) Chloroplast symbiosis and development of the ascoglossan opistobranch Elysia chlorotica. PhD thesis, Northeastern University, Boston, MA

  61. West HH, Harrigan J (1979) Symbiosis and development in two populations of Elysia chlorotica. Am Zool 19(3):958

    Google Scholar 

  62. West HH, Harrigan JF, Pierce SK (1984) Hybridization of two populations of a marine opisthobranch with different developmental patterns. Veliger 26(3):199–206

    Google Scholar 

  63. Worful JM (2008) Elysia chlorotica (Gould, 1870): towards the development of a novel system for the elucidation of horizontal gene transfer, invertebrate developmental biology and secondary metabolites. M.S Thesis, University of Maine

  64. Yamamoto YY, Yusa Y, Yamamoto S, Hirano Y, Hirano Y, Motomura T, Tanemura T, Obokata J (2009) Identification of photosynthetic sacoglossans from Japan. Encocytobiosis Cell Res 19:112–119

    Google Scholar 

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Acknowledgements

We would like to acknowledge Geoffry Davis who aided in the E. chlorotica culture, Kathryn Dutil who maintained the V. litorea cultures, and Søren Hanson who provided the I. galbana cultures. We would also like to thank the reviewers of this paper for providing constructive suggestions. This research was supported by the National Science Foundation (grant IOS-0726178 to M.E.R.). This is Maine Agricultural and Forest Experiment Station Publication Number 3297, Hatch Project no. ME08361-08MRF (NC 1168).

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Correspondence to Karen N. Pelletreau.

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Pelletreau, K.N., Worful, J.M., Sarver, K.E. et al. Laboratory culturing of Elysia chlorotica reveals a shift from transient to permanent kleptoplasty. Symbiosis 58, 221–232 (2012). https://doi.org/10.1007/s13199-012-0192-0

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Keywords

  • Elysia chlorotica
  • Vaucheria litorea
  • Kleptoplasty
  • Symbiosis
  • Invertebrate development