Planta

, Volume 237, Issue 2, pp 559–572 | Cite as

What remains after 2 months of starvation? Analysis of sequestered algae in a photosynthetic slug, Plakobranchus ocellatus (Sacoglossa, Opisthobranchia), by barcoding

  • Gregor Christa
  • Lily Wescott
  • Till F. Schäberle
  • Gabriele M. König
  • Heike Wägele
Original Article

Abstract

The sacoglossan sea slug, Plakobranchus ocellatus, is a so-called long-term retention form that incorporates chloroplasts for several months and thus is able to starve while maintaining photosynthetic activity. Little is known regarding the taxonomy and food sources of this sacoglossan, but it is suggested that P. ocellatus is a species complex and feeds on a broad variety of Ulvophyceae. In particular, we analysed specimens from the Philippines and starved them under various light conditions (high light, low light and darkness) and identified the species of algal food sources depending on starvation time and light treatment by means of DNA-barcoding using for the first time the combination of two algal chloroplast markers, rbcL and tufA. Comparison of available CO1 and 16S sequences of specimens from various localities indicate a species complex with likely four distinct clades, but food analyses do not indicate an ecological separation of the investigated clades into differing foraging strategies. The combined results from both algal markers suggest that, in general, P. ocellatus has a broad food spectrum, including members of the genera Halimeda, Caulerpa, Udotea, Acetabularia and further unidentified algae, with an emphasis on H. macroloba. Independent of the duration of starvation and light exposure, this algal species and a further unidentified Halimeda species seem to be the main food source of P. ocellatus from the Philippines. It is shown here that at least two (or possibly three) barcode markers are required to cover the entire food spectrum in future analyses of Sacoglossa.

Keywords

Chlorophyta DNA-barcoding Kleptoplasty Photosynthesis Rbcl TufA 

Abbreviations

DT

Dark treatment

LT

Low light intensity treatment

HT

High light intensity treatment

LTR

Long-term retention of chloroplasts

References

  1. Agardh CA (1873) Till algernes systematik. Nya bidrag. Lunds Universitets Års-Skrift. Afd Mathematik Naturvetenskap 9:1–71Google Scholar
  2. Bhattacharya D, Friedl T, Damberger S (1996) Nuclear-encoded rDNA group I introns: origins and phylogenetic relationships of insection site lineages in the green algae. Mol Biol Evol 13:978–989PubMedCrossRefGoogle Scholar
  3. CBOL Plant working group, comm. by Janzen DH (2009) A DNA barcode for land plants. Proc Natl Acad Sci USA 106:12794–12797CrossRefGoogle Scholar
  4. Curtis NE, Massey SE, Pierce SK (2006) The symbiotic chloroplasts in the sacoglossan Elysia clarki are from several algal species. Invert Biol 125:336–345CrossRefGoogle Scholar
  5. Evertsen J, Burghardt I, Johnsen G, Wägele H (2007) Retention of functional chloroplasts in some sacoglossans from the Indo-Pacific and Mediterranean. Mar Biol 151:2159–2166CrossRefGoogle Scholar
  6. Famá P, Wysor B, Kooistra WH, Zuccarello G (2002) Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. J Phycol 38:1040–1050CrossRefGoogle Scholar
  7. Giménez-Casalduero F, Muniain C (2008) The role of kleptoplasts in the survival rates of Elysia timida (Risso 1818): (Sacoglossa: Opisthobranchia) during periods of food shortage. J Exp Mar Biol Ecol 357:181–187CrossRefGoogle Scholar
  8. Giménez-Casalduero F, Muniain C, González-Wangüemert M, Garrote-Moreno A (2011) Elysia timida (Risso, 1818) three decades of research. Anim Biodiv Conserv 34:217–227Google Scholar
  9. Gould AA (1852) United States exploring expedition. During the years 1838 (1839), 1840, 1841, 1842. Under the command of Charles Wilkes, U.S.N. vol. XII: Mollusca & Shells. Sherman, Philadelphia, p 510Google Scholar
  10. Gould AA (1870) Report on the invertebrata of Massachusetts. 2nd edn, comprising the Mollusca. Wright and Potter, Boston, p 524Google Scholar
  11. Green BJ, Li W-Y, 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 124:331–342PubMedCrossRefGoogle Scholar
  12. Green BJ, Fox TC, Rumpho ME (2005) Stability of isolated algal chloroplasts that participate in a unique mollusc/kleptoplast association. Symbiosis 40:31–40Google Scholar
  13. Hajibabaei M, Smitj MA, Janzen DH, Rodriguez JJ, Whitfield JB, Hebert PDN (2006) A minimalist barcode can identify a specimen whose DNA is degraded. Mol Ecol Notes 6:959–964CrossRefGoogle Scholar
  14. Händeler K, Wägele H (2007) Preliminary study on molecular phylogeny of Sacoglossa and a compilation of their food organisms. Bonn Zool Beitr 3(4):231–254Google Scholar
  15. Händeler K, Grzymbowski Y, Krug JP, Wägele H (2009) Functional chloroplasts in metazoan cells—a unique evolutionary strategy in animal life. Front Zool 6:28PubMedCrossRefGoogle Scholar
  16. Händeler K, Wägele H, Wahrmund U, Rüdinger M, Knoop V (2010) Slugs’ last meals: molecular identification of sequestered chloroplasts from different algal origins in Sacoglossa (Opisthobranchia, Gastropoda). Mol Ecol Res 10:968–978CrossRefGoogle Scholar
  17. Hanten JJ, Pierce SK (2001) Synthesis of several light-harvesting complex I polypeptides is blocked by cycloheximide in symbiotic chloroplast in the sea slug, Elysia chlorotica (Gould): a case for horizontal gene transfer between alga and animal? Biol Bull 201:34PubMedCrossRefGoogle Scholar
  18. Hasselt JC (1824) Extrait d’une lettre du Dr. J. C. van Hasselt au Prof. van Swinderen sur mollusques de Java (traduit de l’Allgem. konst en letterbode, 1824, nos. 2, 3, 4) Tjuringe (ile Java) le 25 Mai 1823 (1). Bull Sci Nat Geol 3:237–248Google Scholar
  19. Haugen P, Simon DM, Bhattacharya D (2005) The natural history of group I introns. Trends Genet 21:111–119PubMedCrossRefGoogle Scholar
  20. Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B 270:313–321CrossRefGoogle Scholar
  21. Hebert PDN, Penton EH, Burns JM, Janzen DH, Hallwachs W (2004) Ten species in one: DNA barcoding reveals cryptic species in the neotropical skipper butterfly Astraptes fulgerator. Proc Natl Acad Sci USA 101:14812–14817PubMedCrossRefGoogle Scholar
  22. Hirose E (2005) Digestive system of the sacoglossan Plakobranchus ocellatus (Gastropoda: Opisthobranchia): light- and electron-microscopic observations with remarks on chloroplast retention. Zool Sci 22:905–916PubMedCrossRefGoogle Scholar
  23. Huelsken T, Wägele H, Peters B, Mather A, Hollmann M (2011) Molecular analysis of adults and egg masses reveals two independent lineages within the infaunal gastropod Naticarius onca (Röding 1798) (Caenogastropoda: Naticidae). Molluscan Res 31:141–151Google Scholar
  24. Jensen KR (1980) A review of sacoglossan diets, with comparative notes on radula and buccal anatomy. Malacol Rev 13:55–77Google Scholar
  25. Jensen KR (1992) Anatomy of some Indo-Pacific Elysiidae (Opisthobranchia: Sacoglossa (=Ascoglossa)), with a discussion of the generic division and phylogeny. J Molluscan Stud 58:257–296CrossRefGoogle Scholar
  26. Jensen KR (1996) Phylogenetic systematics and classification of the Sacoglossa (Mollusca, Gastropoda, Opisthobranchia). Phil Trans R Soc Lond B 351:91–122CrossRefGoogle Scholar
  27. Jensen KR (1997) Evolution of the Sacoglossa (Mollusca, Opisthobranchia) and the ecological associations with their food plants. Evol Ecol 11:301–335CrossRefGoogle Scholar
  28. Kawaguti S, Yamasu T (1965) Electron microscopy on the symbiosis between an elysioid gastropod and chloroplasts of a green alga. Biol J Okayama Univ 11:57–65Google Scholar
  29. Kosakovsky Pond SL, Frost SDW, Muse SV (2005) HyPhy: hypothesis testing using phylogenies. Bioinformatics 21:676–679CrossRefGoogle Scholar
  30. Maeda T, Kajita T, Maruyama T, Hirano Y (2010) Molecular phylogeny of the Sacoglossa, with a discussion of gain and loss of kleptoplasty in the evolution of the group. Biol Bull 219:17–26PubMedGoogle Scholar
  31. Maeda T, Hirose E, Chikaraishi Y, Kawato M, Takishita K, Yoshida T, Iwai K, Maruyama T (2012) Algivore or phototroph? Plakobranchus ocellatus (Gastropoda) continuously acquires kleptoplasts and nutrition from multiple algal species in nature. PloS One 7:e42024. doi:10.1371/journal.pone.0042024
  32. Marín A, Ros J (1992) Dynamics of a peculiar plant-herbivore relationship: the photosynthetic ascoglossan Elysia timida and the chlorophycean Acetabularia acetabulum. Mar Biol 112:677–682CrossRefGoogle Scholar
  33. Mujer 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 93:12333–12338PubMedCrossRefGoogle Scholar
  34. O’Kelly CJ, Bellows WK, Wysor B (2004) Phylogenetic position of Bolbocoleon piliferum (Ulvophyceae, Chlorophyta): evidence from reproduction, zoospore and gamete ultrastructure, and small subunit rRNA gene sequences. J Phycol 40:209–222CrossRefGoogle Scholar
  35. Pelletreau KN, Bhattacharya D, Price DC, Worful JM, Moustafa A, Rumpho ME (2011) Sea slug kleptoplasty and plastid maintenance in a metazoan. Plant Physiol 155:1561–1565PubMedCrossRefGoogle Scholar
  36. Pierce SK, Massey SE, Hanten JJ, Curtis NE (2003) Horizontal transfer of functional nuclear genes between multicellular organisms. Biol Bull 204:237–240PubMedCrossRefGoogle Scholar
  37. Pierce SK, Curtis NE, Massey SE, Bass AL, Karl SA, Finney CM (2006) A morphological and molecular comparison between Elysia crispata and a new species of kleptoplastic sacoglossan sea slug (Gastropoda: Opisthobranchia) from the Florida Keys, USA. Moll Res 26:23–38Google Scholar
  38. Pierce SK, Fang X, Schwartz JA, Jiang X, Zhao W, Curtis NE, Kocot K, Yang B, Wang J (2012) Transcriptomic evidence for the expression of horizontally transferred algal nuclear genes in the photosynthetic sea slug, Elysia chlorotica. Mol Biol Evol 29:1545–1556PubMedCrossRefGoogle Scholar
  39. Pombert JF, Otis C, Lemieux C, Turmel M (2005) The chloroplast genome sequence of the green alga Pseudendoclonium akinetum (Ulvophyceae) reveals unusual structural features and new insights into the branching order of chlorophyte lineages. Mol Biol Evol 22:1903–1918PubMedCrossRefGoogle Scholar
  40. Pombert JF, Lemieux C, Turmel M (2006) The complete chloroplast DNA sequence of the greenalga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes. BMC Biol 4:3PubMedCrossRefGoogle Scholar
  41. Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256PubMedCrossRefGoogle Scholar
  42. Risso A (1818) Mémoire sur quelques gastéropodes nouveaux, nudibranches et tectibranches observés dans la Mer de Nice. J Phys Chim Hist Nat Arts 87:368–377Google Scholar
  43. Rudman WB 1998 Plakobranchus ocellatus van Hasselt, 1824. Sea Slug Forum. Australian Museum, Sydney. http://www.seaslugforum.net/factsheet/placocel. Accessed 26 July 2012
  44. 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 104:303–312PubMedCrossRefGoogle Scholar
  45. Rumpho ME, Worful JM, Lee J, Kannan K, Tylor 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 105:17867–17871PubMedCrossRefGoogle Scholar
  46. Rumpho ME, Pelletreau KN, Moustafa A, Bhattacharya D (2011) The making of a photosynthetic animal. J Exp Biol 214:303–311PubMedCrossRefGoogle Scholar
  47. Saunders GW, Kucera H (2010) An evaluation of rbcL, tufA, UPA, LSU and ITS as DNA barcode markers for the marine green macroalgae. Crypt Algol 31:487–538Google Scholar
  48. Schmitt V, Wägele H (2011) Behavioral adaptations in relation to long-term retention of endosymbiotic chloroplasts in the sea slug Elysia timida (Opisthobranchia, Sacoglossa). Thalassas 27:225–238Google Scholar
  49. Silva PC (1952) A review of nomenclatural conservation in the algae from the point of view of the type method. Univ Calif Publ Botany 25:241–323Google Scholar
  50. Stamatakis A, Hoover P, Rougemont J (2008) A fast bootstrapping algorithm for the RAxML Web-Servers. Syst Biol 57:758–771PubMedCrossRefGoogle Scholar
  51. Timmis JN, Ayliffe MA, Huang CY, Martin W (2004) Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5:123–135PubMedCrossRefGoogle Scholar
  52. Trowbridge CD, Hirano YM, Hirano YJ (2011) Inventory of Japanese sacoglossan opisthobranchs: historical review, current records, and unresolved issues. Am Malacol Bull 29:1–22CrossRefGoogle Scholar
  53. Verbruggen H, De Clerck O et al (2005) Molecular and morphometric data pinpoint species boundaries in Halimeda section Rhipsalis (Bryopsidales, Chlorophyta). J Phycol 4:606–621CrossRefGoogle Scholar
  54. Verbruggen H, Ashworth M, LoDuca ST, Vlaeminck C, Cocquyt E, Sauvage T, Zechman FW, Littler DS, Littler MM, Leliaert F, De Clerck O (2009) A multi-locus time-calibrated phylogeny of the siphonous green algae. Mol Phyl Evol 50:642–653CrossRefGoogle Scholar
  55. Vieira S, Calado G, Coelho H, Serôdio J (2009) Effects of light exposure on the retention of kleptoplastic photosynthetic activity in the sacoglossan mollusc Elysia viridis. Mar Biol 156:1007–1020CrossRefGoogle Scholar
  56. Wägele H, Martin W (2013) Endosymbioses in sacoglossan seaslugs: Photosynthetic animals that keep stolen plastids without borrowing genes. In: Löffelhardt W (ed) Endosymbiosis. Springer, HeidelbergGoogle Scholar
  57. Wägele H, Stemmer K, Burghardt I, Händeler K (2010) Two new sacoglossan sea slug species (Opisthobranchia, Gastropoda): Ercolania annelyleorum sp. nov. (Limapontioidea) and Elysia asbecki sp. nov. (Plakobranchoidea) with notes on anatomy, histology and biology. Zootaxa 2676:1–28Google Scholar
  58. Wägele H, Deusch O, Händeler K, Martin R, Schmitt V, Christa G, Pinzger B, Gould SB, Dagan T, Klussmann-Kolb A, Martin W (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. Mol Biol Evol 28:699–706PubMedCrossRefGoogle Scholar
  59. Weigand AM, Jochum A, Pfenninger M, Steinke D, Klussmann-Kolb A (2011) A new approach to an old conundrum—DNA barcoding sheds new light on phenotypic plasticity and morphological stasis in microsnails (Gastropoda, Pulmonata, Carychiidae) Mol. Ecol Res 11:255–256CrossRefGoogle Scholar
  60. Yamamoto YY, Yusa Y, Yamamoto S, Hirano MY, Hirano YJ, Motomura T, Tanemura T, Obokata J (2009) Identification of photosynthetic sacoglossans from Japan. Endocyt Cell Res 19:112–119Google Scholar
  61. Yamamoto S, Hirano YM, Hirano YJ, Trowbridge CD, Akimoto A, Sakai A, Yusa Y (2012) Effects of photosynthesis on the survival and weight retention of two kleptoplastic sacoglossan opisthobranchs. J Mar Biol Assoc UK. doi:10.1017/S0025315412000628 Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Gregor Christa
    • 1
  • Lily Wescott
    • 1
  • Till F. Schäberle
    • 2
  • Gabriele M. König
    • 2
  • Heike Wägele
    • 1
  1. 1.Forschungsmuseum Alexander KoenigBonnGermany
  2. 2.Institute for Pharmaceutical BiologyUniversity of BonnBonnGermany

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