Skip to main content
Log in

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

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

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.

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

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

DT:

Dark treatment

LT:

Low light intensity treatment

HT:

High light intensity treatment

LTR:

Long-term retention of chloroplasts

References

  • Agardh CA (1873) Till algernes systematik. Nya bidrag. Lunds Universitets Års-Skrift. Afd Mathematik Naturvetenskap 9:1–71

    Google Scholar 

  • 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 13:978–989

    Article  PubMed  CAS  Google Scholar 

  • CBOL Plant working group, comm. by Janzen DH (2009) A DNA barcode for land plants. Proc Natl Acad Sci USA 106:12794–12797

    Article  Google Scholar 

  • Curtis NE, Massey SE, Pierce SK (2006) The symbiotic chloroplasts in the sacoglossan Elysia clarki are from several algal species. Invert Biol 125:336–345

    Article  Google Scholar 

  • 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 

  • Famá P, Wysor B, Kooistra WH, Zuccarello G (2002) Molecular phylogeny of the genus Caulerpa (Caulerpales, Chlorophyta) inferred from chloroplast tufA gene. J Phycol 38:1040–1050

    Article  Google Scholar 

  • 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 357:181–187

    Article  Google Scholar 

  • Giménez-Casalduero F, Muniain C, González-Wangüemert M, Garrote-Moreno A (2011) Elysia timida (Risso, 1818) three decades of research. Anim Biodiv Conserv 34:217–227

    Google Scholar 

  • 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 510

    Google Scholar 

  • Gould AA (1870) Report on the invertebrata of Massachusetts. 2nd edn, comprising the Mollusca. Wright and Potter, Boston, p 524

    Google Scholar 

  • 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 124:331–342

    Article  PubMed  CAS  Google Scholar 

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

    Google Scholar 

  • 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 6:959–964

    Article  CAS  Google Scholar 

  • Händeler K, Wägele H (2007) Preliminary study on molecular phylogeny of Sacoglossa and a compilation of their food organisms. Bonn Zool Beitr 3(4):231–254

    Google Scholar 

  • Händeler K, Grzymbowski Y, Krug JP, Wägele H (2009) Functional chloroplasts in metazoan cells—a unique evolutionary strategy in animal life. Front Zool 6:28

    Article  PubMed  Google Scholar 

  • 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 10:968–978

    Article  Google Scholar 

  • 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 201:34

    Article  PubMed  CAS  Google Scholar 

  • 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 3:237–248

    Google Scholar 

  • Haugen P, Simon DM, Bhattacharya D (2005) The natural history of group I introns. Trends Genet 21:111–119

    Article  PubMed  CAS  Google Scholar 

  • Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B 270:313–321

    Article  CAS  Google Scholar 

  • 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 101:14812–14817

    Article  PubMed  CAS  Google Scholar 

  • Hirose E (2005) Digestive system of the sacoglossan Plakobranchus ocellatus (Gastropoda: Opisthobranchia): light- and electron-microscopic observations with remarks on chloroplast retention. Zool Sci 22:905–916

    Article  PubMed  Google Scholar 

  • 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 31:141–151

    Google Scholar 

  • Jensen KR (1980) A review of sacoglossan diets, with comparative notes on radula and buccal anatomy. Malacol Rev 13:55–77

    Google Scholar 

  • Jensen KR (1992) Anatomy of some Indo-Pacific Elysiidae (Opisthobranchia: Sacoglossa (=Ascoglossa)), with a discussion of the generic division and phylogeny. J Molluscan Stud 58:257–296

    Article  Google Scholar 

  • Jensen KR (1996) Phylogenetic systematics and classification of the Sacoglossa (Mollusca, Gastropoda, Opisthobranchia). Phil Trans R Soc Lond B 351:91–122

    Article  Google Scholar 

  • Jensen KR (1997) Evolution of the Sacoglossa (Mollusca, Opisthobranchia) and the ecological associations with their food plants. Evol Ecol 11:301–335

    Article  Google Scholar 

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

    Google Scholar 

  • Kosakovsky Pond SL, Frost SDW, Muse SV (2005) HyPhy: hypothesis testing using phylogenies. Bioinformatics 21:676–679

    Article  Google Scholar 

  • 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 219:17–26

    PubMed  CAS  Google Scholar 

  • 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

  • Marín A, Ros J (1992) Dynamics of a peculiar plant-herbivore relationship: the photosynthetic ascoglossan Elysia timida and the chlorophycean Acetabularia acetabulum. Mar Biol 112:677–682

    Article  Google Scholar 

  • 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 93:12333–12338

    Article  PubMed  CAS  Google Scholar 

  • 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 40:209–222

    Article  Google Scholar 

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

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • 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 26:23–38

    CAS  Google Scholar 

  • 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 29:1545–1556

    Article  PubMed  CAS  Google Scholar 

  • 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 22:1903–1918

    Article  PubMed  CAS  Google Scholar 

  • 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 4:3

    Article  PubMed  Google Scholar 

  • Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256

    Article  PubMed  CAS  Google Scholar 

  • Risso A (1818) Mémoire sur quelques gastéropodes nouveaux, nudibranches et tectibranches observés dans la Mer de Nice. J Phys Chim Hist Nat Arts 87:368–377

    Google Scholar 

  • Rudman WB 1998 Plakobranchus ocellatus van Hasselt, 1824. Sea Slug Forum. Australian Museum, Sydney. http://www.seaslugforum.net/factsheet/placocel. Accessed 26 July 2012

  • 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 104:303–312

    Article  PubMed  CAS  Google Scholar 

  • 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 105:17867–17871

    Article  PubMed  CAS  Google Scholar 

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

    Article  PubMed  Google Scholar 

  • 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 31:487–538

    Google Scholar 

  • 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 27:225–238

    Google Scholar 

  • Silva PC (1952) A review of nomenclatural conservation in the algae from the point of view of the type method. Univ Calif Publ Botany 25:241–323

    Google Scholar 

  • Stamatakis A, Hoover P, Rougemont J (2008) A fast bootstrapping algorithm for the RAxML Web-Servers. Syst Biol 57:758–771

    Article  PubMed  Google Scholar 

  • Timmis JN, Ayliffe MA, Huang CY, Martin W (2004) Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5:123–135

    Article  PubMed  CAS  Google Scholar 

  • Trowbridge CD, Hirano YM, Hirano YJ (2011) Inventory of Japanese sacoglossan opisthobranchs: historical review, current records, and unresolved issues. Am Malacol Bull 29:1–22

    Article  Google Scholar 

  • Verbruggen H, De Clerck O et al (2005) Molecular and morphometric data pinpoint species boundaries in Halimeda section Rhipsalis (Bryopsidales, Chlorophyta). J Phycol 4:606–621

    Article  Google Scholar 

  • 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 50:642–653

    Article  CAS  Google Scholar 

  • 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 156:1007–1020

    Article  CAS  Google Scholar 

  • Wägele H, Martin W (2013) Endosymbioses in sacoglossan seaslugs: Photosynthetic animals that keep stolen plastids without borrowing genes. In: Löffelhardt W (ed) Endosymbiosis. Springer, Heidelberg

    Google Scholar 

  • 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 2676:1–28

    Google Scholar 

  • 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 28:699–706

    Article  PubMed  Google Scholar 

  • 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 11:255–256

    Article  Google Scholar 

  • 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 19:112–119

    Google Scholar 

  • 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 

Download references

Acknowledgments

We thank Frank Richter (Chemnitz, Germany) for his technical support. Conxita Avila (Barcelona, Spain) kindly provided the 2 pictures of the slugs from Guam, Katharina Händeler (formerly Bonn, Germany) kindly provided a CO1 slug sequence from Guam. Claudia Etzbauer and Emilie Goralski (Bonn, Germany) helped in the molecular labs. Ingolf Rick (Bonn, Germany) kindly measured the spectrum of the Androv lamp used in the light treatment experiments. We thank the German Science Foundation (DFG) for financial support to HW (Wa 618/12). GC was partly financed by an ERC grant to W. Martin (Düsseldorf, Germany): Networkorigins Proj. Ref. 232975.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Heike Wägele.

Additional information

A contribution to the Special Issue on Evolution and Biogenesis of Chloroplasts and Mitochondria.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Christa, G., Wescott, L., Schäberle, T.F. et al. What remains after 2 months of starvation? Analysis of sequestered algae in a photosynthetic slug, Plakobranchus ocellatus (Sacoglossa, Opisthobranchia), by barcoding. Planta 237, 559–572 (2013). https://doi.org/10.1007/s00425-012-1788-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-012-1788-6

Keywords

Navigation