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.
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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
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
CBOL Plant working group, comm. by Janzen DH (2009) A DNA barcode for land plants. Proc Natl Acad Sci USA 106:12794–12797
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
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
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
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
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
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
Gould AA (1870) Report on the invertebrata of Massachusetts. 2nd edn, comprising the Mollusca. Wright and Potter, Boston, p 524
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
Green BJ, Fox TC, Rumpho ME (2005) Stability of isolated algal chloroplasts that participate in a unique mollusc/kleptoplast association. Symbiosis 40:31–40
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
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
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
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
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
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
Haugen P, Simon DM, Bhattacharya D (2005) The natural history of group I introns. Trends Genet 21:111–119
Hebert PDN, Cywinska A, Ball SL, de Waard JR (2003) Biological identifications through DNA barcodes. Proc R Soc Lond Ser B 270:313–321
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
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
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
Jensen KR (1980) A review of sacoglossan diets, with comparative notes on radula and buccal anatomy. Malacol Rev 13:55–77
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
Jensen KR (1996) Phylogenetic systematics and classification of the Sacoglossa (Mollusca, Gastropoda, Opisthobranchia). Phil Trans R Soc Lond B 351:91–122
Jensen KR (1997) Evolution of the Sacoglossa (Mollusca, Opisthobranchia) and the ecological associations with their food plants. Evol Ecol 11:301–335
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
Kosakovsky Pond SL, Frost SDW, Muse SV (2005) HyPhy: hypothesis testing using phylogenies. Bioinformatics 21:676–679
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
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
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
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
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
Pierce SK, Massey SE, Hanten JJ, Curtis NE (2003) Horizontal transfer of functional nuclear genes between multicellular organisms. Biol Bull 204:237–240
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
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
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
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
Posada D (2008) jModelTest: phylogenetic model averaging. Mol Biol Evol 25:1253–1256
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
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
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
Rumpho ME, Pelletreau KN, Moustafa A, Bhattacharya D (2011) The making of a photosynthetic animal. J Exp Biol 214:303–311
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
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
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
Stamatakis A, Hoover P, Rougemont J (2008) A fast bootstrapping algorithm for the RAxML Web-Servers. Syst Biol 57:758–771
Timmis JN, Ayliffe MA, Huang CY, Martin W (2004) Endosymbiotic gene transfer: organelle genomes forge eukaryotic chromosomes. Nat Rev Genet 5:123–135
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
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
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
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
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
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
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
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
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
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
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.
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A contribution to the Special Issue on Evolution and Biogenesis of Chloroplasts and Mitochondria.
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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
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DOI: https://doi.org/10.1007/s00425-012-1788-6