Marine Biotechnology

, Volume 17, Issue 4, pp 377–385 | Cite as

Diversity of Actinobacteria Associated with the Marine Ascidian Eudistoma toealensis

  • Georg Steinert
  • Michael W. Taylor
  • Peter J. Schupp
Short Communication


Ascidians have yielded a wide variety of bioactive natural products. The colonial ascidian Eudistoma toealensis from Micronesia has been identified as the source of a series of staurosporine derivatives, though the exact origin of these derivatives is still unknown. To identify known staurosporine-producing microbes associated with E. toealensis, we analyzed with 16S rRNA gene tag pyrosequencing the overall bacterial community and focused on potential symbiotic bacteria already known from other ascidians or other marine hosts, such as sponges. The described microbiota was one of very high diversity, comprising 43 phyla: two from archaea, 34 described bacterial phyla, and seven candidate bacterial phyla. Many bacteria, which are renowned community members of other ascidians and marine holobionts, such as sponges and corals, were also part of the E. toealensis microbial community. Furthermore, two known producers of indolocarbazoles, Salinispora and Verrucosispora, were found with high abundance exclusively in the ascidian tissue, suggesting that microbial symbionts and not the organism itself may be the true producers of the staurosporines in E. toealensis.


Ascidian Actinobacteria Eudistoma toealensis Microbial diversity Symbiosis 16S rRNA 



PJS acknowledges funding by NIH MBRS SCORE grant S06-GM-44796. GS acknowledges funding for microbial analyses at the University of Auckland in the authors’ laboratory by the German Academic Exchange Service (DAAD) short-term fellowship ‘Microbial Symbiosis and Diversity in Marine Sponges’ from February 2013 to June 2013. We would like to thank Michael Hoggard (University of Auckland, NZ) for additional sample work.

Supplementary material

10126_2015_9622_MOESM1_ESM.png (1.3 mb)
Suppl. Figure 1 Taxonomic breakdown per sample at phylum level—showing all available taxonomic groups. (PNG 1292 kb)
10126_2015_9622_MOESM2_ESM.png (245 kb)
Suppl. Figure 2 nonmetric multidimensional scaling (nMDS) for all 0.03 OTUs and Actinobacteria 0.03 OTUs for three treatments: source—A = EtCI (blue squares), B = EtPI (blue triangles), C = rootPI (red circles); habitat—ascidian or environmental; location—Chuuk or Pohnpei Island. Shown p values are from ‘Permutational Multivariate Analysis of Variance Using Distance Matrices’ (adonis) analysis. Shown ellipses are based on the treatments (A, B, C) used for the adonis hypothesis test. (PNG 244 kb)
10126_2015_9622_MOESM3_ESM.xlsx (19 kb)
Suppl. Table 1 Fingerprint phyla—raw table for Fig. 1 and Suppl. Figure 1. (XLSX 18 kb)
10126_2015_9622_MOESM4_ESM.xlsx (90 kb)
Suppl. Table 2 Fingerprint full resolution—raw table for nMDS and Suppl. Table 3 (XLSX 89 kb)
10126_2015_9622_MOESM5_ESM.xlsx (18 kb)
Suppl. Table 3 Actinobacteria diversity—raw table for Actinobacteria heatmap, extracted raw data from Suppl. Table 2 with additional frequency information (XLSX 17 kb)


  1. Behrendt L, Larkum AWD, Trampe E et al (2012) Microbial diversity of biofilm communities in microniches associated with the didemnid ascidian Lissoclinum patella. ISME J 6:1222–1237. doi: 10.1038/ismej.2011.181 PubMedCentralPubMedCrossRefGoogle Scholar
  2. Blunt JW, Copp BR, Keyzers RA et al (2012) Marine natural products. Nat Prod Rep 29:144–222. doi: 10.1039/c2np00090c PubMedCrossRefGoogle Scholar
  3. Blunt JW, Copp BR, Keyzers RA et al (2013) Marine natural products. Nat Prod Rep 30:237–323. doi: 10.1039/c2np20112g PubMedCrossRefGoogle Scholar
  4. Bollmann A, Palumbo AV, Lewis K, Epstein SS (2010) Isolation and physiology of bacteria from contaminated subsurface sediments. Appl Environ Microbiol 76:7413–7419. doi: 10.1128/AEM. 00376-10 PubMedCentralPubMedCrossRefGoogle Scholar
  5. Brück WM, Sennett SH, Pomponi SA et al (2008) Identification of the bacterial symbiont Entotheonella sp. in the mesohyl of the marine sponge Discodermia sp. ISME J 2:335–339. doi: 10.1038/ismej.2007.91 PubMedCrossRefGoogle Scholar
  6. Donia MS, Fricke WF, Partensky F et al (2011) Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis. Proc Natl Acad Sci U S A 108:E1423–E1432. doi: 10.1073/pnas.1111712108 PubMedCentralPubMedCrossRefGoogle Scholar
  7. Du Z, Zhang W, Xia H et al (2010) Isolation and diversity analysis of heterotrophic bacteria associated with sea anemones. Acta Oceanol Sin 29:62–69. doi: 10.1007/s13131-010-0023-1 CrossRefGoogle Scholar
  8. Erwin PM, Carmen Pineda M, Webster N et al (2013) Small core communities and high variability in bacteria associated with the introduced ascidian Styela plicata. Symbiosis 59:35–46. doi: 10.1007/s13199-012-0204-0 CrossRefGoogle Scholar
  9. Erwin PM, Pineda MC, Webster N et al (2014) Down under the tunic: bacterial biodiversity hotspots and widespread ammonia-oxidizing archaea in coral reef ascidians. ISME J 8:575–588. doi: 10.1038/ismej.2013.188 PubMedCentralPubMedCrossRefGoogle Scholar
  10. Freel KC, Nam S-J, Fenical W, Jensen PR (2011) Evolution of secondary metabolite genes in three closely related marine actinomycete species. Appl Environ Microbiol 77:7261–7270. doi: 10.1128/AEM. 05943-11 PubMedCentralPubMedCrossRefGoogle Scholar
  11. Gavrish E, Bollmann A, Epstein S, Lewis K (2008) A trap for in situ cultivation of filamentous Actinobacteria. J Microbiol Methods 72:257–262. doi: 10.1016/j.mimet.2007.12.009 PubMedCentralPubMedCrossRefGoogle Scholar
  12. Grigioni S, Boucher-Rodoni R, Demarta A et al (2000) Phylogenetic characterisation of bacterial symbionts in the accessory nidamental glands of the sepioid Sepia officinalis (Cephalopoda: Decapoda). Mar Biol 136:217–222CrossRefGoogle Scholar
  13. Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55:167–177. doi: 10.1111/j.1574-6941.2005.00046.x PubMedCrossRefGoogle Scholar
  14. Jensen PR, Williams PG, Oh D-C et al (2007) Species-specific secondary metabolite production in marine actinomycetes of the genus Salinispora. Appl Environ Microbiol 73:1146–1152. doi: 10.1128/AEM. 01891-06 PubMedCentralPubMedCrossRefGoogle Scholar
  15. Jiang S, Sun W, Chen M et al (2007) Diversity of culturable actinobacteria isolated from marine sponge Haliclona sp. Antonie Van Leeuwenhoek 92:405–416. doi: 10.1007/s10482-007-9169-z PubMedCrossRefGoogle Scholar
  16. Jimenez P, Ferreira E (2013) Cytotoxicity of actinomycetes associated with the ascidian Eudistoma vannamei (Millar, 1977), endemic of northeastern coast of Brazil. Lat Am J Aquat Res 41:335–343. doi: 10.3856/vol41-issue2-fulltext-12 Google Scholar
  17. Jimenez PC, Wilke DV, Ferreira EG et al (2012) Structure elucidation and anticancer activity of 7-oxostaurosporine derivatives from the Brazilian endemic tunicate Eudistoma vannamei. Mar Drugs 10:1092–1102. doi: 10.3390/md10051092 PubMedCentralPubMedCrossRefGoogle Scholar
  18. Joachimiak MP, Weisman JL, May BC (2006) JColorGrid: software for the visualization of biological measurements. BMC Bioinforma 7:225. doi: 10.1186/1471-2105-7-225 CrossRefGoogle Scholar
  19. Khan ST, Komaki H, Motohashi K et al (2011) Streptomyces associated with a marine sponge Haliclona sp.; biosynthetic genes for secondary metabolites and products. Environ Microbiol 13:391–403. doi: 10.1111/j.1462-2920.2010.02337.x PubMedCrossRefGoogle Scholar
  20. Khan ST, Takagi M, Shin-ya K (2012) Actinobacteria associated with the marine sponges Cinachyra sp., Petrosia sp., and Ulosa sp. and their culturability. Microbes Environ 27:99–104. doi: 10.1264/jsme2.ME11270 PubMedCentralPubMedCrossRefGoogle Scholar
  21. Kim T, Garson M, Fuerst J (2005) Marine actinomycetes related to the “Salinospora” group from the Great Barrier Reef sponge Pseudoceratina clavata. Environ Microbiol 7:509–518. doi: 10.1111/j.1462-2920.2004.00716.x PubMedCrossRefGoogle Scholar
  22. Krupke A, Lavik G, Halm H et al (2014) Distribution of a consortium between unicellular algae and the N2 fixing cyanobacterium UCYN-A in the North Atlantic Ocean. Environ Microbiol 1–42. doi: 10.1111/1462-2920.12431
  23. Lafi FF, Garson MJ, Fuerst JA (2005) Culturable bacterial symbionts isolated from two distinct sponge species (Pseudoceratina clavata and Rhabdastrella globostellata) from the Great Barrier Reef display similar phylogenetic diversity. Microb Ecol 50:213–220. doi: 10.1007/s00248-004-0202-8 PubMedCrossRefGoogle Scholar
  24. López-Legentil S, Song B, Bosch M et al (2011) Cyanobacterial diversity and a new acaryochloris-like symbiont from Bahamian sea-squirts. PLoS One 6:e23938. doi: 10.1371/journal.pone.0023938 PubMedCentralPubMedCrossRefGoogle Scholar
  25. Martínez-García M, Díaz-Valdés M, Wanner G et al (2007) Microbial community associated with the colonial ascidian Cystodytes dellechiajei. Environ Microbiol 9:521–534. doi: 10.1111/j.1462-2920.2006.01170.x PubMedCrossRefGoogle Scholar
  26. Martínez-García M, Stief P, Díaz-Valdés M et al (2008) Ammonia-oxidizing Crenarchaeota and nitrification inside the tissue of a colonial ascidian. Environ Microbiol 10:2991–3001. doi: 10.1111/j.1462-2920.2008.01761.x PubMedCrossRefGoogle Scholar
  27. Moss C, Green DH, Pérez B et al (2003) Intracellular bacteria associated with the ascidian Ecteinascidia turbinata: phylogenetic and in situ hybridisation analysis. Mar Biol 143:99–110. doi: 10.1007/s00227-003-1060-5 CrossRefGoogle Scholar
  28. Muscholl-Silberhorn A, Thiel V, Imhoff JF (2008) Abundance and bioactivity of cultured sponge-associated bacteria from the Mediterranean sea. Microb Ecol 55:94–106. doi: 10.1007/s00248-007-9255-9 PubMedCrossRefGoogle Scholar
  29. Oksanen J, Blanchet FG, Kindt R et al (2011) Vegan: community ecology package. In: R Packag. version 2.0-2 ( Accessed 19 Sep 2013) 
  30. Ondov BD, Bergman NH, Phillippy AM (2011) Interactive metagenomic visualization in a Web browser. BMC Bioinforma 12:385. doi: 10.1186/1471-2105-12-385 CrossRefGoogle Scholar
  31. Pérez-Matos AE, Rosado W, Govind NS (2007) Bacterial diversity associated with the Caribbean tunicate Ecteinascidia turbinata. Antonie Van Leeuwenhoek 92:155–164. doi: 10.1007/s10482-007-9143-9 PubMedCrossRefGoogle Scholar
  32. Piel J (2009) Metabolites from symbiotic bacteria. Nat Prod Rep 26:338–362. doi: 10.1039/b703499g PubMedCrossRefGoogle Scholar
  33. Proksch P, Edrada-Ebel R, Ebel R (2003) Drugs from the sea—opportunities and obstacles. Mar Drugs 1:5–17PubMedCentralCrossRefGoogle Scholar
  34. Quast C, Pruesse E, Yilmaz P et al (2013) The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res 41:D590–D596. doi: 10.1093/nar/gks1219 PubMedCentralPubMedCrossRefGoogle Scholar
  35. Quince C, Lanzen A, Davenport RJ, Turnbaugh PJ (2011) Removing noise from pyrosequenced amplicons. BMC Bioinforma 12:38. doi: 10.1186/1471-2105-12-38 CrossRefGoogle Scholar
  36. R Develpoment Core Team (2013) R: a language and environment for statistical computing. In: R Found. Stat. Comput. Vienna, Austria. Accessed 22 Oct 2013
  37. Rath CM, Janto B, Earl J et al (2011) Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic natural product ET-743. ACS Chem Biol 6:1244–1256. doi: 10.1021/cb200244t PubMedCentralPubMedCrossRefGoogle Scholar
  38. Reveillaud J, Maignien L, Eren MA et al (2014) Host-specificity among abundant and rare taxa in the sponge microbiome. ISME J 8:1198–1209. doi: 10.1038/ismej.2013.227 PubMedCentralPubMedCrossRefGoogle Scholar
  39. Sánchez C, Méndez C, Salas JA (2006) Indolocarbazole natural products: occurrence, biosynthesis, and biological activity. Nat Prod Rep 23:1007–1045. doi: 10.1039/b601930g PubMedCrossRefGoogle Scholar
  40. Schloss PD, Westcott SL, Ryabin T et al (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75:7537–7541. doi: 10.1128/AEM. 01541-09 PubMedCentralPubMedCrossRefGoogle Scholar
  41. Schloss PD, Gevers D, Westcott SL (2011) Reducing the effects of PCR amplification and sequencing artifacts on 16S rRNA-based studies. PLoS One 6:e27310. doi: 10.1371/journal.pone.0027310 PubMedCentralPubMedCrossRefGoogle Scholar
  42. Schmidt EW, Donia MS (2010) Life in cellulose houses: symbiotic bacterial biosynthesis of ascidian drugs and drug leads. Curr Opin Biotechnol 21:827–833. doi: 10.1016/j.copbio.2010.10.006 PubMedCentralPubMedCrossRefGoogle Scholar
  43. Schmidt EW, Obraztsova AY, Davidson SK et al (2000) Identification of the antifungal peptide-containing symbiont of the marine sponge Theonella swinhoei as a novel δ-proteobacterium, “Candidatus Entotheonella palauensis.”. Mar Biol 136:969–977. doi: 10.1007/s002270000273 CrossRefGoogle Scholar
  44. Schmitt S, Tsai P, Bell J et al (2012) Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J 6:564–576. doi: 10.1038/ismej.2011.116 PubMedCentralPubMedCrossRefGoogle Scholar
  45. Schupp PJ, Eder C, Proksch P et al (1999) Staurosporine derivatives from the ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. J Nat Prod 62:959–962. doi: 10.1021/np980527d PubMedCrossRefGoogle Scholar
  46. Schupp PJ, Steube K, Meyer C, Proksch P (2001) Anti-proliferative effects of new staurosporine derivatives isolated from a marine ascidian and its predatory flatworm. Cancer Lett 174:165–172PubMedCrossRefGoogle Scholar
  47. Schupp PJ, Proksch P, Wray V (2002) Further new staurosporine derivatives from the ascidian Eudistoma toealensis and its predatory flatworm Pseudoceros sp. J Nat Prod 65:295–298PubMedCrossRefGoogle Scholar
  48. Schupp PJ, Kohlert-Schupp C, Yoshida WY, Hemscheidt TK (2009) Structure of pseudocerosine, an indolic azafulvene alkaloid from the flatworm Pseudoceros indicus. Org Lett 11:1111–1114. doi: 10.1021/ol8027785 PubMedCentralPubMedCrossRefGoogle Scholar
  49. Sfanos K, Harmody D, Dang P et al (2005) A molecular systematic survey of cultured microbial associates of deep-water marine invertebrates. Syst Appl Microbiol 28:242–264. doi: 10.1016/j.syapm.2004.12.002 PubMedCrossRefGoogle Scholar
  50. Simister RL, Deines P, Botté ES et al (2012a) Sponge-specific clusters revisited: a comprehensive phylogeny of sponge-associated microorganisms. Environ Microbiol 14:517–524. doi: 10.1111/j.1462-2920.2011.02664.x PubMedCrossRefGoogle Scholar
  51. Simister RL, Taylor MW, Tsai P et al (2012b) Thermal stress responses in the bacterial biosphere of the Great Barrier Reef sponge, Rhopaloeides odorabile. Environ Microbiol 14:3232–3246. doi: 10.1111/1462-2920.12010 PubMedCrossRefGoogle Scholar
  52. Sun W, Dai S, Jiang S et al (2010) Culture-dependent and culture-independent diversity of Actinobacteria associated with the marine sponge Hymeniacidon perleve from the South China Sea. Antonie Van Leeuwenhoek 98:65–75. doi: 10.1007/s10482-010-9430-8 PubMedCrossRefGoogle Scholar
  53. Tamaoki T, Nomoto H, Takahashi I (1986) Staurosporine, a potent inhibitor of phospholipid Ca++ dependent protein kinase. Biochem Biophys Res Commun 135:397–402PubMedCrossRefGoogle Scholar
  54. Taylor MW, Schupp PJ, Dahllöf I et al (2004) Host specificity in marine sponge-associated bacteria, and potential implications for marine microbial diversity. Environ Microbiol 6:121–130. doi: 10.1046/j.1462-2920.2003.00545.x PubMedCrossRefGoogle Scholar
  55. Taylor MW, Radax R, Steger D, Wagner M (2007) Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol Mol Biol Rev 71:295–347. doi: 10.1128/MMBR. 00040-06 PubMedCentralPubMedCrossRefGoogle Scholar
  56. Taylor MW, Tsai P, Simister RL et al (2013) “Sponge-specific” bacteria are widespread (but rare) in diverse marine environments. ISME J 7:438–443. doi: 10.1038/ismej.2012.111 PubMedCentralPubMedCrossRefGoogle Scholar
  57. Udwary DW, Zeigler L, Asolkar RN et al (2007) Genome sequencing reveals complex secondary metabolome in the marine actinomycete Salinispora tropica. Proc Natl Acad Sci U S A 104:10376–10381. doi: 10.1073/pnas.0700962104 PubMedCentralPubMedCrossRefGoogle Scholar
  58. Waite DW, Deines P, Taylor MW (2012) Gut microbiome of the critically endangered New Zealand parrot, the kakapo (Strigops habroptilus). PLoS One 7:e35803. doi: 10.1371/journal.pone.0035803 PubMedCentralPubMedCrossRefGoogle Scholar
  59. Webster NS, Taylor MW (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microbiol 14:335–346. doi: 10.1111/j.1462-2920.2011.02460.x PubMedCrossRefGoogle Scholar
  60. Wicke C, Hüners M, Wray V et al (2000) Production and structure elucidation of glycoglycerolipids from a marine sponge-associated microbacterium species. J Nat Prod 63:621–626PubMedCrossRefGoogle Scholar
  61. Wilson MC, Mori T, Rückert C et al (2014) An environmental bacterial taxon with a large and distinct metabolic repertoire. Nature 506:58–62. doi: 10.1038/nature12959 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Georg Steinert
    • 1
  • Michael W. Taylor
    • 2
  • Peter J. Schupp
    • 1
  1. 1.Institute for the Chemistry and Biology of the Marine EnvironmentUniversity of OldenburgOldenburgGermany
  2. 2.Centre for Microbial Innovation, School of Biological SciencesUniversity of AucklandAucklandNew Zealand

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