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Deep Sequencing of Myxilla (Ectyomyxilla) methanophila, an Epibiotic Sponge on Cold-Seep Tubeworms, Reveals Methylotrophic, Thiotrophic, and Putative Hydrocarbon-Degrading Microbial Associations

  • Invertebrate Microbiology
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Abstract

The encrusting sponge Myxilla (Ectyomyxilla) methanophila (Poecilosclerida: Myxillidae) is an epibiont on vestimentiferan tubeworms at hydrocarbon seeps on the upper Louisiana slope of the Gulf of Mexico. It has long been suggested that this sponge harbors methylotrophic bacteria due to its low δ 13C value and high methanol dehydrogenase activity, yet the full community of microbial associations in M. methanophila remained uncharacterized. In this study, we sequenced 16S rRNA genes representing the microbial community in M. methanophila collected from two hydrocarbon-seep sites (GC234 and Bush Hill) using both Sanger sequencing and next-generation 454 pyrosequencing technologies. Additionally, we compared the microbial community in M. methanophila to that of the biofilm collected from the associated tubeworm. Our results revealed that the microbial diversity in the sponges from both sites was low but the community structure was largely similar, showing a high proportion of methylotrophic bacteria of the genus Methylohalomonas and polycyclic aromatic hydrocarbon (PAH)-degrading bacteria of the genera Cycloclasticus and Neptunomonas. Furthermore, the sponge microbial clone library revealed the dominance of thioautotrophic gammaproteobacterial symbionts in M. methanophila. In contrast, the biofilm communities on the tubeworms were more diverse and dominated by the chemoorganotrophic Moritella at GC234 and methylotrophic Methylomonas and Methylohalomonas at Bush Hill. Overall, our study provides evidence to support previous suggestion that M. methanophila harbors methylotrophic symbionts and also reveals the association of PAH-degrading and thioautotrophic microbes in the sponge.

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References

  1. Hentschel U, Schmid M, Wanger M, Fieseler L, Gernert C, Hacker J (2001) Isolation and phylogenetic analysis of bacteria with antimicrobial activities from the Mediterranean sponges Aplysina aerophoba and Aplysina cavernicola. FEMS Microb Ecol 35:305–312

    Article  CAS  Google Scholar 

  2. Thacker RW, Starnes S (2003) Host specificity of the symbiotic cyanobacterium Oscillatoria spongeliae in marine sponge, Dysides spp. Mar Biol 142:643–648

    CAS  Google Scholar 

  3. Margot H, Acebal C, Toril E, Amils R, Fernandez Puentes J (2002) Consistent association of crenarchaeal Archaea with sponges of the genus Axinella. Mar Biol 140(4):739–745

    Article  CAS  Google Scholar 

  4. Vacelet J (1982) Algal-sponge symbioses in the coral reef of New Caledonia: a morphological study. In: Gomez ED, Birkeland CE (eds) Proceedings of the 4th International Coral Reef Symposium, vol. 2 Marine Sciences Center. University of the Philippines, Manila, pp 713–719

    Google Scholar 

  5. Garson MG, Flowers AE, Webb RI, Charan RD, McCaffrey EJ (1998) A sponge/dinoflagellate association in the haposclerid sponge Haliclona sp.: cellular origin of cytotoxic alkaloids by Percoll density gradient fractionation. Cell Tissue Res 293:365–373

    Article  PubMed  CAS  Google Scholar 

  6. Maldonado M, Cortadellas N, Trillas MI, Rützler K (2005) Endosymbiotic yeast maternally transmitted in a marine sponge. Biol Bull 209:94–106

    Article  PubMed  CAS  Google Scholar 

  7. Hentschel U, Fieseler L, Wehrl M, Gernert C, Steinert M, Hacker J et al (2003) Microbial diversity of marine sponges. In: Müller WEG (ed) Sponges (Porifera). Springer, Heidelberg, pp 59–83

    Chapter  Google Scholar 

  8. Vacelet J, Donadey C (1977) Electron microscope study of the association between some sponges and bacteria. J Exp Mar Biol Ecol 30:301–314

    Article  Google Scholar 

  9. Reiswig HM (1975) Bacteria as food for temperate-water marine sponges. Can J Zool 53:493–502

    Article  Google Scholar 

  10. Arillo A, Bavestrello G, Burlando B, Sara M (1993) Metabolic integration between symbiotic cyanobacteria and sponges—a possible mechanism. Mar Biol 117:159–162

    Article  CAS  Google Scholar 

  11. Taylor MW, Radax R, Steger D, Wagner M (2007) Sponge associated microorganisms: evolution, ecology, and biotechnological potential. Microb Mol Biol Rev 71:295–347

    Article  CAS  Google Scholar 

  12. Wilkinson CR, Garrone R (1980) Nutrition of marine sponges. Involvement of symbiotic bacteria in the uptake of dissolved carbon. In: Smith DC, Tiffon Y (eds) Nutrition in the lower metazoan. Pergamon, Oxford, pp 157–161

    Google Scholar 

  13. Wilkinson CR, Garrone R, Herbage D (1979) Sponge collagen degradation in vitro by sponge-specific bacteria. Colloq Int CNRS 291:361–364

    CAS  Google Scholar 

  14. Beer S, Ilan M (1998) In situ measurements of photosynthetic irradiance responses of two Red Sea sponges growing under dim light conditions. Mar Biol 131:613–617

    Article  Google Scholar 

  15. Wilkinson CR (1978) Microbial associations in sponges. I. Ecology, physiology and microbial populations of coral reef sponges. Mar Biol 49:161–167

    Article  Google Scholar 

  16. Hentschel U, Hopke J, Horn M, Friedrich AB, Wagner M, Hacker J et al (2002) Molecular evidence for a uniform microbial community in sponges from different oceans. Appl Environ Microbiol 68:4431–4440

    Article  PubMed  CAS  Google Scholar 

  17. Lee OO, Wong YH, Qian PY (2009) Inter- and intra-specific variations of bacterial communities associated with marine sponges from San Juan Island, Washington. Appl Environ Microbiol 75:3513–3521

    Article  PubMed  CAS  Google Scholar 

  18. Lee OO, Wang Y, Yang J, Lafi FF, Al-Suwailem A, Qian PY (2011) Pyrosequencing reveals highly diverse and species-specific microbial communities in sponges from the Red Sea. ISME J 5:650–664

    Article  PubMed  CAS  Google Scholar 

  19. Wilkinson CR, Nowak M, Austin B, Colwell RR (1981) Specificity of bacterial symbionts in Mediterranean and Great Barrier Reef sponges. Microb Ecol 7(1):13–21

    Article  Google Scholar 

  20. Vacelet J, Boury-Esnault N (2002) A new species of carnivorous deep-sea sponge (Demospongiae, Cladorhizidae) associated with methanotrophic bacteria. Cah Biol Mar 43:141–148

    Google Scholar 

  21. Vacelet J, Boury-Esnault N, Filamedioni A, Fisher CR (1995) A methanotrophic carnivorous sponge. Nature 377:296–296

    Article  CAS  Google Scholar 

  22. Vacelet J, Fialamedioni A, Fisher CR, Boury-Esnault N (1996) Symbiosis between methane-oxidizing bacteria and a deep-sea carnivorous cladorhizid sponge. Mar Ecol Prog Ser 145:77–85

    Article  Google Scholar 

  23. Nishijima M, Lindsay DJ, Hata J, Nakamura A, Kasai H, Ise Y, Fisher C, Fukiwara Y, Kawato M, Maruyama T (2010) Association of thioautotrophic bacteria with deep-sea sponges. Mar Biotechnol 12:253–260

    Article  PubMed  CAS  Google Scholar 

  24. Harrison FW, Gardiner SL, Ruetzler K, Fisher CR (1994) On the occurrence of endosymbiotic bacteria in a new species of sponge from hydrocarbon seep communities in the Gulf of Mexico. Trans Amer Microsc Soc 113:419–420

    Google Scholar 

  25. Maldonado M, Young CM (1998) A new species of poecilosclerid sponge (Porifera) from bathyal methane seeps in the Gulf of Mexico. J Mar Biol Assoc UK 78:795–806

    Article  Google Scholar 

  26. MacDonald IR, Boland GS, Baker JS, Brooks JM, Kennicutt MC II, Bidigare RR (1989) Gulf of Mexico hydrocarbon seep communities. II Spatial distribution of seep organisms and hydrocarbons at Bush Hill Mar Biol 101:235–247

    CAS  Google Scholar 

  27. Zhou J, Bruns MA, Tiedje JM (1996) DNA recovery from soils of diverse composition. Appl Environ Microbiol 62:316–322

    PubMed  CAS  Google Scholar 

  28. Baker GC, Smith JJ, Cowan DA (2003) Review and re-analysis of domain-specific 16S primers. J Microb Methods 55:541–555

    Article  CAS  Google Scholar 

  29. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, Fierer N, Pena AG, Goodrich JK, Gordon JI, Huttley GA, Kelley ST, Knights D, Koenig JE, Ley RE, Lozupone CA, McDonald D, Muegge BD, Pirrung M, Reeder J, Sevinsky JR, Turnbaugh PJ, Walters WA, Widmann J, Yatsunenko T, Zaneveld J, Knight R (2010) QIIME allows analysis of high-throughput community sequencing data. Nat Methods 7:335–336

    Article  PubMed  CAS  Google Scholar 

  30. Reeder J, Knight R (2010) Rapidly denoising pyrosequencing amplicon reads by exploiting rank-abundance distributions. Nat Met 7(9):668–669

    Article  CAS  Google Scholar 

  31. Haas BJ, Gevers D, Earl AM, Feldgarden M, Ward DV, Giannoukos G, Ciulla D, Tabbaa D, Highlander SK, Sodergren E, Methé B, DeSantis TZ, Petrosino JF, Knight R, Birren BW (2011) Chimeric 16S rRNA sequence formation and detection in Sanger and 454-pyrosequenced PCR amplicons. Gen Res 21(3):494–504

    Article  CAS  Google Scholar 

  32. Edgar RC (2010) Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26(19):2460–2461

    Article  PubMed  CAS  Google Scholar 

  33. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32:1792–1797

    Article  PubMed  CAS  Google Scholar 

  34. Lozupone C, Knight R (2005) UniFrac: a new phylogenetic method for comparing microbial communities. Appl Environ Microbiol 71:8228–8235

    Article  PubMed  CAS  Google Scholar 

  35. Lozupone C, Hamady M, Knight R (2006) UniFrac—an online tool for comparing microbial community diversity in a phylogenetic context. BMC Bioinforma 7:371

    Article  Google Scholar 

  36. Amann RI (1995) Fluorescently labeled, ribosomal-RNA-targeted oligonucleotide probes in the study of microbial ecology. Mol Ecol 4:543–553

    Article  CAS  Google Scholar 

  37. Lane DJ (1991) 16S/23S rRNA sequencing. In: Stackebrandt E, Goodfellow M (eds) Nucleic acid techniques in bacterial systematics. Wiley, New York, pp 115–148

    Google Scholar 

  38. Ashelford KE, Chuzhanova NA, Fry JC, Jones AJ, Weightman AJ (2005) At least 1 in 20 16S rRNA sequence records currently held in public repositories is estimated to contain substantial anomalies. Appl Environ Microbiol 71:7724–7736

    Article  PubMed  CAS  Google Scholar 

  39. Schloss PD, Handelsman J (2005) Introducing DOTUR, a computer program for defining operational taxonomic units and estimating species richness. Appl Environ Microbiol 71:1501–1506

    Article  PubMed  CAS  Google Scholar 

  40. Singleton DR, Furlong MA, Rathbun SL, Whitman WB (2001) Quantitative comparisons of 16S rDNA sequence libraries from environmental samples. Appl Environ Microbiol 67:4373–4376

    Article  Google Scholar 

  41. Webster NS, Hill RT (2001) The culturable microbial community of the Great Barrier Reef sponge Rhopaloeides odorabile is dominated by α-proteobacterium. Mar Biol 138:843–851

    Article  CAS  Google Scholar 

  42. Reiswig HM (1981) Partial carbon and energy budgets of the bacteriosponge Verongia fistulans (Ponfera: Demospongiae) in Barbados. PSZN Mar Ecol 2:273–293

    Article  CAS  Google Scholar 

  43. Hentschel U, Usher KM, Taylor MW (2006) Marine sponge as microbial fermenters. FEMS Microb Ecol 55(2):167–177

    Article  CAS  Google Scholar 

  44. Schmitt S, Hentschel U, Taylor MW (2011) Deep sequencing reveals diversity and community structure of complex microbiota in five Mediterranean sponges. Hydrobiologia. doi:10.1007/s10750-011-0799-9

  45. Schmitt S, Tsai P, Bell J, Fromont J, Ilan M, Lindquist N, Perez T, Rodrigo A, Peter J, Schupp PJ, Vacelet J, Webster N, Hentschel U, Taylor MW (2012) Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges. ISME J 6:564–576

    Article  PubMed  CAS  Google Scholar 

  46. Webster NS, Taylor MW, Behnam F, Lücker S, Rattei T, Whalan S et al (2010) Deep sequencing reveals exceptional diversity and modes of transmission for bacterial sponge symbionts. Environ Microb 12:2070–2082

    CAS  Google Scholar 

  47. Webster NS, Taylor MW (2012) Marine sponges and their microbial symbionts: love and other relationships. Environ Microb 14(2):335–346

    Article  CAS  Google Scholar 

  48. Fieseler L, Horn M, Wagner M, Hentschel U (2004) Discovery of the novel candidate phylum “Poribacteria” in marine sponges. Appl Environ Microbiol 70(6):3724–3732

    Article  PubMed  CAS  Google Scholar 

  49. Lafi FF, Fuerst JA, Fieseler L, Engels C, Goh WWL, Hentschel U (2009) Widespread distribution of poribacteria in demospongiae. Appl Environ Microb 75(17):5695–5699

    Article  CAS  Google Scholar 

  50. Pham VD, Hnatow LL, Zhang S, Fallon RD, Sc J, Tomb J, DeLong EF, Keeler SJ (2009) Characterizing microbial diversity in production water from an Alaskan mesothermic petroleum reservoir with two independent molecular methods. Environ Microbiol 11(1):176–187

    Article  PubMed  CAS  Google Scholar 

  51. Meyer B, Kuever J (2008) Phylogenetic diversity and spatial distribution of the microbial community associated with the Caribbean deep-water sponge Polymastia cf. corticata by 16S rRNA, aprA, and amoA gene analysis. Microb Ecol 56(2):306–321

    Article  PubMed  CAS  Google Scholar 

  52. Olson JB, McCarthy PJ (2005) Associated bacterial communities of two deep-water sponges. Aquat Microb Ecol 39:47–55

    Article  Google Scholar 

  53. Cavanaugh CM, Wirsen CO, Jannasch HW (1992) Evidence for methylotrophic symbionts in a hydrothermal vent mussel (Bivalvia: Mytilidae) from the Mid-Atlantic Ridge. Appl Environ Microb 58(12):3799–3803

    CAS  Google Scholar 

  54. Childress JJ, Fisher CR, Brooks JM, Kennicutt MC II, Bidigare R, Anderson AE (1986) A methanotrophic marine molluscan (Bivalvia, Mytilidae) symbiosis: mussels fueled by gas. Science 233(4770):1306–1308

    Article  PubMed  CAS  Google Scholar 

  55. Distel DL, Lee HKW, Cavanaugh CM (1995) Intracellular coexistence of methano- and thioautotrophic bacteria in a hydrothermal vent mussel. Proc Natl Acad Sci USA 92:9598–9602

    Article  PubMed  CAS  Google Scholar 

  56. Fisher CR, Brooks JM, Vodenichar JS, Zande JM, Childress JJ, Burke RA Jr (1993) The co-occurrence of methanotrophic and chemoautotrophic sulfur-oxidizing bacterial symbionts in a deep-sea mussel. Mar Ecol 14(4):277–289

    Article  Google Scholar 

  57. Dyksterhouse SE, Gray JP, Herwig RP, Lara JC, Staley JT (1995) Cylcloclasticus pugettii gen. nov., sp. nov., and aromatic hydrocarbon-degrading bacterium from marine sediments. Int J Sys Bacteriol 45(1):116–123

    Article  CAS  Google Scholar 

  58. Geiselbrecht AD, Hedlund BP, Tichi MA, Staley JT (1998) Isolation of marine polycyclic aromatic hydrocarbon (PAH)-degrading Cycloclasticus strains from the Gulf of Mexico and comparison of their PAH degradation ability with that of Puget Sound Cycloclasticus strains. Appl Environ Microbiol 64(12):4703–4710

    PubMed  CAS  Google Scholar 

  59. Kasai Y, Kishira H, Harayama S (2002) Bacteria belonging to the genus Cycloclasticus play a primary role in the degradation of aromatic hydrocarbons released in a marine environment. Appl Environ Microb 68(11):5625–5633

    Article  CAS  Google Scholar 

  60. Niepceron M, Portet-Koltalo F, Merlin C, Motelay-Massei A, Barray S, Bodilis J (2010) Both Cycloclasticus spp. and Pseudomonas spp. as PAH-degrading bacteria in the Seine estuary (France). FEMS Microb Ecol 71:137–147

    Article  CAS  Google Scholar 

  61. Hedlund BP, Geiselbrecht AD, Bair TJ, Staley JT (1999) Polycyclic aromatic hydrocarbon degradation by a new marine bacterium, Neptunomonas naphthovorans gen. nov., sp. nov. Appl Environ Microb 65(1):251–259

    CAS  Google Scholar 

  62. Hedlund BP, Staley JT (2006) Isolation and characterization Pseudoalteromonas strains with divergent polycyclic aromatic hydrocarbon catabolic properties. Environ Microbiol 8:178–182

    Article  PubMed  CAS  Google Scholar 

  63. Zhang XY, Zhang YJ, Yu Y, Li HJ, Gao ZM, Chen XL, Chen B, Zhang YZ (2010) Neptunomonas antarctica sp. nov., isolated from marine sediment. Int J Sys Evol Microb 60(8):1958–1961

    Article  CAS  Google Scholar 

  64. Bergquist DC, Fleckstein C, Szalai EB, Knisel J, Fisher CR (2004) Environment drives physiological variability in the cold seep mussel Bathymodiolus childressi. Limnol Oceanogr 49:706–715

    Article  Google Scholar 

  65. Powell EN, Barber RD, Kennicutt MC II, Ford SE (1999) Influence of parasitism in controlling the health, reproduction and PAH body burden of petroleum seep mussels. Deep-Sea Res 46:2053–2078

    Article  Google Scholar 

  66. Miyazaki M, Nogi Y, Fujiwara Y, Kawato M, Kubokawa K, Horikoshi K (2008) Neptunomonas japonica sp. nov., an Osedax japonicus symbiont-like bacterium isolated from sediment adjacent to sperm whale carcasses off Kagoshima, Japan. Int J Sys Evol Microb 58(4):866–871

    Article  Google Scholar 

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Acknowledgments

We gratefully acknowledge Mr. Rick Webb, at the Centre for Microscopy and Microanalysis (University of Queensland), who performed the TEM. We also thank Dr. Salim Bougouffa (Hong Kong University of Science and Technology) for the useful advice in bioinformatics and data analysis. Ship time and submersible dives were funded by grant number OCE-0527139 from the National Science Foundation to CMY. This research was supported by the National Basic Research Program of China (973 Program, No. 2012CB417304) and an award (SA-C0040/UK-C0016) made by the King Abdullah University of Science and Technology to PYQ. SMA was supported by a Woods Hole Oceanographic Institution postdoctoral scholarship during manuscript preparation.

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  1. Shawn M. Arellano

    Present address: Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA, 02536, USA

Authors and Affiliations

  1. KAUST Global Collaborative Research Program, Division of Life Science, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, SAR, China

    Shawn M. Arellano, On On Lee, Jiangke Yang, Yong Wang & Pei-Yuan Qian

  2. Computational Bioscience Research Center, King Abdullah University of Science and Technology, Thuwal, Jeddah, 23955-6900, Kingdom of Saudi Arabia

    Feras F. Lafi

  3. Oregon Institute of Marine Biology, University of Oregon, P.O. Box 5389, Charleston, OR, 97420, USA

    Craig M. Young

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Shawn M. Arellano and On On Lee equally contributed in this manuscript.

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Fig. S1

Proportion of classified pyrosequencing reads at different taxonomic levels. Classification was performed with the RDP classifier at a confidence threshold of 50 % (GIF 63 kb)

High Resolution (EPS 4432 kb)

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Arellano, S.M., Lee, O.O., Lafi, F.F. et al. Deep Sequencing of Myxilla (Ectyomyxilla) methanophila, an Epibiotic Sponge on Cold-Seep Tubeworms, Reveals Methylotrophic, Thiotrophic, and Putative Hydrocarbon-Degrading Microbial Associations. Microb Ecol 65, 450–461 (2013). https://doi.org/10.1007/s00248-012-0130-y

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