Abstract
The recent application of molecular tools to address associations between bacteria and marine invertebrates has provided access to an immense diversity of unidentified microbes resistant to cultivation. However, the role of bacteria as partners in animal physiology remains unclear and in most cases difficult to investigate in the absence of adequate condition of cell growth and proliferation. In this work, we studied the reservoir of microbes associated with the excretory organs of Nautilus macromphalus as a model. Using the bacterial 16S RNA gene as a marker, we compared three complementary approaches for bacterial detection: bacterial DNA extraction from N. macromphalus tissues (“molecular approach”), strain isolation to provide a bacterial culture collection (“microbiological approach”) and finally, maintenance of N. macromphalus excretory organ cells with associated bacteria (“cellular approach”). Our results stress the potential of the “cellular approach” as a promising new tool as it promotes the detection of as yet uncultured β-proteobacteria and spirochaetes associated with N. macromphalus, and serves as a foundation for future studies describing potential roles that these bacteria may play in Nautilus.
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References
Amann RI, Krumholz L, Stahl DA (1990) Fluorescent-oligonucleotide probing of whole cells for determinative, phylogenetic, and environmental studies in microbiology. J Bacteriol 172:762–770
Barbieri E, Paster BJ, Hughes D, Zurek L, Moser DP, Teske A, Sogin ML (2001) Phylogenetic characterization of epibiotic bacteria in the accessory nidamental gland and egg of the squid Loligo pealei (Cephalopoda: Loliginidae). Environ Microbiol 3:151–167
Bloodgood RA (1977) The squid accessory nidamental gland, ultrastructure and association with bacteria. Tissue Cell 9:197–208
Boucher-Rodoni R, Mangold K (1994) Ammonia production in cephalopods: physiological and evolutionary aspects. Mar Freshw Behav Physiol 25:53–60
Cole JR, Chai B, Farris RJ, Wang Q, Kulam SA, McGarrell DM, Garrity GM, Tiedje JM (2005) The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis. Nucleic Acids Res 33:294–296
Connon SA, Tovanabootr A, Dolan M, Vergin K, Giovannoni SJ, Semprini L (2005) Bacterial community composition determined by culture-independent and dependent methods during propane-stimulated bioremediation in trichloroethene-contaminated groundwater. Environ Microbiol 7:165–178
Davidson SK, Koropatnick TA, Kossmehl R, Sycuro L, McFall-Ngai MJ (2004) NO means ‘yes’ in the squid-vibrio symbiosis: nitric oxide (NO) during the initial stages of beneficial association. Cell Microbiol 6(12):1139–1151
Domart-Coulon I, Doumenc D, Auzoux-Bordenave S, Le Fichant Y (1994) Identification of media supplements that improve the viability of primary cell cultures of Crassostrea gigas oysters. Cytotechnology 16:109–120
Dubilier N, Mulders C, Ferdelman T, de Beer D, Pernthaler A, Klein M, Wagner M, Erseus C, Thiermann F, Krieger J, Giere O, Amann R (2001) Endosymbiotic sulphate-reducing and sulphide-oxidizing bacteria in an oligochaete worm. Nature 411:298–302
Eilers H, Pernthaler J, Glockner FO, Amann R (2000) Culturability and in situ abundance of pelagic bacteria from the North Sea. Appl Environ Microbiol 66:3044–3051
Frank U, Rabinowitz C, Rinkevich B (1994) In vitro establishment of continuous cell cultures and cell lines from ten colonial cnidarians. Mar Biol 120:491–499
Good IJ (1953) The population frequencies of species and the estimation of population parameters. Biometrica 40:237–264
Grigioni S, Boucher-Rodoni R, Demarta A, Tonolla M, Peduzzi R (2000) Phylogenetic characterisation of bacterial symbionts in the accessory nidamental glands of the sepioid S. officinalis (Cephalopoda: Decapoda). Mar Biol 136:217–222
Hall T (1997–2001) BioEdit. http://www.mbioncsu.edu/BioEdit/bioedit.html
Hentschel U, Usher KM, Taylor MW (2006) Marine sponges as microbial fermenters. FEMS Microbiol Ecol 55(2):167–177
Holmström C, Kjelleberg S (1999) Marine Pseudoalteromonas species are associated with higher organisms and produce biologically active extracellular agents. FEMS Microbiol Ecol 30:285–293
Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20:2317–2319
Kaufman MR, Ikeda Y, Patton C, Van Dykhuizen G, Epel D (1998) Bacterial symbionts colonize the accessory nidamental gland of the squid Loligo opalescens via horizontal transmission. Biol Bull 194:36–43
Kimura H, Sato M, Sasayama Y, Naganuma T (2003) Molecular characterization and in situ localization of endosymbiotic 16S ribosomal RNA and RuBisCO genes in the pogonophoran tissue. Mar Biotechnol 5:261–269
Kushmaro A, Loya Y, Fine M, Rosenberg E (1996) Bacterial infection and coral bleaching. Nature 380:396
Lamarcq LH, McFall-Ngai MJ (1998) Induction of a gradual, reversible morphogenesis of its host’s epithelial brush border by Vibrio fischeri. Infect Immun 66(2):777–785
Marie D, Partensky F, Jacquet S, Vaulot D (1997) Enumeration and cell cycle analysis of natural populations of marine picoplankton by flow cytometry using the nucleic acid stain SYBRgreen I. Appl Environ Microbiol 63:186–193
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxic assays. J Immunol Methods 65:55–63
McFall-Ngai MJ (2002) Unseen forces: the influence of bacteria on animal development. Dev Biol 242:1–14
McFall-Ngai MJ, Ruby EG (1991) Symbiotic recognition and subsequent morphogenesis as early events in an animal–bacterial mutualism. Science 254:1491–1494
Nishiguchi MK, Nair VS (2003) Evolution of symbiosis in the Vibrionaceae: a combined approach using molecules and physiology. Int J Syst Evol Microbiol 53(Pt 6):2019–2026
Nishiguchi MK, Ruby EG, McFall-Ngai MJ (1998) Competitive dominance among strains of luminous bacteria provides an unusual form of evidence for parallel evolution in Sepiolid squid-vibrio symbioses. Appl Environ Microbiol 64(9):3209–3213
Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740
Phillips HJ (1973) Dye exclusion tests for cell viability. In: Kruse PP, Patterson MK (eds) Tissue culture methods and applications. Academic, New York, pp 406–408
Pichon D, Gaia V, Norman MD, Boucher-Rodoni R (2005) Phylogenetic diversity of epibiotic bacteria in the accessory nidamental glands of squids (Cephalopoda: Loliginidae and Idiosepiidae). Mar Biol 147(6):1323–1332
Piel J, Butzke D, Fusetani N, Hui D, Platzer M, Wen G, Matsunaga S (2005) Exploring the chemistry of uncultivated bacterial symbionts: antitumor polyketides of the pederin family. J Nat Prod 68:472–479
Pierantoni U (1917) Organsi luminosi, organi simbiotici e ghiandola nidamentale accessoria nei cefalopodi. Boll Soc Nat Napoli 30:30–36
Rajan TV (2005) The eye does not see what the mind does not know: the bacterium in the worm. Perspect Biol Med 48:31–41
Rieder G, Fischer W, Haas R (2005) Interaction of Helicobacter pylori with host cells: function of secreted and translocated molecules. Curr Opin Microbiol 8(1):67–73
Schipp R, Martin AW, Liebermann H, Magnier Y (1985) Cytomorphology and function of pericardial appendages of Nautilus (Cephalopoda, Tetrabranchiata). Zoomorphology 105:16–29
Schipp R, Chung YS, Arnold JM (1990) Symbiotic bacteria in the coelom of Nautilus (Cephalopoda, Tetrabranchiata). Cell Tissue Res 219:585–604
Schirmer A, Gadkari R, Reeves CD, Ibrahim F, DeLong EF, Hutchinson CR (2005) Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with the marine sponge Discodernia dissolute. Appl Environ Microbiol 71(8):4840–4849
Sritharan V, Barker RH (1991) A simple method for diagnosing M. tuberculosis infection in clinical samples using PCR. Mol Cell Probes 5:385–395
Stackebrandt E, Goebel BM (1994) Taxonomic note: a place for DNA–DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int J Syst Bacteriol 44:846–849
Webster NS, Wilson KJ, Blackall LL, Hill RT (2001) Phylogenetic diversity of bacteria associated with the marine sponge Rhopaloeides odorabile. Appl Environ Microbiol 67:434–444
Wimmer W, Perovic S, Kruse M, Schroder HC, Krasko A, Batel R, Muller WE (1999) Origin of the integrin-mediated signal transduction. Functional studies with cell cultures from the sponge Suberites domuncula. Eur J Biochem 260:156–165
Zar JH (1999) Biostatistical analysis. Prentice-Hall, Englewood Cliffs
Acknowledgments
We thank P. Joannot, S. Loueckote, A. Gerbault and C. Goiran for their help in providing N. macromphalus specimens from New-Caledonia. The help of C. Courties for flow cytometry analysis was highly appreciated. We thank A. Andouche and F. Ponton for helpful comments on this manuscript. This work was supported by a Bonus Quality Research grant from the Muséum National d’Histoire Naturelle (PARIS). The French Ministry for National Education and Research is also acknowledged for providing M. Pernice with a Ph.D. grant. The experiments complied with the current French laws.
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Communicated by S.A. Poulet, Roscoff
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Pernice, M., Pichon, D., Domart-Coulon, I. et al. Primary co-culture as a complementary approach to explore the diversity of bacterial associations in marine invertebrates: the example of Nautilus macromphalus (Cephalopoda: Nautiloidea). Mar Biol 150, 749–757 (2007). https://doi.org/10.1007/s00227-006-0413-2
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DOI: https://doi.org/10.1007/s00227-006-0413-2