Ectosymbioses between invertebrates and sulfur-oxidizing bacteria are widespread in sulfidic marine environments and have evolved independently in several invertebrate phyla. The first example from a freshwater habitat, involving Niphargus ictus amphipods and filamentous Thiothrix ectosymbionts, was recently reported from the sulfide-rich Frasassi caves in Italy. Subsequently, two new Niphargus species, N. frasassianus and N. montanarius, were discovered within Frasassi and found to co-occur with N. ictus. Using a variety of microscopic and molecular techniques, we found that all three Frasassi-dwelling Niphargus species harbor Thiothrix ectosymbionts, which belong to three distinct phylogenetic clades (named T1, T2, and T3). T1 and T3 Thiothrix dominate the N. frasassianus ectosymbiont community, whereas T2 and T3 are prevalent on N. ictus and N. montanarius. Relative distribution patterns of the three ectosymbionts are host species-specific and consistent over different sampling locations and collection years. Free-living counterparts of T1–T3 are rare or absent in Frasassi cave microbial mats, suggesting that ectosymbiont transmission among Niphargus occurs primarily through inter- or intraspecific inoculations. Phylogenetic analyses indicate that the Niphargus-Thiothrix association has evolved independently at least two times. While ectosymbioses with T1 and T2 may have been established within Frasassi, T3 ectosymbionts seem to have been introduced to the cave system by Niphargus.
References
[1]
Sapp J (2004) The dynamics of symbiosis: an historical overview. Botany 82: 1046–1056.
[2]
Dubilier N, Bergin C, Lott C (2008) Symbiotic diversity in marine animals: the art of harnessing chemosynthesis. Nature Rev Microbiol 6: 725–740.
[3]
Goffredi S (2010) Indigenous ectosymbiotic bacteria associated with diverse hydrothermal vent invertebrates. Environ Microbiol Rep 2: 479–488.
[4]
Wahl M, Mark O (1999) The predominantly facultative nature of epibiosis: experimental and observational evidence. Mar Ecol Prog Ser 187: 59–66.
[5]
Polz MF, Distel DL, Zarda B, Amann R, Felbeck H, et al. (1994) Phylogenetic analysis of a highly specific association between ectosymbiotic, sulfur-oxidizing bacteria and a marine nematode. Appl Environ Microbiol 60: 4461–4467.
[6]
Goffredi SK, Warén A, Orphan VJ, Van Dover CL, Vrijenhoek RC (2004) Novel forms of structural integration between microbes and a hydrothermal vent gastropod from the Indian ocean. App Env Microbiol 70: 3082–3090.
[7]
Bayer C, Heindl NR, Rinke C, Lücker S, Ott JA, et al. (2009) Molecular characterization of the symbionts associated with marine nematodes of the genus Robbea. Environ Microbiol Rep 1: 136–144.
[8]
Petersen JM, Ramette A, Lott C, Cambon-Bonavita M-A, Zbinden M, et al. (2010) Dual symbiosis of the vent shrimp Rimicaris exoculata with filamentous gamma-and epsilonproteobacteria at four Mid-Atlantic Ridge hydrothermal vent fields. Environ Microbiol 12: 2204–2218.
[9]
Ruehland C, Dubilier N (2010) Gamma- and epsilonproteobacterial ectosymbionts of a shallow-water marine worm are related to deep-sea hydrothermal vent ectosymbionts. Environ Microbiol 12: 2312–2326.
[10]
Bulgheresi S, Gruber-Vodicka HR, Heindl NR, Dirks U, Kostadinova M, et al. (2011) Sequence variability of the pattern recognition receptor Mermaid mediates specificity of marine nematode symbioses. ISME J 5: 986–998.
[11]
Gillan DC, Dubilier N (2004) Novel epibiotic Thiothrix bacterium on a marine amphipod. Appl Environ Microbiol 70: 3772–3775.
[12]
Dattagupta S, Schaperdoth I, Montanari A, Mariani S, Kita N, et al. (2009) A novel symbiosis between chemoautotrophic bacteria and a freshwater cave amphipod. ISME J 3: 935–943.
[13]
Sarbu SM, Galdenzi S, Menichetti M, Gentile G (2000) Geology and biology of the Frasassi caves in central Italy: an ecological multi-disciplinary study of a hypogenic underground karst system. In: Wilkens H, Culver DC, Humphreys WF, editors. Subterranean Ecosystems. Ecosystems of the World. Elsevier Science: Amsterdam. pp 359–378.
[14]
Macalady JL, Lyon EH, Koffman B, Albertson LK, Meyer K, et al. (2006) Dominant microbial populations in limestone-corroding stream biofilms, Frasassi cave system, Italy. Appl Environ Microbiol 72: 5596–5609.
[15]
Macalady JL, Dattagupta S, Schaperdoth I, Jones DS, Druschel G, et al. (2008) Niche differentiation among sulfur-oxidizing bacterial populations in cave waters. ISME J 2: 590–601.
[16]
Bertolani R, Manicardi GC, Rebecchi L (1994) Faunistic study in the karst complex of Frasassi (Genga, Italy). Int J Speleol 23: 61–77.
[17]
Flot J-F, W？rheide G, Dattagupta S (2010) Unsuspected diversity of Niphargus amphipods in the chemoautotrophic cave ecosystem of Frasassi, central Italy. BMC Evol Biol 10: 171.
[18]
Karaman GS, Borowsky B, Dattagupta S (2010) Two new species of the genus Niphargus Schi？dte, 1849 (Amphipoda, fam. Niphargidae) from the Frasassi cave system in Central Italy. Zootaxa 2439: 35–52.
[19]
Chaston J, Goodrich-Blair H (2010) Common trends in mutualism revealed by model associations between invertebrates and bacteria. FEMS Microbiol Rev 34: 41–58.
[20]
Bright M, Bulgheresi S (2010) A complex journey: transmission of microbial symbionts. Nat Rev Microbiol 8: 218–230.
[21]
Weisburg WG, Barns SM, Pelletier DA, Lane DJ (1991) 16S ribosomal DNA amplification for phylogenetic study. J Bacteriol 173: 697–703.
[22]
Huber T, Faulkner G, Hugenholtz P (2004) Bellerophon: a program to detect chimeric sequences in multiple sequence alignments. Bioinformatics 20: 2317–2319.
[23]
Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215: 403–410.
[24]
Katoh K, Toh H (2010) Parallelization of the MAFFT multiple sequence alignment program. Bioinformatics 26: 1899–1900.
[25]
Katoh K, Toh H (2008) Improved accuracy of multiple ncRNA alignment by incorporating structural information into a MAFFT-based framework. BMC Bioinformatics 9: 212.
[26]
Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, et al. (2009) Introducing mothur: open-source, platform-independent, community-supported software for describing and comparing microbial communities. Appl Environ Microbiol 75: 7537–7541.
[27]
Posada D (2008) JModelTest: Phylogenetic Model Averaging. Mol Biol Evol 25: 1253–1256.
[28]
Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: 696–704.
[29]
Watsuji T, Nakagawa S, Tsuchida S, Toki T, Hirota A, et al. (2010) Diversity and function of epibiotic microbial communities on the galatheid crab, Shinkaia crosnieri. Microbes Environ 25: 288–294.
[30]
Gouy M, Guindon S, Gascuel O (2010) SeaView version 4: a multiplatform graphical user interface for sequence alignment and phylogenetic tree building. Mol Biol Evol 27: 221–224.
[31]
Hugenholtz P, Tyson GW, Blackall LL (2002) Design and evaluation of 16S rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization. In: Aquino de Muro M, Rapley R, editors. Gene Probes: Principles and Protocols. Humana Press: London. pp 29–42.
[32]
Ashelford KE, Weightman AJ, Fry JC (2002) PRIMROSE: a computer program for generating and estimating the phylogenetic range of 16S rRNA oligonucleotide probes and primers in conjunction with the RDP-II database. Nucleic Acids Res 30: 3481–3489.
[33]
Fuchs BM, Gl？ckner FO, Wulf J, Amann R (2000) Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes. Appl Environ Microbiol 66: 3603–3607.
[34]
Amann RI (1995) In situ identification of microorganisms by whole cell hybridization with rRNA-targeted nucleic acid probes. In: Akkerman ADL, van Elsas DJ, de Bruijn FJ, editors. Molecular Microbial Ecology Manual. Kluwer Academic Publishers: Dordrecht. pp 1–15.
[35]
Daims H, Brühl A, Amann R, Schleifer KH, Wagner M (1999) The domain-specific probe EUB338 is insufficient for the detection of all bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22: 434–444.
[36]
Fisher MM, Triplett EW (1999) Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl Environ Microbiol 65: 4630–4636.
[37]
Meziti A, Ramette A, Mente E, Kormas KA (2010) Temporal shifts of the Norway lobster (Nephrops norvegicus) gut bacterial communities. FEMS Microbiol Ecol 74: 472–484.
[38]
Boer SI, Hedtkamp SIC, Van Beusekom JEE, Fuhrman JA, Boetius A, et al. (2009) Time- and sediment depth-related variations in bacterial diversity and community structure in subtidal sands. ISME J 3: 780–791.
[39]
Ramette A (2009) Quantitative community fingerprinting methods for estimating the abundance of operational taxonomic units in natural microbial communities. Appl Environ Microbiol 75: 2495–2505.
[40]
Muyzer G, Hottentrager S, Teske A, Wawer C (1996) Denaturing gradient gel electrophoresis of PCR-amplified 16S rDNA – a new molecular approach to analyze the genetic diversity of mixed microbial communities. In: Akkermans ADL, van Elsas JD, de Bruijn FJ, editors. Molecular microbial ecology manual. Kluwer Academic Publishing: Dordrecht. pp 3.4.4.1–3.4.4.22.
[41]
Cardinale M, Brusetti L, Quatrini P, Borin S, Puglia AM, et al. (2004) Comparison of different primer sets for use in Automated Ribosomal Intergenic Spacer Analysis of complex bacterial communities. Appl Environ Microbiol 70: 6147–6156.
[42]
Bierne N, Tanguy A, Faure M, Faure B, David E, et al. (2007) Mark-recapture cloning: a straightforward and cost-effective cloning method for population genetics of single-copy nuclear DNA sequences in diploids. Mol Ecol Notes 7: 562–566.
[43]
Legendre P, Legendre L (1998) Numerical Ecology. Elsevier science: Amsterdam. 853 p.
[44]
Ramette A (2007) Multivariate analyses in microbial ecology. FEMS Microbiol Ecol 62: 142–160.
[45]
R Development Core Team (2011) R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing: Vienna. ISBN 3-900051-07-0. R Project website. Available: http://www.R-project.org. Accessed 2012 Jan 9.
[46]
Oksanen J, Blanchet FG, Kindt R, Legendre P, Minchin PR, et al.. (2011) vegan: Community Ecology Package. R Project website. Available: http://CRAN.R-project.org/package=vegan. Accessed 2012 Jan 9.
[47]
Moran NA (1996) Accelerated evolution and Muller's rachet in endosymbiotic bacteria. Proc Natl Acad Sci USA 93: 2873–2878.
[48]
Moran NA, Plague GR (2004) Genetic changes following host restriction in bacteria. Curr Opin Genet Dev 14: 627–633.
[49]
Larkin JM, Shinabarger DL (1983) Characterization of Thiothrix nivea. Int J Syst Bacteriol 33: 841–846.
[50]
Chernousova E, Gridneva E, Grabovich M, Dubinina G, Akimov V, et al. (2009) Thiothrix caldifontis sp. nov. and Thiothrix lacustris sp. nov., gammaproteobacteria isolated from sulfide springs. Int J Syst Evol Microbiol 59: 3128–3135.
[51]
Bulgheresi S, Schabussova I, Chen T, Mullin NP, Maizels RM, et al. (2006) A new C-type lectin similar to the human immunoreceptor DC-SIGN mediates symbiont acquisition by a marine nematode. Appl Environ Microbiol 72: 2950–2956.
[52]
Ott J, Bright M, Bulgheresi S (2004) Marine microbial thiotrophic ectosymbioses. Oceanogr Mar Biol 42: 95–118.
[53]
Fi？er C, Sket B, Trontelj P (2008) A phylogenetic perspective on 160 years of troubled taxonomy of Niphargus (Crustacea: Amphipoda). Zool Scripta 37: 665–680.
[54]
V？in？l？ R, Witt J, Grabowski M, Bradbury J, Jazdzewski K, et al. (2008) Global diversity of amphipods (Amphipoda; Crustacea) in freshwater. Hydrobiologia 595: 241–255.
[55]
Sarbu SM, Kane TC, Kinkle BK (1996) A chemoautotrophically based cave ecosystem. Science 272: 1953–1955.
[56]
Unz RF, Head IM (2005) Genus I. Thiothrix Winogradsky 1888. In: Garrity GM, Brenner DJ, Krieg NR, Staley JR., editors. Bergey's Manual of Systematic Bacteriology, Volume Two: The Proteobacteria, Parts A–C. Springer Verlag. 1392 p.
[57]
Brosius J, Dull TJ, Sleeter DD, Noller HF (1981) Gene organization and primary structure of a ribosomal operon from Escherichia coli. J Mol Biol 148: 107–127.
