Intra-host symbiont diversity in eastern Pacific cold seep tubeworms identified by the 16S-V6 region, but undetected by the 16S-V4 region


Autoři: Corinna Breusing aff001;  Maximilian Franke aff003;  Curtis Robert Young aff002
Působiště autorů: Monterey Bay Aquarium Research Institute, Moss Landing, CA, United States of America aff001;  National Oceanography Centre, Southampton, England, United Kingdom aff002;  Max Planck Institute for Marine Microbiology, Bremen, Germany aff003
Vyšlo v časopise: PLoS ONE 15(1)
Kategorie: Research Article
doi: 10.1371/journal.pone.0227053

Souhrn

Vestimentiferan tubeworms are key taxa in deep-sea chemosynthetic habitats worldwide. As adults they obtain their nutrition through their sulfide-oxidizing bacterial endosymbionts, which are acquired from the environment. Although horizontal transmission should favor infections by various symbiotic microbes, the current paradigm holds that every tubeworm harbors only one endosymbiotic 16S rRNA phylotype. Although previous studies based on traditional Sanger sequencing have questioned these findings, population level high-throughput analyses of the symbiont 16S diversity are still missing. To get further insights into the symbiont genetic variation and uncover hitherto hidden diversity we applied state-of-the-art 16S-V4 amplicon sequencing to populations of the co-occurring tubeworm species Lamellibrachia barhami and Escarpia spicata that were collected during E/V Nautilus and R/V Western Flyer cruises to cold seeps in the eastern Pacific Ocean. In agreement with earlier work our sequence data indicated that L. barhami and E. spicata share one monomorphic symbiont phylotype. However, complementary CARD-FISH analyses targeting the 16S-V6 region implied the existence of an additional phylotype in L. barhami. Our results suggest that the V4 region might not be sufficiently variable to investigate diversity in the intra-host symbiont population at least in the analyzed sample set. This is an important finding given that this region has become the standard molecular marker for high-throughput microbiome analyses. Further metagenomic research will be necessary to solve these issues and to uncover symbiont diversity that is hidden below the 16S rRNA level.

Klíčová slova:

Haplotypes – Hydrothermal vents – Nucleotide sequencing – Polymerase chain reaction – Population genetics – Probe hybridization – Ribosomal RNA – Symbiosis


Zdroje

1. Halanych KM. Molecular phylogeny of siboglinid annelids (a.k.a. pogonophorans): a review. Hydrobiologia 2005; 535: 297–307.

2. Bright M, Lallier FH. The biology of vestimentiferan tubeworms. Oceano Mar Biol Ann Rev. 2010; 48: 213–265.

3. Dando PR, Southward AF, Southward EC, Dixon DR, Crawford A, Crawford M. Shipwrecked tube worms. Nature 1992; 356: 667–667.

4. Hughes DJ, Crawford M. A new record of the vestimentiferan Lamellibrachia sp. (Polychaeta: Siboglinidae) from a deep shipwreck in the eastern Mediterranean. Mar Biodivers Rec. 2009; 1: e21.

5. Gambi MC, Schulze A, Amato E. Record of Lamellibrachia sp. (Annelida: Siboglinidae: Vestimentifera) from a deep shipwreck in the western Mediterranean Sea (Italy). Mar Biodivers Rec. 2011; 4: e24.

6. Vrijenhoek RC. Genetics and evolution of deep-sea chemosynthetic bacteria and their invertebrate hosts. In: Kiel S, editor. The Vent and Seep Biota. Netherlands: Springer; 2010. pp. 15–49.

7. Bright M, Sorgo A. Ultrastructural reinvestigation of the trophosome in adults of Riftia pachyptila (Annelida, Siboglinidae). Invert Biol. 2003; 122: 347–368.

8. Southward EC, Schulze A, Gardiner SL. Pogonophora (Annelida): form and function. Hydrobiologia 2005; 535: 227–251.

9. Nussbaumer AD, Fisher CR, Bright M. Horizontal endosymbiont transmission in hydrothermal vent tubeworms. Nature 2006; 441: 345–348. doi: 10.1038/nature04793 16710420

10. Klose J, Polz MF, Wagner M, Schimak MP, Gollner S, Bright M. Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free-living population. PNAS 2015; 112: 11300–11305. doi: 10.1073/pnas.1501160112 26283348

11. Frank SA. Host-symbiont conflict over the mixing of symbiotic lineages. Proc Biol Sci. 1996; 263: 339–344. doi: 10.1098/rspb.1996.0052 8920255

12. McMullin ER, Hourdez S, Schaeffer SW, Fisher CR. Phylogeny and biogeography of deep sea vestimentiferan tubeworms and their bacterial symbionts. Symbiosis 2003; 34: 1–41.

13. Gardebrecht A, Markert S, Sievert SM, Felbeck H, Thürmer A, Albrecht D, et al. Physiological homogeneity among the endosymbionts of Riftia pachyptila and Tevnia jerichonana revealed by proteogenomics. ISME J. 2012; 6: 766–776. doi: 10.1038/ismej.2011.137 22011719

14. Di Meo CA, Wilbur AE, Holben WE, Feldman RA, Vrijenhoek RC, Cary SC. Genetic variation among endosymbionts of widely distributed vestimentiferan tubeworms. Appl Environ Microbiol. 2000; 66: 651–658. doi: 10.1128/aem.66.2.651-658.2000 10653731

15. Vrijenhoek RC, Duhaime M, Jones WJ. Subtype variation among bacterial endosymbionts of tubeworms (Annelida: Siboglinidae) from the Gulf of California. Biol Bull. 2007; 212: 180–184. doi: 10.2307/25066600 17565107

16. Duperron S, De Beer D, Zbinden M, Boetius A, Schipani V, Kahil N, et al. Molecular characterization of bacteria associated with the trophosome and the tube of Lamellibrachia sp., a siboglinid annelid from cold seeps in the eastern Mediterranean. FEMS Microbiol Ecol. 2009; 69: 395–409. doi: 10.1111/j.1574-6941.2009.00724.x 19583785

17. Thiel V, Hügler M, Blümel M, Baumann HI, Gärtner A, Schmaljohann R, et al. Widespread occurrence of two carbon fixation pathways in tubeworm endosymbionts: lessons from hydrothermal vent associated tubeworms from the Mediterranean Sea. Front Microbiol. 2012; 3: 423. doi: 10.3389/fmicb.2012.00423 23248622

18. Zimmermann J, Lott C, Weber M, Ramette A, Bright M, Dubilier N, et al. Dual symbiosis with co‐occurring sulfur‐oxidizing symbionts in vestimentiferan tubeworms from a Mediterranean hydrothermal vent. Environ Microbiol. 2014; 16: 3638–3656. doi: 10.1111/1462-2920.12427 24552661

19. Reveillaud J, Anderson R, Reves-Sohn S, Cavanaugh C, Huber JA. Metagenomic investigation of vestimentiferan tubeworm endosymbionts from Mid-Cayman Rise reveals new insights into metabolism and diversity. Microbiome 2018; 6: 19. doi: 10.1186/s40168-018-0411-x 29374496

20. Perez M, Juniper SK. Insights into symbiont population structure among three vestimentiferan tubeworm host species at eastern Pacific spreading centers. Appl Environ Microbiol. 2016; 82: 5197–5205. doi: 10.1128/AEM.00953-16 27316954

21. Li Y, Liles MR, Halanych KM. Endosymbiont genomes yield clues of tubeworm success. ISME J. 2018; 12: 2785–2795. doi: 10.1038/s41396-018-0220-z 30022157

22. Yang Y, Sun J, Sun Y, Kwan YH, Wong WC, Zhang Y, et al. Genomic, transcriptomic, and proteomic insights into the symbiosis of deep-sea tubeworm holobionts. ISME J. 2019; doi: 10.1038/s41396-019-0520-y 31595051

23. Salathé RM, Vrijenhoek RC. Temporal variation and lack of host specificity among bacterial endosymbionts of Osedax bone worms (Polychaeta: Siboglinidae). BMC Evol Biol. 2012; 12: 189. doi: 10.1186/1471-2148-12-189 23006795

24. Geller J, Meyer C, Parker M, Hawk H. Redesign of PCR primers for mitochondrial cytochrome c oxidase subunit I for marine invertebrates and application in all-taxa biotic surveys. Mol Ecol Resour. 2013; 13: 851–861. doi: 10.1111/1755-0998.12138 23848937

25. Johnson SB, Won YJ, Harvey JB, Vrijenhoek RC. A hybrid zone between Bathymodiolus mussel lineages from eastern Pacific hydrothermal vents. BMC Evol Biol. 2013; 13: 21. doi: 10.1186/1471-2148-13-21 23347448

26. Breusing C, Johnson SB, Tunnicliffe V, Vrijenhoek RC. Population structure and connectivity in Indo-Pacific deep-sea mussels of the Bathymodiolus septemdierum complex. Conserv Genet. 2015; 16: 1415–1430.

27. Caporaso JG, Lauber CL, Walters WA, Berg-Lyons D, Lozupone CA, Turnbaugh PJ, et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. PNAS 2011; 108: 4516–4522. doi: 10.1073/pnas.1000080107 20534432

28. Andrews S. FastQC: a quality control tool for high throughput sequence data. http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ 2010.

29. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 2010; 30: 2114–2120.

30. Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 2010; 26: 2460–2461. doi: 10.1093/bioinformatics/btq461 20709691

31. Caporaso JG, Kuczynski J, Stombaugh J, Bittinger K, Bushman FD, Costello EK, et al. QIIME allows analysis of high-throughput community sequencing data. Nature Methods 2010; 7: 335–336. doi: 10.1038/nmeth.f.303 20383131

32. Eren AM, Maignien L, Sul WJ, Murphy LG, Grim SL, Morrison HG, et al. Oligotyping: Differentiating between closely related microbial taxa using 16S rRNA gene data. Methods Ecol Evol. 2013; 4: 1111–1119.

33. Daims H, Brühl A, Amann R, Schleifer KH, Wagner M. 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. 1999; 22: 434–444. doi: 10.1016/S0723-2020(99)80053-8 10553296

34. Wallner G, Amann R, Beisker W. Optimizing fluorescent in situ-hybridization with rRNA-targeted oligonucleotide probes for flow cytometric identification of microorganisms. Cytometry 1993; 14: 136–143. doi: 10.1002/cyto.990140205 7679962

35. Steedman HF. Polyester wax: a new ribboning embedding medium for histology. Nature 1957; 179: 1345. doi: 10.1038/1791345a0 13451615

36. Pernthaler A, Pernthaler J, Amann R. Fluorescence in situ hybridization and catalyzed reporter deposition for the identification of marine bacteria. Appl Environ Microbiol. 2002; 68: 3094–3101. doi: 10.1128/AEM.68.6.3094-3101.2002 12039771

37. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nature Methods 2012; 9: 676–682. doi: 10.1038/nmeth.2019 22743772

38. Fauvart M, Michiels J. Rhizobial secreted proteins as determinants of host specificity in the rhizobium–legume symbiosis. FEMS Microbiol Lett. 2008; 285: 1–9. doi: 10.1111/j.1574-6968.2008.01254.x 18616593

39. Nyholm SV, Song P, Dang J, Bunce C, Girguis PR. Expression and putative function of innate immunity genes under in situ conditions in the symbiotic hydrothermal vent tubeworm Ridgeia piscesae. PLoS ONE 2012; 7: e38267. doi: 10.1371/journal.pone.0038267 22701617

40. Boetius A. Microfauna–macrofauna interaction in the seafloor: lessons from the tubeworm. PLoS Biology 2005; 3: e102. doi: 10.1371/journal.pbio.0030102 15760275

41. Cordes EE, Arthur MA, Shea K, Arvidson RS, Fisher CR. Modeling the mutualistic interactions between tubeworms and microbial consortia. PLoS Biology 2005; 3: e77. doi: 10.1371/journal.pbio.0030077 15736979

42. Verna C, Ramette A, Wiklund H, Dahlgren TG, Glover AG, Gaill F, et al. High symbiont diversity in the bone‐eating worm Osedax mucofloris from shallow whale‐falls in the North Atlantic. Environ Microbiol. 2010; 12: 2355–2370. doi: 10.1111/j.1462-2920.2010.02299.x 21966925

43. Gage DJ. Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev. 2004; 68: 280–300. doi: 10.1128/MMBR.68.2.280-300.2004 15187185

44. Ikuta T, Takaki Y, Nagai Y, Shimamura S, Tsuda M, Kawagucci S, et al. Heterogeneous composition of key metabolic gene clusters in a vent mussel symbiont population. ISME J. 2016; 10: 990–1001. doi: 10.1038/ismej.2015.176 26418631

45. Chakravorty S, Helb D, Burday M, Connell N, Alland D. A detailed analysis of 16S ribosomal RNA gene segments for the diagnosis of pathogenic bacteria. J. Microbiol Methods. 2007; 69: 330–339. doi: 10.1016/j.mimet.2007.02.005 17391789


Článek vyšel v časopise

PLOS One


2020 Číslo 1