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Journal of Oceanography

, Volume 68, Issue 5, pp 599–613 | Cite as

Diversity analysis of protistan microplankton in Sagami Bay by 18S rRNA gene clone analysis using newly designed PCR primers

  • Sau Pin Kok
  • Tomohiko Kikuchi
  • Tatsuki Toda
  • Norio Kurosawa
Original Article

Abstract

The diversity of protistan microplankton in Sagami Bay was revealed by 18S rRNA gene clone analysis using newly designed PCR primers. PCR amplification consisted of a first reaction targeting the V3–V5 region of the 18S rRNA gene, followed by a nested reaction targeting the V3–V4 region. In total, 629 clones consisting of 108 phylotypes were affiliated with a variety of protistan groups including dinoflagellates, diatoms, prymnesiomonada, chlorophyta, ciliophora, cercozoa, and heterokonta. The dinoflagellate group was detected most frequently and shared approximately 74 % of the total clones. Within this group, approximately half of the clones belonged to the parasitic dinoflagellate Syndiniales group I, which was first reported from Sagami Bay. The genera Woloszynskia, Gonyaulax, Neoceratium, and Karlodinium have not been reported from this bay until now. The second most frequent group was diatoms, which shared approximately 22 % of the total clones. Within this group, highly diverse Thalassiosira phylotypes were detected, and they shared approximately 70 % of the diatom clones. Therefore, highly diverse protists including some candidate groups were successfully detected, indicating that the designed primers and PCR protocol will be useful for molecular diversity analyses of protistan microplankton communities in aquatic environments.

Keywords

Protistan microplankton 18S rDNA Community structure Sagami Bay PCR primer 

Notes

Acknowledgments

We are grateful to Mr. Y. Asakura at the Manazuru Marine Laboratory for Science Education, Yokohama National University, for his support in collecting samples. We also thank Dr. H. Miyaguchi and Dr. K. Watanabe for their advice and support in conducting this study. We also wish to express our sincere thanks to Prof. Dr. S. Taguchi, Soka University, and Dr. K. Hamasaki, Tokyo University, for their valuable suggestions to improve this paper.

References

  1. Adachi M (2000) Phylogenic analysis of phytoplankton. Ocean Mon Mag Special Eds 23:18–23 (in Japanese)Google Scholar
  2. Altschul SF, Gish W, Miller W, Myers EW, Lipman D (1990) Basic local alignment search tool. J Mol Biol 215:403–410. doi: 10.1016/S00-2836(05)80360-2 Google Scholar
  3. Ara K, Hiromi J (2008) Temporal variability and characterization of physicochemical properties in the neritic area of Sagami Bay, Japan. J Oceanogr Soc Japan 64:195–210Google Scholar
  4. Ara K, Fukuyama S, Tashiro M, Hiromi J (2011) Seasonal and year-to-year variability in chlorophyll a and microphytoplankton assemblages for 9 years (2001–2009) in the neritic area of Sagami Bay, Japan. Plankton Benthos Res 6:158–174CrossRefGoogle Scholar
  5. Azam F, Fenchel T, Field JG, Gray JS, Meyer-Reil LA, Thingstad F (1983) The ecological role of water-column microbes in the sea. Mar Ecol Prog Ser 10:257–263. doi: 10.3354/meps010257 CrossRefGoogle Scholar
  6. Baek SH, Shimode S, Kikuchi T (2007) Reproductive ecology of the dominant dinoflagellate Ceratium fusus, in coastal area of Sagami Bay, Japan. J Oceanogr Soc Japan 63:35–45Google Scholar
  7. Behnke A, Barger KJ, Bunge J, Stoeck T (2010) Spatio-temporal variation in protistan communities along as O2/H2S gradient in the anoxic Framvaren Fjord (Norway). FEMS Microbiol Ecol 72:89–102. doi: 10.1111/j.1574-6941.2010.00836.x CrossRefGoogle Scholar
  8. Boon N, De Windt W, Verstraete W, Top EM (2002) Evaluation of nested PCR-DGGE (denaturing gradient gel electrophoresis) with group-specific 16S rRNA primers for the analysis of bacterial communities from different wastewater treatment plants. FEMS Microbiol Ecol 39:101–112. doi: 10.1111/j.1574-6941.2002.tb00911.x Google Scholar
  9. Burton RS (1996) Molecular tools in marine ecology. J Exp Mar Biol Ecol 200:85–101. doi: 10.1016/S0022-0981(96)02641-X CrossRefGoogle Scholar
  10. Caron DA, Lim EL, Dennett MR, Gast RJ, Kosman C, Delong EF (1999) Molecular phylogenetic analysis of the heterotrophic chrysophyte genus Paraphysomonas (Chrysophyceae), and the design of rRNA-targeted oligonucleotide probes for two species. J Phycol 35:824–837. doi: 10.1046/j.1529-8817.1999.3540824.x CrossRefGoogle Scholar
  11. Chambouvet A, Morin P, Marie D, Guillou L (2008) Control of toxic marine dinoflagellate blooms by serial parasitic killers. Science 322:1254–1257. doi: 10.1126/science.1164387 CrossRefGoogle Scholar
  12. Coats DW, Park MG (2002) Parasitism of photosynthetic dinoflagellates by three strains of amoebophrya (dinophyta): parasite survival, infectivity, generation time, and host specificity. J Phycol 38:520–528. doi: 10.1046/j.1529-8817.2002.01200.x Google Scholar
  13. Countway PD, Gast RJ, Savai P, Caron DA (2005) Protistan diversity estimates based on 18S rDNA from seawater incubations in the Western North Atlantic. J Eukaryot Microbiol 52:95–106. doi: 10.1111/j.1550-7408.2005.0502006.x CrossRefGoogle Scholar
  14. Countway PD, Gast RJ, Dennett M, Savai P, Rose JM, Caron DA (2007) Distinct protistan assemblages characterize the euphotic zone and deep-sea (2500 m) of the western North Atlantic (Sargasso Sea and Gulf Stream). Environ Microbiol 9:1219–1232. doi: 10.1111/j.1462-2920.2007.01243.x CrossRefGoogle Scholar
  15. Countway PD, Vigil PD, Schnetzer A, Moorthi SD, Caron DA (2010) Seasonal analysis of protistan community structure and diversity at the USC Microbial Observatory (San Pedro Channel, North Pacific Ocean). Limnol Oceanogr 55:2381–2396. doi: 10.4319/lo.2010.55.6.2381 CrossRefGoogle Scholar
  16. Díez B, Pedros-AHo C, Massana R (2001) Study of genetic diversity of eukaryotic picoplankton in different oceanic regions by small-subunit rRNA gene cloning and sequencing. Appl Environ Microbiol 67:2932–2941. doi: 10.1128/AEM.67.7.2932-2941.2001 CrossRefGoogle Scholar
  17. Edgcomb V, Orsi W, Bunge J, Jeon S, Christen R, Leslin C, Holder M, Taylor GT, Suarez P, Varela R, Epstein S (2011) Protistan microbial observatory in the Cariaco Basin, Caribbean. I. Pyrosequencing vs Sanger insights into species richness. J ISME 5:1344–1356. doi: 10.1038/ismej.2011.6 CrossRefGoogle Scholar
  18. Fitzpatrick E, Caron DA, Schnetzer A (2010) Development and environmental application of a genus-specific quantitative PCR approach for Pseudo-nitzschia sp. Mar Biol 157:1161–1169. doi: 10.1007/s00227-009-1383-y CrossRefGoogle Scholar
  19. Galuzzi L, Penna A, Bertozzini E, Vila M, Garces E, Magnani M (2004) Development of a real-time PCR assay for rapid detection and quantification of Alexandrum minutum (a dinoflagellate). Appl Environ Microbiol 70:1199–1206. doi: 10.1128/AEM.70.2.1199-1206.2004 CrossRefGoogle Scholar
  20. Galuzzi L, Bertozzini E, Del Campo A, Penna A, Bruce IJ, Magnani M (2006) Capture probe conjugated to paramagnetic nanoparticles for purification of Alexandrium species (Dinophyceae) DNA from environmental samples. J Appl Microbiol 101:36–43. doi: 10.1111/j.1365-2672.2006.02952.x CrossRefGoogle Scholar
  21. Gieskes WWC, Kraay GW (1983) Dominance of Cryptophyceae during the phytoplankton spring bloom in the central North Sea detected by HPLC analysis of pigments. Mar Biol 75:179–185. doi: 10.1007/BF00406000 CrossRefGoogle Scholar
  22. Gómez F, Moreira D, López-García P (2010) Neoceratium gen. nov., a new genus for all marine species currently assigned to Ceratium (Dinophyceae). Protist 161:35–54. doi: 10.1016/j.protis.2009.06.004 CrossRefGoogle Scholar
  23. Good IJ (1953) The population frequencies of species and the estimation of population parameters. Biometrika 40:237–264. doi: 10.1093/biomet/40.3.237 Google Scholar
  24. Groisillier A, Massana R, Valentin K, Vaulotl D, Guillou L (2006) Genetic diversity and habitats of two enigmatic marine alveolate lineages. Aquat Microb Ecol 42:277–291. doi: 10.3354/ame042277 CrossRefGoogle Scholar
  25. Guillou L, Viprey M, Chambouvet A, Welsh RM, Kirkham AR, Massana R, Scanlan DJ, Worden AZ (2008) Widespread occurrence and genetic diversity of marine parasitoids belonging to Syndiniales (Alveolata). Environ Microbiol 10:3349–3365. doi: :10.1111/j.1462-2920.2008.01731.x CrossRefGoogle Scholar
  26. Hansen G, Daugbjerg N, Henriksen P (2000) Comparative study of Gymnodinium mikimotoi and Gymnodinium aureolum, comb. nov. (=Gyrodinium aureolum) based on morphology, pigment composition and molecular data. J Phycol 26:394–410. doi: 10.1046/j.1529-8817.2000.99172.x Google Scholar
  27. Harada A, Ohtsuka S, Horiguchi T (2007) Species of the parasitic genus Duboscquella are members of the enigmatic marine alveolate group I. Protist 158:337–347. doi: 10.1016/j.protis.2007.03.005 CrossRefGoogle Scholar
  28. Hashihama F, Horimoto N, Kanda J, Furuya K, Ishimaru T, Saino T (2008) Temporal variation in phytoplankton composition related to water mass properties in the central part of Sagami Bay. J Oceanogr Soc Japan 64:23–37Google Scholar
  29. Hausmann K, Hu lsmann N, Radek R (2003) Protistology, 3rd edn. Schweizerbart’sche Verlagsbuchhandlung, StuttgartGoogle Scholar
  30. Hogetsu K, Taga N (1977) Suruga Bay and Sagami Bay. In: Hogetsu K, Hatanaka M, Hanaoka T, Kawamura T (eds) JIBP synthesis, productivity of biosensors in coastal regions of Japan, vol 14. University of Tokyo Press, Tokyo, pp 31–172Google Scholar
  31. Hosoi-Tanabe S, Sako Y (2005) Species-specific detection and quantification of toxic marine dinoflagellate Alexandrium tamarense and A. catenella by real-time PCR assay. J Mar Biotechnol 7:506–514. doi: 10.1007/s10126-004-418-4 CrossRefGoogle Scholar
  32. Karlson B, Godhe A, Cusack C, Bresnan E (2010) Introduction to methods for quantitative phytoplankton analysis. In: IOC manuals and guides no 55. UNESCO, ParisGoogle Scholar
  33. Lim EE, Amaral LA, Caron DA, DeLong EF (1993) Application of rRNA-based probes for observing marine nano-planktonic protests. Appl Environ Microbiol 59:1647–1655Google Scholar
  34. Lin SJ, Zhang H, Hou YB, Miranda L, Bhattacharya D (2006) Development of a dinoflagellate-oriented PCR primer set leads to detection of picoplanktonic dinoflagellates from Long Island Sound. Appl Environ Microbiol 72:5626–5630. doi: 10.1128/AEM.00586-06 CrossRefGoogle Scholar
  35. López-Gracía P, Rodríguez-Valera F, Pedró-Alió C, Moreira D (2001) Unexpected diversity of small eukaryotes in deep-sea Antarctic plankton. Nature 409:603–607. doi: 10.1038/35054537 CrossRefGoogle Scholar
  36. Margalef R (1958) Information theory in ecology. Gen Syst 3:36–71. doi: 10.1016/S0097-8485(01)0073-0 Google Scholar
  37. Massana R, Guillou L, Díez B, Pedrós-Alió C (2002) Unveiling the organisms behind novel eukaryotic ribosomal DNA sequences from the ocean. Appl Environ Microbiol 68:4554–4558. doi: 10.1128/AEM.68.9.4554-4558.2002 CrossRefGoogle Scholar
  38. Massana R, Castresana J, Balagué V, Guillou L, Romari K, Groisillier A, Valentin K, Pedrós-Alió C (2004) Phylogenetic and ecological analysis of novel marine stramenopiles. Appl Environ Microbiol 70:3258–3534. doi: 10.1128/AEM.70.6.3528-3534.2004 CrossRefGoogle Scholar
  39. Massana R, Guillou L, Terrado R, Forn I, Pedrós-Alió C (2006) Growth of uncultured heterotrophic flagellates in unamended seawater incubations. Aquat Microb Ecol 45:171–180CrossRefGoogle Scholar
  40. Medlin L, Elwood HJ, Stickel S, Sogin ML (1988) The characterization of enzymatically amplified eukaryotic 16S-like rRNA-coding regions. Gene 71:491–499. doi: 10.1016/0378-1119(88)90066-2 CrossRefGoogle Scholar
  41. Mitbavkar S, Saino T, Horimoto N, Kanda J, Ishimaru T (2009) Role of environment and hydrography in determining the picoplankton community structure of Sagami Bay, Japan. J Oceanogr Soc Japan 65:195–208Google Scholar
  42. Miyaguchi H, Kurosawa N, Toda T (2008) Real-time polymerase chain reaction assays for rapid detection and quantification of Noctiluca scintillans zoospore. Mar Biotechnol 10:133–140. doi: 10.1007/s10126-007-9031-3 CrossRefGoogle Scholar
  43. Montsant A, Jabbari K, Maheswari U, Bowler C (2005) Comparative genomics of the pennate diatom Phaeodactylum tricornutum. Plant Physiol 137:500–513CrossRefGoogle Scholar
  44. Moon-van der Staay SY, Watcher RD, Vaulot D (2001) Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature 409:607–610CrossRefGoogle Scholar
  45. Nakata N (1985) Sagami Bay. In: Oceanography Society of Japan (ed) Coastal oceanography of Japanese Islands. Tokai University Press, Tokyo, pp 417–426 (in Japanese)Google Scholar
  46. Neefs JM, Van De Peer Y, De Rijk P, Chappelle S, De Wachter R (1993) Compilation of small subunit RNA structures. Nucleic Acids Res 21:3025–3049. doi: 10.1093./nar/21.13.3025 CrossRefGoogle Scholar
  47. Onoue Y, Toda T, Ban S (2004) Morphological features and hatching pattern of eggs in Acartia steueri (Crustacea, Copepoda) from Sagami Bay, Japan. Hydrobiologia 511:17–24. doi: 10.1023/B:HYDR.0000014013.37891.46 CrossRefGoogle Scholar
  48. Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740. doi: 10.1126/science.276.5313.734 CrossRefGoogle Scholar
  49. Pierre C, Lecossier D, Boussougant Y, Bocart D, Joly V, Yeni P, Hance AJ (1991) Use of a reamplification protocol improves the sensitivity of detection of Mycobacterium tuberculosis in clinical samples by amplification of DNA. J Clin Microbiol 29:712–717Google Scholar
  50. Puig M, Jofre J, Lucena F, Allard A, Wadell G, Girones R (1994) Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Appl Environ Microbiol 60:2963–2970. doi: 10.1016/S0043-1354(97)00365-5 Google Scholar
  51. Rehnstam-Holm AS, Godhe A, Anderson AM (2002) Molecular studies of Dinophysis (Dinophyceae) species from Sweden and North America. Phycologia 41:348–357. doi: 10.216/i0031-8884-41-4-348-1 CrossRefGoogle Scholar
  52. Savin MC, Martin JL, LeGresley M, Giewat M, Rooney-Varga J (2004) Plankton diversity in the Bay of Fundy as measured by morphological and molecular methods. Microb Ecol 48:51–65CrossRefGoogle Scholar
  53. Schnetzer A, Moorthi SD, Countway PD, Gast RJ, Gilg IC, Caron DA (2011) Depth matters: microbial eukaryote diversity and community structure in the eastern North Pacific revealed through environmental gene libraries. Deep Sea Res 58:16–26. doi: 10.1016/j.dsr.2010.10.003 CrossRefGoogle Scholar
  54. Seung YMS, Staay GWM, Guillou L, Vaulot D (2000) Abundance and diversity of prymnesiophytes in the picoplankton community from the equatorial Pacific Ocean inferred from 18S rDNA sequences. Limnol Oceanogr 45:98–109. doi: 10.4319/lo.2000.45.1.0098 CrossRefGoogle Scholar
  55. Sherr EB, Sherr BF, Caron DA, Vaulot D, Worden AZ (2007) Oceanic protist. Oceanography 20:130–134. doi: 10.5670/oceanog.2007.57 CrossRefGoogle Scholar
  56. Shimode S, Baek SH, Ohsone T, Kikuchi T (2009) Long-term monitoring on nutrients and plankton communities in the north western part of Sagami Bay. Gekkan Kaiyo 41:86–97 (in Japanese)Google Scholar
  57. Singleton DR, Furlong MA, Rathbun SL, Whitman WB (2001) Quantitative comparisons of 16S rRNA gene sequence libraries from environmental samples. Appl Environ Microbiol 67:4374–4376. doi: 10.1128/AEM.67.9.4374-4376.2001 CrossRefGoogle Scholar
  58. Smayda TJ (1990) Novel and nuisance phytoplankton blooms in the sea: evidence for a global epidemic. In: Granéli E, Sundström B, Edler L, Anderson DM (eds) Toxic marine phytoplankton. Elsevier, Amsterdam, pp 29–40Google Scholar
  59. Stoeck T, Epstein S (2003) Novel eukaryotic lineages inferred from small-subunit rRNA analyses of oxygen-depleted marine environments. Appl Environ Microbiol 69:2657–2663. doi: 10.1128/AEM.69.5.2657-2663.2003 CrossRefGoogle Scholar
  60. Takishita K, Tsuchiya M, Kawato M, Oguri K, Kitazato H, Maruyama T (2007a) Genetic diversity of microbial eukaryotes in anoxic sediment of the saline meromictic lake Namako-ike (Japan): on the detection of anaerobic or anoxic-tolerant lineages of eukaryotes. Protist 158:51–64. doi: 10.1016/j.protis.2006.07.003 CrossRefGoogle Scholar
  61. Takishita K, Yubuki N, Kakizoe N, Inagaki Y, Maruyama T (2007b) Diversity of microbial eukaryotes in sediments at a deep-sea methane cold seep: surveys of ribosomal DNA libraries from raw sediment samples and two enrichment cultures. Extremophiles 11:563–576CrossRefGoogle Scholar
  62. Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680. doi: 10.1093/nar/22.22.4673 CrossRefGoogle Scholar
  63. Trask BJ, van den Engh GJ, Elgershuizen JHBW (1982) Analysis of phytoplankton by flow cytometry. Cytometry 2:256–264Google Scholar
  64. Yoshiki T, Yamanoha B, Kikuchi T, Shimizu A, Toda T (2008) Hydrostatic pressure-induced apoptosis on nauplii of Calanus sinicus. Mar Biol 156:97–106. doi: 10.1007/s00227-008-1066-0 CrossRefGoogle Scholar

Copyright information

© The Oceanographic Society of Japan and Springer 2012

Authors and Affiliations

  • Sau Pin Kok
    • 1
  • Tomohiko Kikuchi
    • 2
  • Tatsuki Toda
    • 1
  • Norio Kurosawa
    • 1
  1. 1.Department of Environmental Engineering for Symbiosis, Faculty of EngineeringSoka UniversityHachiojiJapan
  2. 2.Department of Hydrographic Science, Faculty of Education and Human SciencesYokohama National UniversityHodogayaJapan

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