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

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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.

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References

  • Adachi M (2000) Phylogenic analysis of phytoplankton. Ocean Mon Mag Special Eds 23:18–23 (in Japanese)

    Google Scholar 

  • 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 

  • Ara K, Hiromi J (2008) Temporal variability and characterization of physicochemical properties in the neritic area of Sagami Bay, Japan. J Oceanogr Soc Japan 64:195–210

    Google Scholar 

  • 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 6:158–174

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 63:35–45

    Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 

  • Burton RS (1996) Molecular tools in marine ecology. J Exp Mar Biol Ecol 200:85–101. doi:10.1016/S0022-0981(96)02641-X

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 

  • 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

    Article  Google Scholar 

  • 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 64:23–37

    Google Scholar 

  • Hausmann K, Hu lsmann N, Radek R (2003) Protistology, 3rd edn. Schweizerbart’sche Verlagsbuchhandlung, Stuttgart

    Google Scholar 

  • 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 31–172

  • 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

    Article  Google Scholar 

  • Karlson B, Godhe A, Cusack C, Bresnan E (2010) Introduction to methods for quantitative phytoplankton analysis. In: IOC manuals and guides no 55. UNESCO, Paris

  • Lim EE, Amaral LA, Caron DA, DeLong EF (1993) Application of rRNA-based probes for observing marine nano-planktonic protests. Appl Environ Microbiol 59:1647–1655

    Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Margalef R (1958) Information theory in ecology. Gen Syst 3:36–71. doi:10.1016/S0097-8485(01)0073-0

    Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Massana R, Guillou L, Terrado R, Forn I, Pedrós-Alió C (2006) Growth of uncultured heterotrophic flagellates in unamended seawater incubations. Aquat Microb Ecol 45:171–180

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 65:195–208

    Google Scholar 

  • 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

    Article  Google Scholar 

  • Montsant A, Jabbari K, Maheswari U, Bowler C (2005) Comparative genomics of the pennate diatom Phaeodactylum tricornutum. Plant Physiol 137:500–513

    Article  Google Scholar 

  • Moon-van der Staay SY, Watcher RD, Vaulot D (2001) Oceanic 18S rDNA sequences from picoplankton reveal unsuspected eukaryotic diversity. Nature 409:607–610

    Article  Google Scholar 

  • 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)

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740. doi:10.1126/science.276.5313.734

    Article  Google Scholar 

  • 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 29:712–717

    Google Scholar 

  • 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 

  • 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

    Article  Google Scholar 

  • 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 48:51–65

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Sherr EB, Sherr BF, Caron DA, Vaulot D, Worden AZ (2007) Oceanic protist. Oceanography 20:130–134. doi:10.5670/oceanog.2007.57

    Article  Google Scholar 

  • 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 

  • 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

    Article  Google Scholar 

  • 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 29–40

  • 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

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • 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 11:563–576

    Article  Google Scholar 

  • 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

    Article  Google Scholar 

  • Trask BJ, van den Engh GJ, Elgershuizen JHBW (1982) Analysis of phytoplankton by flow cytometry. Cytometry 2:256–264

    Google Scholar 

  • 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

    Article  Google Scholar 

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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.

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  1. Department of Environmental Engineering for Symbiosis, Faculty of Engineering, Soka University, 1-236 Tangi-cho, Hachioji, Tokyo, 192-8577, Japan

    Sau Pin Kok, Tatsuki Toda & Norio Kurosawa

  2. Department of Hydrographic Science, Faculty of Education and Human Sciences, Yokohama National University, 79-1 Tokiwadai, Hodogaya, Yokohama, Kanagawa, 240-8501, Japan

    Tomohiko Kikuchi

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  1. Sau Pin Kok
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Kok, S.P., Kikuchi, T., Toda, T. et al. Diversity analysis of protistan microplankton in Sagami Bay by 18S rRNA gene clone analysis using newly designed PCR primers. J Oceanogr 68, 599–613 (2012). https://doi.org/10.1007/s10872-012-0121-0

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