Chinese Journal of Oceanology and Limnology

, Volume 30, Issue 2, pp 256–263

Application of rRNA probes and fluorescence in situ hybridization for rapid detection of the toxic dinoflagellate Alexandrium minutum

  • Xianghai Tang (唐祥海)
  • Rencheng Yu (于仁成)
  • Mingjiang Zhou (周名江)
  • Zhigang Yu (于志刚)


The dinoflagellate Alexandrium minutum is often associated with harmful algal blooms (HABs). This species consists of many strains that differ in their ability to produce toxins but have similar morphology, making identification difficult. In this study, species-specific rRNA probes were designed for whole-cell fluorescence in situ hybridization (FISH) to distinguish A. minutum from two phylogenetic clades. We acquired the complete SSU to LSU rDNA sequences (GenBank accession numbers JF906989-JF906999) of 11 Alexandrium strains and used these to design rRNA targeted oligonucleotide probes. Three ribotype-specific probes, M-GC-1, M-PC-2, and M-PC-3, were designed. The former is specific for the GC clade (“Global clade”) of A. minutum, the majority of which have been found non-toxic, and the latter two are specific for the PSP (paralytic shellfish poisoning)-producing PC clade (“Pacific clade”). The specificity of these three probes was confirmed by FISH. All cells in observed fields of view were fluorescently labeled when probes and target species were incubated under optimized FISH conditions. However, the accessibility of rRNA molecules in ribosomes varied among the probe binding positions. Thus, there was variation in the distribution of positive signals in labeled cells within nucleolus and cytosol (M-GC-1, M-PC-3), or just nucleolus (M-PC-2). Our results provide a methodological basis for studying the biogeography and population dynamics of A. minutum, and providing an early warning of toxic HABs.


fluorescence in situ hybridization (FISH) Alexandrium minutum rRNA probe 


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  1. Adachi M, Sako Y, Ishida Y. 1996a. Analysis of Alexandrium (Dinophyceae) species using sequence of the 5.8S ribosomal DNA and internal transcribed spacer regions. J. Phycol., 32: 424–432.CrossRefGoogle Scholar
  2. Adachi M, Sako Y, Ishida Y. 1996b. Identification of the toxic dinoflagellates Alexandrium catenella and A. tamarense (Dinophyceae) using DNA probes and whole-cell hybridization. J. Phycol., 32: 1 049–1 052.Google Scholar
  3. Ahn S, Kulis D M, Erdner D L et al. 2006. Fiber-optic microarray for simultaneous detection of multiple harmful algal bloom species. Appl. Environ. Microbiol., 72(9): 5 742–5 749.CrossRefGoogle Scholar
  4. Anderson D M, Kulis D M, Keafer B A et al. 1999. Detection of the toxic dinoflagellate Alexandrium fundyense (Dinophyceae) with oligonucleotide and antibody probes: variability in labeling intensity with physiological condition. J. Phycol., 35(4): 870–883.CrossRefGoogle Scholar
  5. Anderson D M, Kulis D, Keafer B A et al. 2005. Identification and enumeration of Alexandrium spp. from the Gulf of Maine using molecular probes. Deep-Sea Res. II, 52: 2 467–2 490.Google Scholar
  6. Balech E. 1995. The Genus Alexandrium Halim (Dinoflagellata). Sherkin Island Marine Station, Cork, Ireland.Google Scholar
  7. Cembella A D. 1998. Ecophysiology and metabolism of paralytic shellfish toxins in marine microalgae. In: Anderson D M, Cembella A D, Hallegraeff G M eds. Physiological Ecology of Harmful Algal Blooms. NATO ASI Series. Springer, Berlin. p.381–403.Google Scholar
  8. Dyhrman S T, Erdner D, Du J L et al. 2006. Molecular quantification of toxic Alexandrium fundyense in the Gulf of Maine using real-time PCR. Harmful Algae, 5(3): 242–250.CrossRefGoogle Scholar
  9. Guilllard R R L, Ryther J H. 1962. Studies of marine planktonic diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can. J. Microl., 8: 229–239.CrossRefGoogle Scholar
  10. Hallegraeff G M. 1993. A review of harmful algal blooms and their apparent global increase. Phycologia, 32: 79–99.CrossRefGoogle Scholar
  11. Hansen G, Daugbjerg N, Franco J M. 2003. Morphology, toxin composition and LSU rDNA phylogeny of Alexandrium minutum (Dinophyceae) from Denmark, with some morphological observations on other European strains. Harmful Algae, 2(4): 317–335.CrossRefGoogle Scholar
  12. Higman W A, Stone D M, Lewis J M. 2001. Sequence comparison of toxic and non-toxic Alexandrium tamarense (Dinophyceae) isolates from UK waters. Phycologia, 40: 256–262.CrossRefGoogle Scholar
  13. Hosoi-Tanabe S, Sako Y. 2005a. Rapid detection of natural cells of Alexandrium tamarense and A. catenella (Dinophyceae) by fluorescence in situ hybridization. Harmful Algae, 4(2): 319–328.CrossRefGoogle Scholar
  14. Hosoi-Tanabe S, Sako Y. 2005b. Species-specific detection and quantification of toxic marine dinoflagellates Alexandrium tamarense and A. catenella by Real-Time PCR assay. Mar. Biotechnol., 7(5): 506–514.CrossRefGoogle Scholar
  15. John U, Cembella A, Hummert C et al. 2003. Discrimination of the toxigenic dinoflagellates Alexandrium tamarense and A. ostenfeldii in co-occurring natural populations from Scottish coastal waters. Eur. J. Phycol., 38(1): 25–40.CrossRefGoogle Scholar
  16. John U, Medlin L K, Groben R. 2005. Development of specific rRNA probes to distinguish between geographic clades of the Alexandrium tamarense species complex. J. Plankton Res., 27(2): 199–204.CrossRefGoogle Scholar
  17. Ki J S, Han M S. 2005. Molecular analysis of complete SSU to LSU rDNA sequence in the harmful dinoflagellate Alexandrium tamarense (Korean isolate, HY970328M). Ocean Sci. J., 40: 155–166.CrossRefGoogle Scholar
  18. Ki J S, Han M S. 2007. Informative characteristics of 12 divergent domains in complete large subunit rDNA sequences from the harmful dinoflagellate genus, Alexandrium (Dinophyceae). J. Eukaryot. Microbiol., 54(2): 210–219.CrossRefGoogle Scholar
  19. Kim C J, Kim C H, Sako Y. 2005. Development of molecular identification method for genus Alexandrium (Dinophyceae) using whole-cell FISH. Mar. Biotechnol., 7(3): 215–222.CrossRefGoogle Scholar
  20. Lilly E L, Halanych K M, Anderson D M. 2005. Phylogeny, biogeography, and species boundaries within the Alexandrium minutum group. Harmful Algae, 4(6): 1 004–1 020.CrossRefGoogle Scholar
  21. McCauley L A R, Erdner D L, Nagai S et al. 2009. Biogeographic analysis of the globally distributed harmful algal bloom species Alexandrium minutum (dinophyceae) based on rRNA gene sequences and microsatellite markers. J. Phycol., 45(2): 454–463.CrossRefGoogle Scholar
  22. Pitcher G C, Cembella A D, Joyce L B et al. 2007. The dinoflagellate Alexandrium minutum in Cape Town harbour (South Africa): Bloom characteristics, phylogenetic analysis and toxin composition. Harmful Algae, 6(6): 823–836.CrossRefGoogle Scholar
  23. Sako Y, Hosoi-Tanabe S, Uchida A. 2004. Fluorescence in situ hybridization using rRNA-targeted probes for simple and rapid identification of the toxic dinoflagellates Alexandrium tamarense and Alexandrium catenella. J. Phycol., 40(3): 598–605.CrossRefGoogle Scholar
  24. Scholin C A, Herzog M, Sogin M et al. 1994. Identification of group- and strain-specific genetic markers for globally distributed Alexandrium (Dinophyceae) species. II. Sequence analysis of a fragment of the large-subunit ribosomal RNA gene. J. Phycol., 30: 999–1 011.CrossRefGoogle Scholar
  25. Scorzetti G, Brand L E, Hitchcock G L et al. 2009. Multiple simultaneous detection of Harmful Algal Blooms (HABs) through a high throughput bead array technology, with potential use in phytoplankton community analysis. Harmful Algae, 8(2): 196–211.CrossRefGoogle Scholar
  26. Tang X H, Yu R C, Yan T et al. 2006. Sequence analysis and molecular phylogenetics of ribosomal RNA gene of Alexandrium (dinophyceae) strains in china. Oceanol. Limnol. Sin., 37(6): 530–535. (in Chinese with English abstract)Google Scholar
  27. Taylor F J R, Fukuyo Y, Larsen J. 1995. Taxonomy of harmful dinoflagellates. In: Hallegraeff G M, Anderson D M, Cembella A D eds. Manual on Harmful Marine Microalgae. IOC Manuals and Guides No.33. IOC of UNESCO. p.283–317.Google Scholar
  28. Touzet N, Davidson K, Pete R et al. 2010. Co-occurrence of the west European (Gr.III) and north American (Gr.I) ribotypes of Alexandrium tamarense (Dinophyceae) in Shetland, Scotland. Protist, 161(3): 370–384.CrossRefGoogle Scholar
  29. Touzet N, Raine R. 2007. Discrimination of Alexandrium andersoni and A. minutum (Dinophyceae) using LSU rRNA-targeted oligonucleotide probes and fluorescent whole-cell hybridisation. Phycologia, 46(2): 168–177.CrossRefGoogle Scholar
  30. Turner J T, Doucette G J, Powell C L et al. 2000. Accumulation of red tide toxins in larger size fractions of zooplankton assemblages from Massachusetts Bay, USA. Mar. Ecol. Prog. Ser., 203: 95–107.CrossRefGoogle Scholar
  31. Wang D Z, Jiang T J, Hsieh D P H. 2005. Toxin composition variations in cultures of Alexandrium species isolated from the coastal waters of southern China. HarmfuI Algae, 4(1): l09–121.Google Scholar
  32. Wang Z H, Matsuoka K, Qi Y Z et al. 2004. Dinoflagellate cysts in recent sediments from Chinese coastal waters. Mar. Ecol., 25(4): 289–311.CrossRefGoogle Scholar
  33. Yu R C, Hummert C, Luekas B et al. 1998. Analysis of PSP toxins in algae and shellfish samples from China using a modified HPLC method. Chromatographia, 48(9): 67l–677.CrossRefGoogle Scholar
  34. Yu R C, Tang X H, Zhang Q C et al. 2006. Application of fluorescence in-situ hybridization (FISH) method to detect “tamarense/catenella species complex” (“Temperate Asian” ribotype) in Genus Alexandrium along Chinese coast. Acta Sci. Circumst., 26(4): 646–651. (in Chinese with English abstract)Google Scholar
  35. Zhen Y, Mi T Z, Yu Z G. 2009. Detection of several harmful algal species by sandwich hybridization integrated with a nuclease protection assay. Harmful Algae, 8(5): 651–657.CrossRefGoogle Scholar
  36. Zhou M J, Li J, Luekas B et al. 1999. A recent shellfish toxin investigation in China. Mar. Pollut. Bull., 39(1): 33l–334.CrossRefGoogle Scholar
  37. Zhou M J, Zhu M Y, Zhang J. 2001. Status of harmful algal blooms and related research activities in China. Chin. Bull. Life Sci., 13(2): 54–59. (in Chinese with English abstract)Google Scholar

Copyright information

© Chinese Society for Oceanology and Limnology, Science Press and Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  • Xianghai Tang (唐祥海)
    • 1
  • Rencheng Yu (于仁成)
    • 2
  • Mingjiang Zhou (周名江)
    • 2
  • Zhigang Yu (于志刚)
    • 1
  1. 1.Key Laboratory of Marine Chemistry Theory and Technology, Ministry of EducationOcean University of ChinaQingdaoChina
  2. 2.Key Laboratory of Marine Ecology and Environmental Sciences, Institute of OceanologyChinese Academy of SciencesQingdaoChina

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