, Volume 83, Issue 3, pp 220–226 | Cite as

Characterization, identification, and screening for tetrodotoxin production by bacteria associated with the ribbon worm (Nemertea) Cephalotrix simula (Ivata, 1952)

  • I. A. Beleneva
  • T. Yu. Magarlamov
  • A. D. Kukhlevsky
Experimental Articles


The taxonomic composition of bacteria associated with the proboscis worm Cephalothrix simula was studied and screening of the tetrodotoxin (TTX)-producing bacteria was carried out using confocal laser scanning microscopy and polyclonal antibodies. Bacterial isolates were identified using the 16S rRNA gene sequencing and phenotypic characteristics. A Bacillus species was found to be responsible for tetrodotoxin production in proboscis worm C. simula. Vibrio spp. dominated the associated microflora (68.18% of the total number of isolates). The analysis of sensitivity of 16 strains to antibiotics of various classes revealed multiple resistance to three or more antibiotics in all the studied isolates. Poor growth of most of the isolates on all the laboratory media was an indirect confirmation of the symbiotic relationships between the micro- and macroorganisms.


marine heterotrophic bacteria associations nemerteans tetrodotoxin production 


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  1. 1.
    Chernyshev, A.V., Sravnitel’naya morfologiya, sistematika i filogeniya nemertin (Comparative Morphology, Systematics, and Phylogeny of Proboscis Worms), Vladivostok: Dal’nauka, 2011.Google Scholar
  2. 2.
    Hwang, D.F., Arakawa, O., Saito, T., Noguchi, T., Simidu, U., Tsukamoto, K., Shida, Y., and Hashimoto, K., Tetrodotoxin-producing bacteria from the blue-ringed octopus Octopus maculosus, Mar. Biol., 1989, vol. 100, no. 3, pp. 327–332.CrossRefGoogle Scholar
  3. 3.
    Noguchi, T., Uzu, A., Koyama, K., and Hashimoto, K., Occurrence of tetrodotoxin as the major toxin in xanthid crab Atergatis floridus, Bull. Japan. Soc. Sci. Fish, 1983, vol. 49, pp. 1887–1892.CrossRefGoogle Scholar
  4. 4.
    Maruyama, J., Noguchi, T., Jeon, J.K., Harada, T., and Hashimoto, K., Occurrence of tetrodotoxin in the star-fish Astropecten latespinosus, Experientia, 1984, vol. 40, pp. 1395–1396.CrossRefGoogle Scholar
  5. 5.
    Thuesen, E.V. and Kogure, K., Bacterial production of tetrodotoxin in four species of Chaetognatha, Biol. Bull., 1989, vol. 176, pp. 191–194.CrossRefGoogle Scholar
  6. 6.
    Kanoh, S., Distribution of tetrodotoxin in vertebrates, in Recent Advances in Tetrodotoxin Research, Hashimoto, K., Ed., Tokyo: Koseisha-Koseikaku, 1988, pp. 32–44.Google Scholar
  7. 7.
    Noguchi, T., Jeon, J.K., Arakawa, O., Sugita, H., Deguchi, Y., Shida, Y., and Hashimoto, K., Occurrence of tetrodotoxin and anhydrotetrodotoxin in Vibrio sp. isolated from the intestines of a xanthid crab, Atergatis floridus, J. Biochem., 1986, vol. 99, pp. 311–314.Google Scholar
  8. 8.
    Yu, V.C-H., Yu, P.H-F., Ho, K-C., and Lee F.W.-F., Isolation and identification of a new tetrodotoxin-producing bacterial species, Raoultella terrigena, from Hong Kong marine puffer fish Takifugu niphobles, Mar. Drugs, 2011, vol. 9, pp. 2384–2396.PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Carrol, S., McEvoy, E.G., and Gibson, R., The production of tetrodotoxin-like substances by nemertean worms in conjunction with bacterias, J. Exp. Mar. Biol. Ecol., 2003, vol. 288, pp. 51–63.CrossRefGoogle Scholar
  10. 10.
    Youschimizu, M. and Kimura, T., Study on the intestinal microflora of salmonids, Fish Pathol., 1976, vol. 10, pp. 243–259.CrossRefGoogle Scholar
  11. 11.
    Beleneva, I.A., Kukhlevsky, A.D., and Kharchenko, U.V., Antimicrobial activity of heterotrophic bacterial strains of marine origin, Jundishapur J. Microbiol., 2013, vol. 6, no. 2, pp. 166–175.Google Scholar
  12. 12.
    Smibert, R.M. and Krieg, N.R., Phenotypic characterization, in Methods for General and Molecular Bacteriology, Gerhardt, P., Murray, R.G.E., Wood, W.A. and Krieg, N.R., Eds., Washington: D.C: American Soc. Microbiol., 1994, pp. 607–655.Google Scholar
  13. 13.
    Sambrook, J.F., Fristch, E.F., and Maniatis, T., Molecular Cloning: A Laboratory Manual, 2nd ed., New York: Cold Sprong Harbor Lab. Press, 1989.Google Scholar
  14. 14.
    Beleneva, I.A. and Kukhlevskii, A.D., Characterization of Vibrio gigantis and Vibrio pomeroyi isolated from invertebrates of Peter the Great Bay, Sea of Japan, Microbiology (Moscow), 2010, vol. 79, no. 3, pp. 402–407.CrossRefGoogle Scholar
  15. 15.
    Venkateswaran, K., Moser, D.P., Dollhopf, M.E., Lies, D.P., Saffarini, D.A., MacGregor, B. J., Ringelberg, D. B., White, D. C., Nishijima, M., Sano, H., Burghardt, J., Stackebrandt, E., and Nealson, K.H., Polyphasic taxonomy of the genus Shewanella and description of Shewanella oneidensis sp. nov., Int. J. Syst. Bacteriol., 1999, vol. 49, pp. 705–724.PubMedCrossRefGoogle Scholar
  16. 16.
    Giovannoni, S. and Rappé, M., Evolution, diversity, and molecular ecology of marine prokaryotes, in Microbial Ecology of the Ocean, Kirchman, D.L., Ed., New York: Wiley-Liss, 2000, pp. 47–84.Google Scholar
  17. 17.
    Thompson, F.L., Iida, T., and Swings, J., Biodiversity of vibrios, Microbiol. Mol. Biol. Rev., 2004, vol. 68, no. 3, pp. 403–431.PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Wu, Z., Yang, Y., Xie, L., Xia, G., Hu, J., Wang, S., and Zhang, R., Toxicity and distribution of tetrodotoxin-producing bacteria in puffer fish Fugu rubripes collected from the Bohai Sea of China, Toxicon, 2005, vol. 46, no. 4, pp. 471–476.PubMedCrossRefGoogle Scholar
  19. 19.
    Wang, J. and Fan, Y., Isolation and characterization of a Bacillus species capable of producing tetrodotoxin from puffer fish Fugu obscurus, J. Microbiol. Biotechnol., 2010, vol. 26, pp. 1755–1760.CrossRefGoogle Scholar
  20. 20.
    Long, R. and Azam, F., Antagonistic interactions among marine pelagic bacteria, Appl. Environ. Microbiol., 2001, vol. 67, no. 11, pp. 4975–4983.PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Suzuki, S., Tetracycline resistance gene in Asian aquatic environments, in Interdisciplinary Studies on Environmental Chemistry-Biological Responses to Contaminants, Hamamura, N., Suzuki, S. Mendo, S. Barroso, C.M., Iwata, H., and Tanabe, S., Eds., Tokyo: TERRAPUB, 2010, pp. 1–8.Google Scholar
  22. 22.
    Mindlin, S.Z., Petrova, M.A., Bass, I.A., and Gorlenko, Zh.M., Origin, evolution, and migration of drug resistance genes, Russ. J. Genet., 2006, vol. 42, no. 11, pp. 1257–1271.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2014

Authors and Affiliations

  • I. A. Beleneva
    • 1
    • 2
  • T. Yu. Magarlamov
    • 1
    • 2
  • A. D. Kukhlevsky
    • 1
    • 2
  1. 1.A.V. Zhirmunsky Institute of Marine Biology, Far Eastern BranchRussian Academy of SciencesVladivostokRussia
  2. 2.Far Eastern Federal UniversityVladivostokRussia

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