Journal of Chemical Ecology

, Volume 38, Issue 11, pp 1342–1350 | Cite as

Depuration of Tetrodotoxin and Changes in Bacterial Communities in Pleurobranchea maculata Adults and Egg Masses Maintained in Captivity

  • Susanna A. Wood
  • Margaux Casas
  • David I. Taylor
  • Paul McNabb
  • Lauren Salvitti
  • Shaun Ogilvie
  • S. Craig Cary


Depuration of tetrodotoxin (TTX) was investigated in adult grey side-gilled sea slugs, Pleurobranchaea maculata, maintained in captivity on a TTX-free diet. Three adults were harvested every 21 days for 126 days, and TTX concentrations were measured in organs/tissues and egg masses. Automated rRNA intergenic spacer analysis (ARISA) was used to investigate bacterial community structure in selected samples. Linear modeling of adult data demonstrated a decline (P < 0.001) in average total TTX concentrations over time. Temporal data obtained from a wild population showed similar depuration rates, indicating that once adults reach a certain size, or sexual maturity, TTX is no longer produced or acquired substantially. Depuration rates differed among organs, with concentrations in the heart declining the fastest. The gonads had the slowest and least significant depuration rate indicating, at most, weak depuration of this tissue. There was a strong correlation (R 2 = 0.66) between TTX concentrations in the first-laid egg masses and total TTX in the corresponding adult. These data suggest that adult P. maculata transfer TTX to their offspring, and presumably that functions as a chemical defense. ARISA data showed a shift in bacterial community structure within 3 weeks of introduction to captivity. Based on the combined data, the exact origin of TTX in P. maculata is unclear, with evidence both in favor and against a dietary source, and endogenous or bacterial production.


Chemical defense Depuration Egg mass toxicity Pleurobranchaea maculata Tetrodotoxin 



This research was funded by the Marsden fund of the Royal Society of New Zealand (UOW1002) and Ngā Pae o Te Māramatanga (Project: 10RF18). We thank the Auckland Harbour Master and Jarrod Walker from the Auckland Council for assistance during P. maculata collections. P.M. is supported by a New Zealand Ministry for Science and Innovation Te Tipu Pūtaiao Ph.D. fellowship (CAWX0905). We are grateful to Janet Adamson (Cawthron) for technical assistance and Eric Bottos (Waikato University) and Eric Goodwin (Cawthron) for assistance with statistical analysis.


  1. Abdo, Z., Schüette, U., Bent, S., Williams, C., Forney, L., and Joyce, P. 2006. Statistical methods for characterizing diversity of microbial communities by analysis of terminal restriction fragment length polymorphisms of 16S rRNA genes. Environ. Microbiol. 8:929–938.PubMedCrossRefGoogle Scholar
  2. Dhanasiri, A. K., Brunvold, L., Brinchmann, M. F., Korsnes, K., Bergh, Ø., and Kiron, V. 2011. Changes in the intestinal microbiota of wild atlantic cod Gadus morhua L. upon captive rearing. Microbial. Ecol. 61:20–30.CrossRefGoogle Scholar
  3. Cardalll, B. L., Brodie JR, E. D., Brodie III, E. D., and Hanifin, C. T. 2004. Secretion and regeneration of tetrodotoxin in the rough-skin newt (Taricha granulosa). Toxicon 44:933–938.CrossRefGoogle Scholar
  4. Cardinale, M., Brusetti, L., Quatrini, P., Borin, S., Puglia, A., Rizzi, A., Zanardini, E., Sorlini, C., Corselli, C., and Daffonchio, D. 2004. Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl. Environ. Microbiol. 70:6147–6156.PubMedCrossRefGoogle Scholar
  5. Chau, R., Kalaitzis, J. A., and Neilan, B. A. 2011. On the origins and biosynthesis of tetrodotoxin. Aquat. Toxicol. 104:61–72.PubMedCrossRefGoogle Scholar
  6. Dao, H. V., Yoshinobu, T., Sato, S., Fukuyo, Y., and Kodama, M. 2009. Frequent occurrence of the tetrodotoxin-bearing horseshoe crab Carcinoscorpius rotundicauda in Vietnam. Fish. Sci. 75:435–438.CrossRefGoogle Scholar
  7. Fisher, M. M. and Triplett, E. W. 1999. Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl. Environ. Microbiol. 65:4630–4636.PubMedGoogle Scholar
  8. Gibson, G. D. 2003. Larval development and metamorphosis in Pleurobranchaea maculata, with a review of development in the Notaspidea (Opisthobranchia). Biol. Bull. (Woods Hole) 205:121–132.CrossRefGoogle Scholar
  9. Hanifin, C. T. 2010. The chemical and evolutionary ecology of tetrodotoxin (TTX) toxicity in terrestrial vertebrates. Mar. Drugs 8:577–593.PubMedCrossRefGoogle Scholar
  10. Hanifin, C. T., Brodie III, E. D., and Brodie Jr., E. D. 2002. Tetrodotoxin levels of the rough-skin newt, Taricha granulosa, increase in long-term captivity. Toxicon 40:1149–1153.PubMedCrossRefGoogle Scholar
  11. Hanifin, C. T., Brodie III, E. D., and Brodie Jr., E. D. 2003. Tetrodotoxin levels in eggs of the rough-skin newt, Taricha granulosa, are correlated with female toxicity. J. Chem. Ecol. 25:1729–1739.Google Scholar
  12. Hanifin, C. T., Yotu-Yamashita, M., Yasumoto, T., Brodie III, E. D., and Brodie JR, E. D. 1999. Toxicity of dangerous prey: Variation of tetrodotoxin levels within and among populations of the newt Taricha granulosa. J. Chem. Ecol. 25:2161–2175.CrossRefGoogle Scholar
  13. Hwang, D. E., Lu, S. C., and Jeng, S. S. 1991. Occurrence of tetrodotoxin in the gastropods Rapana rapiformis and R. venosa venosa. Mar. Biol. 111:65–69.CrossRefGoogle Scholar
  14. Isaacs, L. T., Kan, J., Nguyen, L., Videau, P., Anderson, M. A., Wright, T. L., and Hill, R. T. 2009. Comparison of the bacterial communities of wild and captive sponge Clathria prolifera from the Chesapeake Bay. Mar. Biotechnol. 6:758–70.CrossRefGoogle Scholar
  15. Lehman, E. M., Brodie, E. D. J. R., and Brodie III, E. D. 2004. No evidence for an endosymbiotic bacterial origin of tetrodotoxin in the newt Taricha granulosa. Toxicon 44:243–249.PubMedCrossRefGoogle Scholar
  16. Matsui, T., Yamamori, K., Furukawa, K., and Kono, M. 2000. Purification and some properties of a tetrodotoxin binding protein from the blood plasma of kusafugu, Takifugu niphobles. Toxicon 38:463–468.PubMedCrossRefGoogle Scholar
  17. Matsumura, K. 2001. No ability to produce tetrodotoxin in bacteria. Appl. Environ. Microbiol. 67:2393–2394.PubMedCrossRefGoogle Scholar
  18. Matsumura, K. 1995. Re-examination of tetrodotoxin production by bacteria. Appl. Environ. Microbiol. 61:3468–3470.PubMedGoogle Scholar
  19. McNabb, P., Selwood, A. I., Munday, R., Wood, S. A., Taylor, D. I., Mackenzie, L. A., van Ginkel, R., Rhodes, L. L., Cornelisen, C., Heasman, K., Holland, P. T., and King, C. 2010. Detection of tetrodotoxin from the grey side-gilled sea slug - Pleurobranchaea maculata, and associated dog neurotoxicosis on beaches adjacent to the Hauraki Gulf, Auckland, New Zealand. Toxicon 56:466–473.PubMedCrossRefGoogle Scholar
  20. Nagashima, Y., Mataki, I., Toyoda, M., Nakajima, H., Tsumotoo, K., Shimakura, K., and Shioma, K. 2010. Change in tetrodotoxin content of the puffer fish Takifugu rubripes during seed production from fertilized eggs to juveniles. Food Hyg. Saf. Sci. 51:48–51.CrossRefGoogle Scholar
  21. Noguchi, T. and Arakawa, O. 2008. Tetrodotoxin – distribution and accumulation in aquatic organisms, and cases of human intoxication. Mar. Drugs 6:220–242.PubMedCrossRefGoogle Scholar
  22. Noguchi, T., Arakawa, O., and Takatani, T. 2006. Toxicity of pufferfish Takifugu rubripes cultured in net cages at sea or aquaria on land. Comp. Biochem. Phys. D. 153–157.Google Scholar
  23. Noguchi, T. and Ebesu, J. S. M. 2001. Puffer poisoning: epidemiology and treatment. J. Toxicol. Toxin Rev. 20:1–10.Google Scholar
  24. Pawlik, J. R., Kernan, M. R., Molinski, T. F., Harper, M. K., and Faulkner, D. J. 1988. Defensive chemicals of the Spanish dancer nudibranch Hexabranchus sanguineus and its egg ribbons: macrolides derived from a sponge diet. J. Exp. Mar. Biol. Ecol. 119:99–109.CrossRefGoogle Scholar
  25. Schroeder, F. C., Gonzalez, A., Eisner, T., and Meinwald, J. 1999. Miriamin, a defensive diterpene from the eggs of a land slug (Arion sp.). Proc. Nat. Acad. Sci. USA 96:13620–13625.PubMedCrossRefGoogle Scholar
  26. Snedecor, G. W. and Cochran, W. O. 1980. pp. 503, Statistical methods. Iowa State University Press, Ames.Google Scholar
  27. Willan, R. C. 1984. A review of diets in the Notaspidea (Mollusca: Opisthobranchia). J. Mal. Soc. Aust. 6:125–142.Google Scholar
  28. Williams, B. L. and Caldwell, R. L. 2009. Intra‑organismal distribution of tetrodotoxin in two species of blue‑ringed octopuses (Hapalochlaena fasciata and H. lunulata). Toxicon 54:345–353.PubMedCrossRefGoogle Scholar
  29. Williams, B. L., Hanifin, C. T., Brodie Jr., E. D., and Caldwell, R. L. 2011. Ontogenty of tetrodtoxin levels in blue-ringed octopuses: maternal investment and apparent independent production in offspring of Hapalochlaena lunulata. J. Chem. Ecol. 37:10–17.PubMedCrossRefGoogle Scholar
  30. Wood, S. A., Taylor, D. I., McNabb, P., Walker, J., Adamson, J., and Cary, S. C. 2012. Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations. Mar. Drugs. 10:163–176.PubMedCrossRefGoogle Scholar
  31. Yotsu-Yamashita, M., Sugimoto, A., Terakawa, T., Shoji, Y., Miyazawa, T., and Yasumoto, T. 2001. Purification, characterization, and cDNA cloning of a novel soluble saxitoxin and tetrodotoxin binding protein from plasma of the puffer fish, Fugu pardalis. Eur. J. Biochem. 268:5937–5946.PubMedCrossRefGoogle Scholar
  32. Yotsu-Yamashita, M., Gilhen, J., Russell, R. W., Krysko, K. L., Melaun, C., Kurz, A., Kauferstein, S., Kordis, D., and Mebs, D. 2012. Variability of tetrodotoxin and of its analogues in the red-spotted newt, Notophthalmus viridescens (Amphibia: Urodela: Salamandridae). Toxicon 59:257–264.PubMedCrossRefGoogle Scholar
  33. Yotsu-Yamashita, M., Mebs, D., and Yasumoto, T. 1992. Tetrodotoxin and its analogues in extracts from the toad Atelopus oxyrhynchus (family: bufonidae). Toxicon 30:1489–1492.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2012

Authors and Affiliations

  • Susanna A. Wood
    • 1
    • 2
  • Margaux Casas
    • 1
    • 3
  • David I. Taylor
    • 1
  • Paul McNabb
    • 1
    • 4
  • Lauren Salvitti
    • 2
  • Shaun Ogilvie
    • 1
    • 5
  • S. Craig Cary
    • 2
  1. 1.Cawthron InstituteNelsonNew Zealand
  2. 2.Department of Biological SciencesUniversity of WaikatoHamiltonNew Zealand
  3. 3.Institut Polytechnique LaSalleBeauvaisFrance
  4. 4.Department of ChemistryOtago UniversityDunedinNew Zealand
  5. 5.Eco Research Associates LtdChristchurchNew Zealand

Personalised recommendations