Journal of Chemical Ecology

, Volume 36, Issue 11, pp 1171–1179 | Cite as

The Effect of Sampling Methods on the Apparent Constituents of Ink from the Squid Sepioteuthis australis

  • F. Madaras
  • J. P. Gerber
  • F. Peddie
  • M. J. Kokkinn


Results of experiments conducted on ink recovered from the squid Sepioteuthis australis indicate that there is no epinephrine or protein naturally present in the ink as it would be ejected in vivo. Protein content was effectively zero when ink was syringed from the duct end of the ink sac of freshly killed animals. By contrast, there were proteins in samples collected from dead specimens where ink was collected by a stripping method. From these samples, a single large molecular weight protein was identified as having tyrosinase activity. Digestion of syringed ink did not yield signs of melanin-bound proteins. Analysis of supernatants after centrifugation of squid ink consistently revealed the presence of DOPA, dopamine, and taurine, whereas epinephrine and nor-epinephrine were recorded from what was believed to be contaminated ink. Histological investigations of the ink sac revealed a compartmentalised glandular structure distal to the duct end. Closer observation of the glandular tissue showed that compartments increased in size as they matured and moved further into the lumen. It was concluded that the presence of epinephrine and tyrosinase (or a related protein) in the ink of S. australis could be attributed to rupturing of basal glandular compartments or contamination from other sources during the extraction process.

Key Words

Sepioteuthis australis Ink sac Ink Protein Tyrosinase DOPA Dopamine Taurine 



We thank all those who accompanied us on squid fishing expeditions. Andrew Beck and Shaun O’Sullivan produced high quality sections of ink sac tissue. Miguel De Barros Lopez gave us invaluable advice and guidance. The School of Pharmacy and Medical Sciences, through Allan Evans, gave us material support and encouragement without which we could not have conducted this work.


  1. Bakhayokho, M. 1983. Biology of the cuttlefish Sepia officinalis hierredda off the Senegalese coast. pp. 204–263 in J.F. CADDY (ed.) Advances in Assessment of World Cephalopod Resources. FAO Fisheries Technical Paper. 231.Google Scholar
  2. Bush, S. L., and Robison, B. H. 2007. Ink utilization by mesopelagic squid. Mar. Biol. 152:485–494.CrossRefGoogle Scholar
  3. Derby, C. D. 2007. Escape by inking and secreting: marine molluscs avoid predators through a rich array of chemicals and mechanisms. Biol. Bull. 213:274–289.CrossRefPubMedGoogle Scholar
  4. Derby, C. D., Kicklighter, C. E., Johnson, P. M., and Zhang, X. 2007. Chemical Composition of inks of diverse marine molluscs suggests convergent chemical defenses. J. Chem. Ecol. 33:1105–1113CrossRefPubMedGoogle Scholar
  5. Fiore, G., Poli, A., Di Cosmo, A., D’ischia, M., and Palumbo, A. 2004. Dopamine in the ink defence system of Sepia officinalis: biosynthesis, vesicular compartmentation in mature ink gland cells, nitric oxide (NO)/cGMP-induced depletion and fate in secreted ink. Biochem. J. 378:785–791.CrossRefPubMedGoogle Scholar
  6. Fling, M., Horowitz, N. H., and Heinemann, S. F. 1963. The isolation and properties of crystalline tyrosinase from Neurospora. J. Biol. Chem. 238:2045–2053.PubMedGoogle Scholar
  7. Fox, D. L. 1976. Animal Biochromes and Structural Colors. University of California Press, Berkeley. pp. 215–240.Google Scholar
  8. Gilly, W. F., and Lucero, M. T. 1992. Behavioral responses to chemical stimulation of the olfactory organ in the squid Loligo opalescens. J. Exp. Biol. 162:209–229.Google Scholar
  9. Jaenicke, E., and Decker, H. 2004. Conversion of crustacean hemocyanin to catecholoxidase. Micron. 35:89–90.CrossRefPubMedGoogle Scholar
  10. Juorio, A. V. 1971. Catecholamines and 5-hydroxytryptamine in nervous tissue of cephalopods. J. Physiol. 216:213–226.PubMedGoogle Scholar
  11. Lacoste, A., Malham, S. K., Cueff, A., Jalabert, F., Gélébart, F., and Poulet, S. A. 2001. Evidence for a form of adrenergic response to stress in the mollusc Crassostrea gigas. J. Exp. Biol. 204:1247–1255.PubMedGoogle Scholar
  12. Lei, M., Wang, J., Wang, Y., Pang, L., Wang, Y., Xu, W., and Xue, C. 2007. Study of the radio-protective effect of cuttlefish ink on hemopoietic injury. Asia Pac. J. Clin. Nutr. 16:239–243.PubMedGoogle Scholar
  13. Linh Tran, M., Powell, B. J., and Meredith, P. 2006. Chemical and structural disorder in eumelanins: A possible explanation for broadband absorbance. Biophys J. 90:743–752.CrossRefPubMedGoogle Scholar
  14. Liu, Y., and Simon, J. D. 2003. The effect of preparation procedures on the morphology of melanin from the ink sac of Sepia officinalis. Pigment Cell Res. 16:72–80.CrossRefPubMedGoogle Scholar
  15. Lucerno, M., Farrington, H., and Gilly, W. F. 1994. Quantification of L-Dopa and dopamine in squid ink: Implications for chemoreception. Biol. Bull. 187: 55–63.CrossRefGoogle Scholar
  16. Macginitie, G., and Macginitie, N. 1968. Natural History of Marine Animals, 2nd ed. McGraw-Hill, New York. pp. 395–397.Google Scholar
  17. Madaras, F., Mirtschin, J. P., and Kuchel, T. 2005. Antivenom development in Australia. Toxin Reviews 24:79–94.CrossRefGoogle Scholar
  18. Meng, S., and Kaxiras, E. 2008. Theoretical models of eumelanin protomolecules and their optical properties. Biophy. J. 94:2095–2105CrossRefGoogle Scholar
  19. Meredith, P., Powell, B. J., Riesz, J., Nighswander-Rempel, S. P., Pederson, M. R., and Moore, E. G. 2006. Towards structure-property-function relationships for eumelanin. Soft Matter 2:37–44.CrossRefGoogle Scholar
  20. Messenger, J. B., Nixon, M., and Ryan, K. P. 1985. Magnesium chloride as an anaesthetic for cephalopods. Comp. Biochem. Physiol. C. 82:203–205.CrossRefPubMedGoogle Scholar
  21. Naraoka,T., Uchisawa, H., Mori, H., Chiba, S., and Kimura, A. 2000. Tyrosinase activity in antitumor compounds from squid ink. Food Sci. Technol. Res. 6:171–175.CrossRefGoogle Scholar
  22. Ortonne, J. P., Voul, O. T., Khatchadourian, C., Palumbo, A., and Prota, G. 1981. A re-examination of melanogenesis in the ink gland of Cephalopods. pp. 49–57 in M. Seiji (ed.). Pigment Cell 1981: Phenotypic Expression in Pigment Cells. University of Tokyo Press.Google Scholar
  23. Palumbo, A. 2003. Melanogenesis in the ink gland of Sepia officinalis. Pigment Cell Res. 16:517–522.CrossRefPubMedGoogle Scholar
  24. Palumbo, A., Di Cosmo, A., Gesualdo, I., and Hearing, V. J. 1997. Subcellular localization and function of melanogenic enzymes in the ink gland of Sepia officinalis. Biochem. J. 323:749–756.PubMedGoogle Scholar
  25. Prota, G. 2000. Melanins, melanogenesis and melanocytes: Looking at their functional significance from a chemist’s viewpoint. Pigment Cell Res. 13:283–293.CrossRefPubMedGoogle Scholar
  26. Prota, G., Ortonne, J. P., Voulot, C., Khatchadourian, C., Nardi, G., and Palumbo, A. 1981. Occurrence and properties of tyrosinase in the ejected ink of cephalopods. Comp. Biochem. Physiol. Part B 68:415–419.CrossRefGoogle Scholar
  27. Roper, C. F. E., Sweeney, M. J., and Nauen, C. E. 1984. Cephalopods of the World. An Annotated and Illustrated Catalogue of Species of interest to Fisheries. FAO Species Catalogue, No. 125 3:47–49.Google Scholar
  28. Russo, G. L., De Nisco, E., Fiore, G., Di Donato, P., D’ischia, M., and Palumbo, A. 2003. Toxicity of melanin-free ink of Sepia officinalis to transformed cell lines: identification of the active factor as tyrosinase. Biochem. Biophys. Res. Commun. 308:293–299.CrossRefPubMedGoogle Scholar
  29. Schoot Uiterkamp, A. J. M., and Mason, H. S. 1973. Magnetic dipole-dipole coupled Cu(II) pairs in nitric oxide-treated tyrosinase: A structural relationship between the active sites of tyrosinase and hemocyanin. Proc. Nat. Acad. Sci. USA 70:993–996.CrossRefGoogle Scholar
  30. Schraermeyer, U. 1994. Fine structure of melanogenesis in the ink sac of Sepia officinalis. Pigment Cell Res. 7:52–60.CrossRefPubMedGoogle Scholar
  31. Springer, J., Ruth, P., Beuerlein, K., Palus, P., Schipp, R., and Westermann, B. 2005. Distribution and function of biogenic amines in the heart of Nautilus pompilius L. (Cephalopoda, Tetrabranchiata). J. Molec. Histol. 36:345–353.CrossRefGoogle Scholar
  32. Takaya, Y., Uchisawa, H., Matsue, H., Okuzaki, B., Narumi, Sasaki, F. J., and Ishida K. 1994. An investigation of the antitumor peptidoglycan fraction from squid ink. Biol. Pharm. Bull. 17: 846–849.PubMedGoogle Scholar
  33. Wang, C., Fan, X., Yu, H., and Miao, M. 2008. Histology of the ink sac of Sepiella maindroni and ultrastructure of the ink formation. Current Zool. 54:366–372.Google Scholar
  34. Winstanley, R. H., Potter, M. A., and Caton, A. E. 1983. Australian cephalopod resources. Memoirs Natl Museum Victoria 44:243–253.Google Scholar
  35. Wood, J. B., Maynard, A. E., Lawlor, A. G., Sawyer, E. K., Simmons, D. M., Pennoyer, K. E., and Derby, C. D. 2010. Caribbean reef squid, Sepioteuthis sepioidea, use ink as a defense against predatory French grunts, Haemulon flavolineatum. J. Exp. Mar. Biol. Ecol. 388:20–27.CrossRefGoogle Scholar
  36. Wood, J. B., Pennoyer, K. E., and Derby, C. D. 2008. Ink is a conspecific alarm cue in the Caribbean reef squid, Sepioteuthis sepioidea. J. Exp. Mar. Biol. Ecol. 367:11–16.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • F. Madaras
    • 1
  • J. P. Gerber
    • 1
  • F. Peddie
    • 1
  • M. J. Kokkinn
    • 1
  1. 1.School of Pharmacy and Medical SciencesThe University of South AustraliaAdelaideAustralia

Personalised recommendations