Approaches to a Molecular Identification of Sex Pheromones in Blue Crabs



Molecular identification of sex pheromones in marine crustaceans has proven to be very difficult, and so far no unequivocal identification for any decapod crustacean has been published. Some of these difficulties are common to other animals – pheromones are often blends of molecules at low concentrations. Some difficulties are more specific to marine crustaceans – pheromones are often small and polar molecules that are difficult to separate from salts in their source (often urine) or carrier medium (sea water). These difficulties led us to take on new approaches as we searched for sex pheromones in the blue crab Callinectes sapidus. Premolt pubertal female blue crabs that are ready to mate release a pheromone in their urine. This pheromone is detected by male crabs using specific chemical sensors – aesthetasc sensilla on the antennules. Male blue crabs respond to the pheromone with courtship stationary paddling, a distinctive behavior that is useful in bioassays for pheromone identification. We used bioassay-guided fractionation to demonstrate that the pheromone of female blue crabs is of low molecular mass (<1,000 Da) and possibly a mixture. We used liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance, biomarker targeting, and metabolomics approaches to isolate molecules specific to premolt pubertal females and that are thus candidate pheromones. Our working hypothesis is that female blue crabs release a species-specific sex pheromone in their urine that is composed of two functional classes of molecules, both of which are small and polar. One class distinguishes females from males and thus is a sex-specific signal, and a second class distinguishes blue crabs from other species and thus constitutes a species-specific signal.


High Performance Liquid Chromatography Blue Crab Decapod Crustacean Target Animal Female Crab 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



We thank our colleagues who have contributed in many ways to our work, in particular Barry Ache, Peter Anderson, Todd Barsby, Norman Byrd, Sekar Chandrasekaran, Markus Germann, Richard Gleeson, John Glushka, Yuji Kakazu, Julia Kubanek, Koichi Matsumura, Dell Allen Newman, Dan Rittschof, Tomoyoshi Soga, and Siming Wang. Funding was provided by NSF grants IBN 0077474, IBN-0324435, IBN-0614685, Japan Society for the Promotion of Science Postdoctoral Fellowship for Research Abroad, Brains & Behavior Program, and Center for Behavioral Neuroscience through the STC Program of NSF under Agreement No. IBN-9876754.


  1. Asai N, Fusetani N, Matsumaga S, Sasaki J (2000) Sex pheromones of the hair crab Erimacrus isenbeckii. Part 1: isolation and structures of novel ceramides. Tetrahedron 56:9895–9899CrossRefGoogle Scholar
  2. Asai N, Fusetani N, Matsunaga S (2001) Sex pheromones of the hair crab Erimacrus isenbeckii. II. Synthesis of ceramides. J Nat Prod 64:1210–1215CrossRefPubMedGoogle Scholar
  3. Atema J, Steinbach MA (2007) Chemical communication in the social behavior of the lobster, Homarus americanus, and other decapod Crustacea. In: Duffy E, Thiel M (eds) Ecology and evolution of social behavior: crustaceans as model systems. Oxford University Press, Oxford, pp 115–144CrossRefGoogle Scholar
  4. Baldwin J, Johnsen S (2009) The importance of color in mate choice of the blue crab Callinectes sapidus. J Exp Biol 212:3762–3768CrossRefPubMedGoogle Scholar
  5. Bradbury JW, Vehrencamp SL (1998) Principles of animal communication. Sinauer Associates, SunderlandGoogle Scholar
  6. Bublitz R, Sainte-Marie B, Newcomb-Hodgetts C, Fletcher N, Smith M, Hardege JD (2008) Interspecific activity of the sex pheromone of the European shore crab (Carcinus maenas). Behaviour 145:1465–1478CrossRefGoogle Scholar
  7. Bushmann PJ (1999) Concurrent signals and behavioral plasticity in blue crab (Callinectes sapidus Rathbun) courtship. Biol Bull 197:63–71CrossRefGoogle Scholar
  8. Caskey JL, Bauer RT (2005) Behavioral tests for a possible contact pheromone in the caridean shrimp Palaemonetes pugio. J Crust Biol 25:571–576CrossRefGoogle Scholar
  9. Cate HS, Gleeson RA, Derby CD (1999) Activity-dependent labeling of the olfactory organ of blue crabs suggests that pheromone-sensitive and food-odor sensitive receptor neurons are packaged together in aesthetasc sensilla. Chem Senses 24:559Google Scholar
  10. Díaz ER, Thiel M (2004) Chemical and visual communication during mate searching in the rock shrimp. Biol Bull 206:134–143CrossRefPubMedGoogle Scholar
  11. Dickman BD, Webster DR, Page JL, Weissburg MJ (2009) Three-dimensional odorant concentration measurements around actively tracking blue crabs. Limnol Oceanogr Methods 7:96–108Google Scholar
  12. Dreanno C, Matsumura K, Dohmae N, Takio K, Hirota H, Kirby RR, Clare AS (2006a) An α2-macroglobulin-like protein is the cue to gregarious settlement of the barnacle Balanus amphitrite. Proc Natl Acad Sci USA 103:14396–14401CrossRefPubMedGoogle Scholar
  13. Dreanno C, Kirby RR, Clare AS (2006b) Smelly feet are not always a bad thing: the relationship between cyprid footprint protein and the barnacle settlement pheromone. Biol Lett 2:423–425CrossRefPubMedGoogle Scholar
  14. Dreanno C, Kirby RR, Clare AS (2007) Involvement of the barnacle settlement-inducing protein complex (SIPC) in species recognition at settlement. J Exp Mar Biol Ecol 351:276–282CrossRefGoogle Scholar
  15. Fields DM, Weissburg MJ, Browman HI (2007) Chemoreception in the salmon louse Lepeophtheirus salmonis: an electrophysiology approach. Dis Aquat Org 78:161–168CrossRefPubMedGoogle Scholar
  16. Findlay J, Levvy GA, Marsh CA (1958) Inhibition of glycosidases by aldonolactones of corresponding configuration. 2. Inhibitors of β-N-acetylglucosaminidase. Biochem J 69:467–476PubMedGoogle Scholar
  17. Gleeson RA (1980) Pheromone communication in the reproductive behavior of the blue crab, Callinectes sapidus. Mar Behav Physiol 7:119–134CrossRefGoogle Scholar
  18. Gleeson RA (1982) Morphological and behavioral identification of the sensory structures mediating pheromone reception in the blue crab Callinectes sapidus. Biol Bull 163:162–171CrossRefGoogle Scholar
  19. Gleeson RA (1991) Intrinsic factors mediating pheromone communication in the blue crab, Callinectes sapidus. In: Martin JW, Bauer RT (eds) Crustacean sexual biology. Columbia University Press, New York, pp 17–32Google Scholar
  20. Gleeson RA, Adams MA, Smith AB III (1984) Characterization of a sex pheromone in the blue crab, Callinectes sapidus: crustecdysone studies. J Chem Ecol 10:913–921CrossRefGoogle Scholar
  21. Gleeson RA, Adams MA, Smith AB III (1987) Hormonal modulation of pheromone-mediated behavior in a crustacean. Biol Bull 172:1–9CrossRefGoogle Scholar
  22. Gleeson RA, McDowell LM, Aldrich HC (1996) Structure of the aesthetasc (olfactory) sensilla of the blue crab. Callinectes sapidus: transformations as a function of salinity. Cell Tissue Res 284:279–288CrossRefGoogle Scholar
  23. Hardege JD, Jennings A, Hayden D, Muller CT, Pascoe D, Bentley MG, Clare AS (2002) Novel behavioral assay and partial purification of a female-derived sex pheromone in Carcinus maenas. Mar Ecol Prog Ser 244:179–189CrossRefGoogle Scholar
  24. Hay ME, Stachowicz JJ, Cruz-Rivera E, Bullard S, Deal MS, Lindquist N (1998) Bioassays with marine and freshwater macroorganisms. In: Haynes KF, Millars JG (eds) Methods in chemical ecology, vol. 2, bioassay methods. Chapman and Hall, New York, pp 39–141Google Scholar
  25. Hayden D, Jenning A, Müller C, Pascoe D, Bublitz R, Webb H, Breithaupt T, Watkins L, Hardege JD (2007) Sex-specific mediation of foraging in the shore crab, Carcinus maenas. Horm Behav 52:162–168CrossRefPubMedGoogle Scholar
  26. Haynes KF, Millars JG (eds) (1998) Methods in chemical ecology, vol. 1, chemical methods. Chapman and Hall, New YorkGoogle Scholar
  27. Ingvarsdóttir A, Birkett MA, Duce I, Genna RL, Mordue W, Pickett JA, Wadhams LJ, Mordue (Luntz) AJ (2002a) Semiochemical strategies for sea louse control: host location cues. Pest Manag Sci 58:537–545CrossRefPubMedGoogle Scholar
  28. Ingvarsdóttir A, Birkett MA, Duce I, Mordue W, Pickett JA, Wadhams LJ, Mordue (Luntz) AJ (2002b) Role of semiochemicals in mate location by parasitic sea louse, Lepeophtheirus salmonis. J Chem Ecol 28:2107–2117CrossRefPubMedGoogle Scholar
  29. Jivoff P, Hines AH, Quackenbush LS (2007) Reproduction biology and embryonic development. In: Kennedy VS, Cronin LE (eds) The blue crab Callinectes sapidus. Maryland Sea Grant, College ParkGoogle Scholar
  30. Kamio M (2009) Toward identifying sex pheromones in blue crabs: using biomarker targeting within the context of evolutionary chemical ecology. Ann NY Acad Sci 1170:456–461CrossRefPubMedGoogle Scholar
  31. Kamio M, Matsunaga S, Fusetani N (2002) Copulation pheromone in the crab, Telmessus cheiragonus (Brachyura: Decapoda). Mar Ecol Prog Ser 234:183–190CrossRefGoogle Scholar
  32. Kamio M, Matsunaga S, Fusetani N (2003) Observation on the mating behaviors of the helmet crab Telmessus cheiragonus (Brachyura: Cheiragonidae). J Mar Biol Assoc UK 83:1007–1013CrossRefGoogle Scholar
  33. Kamio M, Reidenbach M, Derby CD (2008) To paddle or not: determinants and consequences of courtship display by male blue crabs, Callinectes sapidus. J Exp Biol 211:1243–1248CrossRefPubMedGoogle Scholar
  34. Kennedy VS, Cronin LE (2007) The blue crab: Callinectes sapidus. University of Maryland Sea Grant Press, College Park, 800 ppGoogle Scholar
  35. Kittredge JS, Terry M, Takahashi FT (1971) Sex pheromone activity of the molting hormone, crustecdysone, on male crabs. Fish Bull 69:337–343Google Scholar
  36. Kleps RA, Myers TC, Lipcius RN, Henderson TO (2007) A sex-specific metabolite identified in a marine invertebrate utilizing Phosphorus-31 nuclear magnetic resonance. PLoS One 2:e780CrossRefPubMedGoogle Scholar
  37. Koehn FE, Carter GT (2005) The evolving role of natural products in drug discovery. Nat Rev Drug Discov 4:206–220CrossRefPubMedGoogle Scholar
  38. Legler G, Lüllau E, Kappes E, Kastenholz F (1991) Bovine N-acetyl-β-D-glucosaminidase: affinity purification and characterization of its active site with nitrogen containing analogs of N-acetylglucosamine. Biochim Biophys Acta 1080:89–95PubMedGoogle Scholar
  39. Lin DY, Zhang S-Z, Block E, Katz LC (2005) Encoding social signals in the mouse main olfactory bulb. Nature 434:470–477CrossRefPubMedGoogle Scholar
  40. Merzendorfer H, Zimoch L (2003) Chitin metabolism in insects: structure, function and regulation of chitin synthases and chitinases. J Exp Biol 206:4393–4412CrossRefPubMedGoogle Scholar
  41. Nojima S, Schal C, Webster FX, Santangelo RG, Roelofs WL (2005) Identification of the sex pheromone of the German cockroach, Blattella germanica. Science 307:1104–1106CrossRefPubMedGoogle Scholar
  42. Rittschof D (2005) Male blue crab pheromone originates in semen. Chem Senses 30:A144CrossRefGoogle Scholar
  43. Sarker SD, Latif Z, Gray A (2006) Natural products isolation, 2nd edn. Humana Press, TotowaGoogle Scholar
  44. Schmidt M, Ache BW (1996) Processing of antennular input in the brain of the spiny lobster. Panulirus argus. II. The olfactory pathway. J Comp Physiol A 178:605–628CrossRefGoogle Scholar
  45. Shabani S, Kamio M, Derby CD (2009) Spiny lobsters use urine-borne signals to communicate social status. J Exp Biol 212:2464–2474CrossRefPubMedGoogle Scholar
  46. Soga T, Baran R, Suematsu M, Ueno Y, Ikeda S, Sakurakawa T, Kakazu Y, Ishikawa T, Robert M, Nishioka T, Tomita M (2006) Differential metabolomics reveals ophthalmic acid as an oxidative stress biomarker indicating hepatic glutathione consumption. J Biol Chem 281:16768–16776CrossRefPubMedGoogle Scholar
  47. Sorensen PW, Hoye TR (2007) A critical review of the discovery and application of a migratory pheromone in an invasive fish, the sea lamprey Petromyzon marinus L. J Fish Biol 71:100–114CrossRefGoogle Scholar
  48. Teytaud AR (1971) The laboratory studies of sex recognition in the blue crab, Callinectes sapidus Rathbun. Sea Grant Technical Bulletin, University of Miami Sea Grant Program 15, pp 1–63Google Scholar
  49. Ting JH, Snell TW (2003) Purification and sequencing of a mate-recognition protein from the copepod Tigriopus japonicus. Mar Biol 143:1–8CrossRefGoogle Scholar
  50. Warner WW (1976) Beautiful swimmers: watermen, crabs and the Chesapeake Bay. Little, Brown, BostonGoogle Scholar
  51. Wyatt TD (2003) Pheromones and animal behaviour: communication by smell and taste. Cambridge University Press, CambridgeCrossRefGoogle Scholar
  52. Yambe H, Kitamura S, Kamio M, Yamada M, Matsunaga S, Fusetani N, Yamazaki F (2006) L-Kynurenine, an amino acid identified as a sex pheromone in the urine of ovulated female masu salmon. Proc Natl Acad Sci USA 103:15370–15374CrossRefPubMedGoogle Scholar
  53. Yem DW, Wu HC (1976) Purification and properties of β-N-D-acetylglucosaminidase from Escherichia coli. J Bacteriol 125:324–331PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of Ocean ScienceTokyo University of Marine Science and TechnologyTokyoJapan

Personalised recommendations