Analysis of Ascarosides from Caenorhabditis elegans Using Mass Spectrometry and NMR Spectroscopy

  • Xinxing Zhang
  • Jaime H. Noguez
  • Yue Zhou
  • Rebecca A. Butcher
Part of the Methods in Molecular Biology book series (MIMB, volume 1068)


The nematode Caenorhabditis elegans secretes a family of water-soluble small molecules, known as the ascarosides, into its environment and uses these ascarosides in chemical communication. The ascarosides are derivatives of the 3,6-dideoxysugar ascarylose, modified with different fatty acid-derived side chains. C. elegans uses specific ascarosides, which are together known as the dauer pheromone, to trigger entry into the stress-resistant dauer larval stage. In addition, C. elegans uses specific ascarosides to control certain behaviors, including mating attraction, aggregation, and avoidance. Although in general the concentration of the ascarosides in the environment increases with population density, C. elegans can vary the types and amounts of ascarosides that it secretes depending on the culture conditions under which it has been grown and its developmental history. Here, we describe how to grow high-density worm cultures and the bacterial food for those cultures, as well as how to extract the culture medium to generate a crude pheromone extract. Then, we discuss how to analyze the types and amounts of ascarosides in that extract using mass spectrometry and NMR spectroscopy.

Key words

Dauer Ascarosides Pheromone C. elegans Mass spectrometry NMR spectroscopy dqf-COSY 



Funding for this work was provided by the National Institutes of Health (GM87533 to R.A.B.) and the Human Frontiers Science Program (RGY0042/2010 subcontract to R.A.B.). Our work on the triple quadrupole mass spectrometer was supported in part by the National Institutes of Health and National Center for Research Resources CTSA grant 1UL 1RR029890. We would like to thank Dr. Tim Garrett (University of Florida) for helpful suggestions on the LC-MS/MS parameters and Dr. Ion Ghiviriga (University of Florida) for helpful suggestions on the NMR parameters. We would like to thank Prof. Justin R. Ragains (Louisiana State University) for providing the synthetic ascaroside standards that are used in our work.


  1. 1.
    Jeong PY, Jung M, Yim YH et al (2005) Chemical structure and biological activity of the Caenorhabditis elegans dauer-inducing pheromone. Nature 433:541–545PubMedCrossRefGoogle Scholar
  2. 2.
    Butcher RA, Fujita M, Schroeder FC et al (2007) Small-molecule pheromones that control dauer development in Caenorhabditis elegans. Nat Chem Biol 3:420–422PubMedCrossRefGoogle Scholar
  3. 3.
    Butcher RA, Ragains JR, Kim E et al (2008) A potent dauer pheromone component in C. elegans that acts synergistically with other components. Proc Natl Acad Sci USA 105:14288–14292PubMedCrossRefGoogle Scholar
  4. 4.
    Srinivasan J, Kaplan F, Ajredini R et al (2008) A blend of small molecules regulates both mating and development in Caenorhabditis elegans. Nature 454:1115–1118PubMedCrossRefGoogle Scholar
  5. 5.
    Pungaliya C, Srinivasan J, Fox BW et al (2009) A shortcut to identifying small molecule signals that regulate behavior and development in Caenorhabditis elegans. Proc Natl Acad Sci USA 106(19):7708–7713PubMedCrossRefGoogle Scholar
  6. 6.
    Butcher RA, Ragains JR, Clardy J (2009) An indole-containing dauer pheromone component with unusual dauer inhibitory activity at higher concentrations. Org Lett 11:3100–3103PubMedCrossRefGoogle Scholar
  7. 7.
    von Reuss SH, Bose N, Srinivasan J et al (2012) Comparative metabolomics reveals biogenesis of ascarosides, a modular library of small-molecule signals in C. elegans. J Am Chem Soc 134:1817–1824CrossRefGoogle Scholar
  8. 8.
    Srinivasan J, von Reuss SH, Bose N et al (2012) A modular library of small molecule signals regulates social behaviors in Caenorhabditis elegans. PLoS Biol 10:e1001237PubMedCrossRefGoogle Scholar
  9. 9.
    Artyukhin AB, Yim JJ, Srinivasan J et al (2013) Succinylated octopamine ascarosides and a new pathway of biogenic amine metabolism in C. elegans. J Biol Chem 288:18778–18783Google Scholar
  10. 10.
    Butcher RA, Ragains JR, Li W et al (2009) Biosynthesis of the Caenorhabditis elegans dauer pheromone. Proc Natl Acad Sci USA 106:1875–1879PubMedCrossRefGoogle Scholar
  11. 11.
    Kaplan F, Srinivasan J, Mahanti P et al (2011) Ascaroside expression in Caenorhabditis elegans is strongly dependent on diet and developmental stage. PLoS One 6:e17804PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Xinxing Zhang
    • 1
  • Jaime H. Noguez
    • 1
  • Yue Zhou
    • 1
  • Rebecca A. Butcher
    • 1
  1. 1.Department of ChemistryUniversity of FloridaGainesvilleUSA

Personalised recommendations