Detection of very long-chain hydrocarbons by laser mass spectrometry reveals novel species-, sex-, and age-dependent differences in the cuticular profiles of three Nasonia species

  • Tanja Bien
  • Jürgen GadauEmail author
  • Andreas Schnapp
  • Joanne Y. Yew
  • Christian Sievert
  • Klaus DreisewerdEmail author
Research Paper


Long-chain cuticular hydrocarbons (CHC) are key components of chemical communication in many insects. The parasitoid jewel wasps from the genus Nasonia use their CHC profile as sex pheromone and for species recognition. The standard analytical tool to analyze CHC is gas chromatography coupled with mass spectrometric detection (GC/MS). This method reliably identifies short- to long-chain alkanes and alkenes, but CHC with more than 40 carbon atoms are usually not detected. Here, we applied two laser mass spectrometry (MS) techniques, namely direct laser desorption/ionization (d)LDI and silver-assisted (Ag-)LDI MS, respectively, to analyze CHC profiles of N. vitripennis, N. giraulti, and N. longicornis directly from the cuticle or extracts. Furthermore, we applied direct analysis in real-time (DART) MS as another orthogonal technique for extracts. The three methods corroborated previous results based on GC/MS, i.e., the production of CHC with carbon numbers between C25 and C40. However, we discovered a novel series of long-chain CHC ranging from C41 to C51/C52. Additionally, several previously unreported singly and doubly unsaturated alkenes in the C31-C39 range were found. Use of principal component analysis (PCA) revealed that the composition of the newly discovered CHC varies significantly between species, sex, and age of the animals. Our study adds to the growing literature on the presence of very long-chain CHC in insects and hints at putative roles in insect communication.

Graphical abstract


Laser mass spectrometry Nasonia Long-chain cuticular hydrocarbons Principal component analysis DART 



We thank Jens Soltwisch for the help with the oTOF mass spectrometer. Financial support by the German Research Foundation (DFG; grant DR 416/10-1 to K.D.), the Department of Defense US Army Research Laboratory (grant W911NF-16-1-0216 to J.Y.Y.), and the University of Münster for travel subsidies within their Internationalisierungsstrategie (to K.D.) is gratefully acknowledged.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

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  1. 1.
    Lockey KH. Lipids of the insect cuticle: origin, composition and function. Comp Biochem Physiol B Biochem Mol Biol. 1998;89:595–645.CrossRefGoogle Scholar
  2. 2.
    Howard RW. Cuticular hydrocarbons and chemical communication. In: Stanley-Samuelson DW, Nelson DR, editors. Insect lipids: chemistry, biochemistry and biology. Lincoln: University of Nebraska Press; 1993.Google Scholar
  3. 3.
    Gibbs AG. Water-proofing properties of cuticular lipids. Am Zool. 1998;38:471–82.CrossRefGoogle Scholar
  4. 4.
    Blomquist GJ, Bagnères AG. Insect hydrocarbons: biology, biochemistry, and chemical ecology. Cambridge: Cambridge University Press; 2010.CrossRefGoogle Scholar
  5. 5.
    Wyatt TD. Pheromones and animal behavior: chemical signals and signatures. 2nd ed. Cambridge: Cambridge University Press; 2014.Google Scholar
  6. 6.
    Yew JY, Chung H. Insect pheromones: an overview of function, form, and discovery. Prog Lipid Res. 2015;59:88–105.CrossRefGoogle Scholar
  7. 7.
    Stanley-Samuelson DW, Jurenka RA, Cripps C, Blomquist GJ. Fatty acids in insects: composition, metabolism, and biological significance. Arch Insect Biochem Physiol. 1988;9:1–33.CrossRefGoogle Scholar
  8. 8.
    Fröhlich B, Riederer M, Tautz J. Honeybees discriminate cuticular waxes based on esters and polar components. Apidologie. 2001;32:265–74.CrossRefGoogle Scholar
  9. 9.
    Yew JY, Dreisewerd K, Luftmann H, Müthing J, Pohlentz G, Kravitz EA. A new male sex pheromone and novel cuticular cues for chemical communication in Drosophila. Curr Biol. 2009;19:1245–54.CrossRefGoogle Scholar
  10. 10.
    Yew JY, Dreisewerd K, De Oliveira CC, Etges WJ. Male-specific transfer and fine scale spatial differences of newly identified cuticular hydrocarbons and triacylglycerides in a Drosophila species pair. PLoS One. 2011;6:e16898.CrossRefGoogle Scholar
  11. 11.
    Chin JSR, Ellis SR, Pham HT, Blanksby SJ, Mori K, Koh QL, et al. Sex-specific triacylglycerides are widely conserved in Drosophila and mediate mating behavior. Elife. 2014;3:e01751.CrossRefGoogle Scholar
  12. 12.
    Shikichi Y, Shankar S, Yew JY, Mori K. Synthesis and bioassay of the eight analogues of male pheromone CH503 (3-Acetoxy-11,19-octacosadien-1-ol) of the Drosophila melanogaster fruit fly. Biosci Biotechnol Biochem. 2013;77:1931–8.CrossRefGoogle Scholar
  13. 13.
    Amirav A, Gordin A, Poliak M, Fialkov. Gas chromatography mass spectrometry with supersonic molecular beams. J Mass Spectrom 2008;43:141–163.Google Scholar
  14. 14.
    Lu B, Zelle KM, Seltzer R, Hefetz A, Ben-Shahar Y. Feminization of pheromone-sensing neurons affects mating decisions in Drosophila males. Biol Open. 2014;3:152–60.CrossRefGoogle Scholar
  15. 15.
    Cvačka J, Jiroš P, Šobotník J, Hanus R, Svatoš A. Analysis of insect cuticular hydrocarbons using matrix-assisted laser desorption/ionization mass spectrometry. J Chem Ecol. 2006;32:409–34.CrossRefGoogle Scholar
  16. 16.
    Kaftan F, Vrkoslav V, Kynast P, Kulkarni P, Böcker S, Cvačka J, et al. Mass spectrometry imaging of surface lipids on intact Drosophila melanogaster flies. J Mass Spectrom. 2014;49:223–32.CrossRefGoogle Scholar
  17. 17.
    Horká P, Vrkoslav V, Hanus R, Pecková K, Cvačka J. New MALDI matrices based on lithium salts for the analysis of hydrocarbons and wax esters. J Mass Spectrom. 2014;49:628–38.CrossRefGoogle Scholar
  18. 18.
    Yew JY, Cody RB, Kravitz EA. Cuticular hydrocarbon analysis of an awake behaving fly using direct analysis in real-time time-of-flight mass spectrometry. Proc Natl Acad Sci U S A. 2008;105:7135–40.CrossRefGoogle Scholar
  19. 19.
    Gross J. Direct analysis in real time—a critical review on DART-MS. Anal Bioanal Chem. 2014;406:63–80.CrossRefGoogle Scholar
  20. 20.
    Yang ZH, Attygalle AB. Aliphatic hydrocarbon spectra by helium ionization mass spectrometry (HIMS) on a modified atmospheric-pressure source designed for electrospray ionization. J Am Soc Mass Spectrom. 2011;22:1395–402.CrossRefGoogle Scholar
  21. 21.
    Cody RB, Dane AJ. Soft ionization of saturated hydrocarbons, alcohols and nonpolar compounds by negative-ion direct analysis in real-time mass spectrometry. J Am Soc Mass Spectrom. 2013;24:329–34.CrossRefGoogle Scholar
  22. 22.
    Ng SH, Shankar S, Shikichi Y, Akasaka K, Mori K, Yew JY. Pheromone evolution and sexual behavior in Drosophila are shaped by male sensory exploitation of other males. Proc Natl Acad Sci U S A. 2014;111:3056–61.CrossRefGoogle Scholar
  23. 23.
    Yew JY, Soltwisch J, Pirkl A, Dreisewerd K. Direct laser desorption ionization of endogenous and exogenous compounds from insect cuticles: practical and methodologic aspects. J Am Soc Mass Spectrom. 2011;22:1273–84.CrossRefGoogle Scholar
  24. 24.
    Sherrod SD, Diaz AJ, Russell WK, Cremer P, Russell DH. Silver nanoparticles as selective ionization probes for analysis of olefins by mass spectrometry. Anal Chem. 2008;80:6796–9.CrossRefGoogle Scholar
  25. 25.
    Sekuła J, Niziol J, Rode W, Ruman T. Silver nanostructures in laser desorption/ionization mass spectrometry and mass spectrometry imaging. Analyst. 2015;140:6195–209.CrossRefGoogle Scholar
  26. 26.
    Schnapp A, Niehoff AC, Koch A, Dreisewerd K. Laser desorption/ionization mass spectrometry of lipids using etched silver substrates. Methods. 2016;104:194–203.CrossRefGoogle Scholar
  27. 27.
    Carlson DA, Geden CJ, Bernier UR. Identification of pupal exuviae of Nasonia vitripennis and Muscidifurax raptorellus parasitoids using cuticular hydrocarbons. Biol Control. 1999;15:97–106.CrossRefGoogle Scholar
  28. 28.
    Steiner S, Mumm R, Ruther J. Courtship pheromones in parasitic wasps: comparison of bioactive and inactive hydrocarbon profiles by multivariate statistical methods. J Chem Ecol. 2007;33:825–38.CrossRefGoogle Scholar
  29. 29.
    Niehuis O, Büllesbach J, Judson AK, Schmitt T, Gadau J. Genetics of cuticular hydrocarbon differences between males of the parasitoid wasps Nasonia giraulti and Nasonia vitripennis. Heredity. 2011;107:61–70.CrossRefGoogle Scholar
  30. 30.
    Giesbers MCWG, Gerritsma S, Büllesbach J, Diao W, Pannebakker BA, van de Zande L, et al. Prezygotic isolation in the parasitoid wasp genus Nasonia. Speciation: natural processes, genetics and biodiversity. Hauppauge: Nova Science Publishers; 2013.Google Scholar
  31. 31.
    Büllesbach J, Gadau J, Beukeboom LW, Echinger F, Raychoudhury R, Werren JH, et al. Cuticular hydrocarbon divergence in the jewel wasp Nasonia: evolutionary shifts in chemical communication channels? J Evol Biol. 2013;26:2467–78.CrossRefGoogle Scholar
  32. 32.
    Büllesbach J, Greim C, Raychoudhury R, Schmitt T. Asymmetric assortative mating behaviour reflects incomplete pre-zygotic isolation in the Nasonia species complex. Ethology. 2014;120:834–43.CrossRefGoogle Scholar
  33. 33.
    Mair MM, Kmezic V, Huber S, Pannebakker BA, Ruther J. The chemical basis of mate recognition in two parasitoid wasp species of the genus Nasonia. Entomol Exp Appl. 2017;164:1–15.CrossRefGoogle Scholar
  34. 34.
    Steiner S, Hermann N, Ruther J. Characterization of a female-produced courtship pheromone in the parasitoid Nasonia vitripennis. J Chem Ecol. 2006;32:1687–702.CrossRefGoogle Scholar
  35. 35.
    Büllesbach J, Vetter SG, Schmitt T. Differences in the reliance on cuticular hydrocarbons as sexual signaling and species discrimination cues in parasitoid wasps. Front Zool. 2018;15:22.CrossRefGoogle Scholar
  36. 36.
    Ruther J, Stahl LM, Steiner S, Garbe LA, Tolasch TA. Male sex pheromone in a parasitic wasp and control of the behavioral response by the female’s mating status. J Exp Biol. 2007;210:2163–9.CrossRefGoogle Scholar
  37. 37.
    Niehuis O, Büllesbach J, Gibson JD, Pothmann D, Hanner C, Mutti NS, et al. Behavioural and genetic analyses of Nasonia shed light on the evolution of sex pheromones. Nature. 2013;494:345–8.CrossRefGoogle Scholar
  38. 38.
    Blaul B, Steinbauer R, Merkl P, Merkl R, Tschochner H, Ruther J. Oleic acid is a precursor of linoleic acid and the male sex pheromone in Nasonia vitripennis. Insect Biochem Mol Biol. 2014;51:33–40.CrossRefGoogle Scholar
  39. 39.
    Soltwisch J, Souady J, Berkenkamp S, Dreisewerd K. Effect of gas pressure and gas type on the fragmentation of peptide and oligosaccharide ions generated in an elevated pressure UV/IR-MALDI ion source coupled to an orthogonal time-of-flight mass spectrometer. Anal Chem. 2009;81:2921–34.CrossRefGoogle Scholar
  40. 40.
    Pirkl A, Meier M, Popkova J, Letzel M, Schnapp A, Schiller J, et al. Analysis of free fatty acids by ultraviolet laser desorption ionization mass spectrometry using insect wings as hydrophobic sample substrate. Anal Chem. 2014;86:10763–71.CrossRefGoogle Scholar
  41. 41.
    Kühbandner S, Ruther JJ. Solid phase micro-extraction (SPME) with in situ transesterification: An easy method for the detection of non-volatile fatty acid derivatives on the insect cuticle. J Chem Ecol 2015;41:584–592.Google Scholar
  42. 42.
    Thompson SN, Barlow JS. The fatty acid composition of parasitic hymenoptera and its possible biological significance. Ann Entomol Soc Am. 1974;67:627–32.CrossRefGoogle Scholar
  43. 43.
    Brandstetter B, Ruther J. An insect with a delta-12 desaturase, the jewel wasp Nasonia vitripennis, benefits from nutritional supply with linoleic acid. Naturwissenschaften. 2016;103:40.CrossRefGoogle Scholar
  44. 44.
    Ruther J, Thal K, Steiner S. Pheromone communication in Nasonia vitripennis: abdominal sex attractant mediates site fidelity of releasing males. J Chem Ecol. 2011;37:161–5.CrossRefGoogle Scholar
  45. 45.
    Drijfhout F, Kather R, Martin S. The role of cuticular hydrocarbons in insects. In: Zhang W, Liu H, editors. Behavioral and chemical ecologoly. Hauppauge: Nova Science Publishers; 2010. p. 91–114.Google Scholar
  46. 46.
    Jansen J, Pokorny T, Schmitt T. Disentangling the effect of insemination and ovary development on the cuticular hydrocarbon profile in the bumblebee Bombus terrestis (Hymenoptera: Apidae). Apidologie. 2016;47:101–13.CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Tanja Bien
    • 1
    • 2
  • Jürgen Gadau
    • 3
    Email author
  • Andreas Schnapp
    • 1
  • Joanne Y. Yew
    • 4
  • Christian Sievert
    • 3
  • Klaus Dreisewerd
    • 1
    • 2
    Email author
  1. 1.Institute for HygieneUniversity of MünsterMünsterGermany
  2. 2.Interdisciplinary Center for Clinical Research (IZKF)University of MünsterMünsterGermany
  3. 3.Institute for Evolution and BiodiversityUniversity of MünsterMünsterGermany
  4. 4.Pacific Biosciences Research CenterUniversity of Hawai‘i at MānoaHonoluluUSA

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