The Science of Nature

, 104:19 | Cite as

Hydrogen peroxide as a new defensive compound in “benzoyl cyanide” producing polydesmid millipedes

  • Yasumasa Kuwahara
  • Takuya Yamaguchi
  • Yayoi Ichiki
  • Tsutomu Tanabe
  • Yasuhisa Asano
Original Paper


Hydrogen peroxide was newly and simultaneously demonstrated with well-known hydrogen cyanide as a component of defensive secretions of “benzoyl cyanide” producing polydesmid millipedes. Presence of hydrogen peroxide was successively evidenced by Trinder reagent’s spray with colorless as well as oily smears of defensive secretions containing benzoyl cyanide and hydrogen cyanide by alkaline picrate paper treatment. Linear correlation was demonstrated between quantities of hydrogen peroxide and benzoyl cyanide. By qualitative assay, seven benzoyl cyanide containing polydesmidans (six species of adults and one species of a nymph at stadium I) tested positive to Trinder reagent, indicative of the presence of hydrogen peroxide (together with hydrogen cyanide), while two cyanogenic species without benzoyl cyanide exhibited negative responses to the reagent. Two types of millipedes were elucidated as species of cyanogenic Polydesmida.


Polydesmida Benzoyl cyanide Hydrogen peroxide Defensive secretion Millipede Trinder reagent Mandelonitrile oxidase 



The Exploratory Research for Advanced Technology (ERATO) Program of the Japan Science and Technology Agency (JST) supported this work. We thank Prof. DeMar Taylor, University of Tsukuba for his careful and critical reading of the manuscript. We also thank Mr. Takahiro Menda for collecting the millipede R. semicircularis semicircularis in Kumamoto Prefecture.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Aggio JF, Derby CD (2008) Hydrogen peroxide and other components in the ink of sea hares are chemical defenses against predatory spiny lobsters acting through non-antennular chemoreceptors. J Exp Mar Biol Ecol 365:28–34CrossRefGoogle Scholar
  2. Aneshansley DJ, Eisner T, Widom JM, Widom B (1969) Biochemistry at 100 °C: explosive secretory discharge of bombardier beetles (Brachinun). Science 165:61–63CrossRefPubMedGoogle Scholar
  3. Aneshansley DJ, Jones TH, Alsop D, Meinwald J, Eisner T (1983) Thermal concomitants and biochemistry of the explosive discharge mechanism of some little known bombardier beetles. Experientia 39:366–368CrossRefGoogle Scholar
  4. Barham D, Trinder P (1972) An improved colour reagent for the determination of blood glucose by the oxidase system. Analyst 97:142–145CrossRefPubMedGoogle Scholar
  5. Bodner M, Raspotnig G (2012) Millipedes that smell like bugs: (E)-alkenals in the defensive secretion of the julid diplopod Allajulus dicentrus. J Chem Ecol 38:547–556CrossRefPubMedGoogle Scholar
  6. Bodner M, Vagalinski B, Makarov SE, Antić DŽ, Vujisić LV, Leis H-J, Raspotnig G (2016) “Quinone millipedes” reconsidered: evidence for a mosaic-like taxonomic distribution of phenol-based secretions across the Julidae. J Chem Ecol 42:249–258CrossRefPubMedPubMedCentralGoogle Scholar
  7. Dadashipour M, IshidaY YK, Asano Y (2015) Discovery and molecular and biocatalytic properties of hydroxynitrile lyase from an invasive millipede, Chamberlinius hualienensis. Proc Natl Acad Sci U S A 112:10605–10610CrossRefPubMedPubMedCentralGoogle Scholar
  8. Duffey SS, Towers GHN (1978) On the biochemical basis of HCN production in the millipede Harpaphe haydeniana (Xystodesmidae: Polydesmida). Can J Zool 56:7–16CrossRefGoogle Scholar
  9. Duffey SS, Underhill EW, Towers GHN (1974) Intermediates in the biosynthesis of HCN and benzaldehyde by a polydesmid millipede, Harpaphe haydeniana (Wood). Comp Biochem Physiol 47B:753–766Google Scholar
  10. Duffey SS, Blum MS, Fales HM, Evans SL, Roncadri RW, Tiemann DL, Nakagawa Y (1977) Benzoyl cyanide and mandelonitrile benzoate in the defensive secretions of millipedes. J Chem Ecol 3:101–113CrossRefGoogle Scholar
  11. Eisner T, Jones TH, Aneshansley DJ, Tschinkel WR, Silberglied RE, Meinwald J (1977) Chemistry of defensive secretions of bombardier beetles (Brachinini, Metriini, Ozaenini, Paussini). J Insect Physiol 23:1383–1386CrossRefGoogle Scholar
  12. Golovatch SI, Kime RD (2009) Millipede (Diplopoda): a review. Soil Organism 81:565–597Google Scholar
  13. Ishida Y, Kuwahara Y, Dadashipour M, Ina A, Yamaguchi T, Morita M, Ichiki Y, Asano Y (2016) A sacrificial millipede altruistically protects its swarm using a drone blood enzyme, mandelonitrile oxidase. Sci Rep 6:26998. doi: 10.1038/srep26998 CrossRefPubMedPubMedCentralGoogle Scholar
  14. Jones TH, Conner WE, Meinwald J, Eisner HE, Eisner T (1976) Benzoyl cyanide and mandelonitrile in the cyanogenetic secretion of a centipede. J Chem Ecol 2:421–429CrossRefGoogle Scholar
  15. Kuwahara Y (1999) Chemical defenses of millipedes. In: Hidaka T, Matsumoto Y, Honda K, Honda H, Tatsuki S (eds) Environmental entomology, behavior, physiology and chemical ecology. The University of Tokyo Press, Tokyo, Japan , pp 291–298written in JapaneseGoogle Scholar
  16. Kuwahara Y, Ômura H, Tanabe T (2002) 2-Nitroethenylbenzene as natural products in millipede defense secretions. Naturwissenschaften 89:308–310CrossRefPubMedGoogle Scholar
  17. Kuwahara Y, Mori N, Sakuma M, Tanabe T (2003) (1Z)- and (1E)-2-nitroethenylbenzenes, and 2-nitroethylbenzene as natural products in defense secretions of a millipede Thelodesmus armatus Miyosi (Polydesmida: Pyrgodesmidae). Jpn J Environ Entomol Zool 14:149–155Google Scholar
  18. Kuwahara Y, Shimizu N, Sakamoto F, Tanabe T (2008) Mandelonitrile as the defense secretion, and phenylacetonitrile as its biosynthetic precursor in Nedyopus tambanus tambanus (Paradoxomatidae: Polydesmida). Jpn J Environ Entomol Zool 19:79–84Google Scholar
  19. Kuwahara Y, Shimizu N, Tanabe T (2011) Release of hydrogen cyanide via post-secretion Schotten-Baumann reaction in defensive fluids of Polydesmoid millipedes. J Chem Ecol 37:232–238CrossRefPubMedGoogle Scholar
  20. Kuwahara Y, Ichiki Y, Morita M, Tanabe T, Asano Y (2015) Chemical polymorphism in defense secretions during ontogenetic development of the millipede Niponia nodulosa. J Chem Ecol 41:15–21CrossRefPubMedGoogle Scholar
  21. Makarov SE (2015) Diplopoda—Integument. In: Minelli A (ed) Treatise on zoology—anatomy, taxonomy, Biology. The Myriapoda, vol 2. Leiden, Brill, pp 69–99Google Scholar
  22. Maschwitz U, Lauschke U, Wuermli M (1979) Hydrogen cyanide-producing glands in a scolopender, Asanada n. Sp. (Chilopoda, Scolopendridae). J Chem Ecol 5:901–907CrossRefGoogle Scholar
  23. Noguchi S, Mori N, Higa Y, Kuwahara Y (1997) Identification of Nedyopus patrioticus patrioticus (Attems, 1898) (Polydesmida: Paradoxomatidae) secretions as possible defense substances. Appl Entomol Zool 32:447–452Google Scholar
  24. Ômura H, Kuwahara Y, Tanabe T (2002a) 1-octen-3-ol together with geosmin: new secretion compounds from a polydesmid millipede, Niponia nodulosa. J Chem Ecol 28:2601–2612CrossRefPubMedGoogle Scholar
  25. Ômura H, Kuwahara Y, Tanabe T (2002b) Species-specific chemical compositions of defense secretions from Parafontaria tonominea tonominea Attems and Riukiaria semicircularis semicircularis Takakuwa (polydesmida: Xystodesmidae). Appl Entomol Zool 37:73–78CrossRefGoogle Scholar
  26. Schildknecht H, Maschwitz U, Krauss D (1968) Blausaeure im Wehrsekret des Erdlaeufers Pachymerium ferrugineum. Naturwissenschaftern 55:230CrossRefGoogle Scholar
  27. Shear WA (2015) The chemical defenses of millipedes (diplopoda): biochemistry, physiology and ecology. Biochem Syst Ecol 61:78–117CrossRefGoogle Scholar
  28. Shimizu N, Kuwahara Y, Yakumaru R, Tanabe T (2012) n-Hexyl laurate and fourteen related fatty acid esters: new secretory compounds from the julid millipede, Anaulaciulus sp. J Chem Ecol 38:23–28CrossRefPubMedGoogle Scholar
  29. Shinohara K (1999) Life of a Japanese millipede, Niponia nodulosa. Insectarium 18:82–86 (in Japanese)Google Scholar
  30. Stoev P, Enghoff H (2008) A revision of the millipede tribe Apfelbeckiini Verhoeff, 1900 (Diplopoda: Callipodida: Schizopetalidae). Steenstrupia 39:47–66Google Scholar
  31. Taira J, Nakamura K, Higa Y (2003) Identification of secretory compounds from the millipede, Oxidus gracilis C. L. Koch (Polydesmida: Paradoxosomatidae) and their variation in different habitats. Appl Entomol Zool 38:401–404CrossRefGoogle Scholar
  32. Vujisić LV, Vučković IM, Makarov SE, Ilić BS, Antić DŽ, Jadranin MB, Todorović NM, Mrkić IV, Vajs VE, Lučić LR, Ćurčić BPM, Mitić BM (2013) Chemistry of the sternal gland secretion of the Mediterranean centipede Himantarium gabrielis (Linnaeus, 1767) (Chilopoda: Geophilomorpha: Himantariidae). Naturwissenschaften 100:861–870CrossRefPubMedGoogle Scholar
  33. Williams HJ, Edwards TG (1980) Estimation of cyanide with alkaline picrate. J Sci Food Agric 31:15–22CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Yasumasa Kuwahara
    • 1
    • 2
  • Takuya Yamaguchi
    • 1
    • 2
  • Yayoi Ichiki
    • 1
    • 2
  • Tsutomu Tanabe
    • 3
  • Yasuhisa Asano
    • 1
    • 2
  1. 1.Biotechnology Research Center and Department of BiotechnologyToyama Prefectural UniversityImizuJapan
  2. 2.Asano Active Enzyme Molecule Project, JST, ERATOImizuJapan
  3. 3.Faculty of EducationKumamoto UniversityKumamotoJapan

Personalised recommendations