Chemical Defense in Millipedes (Myriapoda, Diplopoda): DoRepresentatives of the Family Blaniulidae Belong to the �Quinone� Clade?

by Ljubodrag V. Vujisica), Dragan Z. Anticb), Ivan M. Vuckovica), Tatjana Lj. Sekulicb), Vladimir T.Tomicb), Boris M. Mandica), Vele V. Tesevica), Bozidar P. M. Curcicb), Vlatka E. Vajsc), and Slobodan

E. Makarov*b)

a) Faculty of Chemistry, University of Belgrade, Studentski trg 12–16, 11000 Belgrade, Serbiab) Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 3, 11000 Belgrade,

Serbia (phone: þ381-11-2187266; fax: þ381-11-2638500; e-mail: slobodan@bio.bg.ac.rs)c) Institute of Chemistry, Technology and Metallurgy, Njegoseva 12, 11000 Belgrade, Serbia

The defensive secretions of two blaniulid millipedes, Nopoiulus kochii and Cibiniulus phlepsii, werecharacterized by GC-FID and GC/MS analyses, which showed the presence of a complex mixture ofbenzoquinones, hydroquinones, and oleates. Altogether, 13 compounds were identified. The majorcompound in the secretions of both analyzed species was 2-methyl-1,4-benzoquinone (toluquinone). Thesecond major constituent in the N. kochii secretion was 2-methyl-3,4-(methylenedioxy)phenol, while inthat of C. phlepsii, it was 2-methoxy-3-methyl-1,4-benzoquinone. The defensive secretion of N. kochiialso showed a high content of hydroquinones (13.5%) in comparison to that of C. phlepsii (0.8%). Hexyloleate and octyl oleate were detected for the first time in defensive millipede fluids. The chemicalcomposition of the defensive secretions supports the chemotaxonomic position of the family Blaniulidaein the �quinone� millipede clade.

Introduction. – One of the conspicuous features of the numerous members of theclass Diplopoda is the presence of a pair of exocrine glands located on thepleurotergites. These glands (repugnatorial glands or ozadenes) produce a variety ofvolatile secretions that are released under the threat of predation. To date,135 millipede species have had their secretions chemically characterized [1]. Speciesbelonging to the order Glomerida and Polyzoniida produce alkaloids, whereas thoseclassed as Julida, Spirobolida, and Spirostreptida produce quinones. Polydesmida areknown as cyanogenic, and Callipodida produce cresols as the main defensivecompounds [1]. The species of other millipede orders are either without chemicaldefenses or their composition is not known.

The family Blaniulidae belongs to the order Julida and includes 60 small, slenderspecies from the Western Palaearctic region [2]. This family is divided into foursubfamilies, Mesoblaniulinae, Choneiulinae, Blaniulinae, and Nopoiulinae [2]. To date,Blaniulus guttulatus (Fabricius, 1798) is the only member of the family Blaniulidaewhose chemical pool of defensive secretions has been analyzed [3]. Generally, withinthe whole order of Julida, the chemical arsenal is known in a small number of species[4], and the chemical data do not fit into phylogenetic lineages.

To provide comparative information on the composition of the defensive secretionsin the family Blaniulidae, and considering that the number of detailed works on thesemiochemistry in members of the order Julida is still scarce, the chemical composition

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� 2014 Verlag Helvetica Chimica Acta AG, Z�rich

of the defensive secretions from ozadenes in two blaniulid species, Cibiniulus phlepsii(Verhoeff, 1897) and Nopoiulus kochii (Gervais, 1847), was presented. Photos of thejuvenile form of the two species are shown in Fig. 1. One of the goals of this study was toconfirm that members of the family Blaniulidae belong to the �quinone� clade ofmillipedes.

Results. – The GC-FID and GC/MS analyses of the CH2Cl2 extracts of the twoblaniulids Cibiniulus phlepsii and Nopoiulus kochii showed the presence of bothquinones and nonquinones (Fig. 2 and Table). Eleven quinones were identified, viz., 2-methyl-1,4-benzoquinone (toluquinone, 1), 2-hydroxy-3-methyl-1,4-benzoquinone (2),

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Fig. 1. Juvenile form of a) Cibiniulus phlepsii (Verhoeff, 1897) from Novi Sad, bank of the RiverDanube (photo M. Sciban) and b) Nopoiulus kochii (Gervais, 1847) from the Velika Balanica Cave,

village of Sicevo, near Nis (photo D. Z. A.)

Table. Chemical Composition Determined by GC-FID and GC/MS Analyses of the Defensive Secretionsof the Blaniulid Millipede Species Nopoiulus kochii and Cibiniulus phlepsii

Compound tR [min]a) Content [%]b)

N. kochii C. phlepsii

2-Methyl-1,4-benzoquinone (1) 8.1 42.0 58.12-Hydroxy-3-methyl-1,4-benzoquinone (2) 9.9 6.5 1.12-Methoxy-3-methyl-1,4-benzoquinone (3) 10.8 4.2 33.62,3-Dimethoxy-1,4-benzoquinone (4) 13.1 – trc)2-Methylhydroquinone (5) 13.3 11.4 –2-Methoxy-3-methylhydroquinone (6) 13.5 – 0.82,3-Dimethoxyhydroquinone (7) 13.8 1.0 –2-Methyl-3,4-(methylenedioxy)phenol (8) 14.0 26.8 4.22,3-Dimethoxy-5-methyl-1,4-benzoquinone (9) 14.4 0.7 –2,3-Dimethoxy-5-methylhydroquinone (10) 14.9 1.1 –2,5-Dimethoxy-3-methyl-1,4-benzoquinone (11)d) 15.6 – 0.7Hexyl oleate (12) 26.4 1.1 0.5Octyl oleate (13) 28.0 5.2 1.0

a) tR: Retention time obtained from GC/MS data. b) Relative content calculated from GC-FID peakareas. c) tr: Trace (< 0.1%). d) Tentatively identified compound.

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Fig. 2. GC-FID Profiles of the CH2Cl2 extracts of two blaniulids, a) Nopoiulus kochii (Gervais, 1847) andb) Cibiniulus phlepsii (Verhoeff, 1897) , and structures of the identified compounds. 2-Methyl-1,4-benzoquinone (¼ toluquinone; 1); 2-hydroxy-3-methyl-1,4-benzoquinone (2); 2-methoxy-3-methyl-1,4-benzoquinone (3); 2,3-dimethoxy-1,4-benzoquinone (4); 2-methylhydroquinone (5); 2-methoxy-3-methylhydroquinone (6); 2,3-dimethoxyhydroquinone (7); 2-methyl-3,4-(methylenedioxy)phenol (8);2,3-dimethoxy-5-methyl-1,4-bezoquinone (9); 2,3-dimethoxy-5-methylhydroquinone (10); 2,5-dime-

thoxy-3-methyl-1,4-benzoquinone (11); hexyl oleate (12); octyl oleate (13).

2-methoxy-3-methyl-1,4-benzoquinone (3), 2,3-dimethoxy-1,4-benzoquinone (4), 2-methylhydroquinone (5), 2-methoxy-3-methylhydroquinone (6), 2,3-dimethoxyhydro-quinone (7), 2-methyl-3,4-(methylenedioxy)phenol (8), 2,3-dimethoxy-5-methyl-1,4-benzoquinone (9), 2,3-dimethoxy-5-methylhydroquinone (10), and 2,5-dimethoxy-3-methyl-1,4-benzoquinone (11), as well as two nonquinones, viz., hexyl oleate (12) andoctyl oleate (13). The major constituents of the defensive secretion of N. kochii werecompounds 1 (relative content of 42.0%), 8 (26.8%), and 5 (11.4%), while in C.phlepsii, the dominant constituents were compounds 1 (58.1%) and 3 (33.6%; Table).The nonquinones 12 and 13 were detected in both species. The relative content of allcompounds is presented in the Table.

Discussion. – To date, 20 quinones and 17 nonquinones have been identified in 26julid species from 18 genera [1] [4]. All analyzed species showed multi-componentsecretion patterns consisting of quinones as the main constituents and different minorcompounds.

The structures of the ozadenes in the two analyzed blaniulids were found to belongto the juliform type. They consisted of a more or less spherical sac and efferent ductopening laterally on the pleurotergites via small ozopores (Fig. 3). As in other juliformmillipedes, the terminal portions of the efferent duct have attached muscles that allowfor the opening and closing of the passage for defensive fluids.

The GC-FID and GC/MS analyses of the defensive secretions of the two blaniulidsrevealed that six benzoquinones, 1 –4, 9, and 11, belonged to the identified components,

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Fig. 3. Structure of the ozadenes in Cibiniulus phlepsii (Verhoeff, 1897). s, Spherical sac; e, efferentduct; m, muscle; o, ozopore; scale line¼0.2 mm.

accounting for 53.4 and 93.5% of the total composition identified in N. kochii and C.phlepsii, respectively (Table). In both species, the dominant benzoquinone wascompound 1, which has also been found to be common in other analyzed julids, but hasbeen detected in smaller relative abundance than in the two blaniulids [5]. It isinteresting to note that the absence of 1 appears to be a characteristic of the wholeAllajulus genus [6]. In C. phlepsii, the second most abundant benzoquinone wascompound 3, which, together with 1, represented 91.7% of all defensive chemicalsidentified in this species. On the other hand, in N. kochii, these two quinones composedonly 46.2% of the identified compounds. It is generally well-known that the mostcommon compounds in juliform millipedes are 1,4-benzoquinone and compounds 1 and3 ; however, 1,4-benzoquinone was not identified in the analyzed species. We do notbelieve that the absence of 1,4-benzoquinone in the defensive fluids of the analyzedblaniulids represents a chemotaxonomical marker at the family level; it is not present inozadenes in several non closely-related julid and parajulid genera [4]. The presence ofother benzoquinones, 2, 4, 9, and 11, in the analyzed species is thought to have nosupraspecific chemotaxonomic or phylogenetic significance, since they are also presentin other analyzed juliform millipedes. The absence of these four benzoquinones in thedefensive fluids of the previously analyzed B. guttulatus is probably due to the lesssensitive analytical methods that were used in earlier research. The benzoquinones(with hydroquinones) are known as strong, irritating, and repellent chemicals toarthropods and mammals, and toxic to a great variety of pathogens and parasites [4].Furthermore, the relative content of compound 8 in the defensive exudate of N. kochiiwas significantly higher than that in C. phlepsii (26.8 vs. 4.2%). According to Wu et al.[7], this could be explained by the cyclodehydrogenation reaction of compound 3.

Non-quinonic compounds in the defensive fluids of julids include esters, alcohols,alkenals, and other compounds [4] [6]. Among the 26 analyzed julids (including the twoblaniulids analyzed here), non-quinonic compounds have been detected in 18 species(69%). Another eight �pure quinone� julid species were analyzed between 1961 and1977 or earlier [4], when the analytical equipment was considerably less sensitive thantoday. Every recent report [4] has noted the presence of nonquinones in the defensivefluids of members of the order Julida. Furthermore, there are sporadical opinionsreporting that non-benzoquinone compounds in other juliform millipedes are probablycuticle-derived components or that the identification of such compounds might only betentative [1] [6] [7]. However, at least seven hydroquinones have been previouslydetected in 13 members of the orders Spirobolida and Spirostreptida, as well asmethylbenzaldehyde, dodecanal, and pyridine in three spirobolid species [4]. A recentreport showed that no differences were found in the composition between theconventional hexane extracts from the whole body and the defensive secretion directlysampled by a glass capillary tube, at least in Anaulaciulus species, which points to theabsence of hexane-extractable compounds on the body surface [8]. We share theopinion that the presence of non-quinonic compounds in the defensive fluids in juliformmillipedes represents a specific or generic feature rather than a sporadic occurrence. Itis reasonable to assume that combinations of quinones and esters, alcohols, or alkenalsprovide a more effective defense than any one compound alone. Indeed, it is knownthat some arthropods with defensive quinones also secrete liquid hydrocarbons, whichmay serve as solvents for quinones that act as irritants. Combinations of a complex

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quinine mixture with non-quinonic compounds influence not only the physicochemicalcharacteristics of the juliform defensive secretions, but also offer a potential for intra-or interspecific communications and may represent a chemotaxonomical marker. Thisis consonant with the finding of Shimizu et al. [8], who reported that some of thenonquinones detected in Anaulaciulus species have been known thus far in otherarthropods as compounds with antimicrobial activity (n-hexyl tridecanoate), asspecies-recognition signals (n-hexyl myristate), or, in combination with othercompounds, as pheromones (n-hexyl laurate, n-hexyl caprate, or n-octyl laurate).Even hexadecyl acetate, which was identified in B. guttulatus, is known as a pheromonein a number of organisms [9]. The discovery of hexyl and octyl oleates in the twoblaniulids (as well as in Diplopoda in general) might also be related to similar biologicalfunctions. For example, it is known that oleic acid has different functions in intraspecificinteractions in many invertebrates and vertebrates [10].

At the species level, the chemical composition of the only previously analyzedblaniulid, B. guttulatus, has been shown to consist of seven major components, i.e., twowith unknown structures, compounds 1 and 3, palmityl acetate, a monounsaturated C16-alcohol acetate, and a monounsaturated C18-alcohol acetate [3]. According to thisfinding [3], we assume that the presence of the two quinones 1 and 3 in the defensivesecretion of B. guttulatus implies that its quinone chemical profile is more similar to thatof C. phlepsii (with a relative content of 1 and 3 of 91.7%), than to that of N. kochii(with a relative content of 1 and 3 of 46.2%; Table). However, the nonquinone chemicalprofile of B. guttulatus differs from those of both of the species analyzed in this study.Furthermore, the presence of octyl oleate at a higher relative content in N. kochii thanin C. phlepsii (Table) might represent a chemotaxonomical marker. Finally, it has beenestablished that hydroquinones are precursors of the corresponding quinones; theirpresence was clearly identified in some julid species, mainly as minor components [7].In C. phlepsii, only one hydroquinone (6) was detected as a minor component (0.8%).However, in N. kochii, hydroquinones were identified at considerable amounts. Indeed,the summed-up content of hydroquinones 5, 7, and 10 was 13.5%, which is comparableonly with the hydroquinone content of the spirobolid Acladocricus setigerus (Silvestri,1897) [7]. Hydroquinones were absent in the previously analyzed B. guttulatus [3].Hence, the high content of hydroquinones chemotaxonomically distinguishes N. kochiifrom the other two blaniulid species.

Conclusions. – Data on the composition of the defensive fluids in the order Julidaare scarce, as less than 4% of the total number of species has been investigated. In thefamily Blaniulidae, the chemical composition has been analyzed in only one species.The GC-FID and GC/MS analyses of the CH2Cl2 extracts of N. kochii and C. phlepsiishowed the presence of 13 compounds, including both quinones and nonquinones. Themajor constituents of the defensive secretion of N. kochii identified were 2-methyl-1,4-benzoquinone (1), 2-methyl-3,4-(methylenedioxy)phenol (8), and 2-methylhydroqui-none (5), while in C. phlepsii, the dominant compounds detected were compound 1 and2-methoxy-3-methyl-1,4-benzoquinone (3). In both species, the nonquinones hexyl andoctyl oleates were identified. The results of this study confirmed that representatives ofthe family Blaniulidae belong to the �quinone� clade of millipedes and that a significantconsistency of quinones exists in juliform millipedes.

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May this study stimulate both chemists and biologists to undertake more detailedresearch into the defensive compounds from a large number of diplopod species, tounderstand the complex functions of these compounds and to propose a phylogenetictree based on chemotaxonomical markers.

This study was supported by the Ministry of Science and Technology of Serbia (grants No. 173038 and172053).

Experimental Part

Collection and Handling of Millipedes. The millipedes were collected during May 2013 in twolocalities in Serbia: Cibiniulus phlepsii in a stump on the bank of the River Danube, near Novi Sad andNopoiulus kochii in the Velika Balanica cave, located in the village Sicevo, near Nis. The millipedes werestored in plastic boxes with a layer of decomposed litter, kept in the laboratory for few days at 108 in thedark and sprayed with H2O every day.

Chemical Extraction. For the collection of defensive secretions, five males, five females, and fivejuveniles (stadium IX) of both species were soaked in 1 ml of CH2Cl2 for 3 min. To avoid the effects ofcomposition-altering oxidation and degradation of compounds, a portion of the extracts was subjected toGC/MS analysis immediately after preparation.

GC-FID and GC/MS Analyses. The GC-FID and GC/MS analyses were performed with an Agilent7890A GC system equipped with a 5975C mass selective detector (MSD), FID, and a HP-5 MS cap.column (30 m�0.25 mm i.d., film thickness 0.25 mm, Agilent Technologies). The oven temp. wasprogrammed isothermal at 408 for 1 min, then rising from 40 to 3158 at 108/min, and finally isothermal at3158 for 6.5 min; injector temp., 2508 ; transfer-line temp., 2808 ; FID temp., 3008 ; ion-source temp., 2308 ;carrier gas, He (1.1 ml/min). Samples (1 ml) were injected using a splitless mode, and the EI mass spectra(70 eV) were acquired over the m/z range 35–550 amu.

Library search and mass spectral deconvolution and extraction were performed using MSDChemStation data-analysis software, ver. E.02.02., integrated with DRS (deconvolution reportedsoftware) and NIST AMDIS (automated mass spectral deconvolution and identification system)software, ver. 2.70. The search was performed with a homemade library, which contains 4,951 spectra, andthe commercially available NIST11 and Willey07 libraries containing more than 500,000 spectra.

The relative contents of the identified compounds were computed from the corresponding GC-FIDpeak areas.

REFERENCES

[1] S. M. Blum, �Chemical Defenses of Arthropods�, Academic Press, New York, 1981; S. S. Duffey,M. S. Blum, H. M. Fales, S. L. Evans, E. W. Roncadori, D. L. Tiemann, Y. Nakagawa, J. Chem. Ecol.1977, 3, 101; T. Eisner, O. D. Alsop, K. Hicks, J. Meinwald, � Handbook of ExperimentalPharmacology: Arthropod Venoms�, Ed. P. Bettini, Springer-Verlag, Heidelberg, Berlin, 1978,Vol. 48; S. E. Makarov, B. P. M. Curcic, V. V. Tesevic, M. B. Jadranin, L. V. Vujisic, S. B. Curcic,B. M. Mandic, T. L. Sekulic, B. M. Mitic, J. Chem. Ecol. 2010, 36, 978; F. W. Wood, P. W. Sorensen,Biochem. Syst. Ecol. 2005, 33, 1077; M. Pavan, �Defensive Secretions of Arthropoda�, EuropeanResearch Office, 1968; S. E. Makarov, B. P. M. Curcic, Lj. V. Vujisic, M. B. Jadranin, V. V. Tesevic,I. M. Vuckovic, T. Lj. Sekulic, S. B. Curcic, B. M. Mitic, Chem. Biodiversity 2011, 8, 1284.

[2] H. Enghoff, Senckenberg. Biol. 1984, 64, 393.[3] J. Weatherston, D. Tyrrell, J. E. Percy, Chem. Phys. Let. 1971, 7, 98.[4] H. Rçper, H. K. Heyns, Z. Naturforsch., C 1977, 32, 61; S. S. Duffey, Ph.D. Thesis, University of

British Columbia, 1974; H. Schildknecht, K. H. Weis, Z. Naturforsch., B 1961, 16, 810; A. Huth,Fragm. Faun. 2000, 43, 191; A. Behal, M. C. Phisalix, C. R. Soc. Biol., Paris 1900, 52, 1036; R. Trave,L. Garantil, M. Pavan, Chim. Ind. (Milan, Italy) 1959, 41, 19; A. F. Kluge, T. Eisner, Ann. Entomol.

CHEMISTRY & BIODIVERSITY – Vol. 11 (2014) 489

Soc. Am. 1971, 64, 314; L. M. Bedoussac, M. E. Favila, R. M. Lopez, Chemoecology 2007, 17, 163; A.Monro, M. Chadha, J. Meinwald, T. Eisner, Ann. Entomol. Soc. Am. 1962, 55, 261; A. B. Attygalle,S.-C. Yu, J. Meinwald, T. Eisner, J. Nat. Prod. 1993, 56, 1700; W. F. Wood, J. Shepherd, B. Chong, J.Meinwald, Nature 1975, 253, 625; J. E. Percy, J. Weatherston, Can. J. Zool. 1971, 49, 278; T. Schmitt,F.-T. Krell, K. E. Linsenmair, J. Chem. Ecol. 2004, 30, 731; M. De Bernardi, G. Mellerio, G. Vidari, P.Vita-Finzi, J.-M. Demange, M. Pavan, Naturwissenschaften 1982, 69, 601; M. Barbier, E. Ledeber,Biokhimiia 1957, 22, 236; Y. Kuwahara, S. Noguchi, N. Mori, Y. Higaa, Jpn. J. Environ. Entomol.Zool. 2002, 13, 117; Y. Kuwahara, H. Omura, T. Tanabe, Naturwissenschaften 2002, 89, 308; J. M.Wheeler, J. Meinwald, J. J. Hurst, T. Eisner, Science 1964, 144, 540; A. G. Arab, G. Zacarin, C. S.Fontanetti, M. I. Camargo-Mathias, M. G. Dos Santos, A. C. Cabrera, Entomotropica 2003, 18, 79; J.Smolanoff, J.-M. Demange, J. Meinwald, T. Eisner, Psyche 1975, 82, 78; R. Deml, A. Huth,Naturwissenschaften 2000, 87, 80; L. A. D. Williams, P. D. A. Singh, L. S. Caleb-Williams,Naturwissenschaften 1977, 84, 143; P. Weldon, J. Aldrich, J. Klun, J. Oliver, M. Debboun,Naturwissenschaften 2003, 90, 301.

[5] L. V. Vujisic, S. E. Makarov, B. P. M. Curcic, B. S. Ilic, V. V. Tesevic, D. M. Go�evac, I. M. Vuckovic,S. B. Curcic, B. Mitic, J. Chem. Ecol. 2011, 37, 1358.

[6] M. Bodner, G. Raspotnig, J. Chem. Ecol. 2012, 38, 547.[7] X. Wu, D. W. Buden, A. Attygalle, Chemoecology 2007, 17, 131.[8] N. Shimizu, Y. Kuwahara, R. Yakumaru, T. Tanabe, J. Chem. Ecol. 2012, 38, 23.[9] Y. H. Xiao, J. X. Zhang, S. Q. Li, Proc. Biol. Sci. 2010, 277, 3009; B. D. Jackson, E. D. Morgan, J. P. J.

Billen, Naturwissenschaften 1990, 77, 187; A. Bertsch, H. Schweer, A. Titze, Z. Naturforsch., C 2004,59, 701; S. Garc�a-Rubio, A. B. Attygalle, P. J. Weldon, J. Meinwald, J. Chem. Ecol. 2002, 28, 769.

[10] A. M. El-Sayed, The Pherobase, 2012 (http://www.pherobase.com).

Received November 13, 2013

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