Jpn. J. Environ. Entomol. Zool. 28（2）：63－69（2017） 環動昆 第 28 巻 第 2 号：63－69（2017） Original Article

Defensive allomone components of the Cryptocorypha kumamotensis (Murakami, 1966) [: Pyrgodesmidae] and two related Cryptocorypha spp.

Yasumasa Kuwahara1 2), Tsutomu Tanabe3) and Yasuhisa Asano1 2)

1) Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan 2) Asano Active Enzyme Molecule Project, JST, ERATO, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan 3) Faculty of Education, Kumamoto University, Kumamoto, Kumamoto 860-8555, Japan

（Received: March 3, 2017；Accepted: April 5, 2017）

Abstract Benzaldehyde was identified as the major component (up to 92.6%) of the defense allomones of three Cryptocorypha spp.; Cryptocorypha kumamotensis (Murakami, 1966) and two related Cryptocorypha sp. 1 and sp. 2. It was the major allomone component in both sexes and in individuals collected from different environments. The other components were mandelonitrile and four related compounds (<7.4%). Polydesmida, known as cyanogenic , produce allomones via the actions of two enzymes; hydroxynitrile lyase and mandelonitrile oxidase, with mandelonitrile as the common substrate. Type A Polydesmida contain only hydroxynitrile lyase and produce benzaldehyde as the major allomone component, while type B Polydesmida have both enzymes and produce benzaldehyde and benzoyl cyanide. Non-cyanogenic Polydesmida are classified as type C. In C. kumamotensis, the next most abundant allomone component was mandelonitrile followed by benzyl alcohol. Likewise, the next most abundant component after benzaldehyde was benzoyl cyanide in Cryptocorypha sp. 1, and benzyl alcohol in Cryptocorypha sp. 2. The differences in allomone composition among the three species were too small to be used for conclusive species identification. In addition, no quantitative difference in allomone composition between sexes was detected. The ratio of benzoyl cyanide (2.11%, maximum among Cryptocorypha spp.) to benzaldehyde (92.63%, minimum) was 0.023, well below that of type B species (0.16–1.37). Therefore, these three Cryptocorypha spp. were classified as type A Polydesmida.

Keywords: defensive allomone, Cryptocorypha sp., Cryptocorypha kumamotensis mandelonitrile, polydesmid compounds

Introduction oxidized to 2 or 4. Compound 5 is also consumed as the substrate of manderonitrile oxidase [MOX, putatively Defensive allomones in most polydesmid millipedes (53 out demonstrated as the post-secretion enzyme in millipedes by of 58 species reported worldwide) are mainly composed of the Kuwahara et al. (2011) and characterized as a flavin enzyme following six compounds (Makarov 2015; Shear 2015); by Ishida et al. (2016)]. Manderonitrile oxidase is responsible benzaldehyde (1), benzyl alcohol (2), benzoyl cyanide (3), for not only the production of 3 but also the subsequent benzoic acid (4), mandelonitrile (5) and mandelonitrile generation of 6 and HCN via the Schotten–Bauman reaction benzoate (6). Compound 5 is derived from L-phenylalanine between 3 and 5 (Kuwahara et al., 2011), together with (Duffey et al., 1974), and is decomposed by hydroxynitrile hydrolytic generation of 4 and HCN from 3. lyase [HNL, demonstrated by Duffey and Towers (1978); Polydesmida millipedes can be classified into three groups characterized by Dadashipour et al. (2015)] to a mixture of on the basis of their defensive allomones (Kuwahara et al. hydrogen cyanide (HCN) and 1. Then, 1 is further reduced or 2011): type A, B, and C. Type A millipedes are those

Corresponding author: [email protected]

- 63 - Kuwahara et al. containing HNL and whose allomone is a mixture of 1 and 5, or only 1 (due to thermal decomposition of 5 during gas chromatograph-mass spectrometry (GC/MS) analysis, as mentioned later). Type B millipedes are those containing both HNL and MOX so that they produce at least 1 and 3, with 2, 4, 5, and 6 as minor constituents. Among the 58 cyanogenic Polydesmida worldwide, 18 are thought to be type A, and the 30 species that contain 6 (down-flow component, more stable than 3) are thought to be type B. Five species in the Polydesmida are non-cyanogenic (Makarov 2015; Shear 2015), and are designated as type C (sensu, Kuwahara et al., 2011). Type B millipedes have recently been found to emit hydrogen peroxide as a component of their defensive allomones (Kuwahara et al., 2017a). It has been noted that allomone compositions are variable, and change during ontogenetic development, as demonstrated in Niponia nodulosa and haplodesmid millipedes (Kuwahara et Fig. 1 Adult Cryptocorypha sp. 1, mating pair (A), non-feeding stadium I nymphs (B) and Cryptocorypha al., 2015; 2017b). Therefore, it is important to investigate the kumamotensis (C, upper; stadium VII nymph, and C, lower; allomone composition of millipedes during their life-cycle, adult). especially in the post-embryonic developmental stadia. Among the 13 Cryptocorypha species described so far, two HNL through simply calculating their reaction products as the are found in Japan; Cryptocorypha kumamotensis and 3/1 ratio. Cryptocorypha japonica (Miyoshi, 1957) (Golovatch and VandenSpiegel, 2015). In the present study, C. kumamotensis, Materials and methods and two unidentified Cryptocorypha sp. 1 (collected in Toyoma) and Cryptocorypha sp. 2 (collected in Kyoto) were Collection and maintenance of Cryptocorypha millipedes examined. The partial allomone composition in adult Adult C. kumamotensis were obtained from litter at Cryptocorypha sp. 2 has been reported previously (Kuwahara 32.8234°N, 130.7377°E (8-Cho-me, Kurokami Chuo-ku et al., 2011), but there have been no previous studies on the Kumamoto, Kumamoto) using a Tullgren funnel, on 24th–26th allomone composition of C. kumamotensis and Cryptocorypha May 2015. Adults and stadium I nymphs of Cryptocorypha sp. sp. 1. The partially examined species (Cryptocorypha sp. 2) is 1 (Fig. 1) were obtained by searching through litter under a a typical HNL species (type A) that does not produce 3, and binocular microscope (Shimadzu Co. Ltd., STZ-168). The litter produces 6 only by shake-disturbance with exogenously added was collected from Yakusyouji-ike Park (36.7000°N, 3 (Kuwahara et al., 2011). 137.0843°E), Imizu Toyama in April 2015, and These Cryptocorypha millipedes have a similar shape to that Ranzyo-no-mori (36.6362°N, 137.0444°E, # in Tables 2 and of Niponia nodulosa, but are smaller (about 5–6 mm in body 3), Tonami Toyama, in April 2015 and May 2016. The length). They are some of the most commonly distributed and millipedes were subsequently maintained in the laboratory for easily collected millipedes from litter under shrubbery 1–4 weeks before gas chromatrography – mass spectrometry surrounding playgrounds in schools, household backyards, and (GC/MS) analyses. The millipedes were kept in plastic Petri parks. The body of C. kumamotensis is reddish brown, while dishes (9.5 cm i.d. x 2.3 cm in height) containing litter from the bodies of the two other Cryptocorypha spp. are yellowish the collection sites on top of moistened filter paper under a brown. The nymphs are easily recognizable in soils because of natural photoperiod with high humidity (ca. 100% RH) at 20C. their creamy pink color and characteristic shape. Similarly, Cryptocorypha sp. 2 as a non-3 producer (Kuwahara In this study, we compared the chemical composition of their et al., 2011) was obtained from the Shimogamo Shrine allomones to define species-specific features among these three (35.0311°N, 135.7715°E) in Kyoto from June to September Cryptocorypha spp., and to determine which type the species 2012 and was reared as described above. The age (growth belong to by evaluating the relative activities of MOX and stage) of each nymph was recognizable by two criteria, 1)

- 64 - Defensive allomones of three pyrgodesmid millipedes number of body segments, and 2) number of leg-pairs Table 1. (Shinohara, 1999). GC-profile of C. kumamotensis Analytical methods As shown in Fig. 2, extracts from male and female showed The GC/MS analyses were conducted as reported previously the same GC profiles; 1 was the most abundant allomone (Kuwahara et al., 2011), using an Agilent 5975C Inert XL component (>92.63% in females and 92.63% in males) and EI/CI MSD instrument with a triple-axis detector at 70 eV. there were three other minor components (2, 3, and 5). There This was coupled to the 7890A GC system equipped with an were no differences in allomone composition between males DB-5MS capillary column (30 m × 0.25 mm; 0.25 µm film (n=2) and females (n=2). The second most abundant thickness, Agilent J & W) operated in the split-less mode at component was 5 (5.7%–7.0%), followed by 2 (<0.4%) and 60C for 2 min, then programmed to increase at 10C /min to then 3 (<0.1%). A stadium VII nymph (n=1) showed a similar 290C, and maintained at that temperature for 5 min. Helium profile, with 1 as the major component (94.12%) and 5 as the was used as the carrier gas at a flow rate of 1.00 ml/min. The second most abundant component (5.67%). As listed in Table GC and GC-MS data were processed using ChemStation 2, all analyses (n=5) showed the same pattern of compound (Agilent Technologies Inc.) with reference to an MS database abundance; 1 > 5 > 2 > 3. Compounds 4 and 6 were not (Wiley 9th/NIST 2011 MS Library; Hewlett Packard Co.). detected. Retention indices (Kováts, 1958) were calculated for these GC conditions as described by Bodner and Raspotnig (2012).

Preparation of hexane extracts After measuring the weight and body length and checking the sex (for adults), each live millipede was immersed in 5–50 µl (as indicated in Table 2) hexane for 3 min for extraction, using a conical bottomed micro glass insert (28.96 mm long, 5.73 mm diameter). An aliquot of the hexane extract (1–5 µl) was immediately subjected to GC/MS analysis. In the case of the first stadium nymph, a group of three individuals was extracted with hexane (5 µl) and the whole 5 µl was used for the GC/MS analysis. Fig. 2 Typical gas-liquid chromatograms of hexane extracts from Cryptocorypha kumamotensis. A: stadium VII nymph, B: female, and C: male. Chemicals *; Phthalate esters and unidentified impurities. For GC/MS identification of allomone components, the following chemicals were obtained from Tokyo Chemical GC-profile of Cryptocorypha sp. 1 Industry or Wako Pure Chemical Industry as authentic The same allomone components were detected in males and compounds; 1, 2, 3, 4 and 5. Compound 6 was prepared from 3 females (Fig. 3). Compound 1 was the most abundant, and 5 as described by Kuwahara et al (2011). followed by 3 (0.6%–2.1%). In adults, the third most abundant

component was 2 (0.1%–0.2%), whereas that in non-feeding Results stadium I nymphs was 5 (1.2%), followed by 2 and 3 (0.5%

and 0.1%, respectively). In adults, 3 was more abundant than 2 Identification of compounds detected in Cryptocorypha (Table 2). Compounds 4 and 6 were sporadically distributed. millipedes The components other than 4 and 6 were ranked, from most to All peaks (=compounds) [1(tR, 5.864 min), 2 (tR, 6.933 min), least abundant, as follows: 1 > 3 > 2 > 5 in adults, and 1 > 5 > , 7.809 min), 4 (t , 8.832 min), 5 (t , 11.208 min) and 6 (t , 3 (tR R R R 2 > 3 in stadium I nymphs. 18.531min)] detected in hexane extracts from the three

Cryptocorypha millipedes were identified by GC/MS by GC-profile of Cryptocorypha sp. 2 comparison with those of purchased or synthesized authentic In the male profile (Fig. 4), compound 5 was the second compounds (Table 1). The retention indices are listed in

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most abundant component (3.48%); therefore, the components were ranked from most to least abundant as follows: 1 > 5 > 2

> 3. In other life stages, including nymph stadia, the order was

1 > 2 > 3 > 5, with 5 at 0.04%–0.01