Jpn. J. Environ. Entomol. Zool. 30（２）：39－49（2019） 環動昆 第 30 巻 第２号：39－49（2019） Original Article

Defensive secretion of gracilis (C.L. Koch) (: ): Juvenile-adult and juvenile-juvenile polymorphism, sexual maturation, and sexual dimorphism

Yasumasa Kuwahara1) 2) 3), Yayoi Ichiki1) 2), Masashi Morita1) 2), Tsutomu Tanabe4), Ryu Nakata5), Naoki Mori5) and Yasuhisa Asano2) 3)*

1) Asano Active Enzyme Molecule Project, JST, ERATO, Kyoto Brunch, Kyoto 602-0841, Japan 2) Biotechnology Research Center and Department of Biotechnology, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan 3) Asano Active Enzyme Molecule Project, JST, ERATO, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan 4) Faculty of Advanced Science and Technology, Kumamoto University, Kumamoto 860-8555, Japan 5) Graduate School of Agriculture, Kyoto University, Kyoto 606-1111, Japan

（Received：October 17, 2018；Accepted: April: 24,2019）

Abstract Defensive allomone composition of the greenhouse Oxidus gracilis (C.L. Koch) was observed to shift depending on ontogenetic development, from six component mixtures (mandelonitrile, benzaldehyde, benzoyl cyanide, benzoic acid, benzyl alcohol, and mandelonitrile benzoate) in juvenile stadia to three (mandelonitrile, benzaldehyde, and benzyl alcohol) in adults, in addition to six phenolic compounds (phenol, p-cresol, 2-methoxyphenol, 2-methoxy-5-methylphenol, 2-methoxy-4-methylphenol, and 2,6-dimethoxyphenol) and n-decanal. In the early half of the juvenile life cycle (stadium I to III or IV), benzoyl cyanide, benzoic acid, and mandelonitrile increased progressively, while benzaldehyde decreased stepwise. On the other hand, in the later stages (stadium IV or V to VII), most of components increased or decreased to reach a plateau, except mandelonitrile that showed an increasing trend. Phenol was a major component among six phenolic compounds that accumulated mostly in adults, and exhibited significant distribution bias toward females in the mating season. No sexual behavior, however, was observed upon exposure to isolated groups of males. On the other hand, unusual accumulation of four phenolic compounds (p-cresol, 2-methoxyphenol, 2-methoxy-5-methylphenol, and 2-methoxy-4-methylphenol) was noted in both sexes during mating season. All the observations were verified by statistical discrimination using non-metric multidimensional scaling of Bray-Curtis dissimilarity matrixes as juvenile-adult polymorphism, juvenile-juvenile polymorphism, sexual maturation, and sexual dimorphism.

Keywords: : defense allomone of Oxidus gracilis, juvenile-adult polymorphism, juvenile-juvenile polymorphism, NMDS using Bray-Curtis dissimilarity matrixes, sexual dimorphism, sexual maturation,

Introduction univoltine. Adult emerge in April and mating pairs can be The Oxidus gracilis (C.L. Koch) observed in September. Duffey et al. (1977) examined (Polydesmida: Paradoxosomatidae), considered native to Japan, allomone composition and identified a mixture of is now distributed worldwide, occurring in the tropics, benzaldehyde (1), phenol (2), guaiacol (2-methoxyphenol) (5), temperate North and South America, and continent wide in benzoic acid (7), mandelonitrile benzoate (13), and ethyl Europe (Blower, 1985; Hoffman, 1999). The species is benzoate, among which 2, 5 and ethyl benzoate have been

*Correspondig author:[email protected]

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Materials and Methods

Species All stadia of (originated from Toyama and Kyoto populations), as mentioned later and listed in Table 1, were prepared using field collected adults and juveniles that were subsequently reared in an incubator (20°C, 100% RH). None of chemical differences between two populations were observed by preliminary GC-MS analyses. Toyama population including mated pairs (obtained as mentioned in Acknowledgments) from Toyama Prefectural University, Toyama, Japan (N36.707°, E137.096°), was maintained in a semi-transparent plastic container Fig. 1 Typical gas liquid chromatography profiles of hexane (polyethylene) with soil (ca 1 cm in depth) and litter from the extracts obtained from Oxidus gracilis. G (upper): M habitat. Parts of mated pairs were sampled and analyzed, as virgin male adult (a week after emergence) and HM (lower): male juvenile at stadium VII. 1: benzaldehyde, mentioned later. Fully developed egg masses, laid by gravid 2: phenol, 3: benzyl alcohol, 4: p-cresol, 5: females into the soil spaces (not a construct so-called 2-methoxyphenol, 6: benzoyl cyanide, 7: benzoic acid, “egg-chamber”), were harvested from the container and 8: 2-methoxy-5-methylphenol, 9: 2-methoxy-4-methylphenol, 10: n-decanal, 11: transferred to a plastic Petri dish (9 cm in diameter) with soil mandelonitrile, 12: 2,6-di-methoxyphenol, 13: and litter. Transferring the egg masses too early caused mold mandelonitrile benzoate. infection, which often disturbed emergence of juveniles. *, Unidentified impurities. Newly emerged juveniles (stadium I) assembled into groups

under casted eggshells by curling their bodies with the head at demonstrated to suppress spore germination (Roncadori et al., the center, and dropped into dormancy within 24 h neither 1985). Taira et al. (2003) also identified a mixture of 1, 2, foraging nor constructing protective devices such as “chamber p-cresol (4), creosol (2-methoxy-4-methylphenol) (9), for ecdysis.” They developed into stadium II after 48 - 72 h of mandelonitrile (11), and benzaldehyde dimethyl acetal as dormancy. Stadia I - III were segregated based on the allomones of the species. Between these two research results, following five conditions (A-, A+, B-, B and C) for GC-MS only two compounds 1 and 2 are common. analyses: (A-) stadium I juveniles just after emergence; (A+) We detected benzoyl cyanide (6) and 13 in the juvenile dormant stadium I juveniles; (B-) non-feeding stadium II stadium (Fig.1-GM) of O. gracilis by gas hromatography-mass juveniles, just after ecdysis; (B) feeding stadium II juveniles; spectrometry (GC-MS) analyses, and this juvenile was and (C) feeding stadium III juveniles. All the other feeding considered as an H2O2 emitter (sensu Kuwahara et al., 2017a). juveniles, stadia IV (D), V (E) and VI (F), were collected in The actual H2O2 emission from adult has, however, not been winter from culture soils of plants that were brought into the demonstrated (Kuwahara et al., 2017a), suggesting the greenhouse to avoid snowfall in Toyama Prefectural possibility of juvenile-adult polymorphism. Therefore, University. allomone compositions of the species were re-investigated Stadium VI juveniles (Kyoto population) were collected in across the ontogenetic development from juveniles (stadium I) March near Shimogamo Shrine, Kyoto Japan (N35.031°, to adults, to evaluate and confirm differences of composition E135.771°) and were raised to stadium VII juveniles (G : with growth. M feeding male juvenile; G : feeding female juvenile) and adults By statistical discrimination using non-metric F (H : virgin male adult; H : virgin female adult) by feeding multidimensional scaling (NMDS) of Bray-Curtis dissimilarity M F litters in an incubator (at 20°C, 100% RH). Both virgin adults matrixes, allomone profiles of all stadia and each condition (H and H ) were subjected to GC-MS analysis a week after demonstrated a different cluster. The results verified four M F their emergence (April 23 - 25). biological phenomena, including the one anticipated Allomone compositions of mated pairs (Toyama population) preliminarily.

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Table 1 Summary of sampled millipedes; stadia, biological conditions, extraction for GC-MS, growth rates, and origins Ext. / Inject. (μ Repeti- Body weight mean Stadium, condition Sampling date Growth rate Origin l, hexane) tion ± SD (mg) A- ; I, just emerged* 5 / 5 Sep. 16 n = 4 nd nd Toyama, ra A+ ; I, dormant* ibid. Sep. 17 n = 5 nd nd ibid B- ; II, non-feeding ibid. Sep. 27 n = 5 nd nd ibid B ; II, feeding 5 / 4 Sep. 22 n = 5 nd nd ibid

Asexual juvenile Asexual C ; III, ibid 10 / 4 Sep. 24 n = 5 nd nd ibid

D ; IV, ibid 30 / 4 Jan. 28 n = 5 nd nd Toyama, field colle E ; V, ibid 50 / 1 Jan. 28 n = 5 2.4 ± 0.5 nd ibid F ; VI, ibid 100 / 1 Jan. 28 n = 5 6.1 ± 0.6 F/E 2.54 ibid GM; VII, male, feeding 200 / 1 Apr. 15 n = 5 15.1 ± 1.3 GM/F 2.48 Kyoto, rais Sexual juvenile Sexual GF; VII,female, ibid ibid. Apr. 15 n = 5 17.8 ± 0.5 GF/F 2.92 ibid HM; Male, virgin, feeding 400 / 1 Apr. 23 - 25 n = 5 29.2 ± 5.1 HM/GM 1.93 ibid H ; Female, virgin, ibid ibid. Apr. 23 - 25 n = 5 42.5 ± 3.1 H /G 2.39 ibid F F F Adult IM ; M ale, mated, ibid ibid. Sep. 3 - 4 n = 10 50.8 ± 16.8** IM/HM 1.74 Toyama, field colle

IF ; Female, mated, ibid ibid. Sep. 3 - 4 n = 10 46.6 ± 6.1# IF/HF 1.10 ibid nd: Not determined, nor available, *: Three individuals were extracted at a time, **: Calculated by 3 repetitions, #: Calculated by four repetitions, ##: Derived from field collected juveniles at stadium VI were examined, after checking the sex (mentioned later) and removed using a 10 l microsyringe and were subjected to forced separation. Both individuals were separately extracted GC-MS analysis. Data are expressed as mean ± SD (n = 4, 5, with hexane and subjected to GC-MS analyses (mentioned or 10). later) within 35 - 40 min interval, to minimize effects after separated. Mated pairs separated naturally before analyses Chemical analysis were excluded for examination. Results were summarized as IM GC-MS spectra were obtained as reported by Kuwahara et al.

(mated male, feeding) and IF (mated female, feeding). (2011) using Agilent 5975C Inert XL EI/CI MSD with a The stadium of each juvenile was determined using two triple-Axis Detector at 70 eV, coupled to a 7890A GC system criteria, 1) the number of body segments and 2) number of leg equipped with a DB-5MS column (30 m × 0.25 mm; film pairs (Shinohara, 1999; Kuwahara et al., 2015), by observing thickness, 0.25 m; Agilent J & W) operated in split-less mode under a binocular microscope (Shimadzu Co. Ltd., STZ-168). at 60C for 2 min, then programmed to increase at 10C/min The second criterion is also useful in determining sexes from up to 290C, which was maintained for 5 min. Helium was juveniles at stadium IV to adults. Males possess a leg-pair less used as the carrier gas at a flow rate of 1.00 ml/min. GC and than that of females corresponding to the same stadium GC-MS data were processed using ChemStation (Agilent (Shinohara, 1999). The missing pair of legs develops gradually Technologies Inc.) with reference to an MS database (Wiley from a pair of mounds to male genital organs in adult. There 9th/NIST 2011 MS Library; Hewlett Packard Co.). Retention are no observable morphological differences between sexes in indices (Kováts, 1958; Van Den Dool and Kratz, 1963) were juveniles from stadium I to III, and therefore, these juveniles calculated under the same GC conditions used this time, as are indicated as asexual. described by Bodner and Raspotnig (2012). GC profiles were provided as reconstructed total ion chromatograms (RIC). In Preparation of the hexane extracts case of the non-resolved peak (5 and 6), relative amounts were Only live millipedes were used for allomone extraction. calculated by selected ion chromatogram (SIC) using each base - After weighing (E - IF), determining stadia and conditions (A - ion (m/z 109 for 5 and m/z 105 for 6, respectively) as a

IF), and checking their sexes (GM - IF), each individual monitor. millipede was dipped in the indicated volume (5 - 400 l) of hexane (Table 1) for 3 min using conical-bottomed micro Chemicals glass inserts (28.96 mm in length, 5.73 mm, for A- - E) or The following chemicals (see Fig. 2) and solvents were glass sample tubes (1 ml, screw-cap vial, for F - IF). Extracts obtained commercially and used as described in the text: three of A- and A+ were prepared using three juveniles as a group, as compounds 1, 2 and 4 from Wako Pure Chemical Industries, an exception. The hexane extracts (1 l for E - IF, 4 l for B - Ltd., Osaka, Japan, six compounds 6 and 8 -12 from Tokyo D, or whole volume up to a 5 l aliquot for A- - B-) were Chemical Industry Co., Ltd., Tokyo, Japan, and three

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subjected to statistical comparison. Vegan package (v2.5-2, Oksanen et al., 2018) in R (v3.5.0, R Core Team, 2018) of RStudio (v1.1.453, RStudio, 2018) was used to perform ordinations on the dataset and subsets via non-metric multidimensional scaling (NMDS) based on a Bray-Curtis dissimilarity matrix (Bray and Curtis, 1957).

Results Fig. 2 Chemical structures of defensive allomones detected in

the millipede Oxidus gracilis. 1: benzaldehyde, 2: phenol, 3: benzyl alcohol, 4: p-cresol, 5: Identification of compounds in hexane extracts from the 2-methoxyphenol, 6: benzoyl cyanide, 7: benzoic acid, millipede 8: 2-methoxy-5-methylphenol, 9: GC profiles of Fig. 1 were composed of 13 peaks 2-methoxy-4-methylphenol, 10: n-decanal, 11: mandelonitrile, 12: 2,6-di-methoxyphenol, 13: (compounds with relative intensity more than 0.1%). Peak 1

mandelonitrile benzoate. (GC-tR 5.54 min), 2 (5.76), 3 (6.63), 4 (7.23), 5 (7.51), 6 (7.52), 7 (8.59), 9 (9.08), 10 (9.24) and 11(10.85) were

Table 2 Gas chromatographic and mass spectral data of compounds from extracts of Oxidus gracilius

Compound identified Peak Retention Mass spectrometric fragmentation (m/z) Standard obtained No. index (RI)* as (Library ID)

1 951 106(M+,100), 105(96), 77(90), 51(32) Benzaldehyde com. (97%)

2 970 94(M+, 100), 66(30) Phenol com. (91%)

3 1041 108(M+, 94), 79(100), 77(63), 51(20) Benzyl alcohol com. (97%)

4 1090 108(M+, 81), 107(100), 90(7), 79(20), 77(22) p -Cresol com. (96%)

5 1113 109(100), 124(M+, 94), 81(44), 65(5), 53(15) 2-Methoxyphenol com. (93%)

6 1114 131(M+,72), 105(100), 77(55), 74(8), 51(23) Benzoyl cyanide com. (94%)

7 1202 122(M+, 85), 105(100), 77(72), 51(35) Benzoic acid com. (96%)

8 1234 138(M+, 91), 123(100), 95(26), 77(12), 67(１4), 55(12), 51(7) 2-Methoxy-5-methylphenol **94%

9 1242 138(M+, 100), 123(93), 95(31), 77(17), 67(１9), 55(14), 51(9) 2-Methoxy-4-methylphenol com. (95%)

10 1256 156(M+, 0), 128(8), 112(39), 82(59), 70(64), 57(100), 43(88) n -Decanal com. (91%)

11 1388 133(M+, 70), 115(39), 105(100), 77(95), 51(45) Mandelonitrile# com. (91%)

12 1411 154(M+, 100), 139(93), 111(49), 96(11), 79(8), 69(7), 53(7) 2,６-Dimethoxyphenol **91% 13 1985 237(M+, 17), 116(39), 105(100), 89(11), 77(25), 51(11) Mandelonitrile benzoate# prepd. (80%)

*: Retention index (RI); calculated by HP-5 column under conditions described in text, as reported (Kováts, 1958; Van Den Dool and Kratz 1963).

**: Library ID supported the compound, not available commercially. #; Absolute structure was determined as “R” by HPLC with chiral column.

compounds 3, 5 and 7 from Nacalai Tesque Inc., Kyoto, Japan. identified as 1 - 7 and 9 -11, respectively (Fig. 2 and Table Compound 13 was prepared as indicated in Kuwahara et al. 2), by comparing the obtained mass spectra and GC-tRs with (2011). Hexane (Wako Pure Chemical Industries, Ltd.) was those of standard compounds available commercially. Peak 13 used without purification. (18.11 min) was identified as the synthesized component 13. Peaks 8 (8.98 min) and 12 (11.13 min) were likewise Statistics elucidated using MS database as 8 and 12. The compounds Relative abundance data (profile) of allomone components were classified into the following three groups: 1) polydesmid - from all extracts (A - IF) using single individual, except cases compounds (hereafter abbreviated as PDs) 1, 3, 6, 7, 11 and - + of A and A (three individuals were used as a group), were 13, 2) phenolic compounds (hereafter abbreviated as

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polymorphism. Its mechanism can be explained by on-off function of mandelonitrile oxidase (MOX)(Kuwahara et al., 2011; Ishida et al., 2016) in addition to hydroxynitrile lyase (HNL)(Duffey and Towers, 1978; Dadashipour et al., 2015), - that is, in juvenile stadia (A - GF) allomone was produced by

both enzymes and in adults (HM - IF) only by HNL, as mentioned later.

Detection of n-decanal (10) in A- The extracts of A- were exceptionally composed of three components (6, 60.3%; 10, 5.2%; and 1, 34.5%), and each of these showed the maximum content (6 and 10) or the minimum (1), when compared among all stadia and conditions. Compound 10 (a new and bioactive compound among - millipedes, mentioned later) decreased from 5.2% in A to Fig. 3 Percent composition of Oxidus gracilis defensive + allomones at all growth stadia, sexes, and conditions. 1.2% in A and then to a trace, but remained detectable in all Compounds in each column are arranged in numerical other stadia. Occurrence of 10 might be one of order from the bottom (1) to the top (13). 1: chemotaxonomic marker feasible for identifying the species benzaldehyde, 2: phenol, 3: benzyl alcohol, 4: p-cresol, 5: 2-methoxyphenol, 6: benzoyl cyanide, 7: benzoic during all juvenile stadia (mentioned later). acid, 8: 2-methoxy-5-methylphenol, 9: 2-methoxy-4-methylphenol, 10: n-decanal, 11: Juvenile–juvenile polymorphism on PD composition mandelonitrile, 12: 2,6-di-methoxyphenol, 13: - mandelonitrile benzoate. Juvenile stadia (A - GF) seemed to be further separable into two phases chemically (Fig. 3); early half of juvenile life cycle + phenolics) 2, 4, 5, 8, 9 and 12, and 3) miscellaneous (10). {A - C (or D)} and later half of juvenile life cycle {D (or C) - - As a result, both GC profiles (Fig. 1) were not the same. GM & GF}, except in the case of A as mentioned above. In the

Compounds 6, 7 and 13 detectable in the juvenile (GM) were early half of juvenile life cycle, the content of 1 decreased + lost or negligibly small in the adult (HM). from A (71.6%) to C (35.0%) steadily, while the other four + PDs 6, 7, 11 and 13 increased from A (24.8%) to C (47.1%); + - + Juvenile-adult polymorphism A (1.7%) to D (18.9%); B (0.7%) to C (5.2%); and A (0.7%) - In order to confirm differences between juveniles (A - GF) to B (1.1%) or C (1.0%), respectively. The highest content of 3 and adults (HM - IF), their compositions were investigated (Fig. was observed in B (1.5%) and C (1.4%), as mentioned above. + 3, Table 3). Juveniles of all stadia older than B- were As a whole, early half of juvenile cycle {A - C (or D)} were consistently composed of six PDs (1, 3, 6, 7, 11 and 13), characterized as a combination of decreases (1 and 3) and - - except GF. A was composed of only two PDs (1 and 6) and B increases (6, 7, 11 and 13). of five PDs (1, 6, 7, 11 and 13), presumably due to immaturity. In the later half of juvenile life cycle, content of 1 increased

Compound 3 was detected in B (1.5%), and then the content from C (35.0%) to reach a plateau during E - GF (66.1 - declined to a trace via D (0.1%) - F (0.2%), except 59.0%). The content of 6 decreased gradually from C (47.1%) non-detectable of GF (Table 3). to GM & GF (19.8% & 18.7%) and that of 7 dropped sharply

Adults (HM - IF) contained only two PDs (1 and 11) as the from D (18.9%) to E - GF (3.2 - 0.9%). The content of 11 major and consistent components, together with sporadic and increased up to GM & GF (10.5% & 12.4%) after remaining trace distribution of 3, 6 and 13. Presence of 6 and 13 in HM & almost unchanged during C - E (5.2 - 3.8%). Compound 13

HF {0.4% & 0.6%, and t (= trace) & t, “&” used hereafter to was constant during D - GM & GF (0.1%). connect a set of male and female at the same condition} were Juvenile stadia are morphologically separable into two - concluded as remnants of the preceding stadia (GM & GF), phases; asexual stadia (A - C) and sexual stadia (D - GM & because adults (HM & HF) were used a week after molt, and GF) (Table 3). Sexual stadia correspond to the periods when these PDs were completely lost in the next stage (IM & IF). secondary male characters become recognizable (Shinohara, Therefore, the phenomenon was concluded as juvenile-adult 1999). The present chemical shift, therefore, largely

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Table 3 Percent composition (mean± SD) of millipede defensive secretion along with ontogenetic development.

Polydesmid compounds (PD) [tR]

Mandelo- Benzalde- Benzoyl Mandelo- Benzyl- Benzoic acid nitrile hyde cyanide nitrile alcohol (3) (7) benzoate (1) (6) (11) (13) Rep. Stadium, condition No. [5.54min] [6.63] [7.52] [8.59] [10.85] [18.11]

A- ; I, just emerged* 4 34.5±12.4 nd 60.3±12.3 nd nd nd A+ ; I, dormant* 5 71.6±15.8 nd 24.8±13.2 1.7±1.9 nd 0.7±0.6 B- ; II, non-feeding 5 64.7±7.0 nd 28.3±7.5 5.2±5.0 0.7±0.4 0.8±0.8 B ; II, feeding 5 51.9±6.9 1.5±0.3 36.9±5.5 6.1±1.7 2.2±1.3 1.1±0.9

Asexual juvenile C ; III, ibid 5 35.0±11.7 1.4±0.2 47.1±12.8 9.8±2.9 5.2±3.0 1.0±0.5 D ; IV, ibid 5 47.1±3.2 0.1±0.1 27.5±1.5 18.9±4.6 5.1±2.7 0.1±0.1 E ; ibid 5 66.1±4.3 0.1±0.0 23.8±3.5 1.4±0.8 3.8±0.7 0.1±0.1

F ; VI, ibid 5 64.7±4.9 0.2±0.2 21.4±3.9 3.2±0.8 4.3±0.8 0.1±0.0

GM; VII, male, feeding 5 59.0±7.2 t 19.8±6.8 2.5±1.5 10.8±3.9 0.1±0.1

Sexual juvenile GF; VII,female, ibid 5 60.3±3.6 nd 18.7±4.5 0.9±0.6 12.4±2.9 0.1±0.1

HM; Male, virgin, feeding 5 77.8±2.9 t 0.4±0.2 nd 15.1±4.1 t

HF; Female, virgin, ibid 5 78.7±6.4 t 0.6±0.8 nd 10.9±4.3 t

Adult IM ; M ale, mated, ibid 10 76.8±7.2 t nd nd 4.3±1.6 nd

IF ; Female, mated, ibid 10 70.2±9.4 t nd nd 3.5±1.2 nd Phenolic compounds (Phenolic) [t ] # R Misc. [tR] 2,6-di- 2-Methoxy- 2-Methoxy- 5- 2-Methoxy-4- Phenol p-Cresol Methoxy- n-Decanal phenol methyl-phenol methyl-phenol (2) (4) phenol (10) (5) (8) (9) Rep. (12) Stadium, condition No. [5.76min] [7.23] [7.51] [8.98] [9.08] [11.13] [9.24] A- ; I, just emerged* 4 nd nd nd nd nd nd 5.2±3.3 A+ ; I, dormant* 5 nd nd nd nd nd nd 1.2±0.8 - B ; II, non-feeding 5 nd nd nd nd 0.3±0.2 nd t B ; II, feeding 5 nd nd nd t t nd 0.3±0.1

Asexual juvenile C ; III, ibid 5 0.1±0.1 nd nd nd 0.1±0.1 nd 0.3±0.1

D ; IV, ibid 5 1.1±1.0 nd nd nd t nd 0.1±0.0 E ; ibid 5 4.6±1.3 t nd nd t nd 0.1±0.0 F ; VI, ibid 5 5.8±2.1 t t 0.1±0.0 0.1±0.0 nd 0.1±0.0

GM; VII, male, feeding 5 6.6±1.2 0.2±0.0 t 0.1±0.1 0.2±0.1 0.6±0.3 0.1±0.0 Sexual juvenile GF; VII,female, ibid 5 6.2±0.7 0.2±0.1 t 0.1±0.0 0.2±0.1 0.8±0.4 0.1±0.0 HM; Male, virgin, feeding 5 5.6±1.7 0.1±0.1 t 0.1±0.1 0.2±0.2 0.7±0.3 t HF; Female, virgin, ibid 5 8.1±3.4 0.1±0.1 0.1±0.1 0.1±0.1 0.3±0.3 1.1±0.7 t

Adult I ; M ale, mated, ibid 10 10.0±3.4 4.9±4.2 0.5±0.2 0.7±0.5 2.6±1.0 0.1±0.1 0.1±0.1 M IF ; Female, mated, ibid 10 16.8±4.7 4.4±4.0 0.5±0.2 0.6±0.5 3.8±0.5 0.1±0.1 0.1±0.0

#: M iscelaneous compound, *: 3 individuals were extracted at a time,

nd: not detected, t: abundance is less than 0.1%.

corresponds to morphological shift from asexual to sexual, but between mate-pairs IM & IF: 16.8% ± 4.7% (n=10) for IF ; and not identical. 10.0% ± 3.4% (n=10) for IM (Table 3). As mentioned later, distinct difference between them was also demonstrated by Sexual differences of phenol (2) content in mated adults NMDS. Similar trends were observed between virgin adults:

Compound 2 showed the largest female-biased distribution 8.1% ± 3.4% (n = 5) for HF; and 5.6% ± 1.7% (n = 5) for HM.

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On the contrary, no such difference was detected between immature juveniles GM (6.6%) and GF (6.2%). None of the other compounds showed such biased distribution between sexes in GM & GF, HM & HF nor IM & IF. There might be a sexual dimorphism for 2, implying the sex pheromone function; however, no noticeable sexual behaviors were displayed upon trial exposure of 2.

Sexual maturation and phenolic compounds

Mated adults (IM or IF) showed the maximum amount of five phenolics: 2 (16.8%), 4 (4.9%), 5 (0.5%), 8 (0.7%) and 9 (3.8%), which started to increase from juveniles (less than

1.0% during D (or C) - GM & GF), except 2, which started to increase from C. On the other hand, phenolics 12 started to Fig. 4 Statistical discrimination of Oxidus gracilis defensive allomone profiles at various growth stadia and accumulate in the last juvenile stadium (G 0.6% and G M F conditions by non-metric multidimensional scaling 0.8%) to reach the maximum in virgin adults (HM 0.7% and HF (NMDS) using Bray-Curtis dissimilarity matrix. - 1.1%) and then decreased to 0.1% in both IM & IF. A : Non-feeding juvenile at stadium I, just after emergence, A+: dormant juvenile at stadium I, B+: The most characteristic change of profiles was observed juvenile at stadium II, before feeding, B: feeding between HM & HF and IM & IF. In addition to drastic decrease juvenile at stadium II, C: feeding juvenile at stadium of PD 11 and phenolics 12, conspicuous increase of five III, D: feeding non-sexed juvenile at stadium IV, E: feeding non-sexed juvenile at stadium V, F: feeding phenolics (2, 4, 5, 8 and 9) was observed between HM and IM; non-sexed juvenile at stadium VI, GM: feeding male and between HF and I , that is, virgin and mated adults F juvenile at stadium VII, GF: feeding female juvenile at indicated different profiles (Table 3 and Fig. 3). stadium VII, HM: virgin male adult, HF: virgin female The non-sexual accumulation of five phenolics (2, 4, 5, 8 adult, IM: mated male adult after forced separation, and IF: mated female adult after forced separation. and 9), decline of PD 11 and of phenolics 12, appeared Two broad dotted lines separate cluster groups between corresponding to the process of sexual maturation or to enforce a (the early half of juvenile life cycle) and b (the later the process of sexual behavior. Therefore, these phenomena of half of juveniles life cycle), and between b and c (adults). Solid line separates cluster groups between non-sexual decrease or increase seem to be a process, not only virgin adults and mated adults. Three thin dotted lines to maintain defense activity by compensating for decrease of between male and female. PD 11 with accumulation of phenolics, but also of sexual maturation. - IF (adult), as separated by two broad dotted lines (Fig. 4). From the result of multidimensional scaling, juvenile-adult Secretion profiles of all growth stadia and conditions polymorphism was clearly evidenced as the shift from a and b - Statistical discrimination of profiles (A - IF) by NMDS to c. Juvenile-juvenile polymorphism was concluded also as using Bray-Curtis dissimilarity matrix was applied to examine the change from a to b, and it was not identical to the separation of clusters from each other. All clusters morphological shift from asexual to sexual juvenile, as - corresponding to each profile (A - IF) were statistically mentioned above. From results of the biological observation discriminated, except a partial overlap between GM and GF (Table 3, Fig. 3), it was hard to elucidate the phenomenon, - (Fig. 4). The result indicated that millipedes use a different because the profile of A looked as an exception and that of D composition of secretions depending upon stadia and was ambiguous regarding the period to which it belongs. conditions. As indicated (Fig. 4), sexual differentiation process was also - Clusters of profiles (A - IF) were largely classified into three concluded among three cluster sets (GM & GF, HM & HF and IM - areas: a) broad distribution of A - D (the early half of the & IF) upon thin dotted line, from partly overlapped clusters juvenile life cycle, composed of asexual stadia at I - III and of (GM and GF) to clearly separated clusters (IM & IF) via sexual stadium at IV), b) densely distributed area of E - GM & in-between clusters (HM & HF). As separated by solid line in

GF (the later half of the juvenile life cycle, sexual juvenile Fig. 4, large separation of clusters was observed between HM stadia at V - VII), and c) dense but clearly separated area of HM & HF and IM & IF. The phenomenon was elucidated as the

- 45 - Kuwahara et al composition change due to sexual maturation from virgin to (Kuwahara et al., 2017a). NMDS using Bray-Curtis mated adult. dissimilarity matrix supported the differences of profiles as clearly separated clusters between adults and juveniles. Discussion Typical juvenile-adult polymorphism of defense allomones are known among families of hemipterans (Aldrich, 1988; A total of 13 defense allomones (six PDs, six phenolics, and Kheyri et al., 2014) and the opisthonotal gland (or oil gland) one miscellaneous, with more than 0.1% relative abundance) secretions among four species of “glandulate Oribatida” (sensu were identified from the greenhouse millipede O. gracilis Norton, 1998) of Arachnida (Shimano et al., 2002; Takada et al., (Table 1). The following two compounds were not confirmed 2005; Raspotnig et al., 2005, 2008). However, similar defense in our results: benzaldehyde dimethyl acetal (Taira et al., 2003) allomone shifts have not been recorded among millipedes, and ethyl benzoate (Duffey et al., 1977; Roncadori et al., except in the following three cases: 1) Japanese polydesmid, 1985). Niponia nodulosa (Polydesmida: Cryptodesmidae) (Kuwahara Benzaldehyde dimethyl acetal may be an artifact derived et al., 2015), in which all four PDs 1, 6, 11 and 13 present in from the reaction between 1 and solvent (Noguchi et al., the early half of the juvenile stadia (I - IV) were replaced by 1997a). Ethyl benzoate (Duffey et al., 1977) is producible from volatile organic compounds (presumably of microbial origins) 6, if ethanol is involved, but was not found in this study. Based in the latter juvenile stadia (V - VII) and adults - a case of on our results, the presence of 7 and 13 without detecting 6 as “juvenile-juvenile polymorphism”; 2) julid diplopod Allajulus in the study by Duffey et al. (1977) are puzzling, because 7 dicentrus (Bodner and Raspotnig, 2012) in which allomone and 13 should accompany 6. MOX functioned to produce 6 components gradually increased with ontogenetic and hydrogen peroxide (Kuwahara et al., 2017a) from 11 as the development; and 3) Nedyopus patrioticus patrioticus substrate, and 6 was then hydrolyzed to 7. Compound 13 was (Polydesmida: Paradoxosomatidae) in which the content ratios a product of the Schotten–Baumann reaction between 6 and 11 of phenolics 1:2:4 differed among juveniles at a certain (Kuwahara et al., 2011). stadium (90:8:2) and adults (28:2:70 in male and 26:4:70 in Compound 3 has been known to occur in only three species, female) (Noguchi et al., 1997b). avalae, B. dadayi and B. troglobius The juvenile-juvenile polymorphism of O. gracilis is the (Polydesmida: ) (Makarov et al., 2010, 2012), second example after Ni. nodulosa (Kuwahara et al., 2015), among 53 species of cyanogenic Polydesmida (Makarov, 2015, where the early half of the juvenile life cycle is from stadium I Shear, 2015), and the present identification is the fourth to IV (three successive asexual stadia and one sexual stadium), example. Its generation and wide distribution in juveniles and and the later half of the juvenile life cycle from stadium V to adults is understandable, if we assume that 3 is a product of VII (three sexual stadia). NMDS using Bray-Curtis HNL and subsequent reduction. dissimilarity matrix clearly supported differences of cluster - Based on the distribution patterns (Table 3), three PDs (6, 7 groups from A to D and from E to GM and GF, which and 13) were recognized as juvenile-specific components as correspond to both periods known in Ni. nodulosa (Kuwahara mentioned above, and products catalyzed by MOX (Kuwahara et al., 2015). et al., 2017a) from 11. Compounds 1 and 3 were produced by Accumulation of 2 became evident in the later juvenile

HNL (Dadashipour et al. 2015) from 11, and subsequent stadia from E to GM & GF and in adults (HM - IF). Its most reduction of 1. Therefore, all six PDs in juveniles were distinct female-biased distribution was noted during the mating concluded as products present in a typical type B millipede season (IF), as mentioned above. Distinct difference of clusters

(Kuwahara et al., 2011), and as those derived from two (IM and IF) was also demonstrated by NMDS using Bray-Curtis enzymes (MOX and HNL) and subsequent reactions dissimilarity matrix, indicative of the sex related compound (Kuwahara et al., 2017a). On the other hand, adult secretions involved. Similar female-biased presence of 2 and adult composed mainly of two PDs (1 and 11, a product by HNL and specific accumulation of 4 have been noted in Ne. patrioticus its substrate), were concluded as those of a typical type A patrioticus (Noguchi et al., 1997b), whereas no sex pheromone millipede (Kuwahara et al., 2011) and, that’s why, hydrogen functions have been observed. In Ne. tambanus mangaesinus peroxide has not been detected from adult extracts (Kuwahara (Polydesmida: Paradoxosomatidae), 1-phenyl-2-pentanone is et al., 2017a). As a result, juvenile-adult polymorphism was also known for female-biased distribution during the mating explainable as a shift from two responsible enzymes to one season (Kuwahara et al., 2017b). At present, no sex pheromone

- 46 - Defensive secretion of Oxidus gracilis were not alwaya the same functions on candidates of female-biased distribution have showed that all profiles demonstrated independent clusters. been demonstrated in any reported species belonging to These changes of compositions elucidated juvenile-adult Paradoxosomatidae. polymorphism, juvenile-juvenile polymorphism, sexual It seems to be common among species belonging to dimorphism, and sexual maturation. Paradoxosomatidae that newly emerged juveniles (stadium I) develop into stadium II without feeding. Juveniles (A- - B-) Acknowledgments seemed to be the most fragile period to thrive, because no protective chambers (or cells) against natural enemies were This work was supported by JST ERATO Asano Active constructed to accomplish subsequent dormancy (A+) and Enzyme Molecule Project (Grant Number JPMJRER1102), ecdysis to stadium II (B-). In O. gracilis, compound 10 {new Japan. This research was also supported in part by a discovery among polydesmid millipedes (Makarov, 2015; grant-in-aid for Scientific Research (S) from The Japan Society Shear, 2015) and the highest content in A- (5.2%)} together for Promotion of Sciences (No. 17H06169) to Y. Asano. We with the high amount of 6 might indicate their function as thank Dr. Kimiyasu Isobe for critical opinion on the defense agents, to overcome the most fragile period of growth, manuscript, and Mrs. Sayaka Shichida of our program for because of its antimicrobial activity (Mahboubi and Feizabadi, collecting Oxidus gracilis millipedes from potted Roman 2009) other than following functions; an aggregation chamomile Chamaemelum nobile plantation in this university. pheromone in codling moth larva, (Jumean et al., 2005), and We would like to thank Editage (www.editage.jp) for English an allomone in the common bed bug (Siljander et al., 2008). language editing. In Ne. tambanus mangaesinus, methyl salicylate has been identified to possess the similar function (Kuwahara et al., References 2017b), and further examples can be found in stadium I juveniles of other millipedes belonging to Paradoxosomatidae. Aldrich, J. A. (1988) Chemical ecology of the heteroptera. The abundance of 10 (5.2% - less than 0.1%) decreased with Annu. Rev. Entomol. 33: 211–238. ontogenetic development but persisted in all subsequent stadia. Blower, J. G. (1985) Millipedes: Keys and notes for the This might be a reliable index for chemo-taxonomically identification of the species. In: “Synopses of British identifying O. gracilis at juvenile stadia, as was proposed for Fauna” (Brill, E. J., ed), pp. 244, Backhuys Publishers, methyl salicylate in Ne. tambanus mangaesinus (Kuwahara et al London. ., 2017b). Bodner, M. and G. Raspotnig (2012) Millipedes that smell like Two phenolics 8 and 12 were firstly identified among bugs: (E)-alkenals in the defensive secretion of the julid species of Polydesmida (Makarov, 2015; Shear, 2015). Four diplopod Allajulus dicentrus. J. Chem. Ecol. 38: phenolics 2, 4, 5 and 9 have been known from the present 547-556. species (Duffey et al., 1977; Roncadori et al., 1985; Taira et al., Bray, J. R. and J. T. Curtis (1957) An ordination of upland 2003). Distinct decrease of PD 11 and the unusual forest communities of southern Wisconsin. Ecol. accumulation of five phenolics 2, 4, 5, 8 and 9 were firstly Monogr. 27: 325-349. noticed in mated adults (IM & IF) when compared with virgin Dadashipour, M., Y. Ishida, K. Yamamoto and Y. Asano (2015) adults (HM & HF). We would like to consider this phenomenon Discovery and molecular and biocatalytic properties of between virgin (HM & HF) and mated adults (IM & IF) as a hydroxynitrile lyase from an invasive millipede, signal of sexual maturation. NMDS using Bray-Curtis hualienensis. Proc. Natl. Acad. Sci. USA dissimilarity matrix showed distinct differences between 112: 10605-10610. clusters of HM & HF and IM & IF to conclude the phenomenon Duffey, S. S. and G. H. N. Towers (1978) On the biochemical of sexual maturation. A similar accumulation of two phenolics basis of HCN production in the millipede 2 and 4 has been recorded in mated adults of Ne. tambanus haydeniana (: Polydesmida). Can. J. mangaesinus when compared with virgin adults (Kuwahara et al., Zool. 56: 7-16. 2017b). Duffey, S. S., M. S. Blum, H. M. Fales, S. L. Evans, R. W. The present defense allomone study revealed that the Roncadri, D. L. Tiemann and Y. Nakagawa (1977) allomone compositions changed depending on the stadium and Benzoyl cyanide and mandelonitrile benzoate in the conditions. NMDS using Bray-Curtis dissimilarity matrix defensive secretions of millipedes. J. Chem. Ecol. 3:

- 47 - Kuwahara et al

101-113. , vol 2”. Minelli, A., (ed), pp. 69-99, Brill, Hoffman, R. L. (1999) Checklist of the millipedes of North and Leiden, Middle America. In; “Virginia Mus. Nat. History Makarov, S. E., B. P. M. Ćurčić, V.V. Teševic, M. B. Jadranin, Special Publications” No.8, pp. 1–584. L. Vujisic, S. B. Ćurčić, B. M. Mandic, T. L. Sekulic Ishida, Y., Y. Kuwahara, M. Dadashipour, A. Ina, T. Yamaguchi, and B. M. Mitić (2010) Defensive secretions in three M. Morita, Y. Ichiki and Y. Asano (2016) A sacrificial species of polydesmids (Diplopoda, Polydesmida, millipede altruistically protects its swarm using a drone Polydesmidae). J. Chem. Ecol. 36: 978–982. blood enzyme, mandelonitrile oxidase. Sci. Rep. 6, Makarov, S. E., L.V. Vujisić, B. P. M. Ćurčić, B. S. Ilić, V. V. 26998 DOI: 10.1038/srep26998. Tešević, V. E. Vajs, I. M. Vučković, B. M. Mitić, L. R. Jumean, Z., R. Gries, T. Unruth, E. Rowland and G. Greis Lučić and I. Z. Ðordević (2012) Chemical defense in (2005) Identification of the larval aggregation the cave-dwelling millipede Brachydesmus troglobius pheromone of codling moth, Cydia pomonella. J. Chem. Daday, 1889 (Diplopoda, Polydesmidae). Int. J. Speleol. Ecol. 31: 911-924. 41: 95–100. doi: 10.5038/1827-806X.41.1.10 Kheyri, H., B. W. Cribb and D. J. Merritt (2014) The Noguchi, S., N. Mori, Y. Higa and Y. Kuwahara (1997a) comparative morphology of epidermal glands in Identification of mandelonitrile as a major secretory Pentatomoidea (Heteroptera). Struct. Dev. compound from Chamberilinius hualienensis Wang 43: 211-219. (Polydesmia: Paradoxomamidae). Jpn. J. Environ. Kováts, E. (1958) Gas-chromatographische charakterisierung Entomol. Zool. 8: 208-214. organischer Verbindungen. Teil 1: Retentionsindices Noguchi, S., N. Mori, Y. Higa and Y. Kuwahara (1997b) aliphatischer halogenide, alkohole, aldehyde und ketone. Identification of Nedyopus patrioticus patrioticus Helv. Chim. Acta 41: 1915-1932. (Attems, 1898)(: Paradoxosomatidae) Kuwahara, Y., N. Shimizu and T. Tanabe (2011) Release of secretions as possible defense substances. Appl. hydrogen cyanide via a post-secretion Entomol. Zool. 32: 447-452. Schotten-Baumann reaction in defensive fluids of Norton, R. A. (1998) Morphological evidence for the polydesmoid millipedes. J. Chem. Ecol. 37: 232-238. evolutionary origin of Astigmata (Acari: Acariformes). Kuwahara, Y., Y. Ichiki, M. Morita, T. Tanabe and Y. Asano Exp. Appl. Acarol. 22: 559–594. (2015) Chemical polymorphism in defense secretions Oksanen, J., B. F. Guillaume, R. Kindt, P. Legendre, P. R. during ontogenetic development of the millipede Minchin, R. B. O'Hara, G. L. Simpson, P. Solymos, M. Niponia nodulosa. J. Chem. Ecol. 41: 15-21. Henry, H. Stevens and H. Wagner (2018) Vegan: Kuwahara, Y., M. Morita, Y. Ichiki, T. Tanabe and Y. Asano Community Ecology Package. (2017b) 1-Phenyl-2-pentanone and methyl salicylate: http://CRAN.R-project.org/package=vegan new defense allomone components and their content Raspotnig, G., G. Krisper and R. Schuster (2005) Ontogenetic shift during ontogenetic development of the millipede changes in the chemistry and morphology of oil glands Nedyopus tambanus mangaesinus (Polydesmida: in Hermannia convexa (Acari: Oribatida). Exp. Appl. Paradoxosomatidae). Appl. Entomol. Zool. 52: 447-455. Acarol. 35: 47–58. Kuwahara, Y., T. Yamaguchi, Y. Ichiki, T. Tanabe and Y. Raspotnig G., R. Kaiser, E. Stabentheiner and H-J. Leis (2008) Asano (2017a). Hydrogen peroxide as a new defensive Chrysomelidial in the opisthonotal glands of the compound in “benzoyl cyanide” producing polydesmid oribatid mite, Oribotritia berlesei. J. Chem. Ecol. 34: millipede. Sci. Nat. 104: 19. DOI: 1081–1088. 10.1007/s00114-017-1435-y. R Core Team (2018) R: a language and environment for Mahboubi, M. and M. M. Feizabadi (2009) Antimicrobial statistical computing. R Foundation for Statistical activity of Ducrosia anethifolia essential oil and main Computing, Vienna, Austria. http://www.R-project.org/. component, decanal against methicilin-resistant and (accessed Nov. 14, 2018) methicilin-susceptible Staphylococcus aureus. J. Ess. Roncadori, R. W., S. S. Duffey and M. S. Blum (1985) Oil. Bear. Pla. 12:574-579. Anti-fungal activity of defensive secretions of certain Makarov, S. E. (2015) Diplopoda - Integument. In: “Treatise millipedes. Mycologia 77: 185–191. on Zoology---anatomy, , biology. The RStudio (2018) RStudio: Integrated development environment

- 48 - Defensive secretion of Oxidus gracilis were not alwaya the same

for R. Boston, MA. http://www.rstudio.org/ Taira, J., K. Nakamura and Y. Higa (2003) Identification of Siljander, E. I., R. Gries, G. Khaskin and D. Gries (2008) secretory compounds from the millipede, Oxidus Identification of the airborne aggregation pheromone of gracilis C. L. Koch (Polydesmida: the common bed bug, Cimex lectularius. J. Chem. Ecol. Paradoxosomatidaee) and their variation in different 34: 708-718. habitats. Appl. Entomol. Zool. 38: 401-404. Shear, W. A. (2015) The chemical defenses of millipedes Takada, W., T. Sakata, S. Shimano, Y. Enami, N. Mori, R. (diplopoda): Biochemistry, physiology and ecology. Nishida and Y. Kuwahara (2005) Schloribatid mites as Biochem. System. Ecol. 61: 78-117. the source of pumiliotoxins in dendrobatid frogs. J. Shimano, S., T. Sakata, Y. Mizutani, Y. Kuwahara and J. Aoki Chem. Ecol. 31: 2403–2415. (2002) Geranial: the alarm pheromone in the nymphal Van Den Dool, H. and P. D. Kratz (1963) A generalization of stage of the oribatid mite, Nothrus palustris. J. Chem. the retention index system including linear temperature Ecol. 28: 1831–1837. programmed gas-liquid partition chromatography. J. Shinohara, K. (1999) Life of a Japanese millipede, Niponia Chromatography 11: 463-471. nodulosa. Insectarium 18: 82-86 (in Japanese).

ヤケヤスデ Oxidus gracilis (C.L. Koch)[ヤケヤスデ科オビヤスデ目]防御分泌物の組

成変化；若虫− 成虫間多型と若虫− 若虫間多型、性成熟と雌雄間二型

1 2 3) 1 3) 1 3) 4) 5) 5) 2 3)* 桑原保正 ・市来弥生 ・森田将史 ・田邉 力 ・中田 隆 ・森 直樹 ・浅野泰久

１）浅野酵素活性分子プロジェクト,JST,ERATO,京都分室 ２）富山県立大学生物工学科、生物化学工学研究センター ３）浅野酵素活性分子プロジェクト,JST,ERATO,富山本部 ４）熊本大学先端科学研究部 ５）京都大学農学研究科

ヤケヤスデ Oxidus gracilis の防御物質は，成虫ではオビヤスデ目化合物３成分（マンデロニトリル，ベンズア ルデヒド，ベンジルアルコール），若虫では同３成分（ベンゾイルシアニド，安息香酸，安息香酸マンデロニトリル） の加わった計６成分，フェノール類６成分（成長と共に増加）と n-デカナール（一様に分布）で構成されていた． さらに，若虫（１〜７令）と成虫（性成熟前後）を精査した結果，若虫成虫間以外にも，若 虫期前期{１〜３令（あ るいは４令まで）と同後期{４あるいは５〜７令}で組成が変化し，成虫では，性成熟前（成虫化後 7 日）と後（交 尾中の雌雄）で顕著に異なること，また７令若虫→成虫化直後→成熟後へとの成長に伴い，フェノールが雌に局在 する傾向の拡大（性的二型）を認めた．これらの相違は NMDS 分析でも確認でき，防御物質の組成は若虫− 成虫間及 び若虫− 若虫間で成分多型，成虫化後− 成熟期の間で性成熟現象及び性的二型を示すことが判った.

*Correspondig author:[email protected]

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