Initial defensive secretory compounds emitted from the live millipede and the induction of apoptotic cell death

The initial defensive secretory compounds emitted from a live millipede have not yet been clarified. This study focused on elucidating the initial secretory compounds emitted from a live millipede. Pre-concentration of the defensive secretory volatile organic compounds (VOC) from the live Polidesmida millipedes, Chamberlinius hualienensis and Oxidus gracilis, was performed using a three-stage VOC concentration technique by an on-line GC/MS system. As a result, the monoterpenes derived from the plant metabolite; i.e., α-pinene, α-thujene, β-pinene, 3-carene, β-myrcene, β-phellandrene, γ-terpinene, o,m,p-cymenes, limonene and camphene were first detected as the initial secretory substances. It was elucidated that some plant monoterpenes have a repellent effect and antifungal and antibacterial actions which are used as defensive substances. In addition, this study also confirmed that these monoterpenes induced apoptotic cell death involved in the induction of the caspase 3/7 activity. The millipede feeds on fallen or withered leaves containing the monoterpenes. Thus, the millipede accumulates the plant defensive secretions in the exocrine defense glands of the body somites, which would be used as against predators.

and C. hualienensis (male, n = 2) and female, n = 7) were put together and transferred to a vial (120 ml) for collecting the initial secretion.

Pre-concentration of volatile secretions.
Pre-concentration of the VOC and subsequent constituent analysis were performed using an ENTECH 7100A (Entech Instruments, Inc., CA, USA) online system gas chromatograph (GC) (Agilent 6890 N, Agilent Technologies, CA, USA) equipped with a mass selective detector (MSD) (5975C inert XL MSD, Agilent Technologies, CA, USA). Briefly, the live millipedes without any plant residues were maintained in a vial at room temperature for 40 min and subsequently pre-concentrated by a three-stage VOC concentration technique involving three types of modules (modules 1, 2, and 3) and a trap tube as previously reported 10 . The atmosphere (100 ml) emitted from the millipedes was absorbed in module 1 using a glass bead/tenax trap at -145 °C for 1 min, then at the heating 10 °C while slowly passing nitrogen gas through module 1. The adsorbed sample was then transferred to module 2 containing a tenax trap pre-chilled at -30 °C. The target compounds were back-flushed at 150 °C for 2.5 min prior to being further focused in a capillary focusing trap in a pre-chilled (-160 °C) module 3 (cryo-focus trap). The compounds were then at the heating 110 °C for injection into the analytical column. Cytotoxicity of initial secretory compounds. The MTT assay was used to determine the cell viability 12,13 .
Caspase activity. Cells were plated at a density of 5 × 10 5 cells/ml on 96-well plates after culturing for 24 h, the test samples were prepared as with the MTT assay and they were treated for 17 h. The caspase activity in the cultured cells was measured using the commercially-available caspase-3/7 assay kit 12,13 . Cells were incubated with 100 μl of proluminescent substrate containing Z-DEVD provided in the kit. Following the caspase cleavage, the luminescence of the reaction products was measured using a microplate reader (GLOMAX MULTI DETECTION SYSTEM, Promega, WI, USA). Caspase activity expressed as % of the control for solvent treated cells instead of test compound.
Statistical analysis. The data are expressed as mean ± SD of four samples. Significant differences between the control and test samples were analyzed by the Student's t-test with the significance at *p < 0.05 and **p < 0.01.

Results
The initial defensive secretory compounds emitted from the live millipedes were detected by the pre-concentration of the secretion using a large volume headspace on-line GC/MS system. The total ion chromatogram (TIC) of the initial secretory compounds emitted from both live millipedes of O. gracilis and C. hualienensis are indicated in Fig. 1a,b, respectively. The identity of the initial secretory compounds from the millipede was carried out by their similarity to commercially available authentic compounds and a library search, resulting in the determination of 11 monoterpenes, i.e., α-pinene; α-thujene; β-pinene; 3-carene; β-myrcene; m-cymene; limonene; β-phellandrene; γ-terpinene; o,m,p-cumenes; camphene including 2 sulfur compounds of dimethyl sulfide and dimethyl disulfide, and 3 furan compounds of 2-methyl furan, 3-methyl furan and ethyl furan. In addition, benzaldehyde, 2-methyl-3-buten-2-ol and methyl thiocyanate were detected from both millipedes using the non-polar column DB-1 (data not shown). The chemical structures of the initial secretory compounds detected in this study depicted in Fig. 2. These initial secretary compounds were different from the main secretory compounds, such as phenols and quinones (benzoquinones and hydroquinones) detected from the soaked millipede using an organic solvent 2 . To explore the other biological activities of the initial defensive secretory compounds, their cytotoxicity was examined using two cell lines of a mouse macrophage-like cell, RAW264.7 and PC12 cells, derived from a pheochromocytoma of the rat adrenal medulla. As a result of the evaluation for the RAW264.7 cells, the monoterpenes of α-pinene, β-pinene, 3-carene, β-phellandrene, γ-terpinene, camphene indicated a high cytotoxicity in a dose dependent manner. These cytotoxic secretory compounds also induced the caspase3/7 activity related to the apoptotic cell death (Fig. 3). www.nature.com/scientificreports/ In addition, the cytotoxicity of the initial secretory compounds was also confirmed using the PC12 cells which are widely utilized for studies of different aspects of neuronal physiology (Fig. 4). The cytotoxicity of the monoterpenes of α-pinene, β-pinene, 3-carene and γ-terpinene indicated a high cytotoxicity involved in the apoptotic cell death with the caspase activation. This result was similar to the result of the RAW264.7 cell treated with the test compound (Fig. 4).

Discussion
In this study first demonstrated the initial secretory substance from the live millipedes Polidesmida millipedes, Chamberlinius hualienensis and Oxidus gracilis as depicted in Fig. 2. These initial secretory compounds, such as monoterpenes, sulfur compounds, and furan compounds were first detected in all species of the millipede 2 . In our previous studies, the secretions of phenol, cresol, benzaldehyde and dimethyl acetal in the methanol-soaked millipedes of both C. hualienensis and O. gracilis have been identified as defensive secretory compounds 7,8 . The secretory compounds detected in the soaked millipede were different from those of the initial secretory compounds emitted from the live millipede. In addition, both millipedes collected the following year at the same place were analyzed and new initial secretory compounds of 3-metyhl-2-butaneone, 2-pentanone, 2-butanone, methanethiol, methoxy benzene, and involving methyl thiocyanate from O. gracilis were detected (data not shown). In our previous studies, the concentrations of the internal elements in the millipede and the soil including leaf litter in the habitat were evaluated. The soil concentration in the habitat reflected the characteristics of the www.nature.com/scientificreports/ internal elements and metal accumulation in both millipedes 14,15 . Therefore, both millipedes used in this study, which were collected in the same habitat environment, may contain the similar initial secretary compounds. The cytotoxicity of the initial defensive secretory compounds was examined using two beneficial cell lines of RAW264.7 macrophages for cell death through the inflammatory response and PC12 cells as index of neuronal toxicity. In previous studies have shown in vitro and in vivo antitumor activity of monoterpenes in essential oils obtained from plants 16 . In this study, the monoterpenes of α-pinene, β-pinene and γ-terpinene indicated a high cytotoxicity in the apoptotic cell death which were also reported antitumor activity, such as leukemia, melanoma and colon cancer 16 . The monoterpenes are widely contained in various plants, and some monoterpenes have a Figure 3. The induction of cytotoxicity with apoptotic cell death due to the initial defensive compounds from the live millipede against RAW 264.7 cells, (a) cytotoxicity of the initial defensive compounds. (b) caspase activity of the initial defensive compounds. Significant difference was determined by t-test. *p < 0.05 and **p < 0.01 for the control cell. www.nature.com/scientificreports/ repellent effect and antifungal and antibacterial action [17][18][19] . The millipede feeds on fallen or withered leaves containing the monoterpenes. Thus, the millipede accumulates the plant defensive secretions in the exocrine defense glands of the body somites. The initial secretory compounds emitted from millipede would be used as initial defensive substances against predators as well as the chemical defense of plants. In addition, it is well known that millipedes discharge defensive odorous secretions during outbreak that cause headaches and unpleasant feelings for humans [7][8][9] . Therefore, the biological activity of the initial secretions may be responsible for causing unpleasant sensations.
In conclusion, this study first elucidated the initial defensive secretory compounds emitted from the live millipedes. In particular, the monoterpenes induced apoptotic cell death. The millipede accumulates these cytotoxic  . The induction of cytotoxicity with apoptotic cell death due to the initial defensive compounds from the live millipede against PC12 cells, (a) cytotoxicity of the initial defensive compounds. (b) caspase activity of the initial defensive compounds. Significant difference was determined by t-test. *p < 0.05 and **p < 0.01 for the control cell.