24th ISCE Annual Meeting Pennsylvania, USA (August 21, 2008) Identification of C21 Type II sex pheromone components and novel C20 and C22 trienyl biosynthetic precursors from a wasp moth, Syntomoides imaon (Arctiidae: Syntominae) Tetsu ANDO et al. Graduate School of BASE, Tokyo University of Agriculture and Technology, Tokyo 184-8588, Japan E-mail: [email protected] Diurnal moths Wasp moth Clearwing moth Pheromone: Type II ? Z3,Z13-18:OAc Hawk moth E10,E12-16:Ald Noctuoidea Bombycoidea Papillionoidea Geometroidea Sesioidea Pyraloidea Hesperioidea Pterophoroidea Zygaenoidea Gelechioidea Tortricoidea Yponomeutoidea Cossoidea Castnioidea Tineoidea Hepialoidea Incurvarioidea Nepticuloidea Ditrysia Eriocranioidea Monotrysia Zeugloptera Dacnonypha Type I pheromone Type II pheromone Paramecoptera Arctiid species and pheromone study Taxonomy Japanese Super-family Family Sub-family species Noctuoidea Notodontidae Lymantriidae Arctiidae Lithosiinae 79 Nolidae Syntominae 4 wasp moth Pantheidae Arctiinae 51 Noctuidae In Syntominae, Type II pheromones (C21 triene and 9,10-epoxide) have been identified from two tropical species, Empyreuma mucro and Syntomeida epilais (no field data) (Descoins et al., 1989). Insects used in this study Syntomoides imaon Cramer (a wasp mimic species) Distributed in Southeast Asia, such as India and Taiwan Found only in the Yonaguni-jima Island in Japan Reared on a semisynthetic diet (16L-8D, 25oC) GC-EAD and GC-MS The pheromone gland extract of S. imaon females was analyzed by GC-EAD and GC-MS equipped with a DB-23 column. GC-EAD (0.5 FE) Mass spectra (1 FE) 79 Comp. I 100 - Comp. II 108 (%) 108 234 Comp. I 67 50 - 55 93 121 EAD 135 79 234 290 0 - 100 200 m/z 300 79 - 91 Comp. II 100 106 91 (%) 234 67 50 - 106 FID 55 119 133 79 234 16 17 18 19 288 Rt (min) 0- 100 200 m/z 300 Comp. I (Z3,Z6,Z9-21:H) Comp. II (1,Z3,Z6,Z9-21:H, 1.5 g/female) Comp. III (Z3,Z6,Z9-20:H) Comp. IV (1,Z3,Z6,Z9-20:H) I : II : III : IV = 32:67:0.6:0.7 Chemical reactions with tetraene Ex. 1) 3,6,9-triene Gland extract (20 FE) dienes tetraene N2H4 + H2O2 / EtOH 65oC, 2 hr monoenes GC-MS analysis TIC 14.0 16.0 18.0 Rt (min) Only one triene Selective reduction at 1-position Ex. 2) I Mass chromatogram at m/z 388 II Gland extract (20 FE) III N H + H O / EtOH 2 4 2 2 m/z 388 65oC, 8 hr 24.5 25.0 25.5 o Rt (min) DMDS / I2 , 40 C, 2 hr I DMDS of 9-21:H (m/z 173, 215) II DMDS of 6-21:H (m/z 131, 257) GC-MS analysis III DMDS of 3-21:H (m/z 89, 299) 1,3,6,9-tetraene Field tests Ordinal wasp moth in Japan Amata fortunei Z3,Z6,Z9-21:H & other components (without tetraene) *Syntomoides imaon Triene Tetraene Total males 0.5 mg 1.0 mg 31 1.0 0 0 0 1.0 0 * Z3,Z6,Z9-21:H 0 0 0 & 1,Z3,Z6,Z9-21:H From 28 June to 5 July, 2007 Future works: the optical mixing ratio, roles of minor C20 compounds and visual cues, and etc. Identification of tetraenes C21 C19 Epirrita autumnata (Geometridae) Operophtera brumata Arctia villica (Arctiidae) O. bruceata (Geometridae) Utetheisa ornatrix (Arctiidae) Biosynthesis of Type II pheromones Confirmed step Hemolymph Unconfirmed step Linolenic acid Z9,Z12,Z15-18:Acid Head PBAN 3 6 Z11,Z14,Z17-20:Acid Ingestion 2 Z3,Z6,Z9-19:H SG PBAN 1 5 Z3,Z6,Z9-19:H Lipophorin OenocyteOenocyte 4 epo3,Z6,Z9-19:H Pheromone gland 1 Specific epoxidation (Miyamoto et al., 1999. IBMB, 29: 63) 2 Transportation in hemolymph (Wei et al., 2003. IBMB, 33: 397) C19 triene & epoxide 3 Mode of action of PBAN (Wei et al., 2004. IBMB, 34: 1215) 4 Lipophorin association (Matsuoka et al., 2006. IBMB, 36: 576) 5 Identification of PBAN (Kawai et al., 2007. IBMB, 37: 330) Geometrid moth 6 Structure of pheromone gland (Fujii et al., 2007. JIP, 53: 312) C21 triene desaturation New subject + tetraene Z9,Z12,Z15-18:Acid Z9,Z12,Z15,17-18:Acid -oxidation Z11,Z14,Z17-20:Acid Z11,Z14,Z17,19-20:Acid Identification of these long-chain fatty Z13,Z16,Z19-22:Acid Z13,Z16,Z19,21-22:Acid acids, candidates of Oenocyte biosynthetic Z3,Z6,Z9-21:H 1,Z3,Z6,Z9-21:H Wasp moth Oenocyte precursors Analysis of fatty acids Abdominal integument with oenocytes and peripheral fat bodies Extraction, basic methanolysis, methylation with CH2N2 Mixture of fatty acid methyl esters (FAMEs) biosynthetic GC-MS X HPLC separation (ODS column) precursors Fractions including a long-chain fatty acid ester (A) HPLC Rts of FAMEs Triene diene monoene saturated 2 4 C16 C17 Column: ODS Eluent: 13 5 C18 5% water C19 in MeOH C20 (1.0 ml/min) C21 10 20 30 40 C22 50 Rt (min) (B) Fractionation of S. imaon FAMEs (1 female) 1 GC-MS 3 Fr. A (14-15 min) C20 trienyl o 1 18:3 2 16:1 C20 tetraenyl 3 18:2 4 16:0 Fr. B (18-20 min) X 5 18:1 5 2 4 Fr. C (20-22 min) C22 trienyl o 210 nm 10 20 30 40 50 Rt (min) GC-MS of HPLC fractions Fr. A (14-15 min) 79 100 - 108 264 20:3 O ester (%) 95 OMe 108 50 - 79 TIC M+ 264 289 320 18 20 22 Rt (min) 0 - 100 200 300 m/z 79 Fr. C (20-22 min) 100 - 108 292 22:3 (%) 95 O ester 108 OMe 50 - 79 TIC M+ 292 317 348 18 20 22 Rt (min) 0 - 100 200 300 m/z Ion fragments lower than m/z 150 in the spectra of the 20:3 and 22:3 esters were identical to those of the ester of linolenic acid (18:3), indicating the same trienyl structure. Relative contents; 16:0 (0.3 mg/female, 100%), 20:3 (0.08%), 22:3 (0.03%) Unsaturated positions of C20 trienoate Diimide reduction Fr. A (14-15 min) Mixture of monoenyl esters 20:3 ester DMDS / I 2 GC-MS analysis DMDS adducts of monoenyl esters (A) (B) (C) m/z 173 173 245 m/z 245 SCH H3CS 11 3 O (A) m/z 213 OMe 213 25 26 27 Rt.(min) m/z 131 131 287 H3CS SCH3 m/z 287 14 OMe (B) m/z 255 255 O 25 26 27 Rt.(min) 89 329 m/z 89 H CS SCH3 O 3 17 m/z 329 (C) OMe m/z 297 297 25 26 27 Rt.(min) Methyl ester of 11,14,17-20:Acid Summery Female wasp moths produce Type II pheromones, a mixture of C21 triene and tetraene to attract males. Fatty acid methyl esters (FAMEs) derived from lipids were fractionated by HPLC. GC-MS analysis revealed novel C20 and C22 trienoates, longer-chain analogs of linolenate. In the another experiment with the geometrid moth, which produces C19 pheromone components, only C20 trienoate was detected. Different systems of the chain elongation might play an important role in developing species-specific communication systems mediated with polyunsaturated hydrocarbons and derivatives, components of Type II lepidopteran sex pheromones. Recent related studies 1) Biosynthesis of tetraene in Utetheisa ornatrix (Arctiidae: Arctiinae) Choi et al., 2007. JCE, 33: 1336 “D4-Z3,Z6,Z9-21:H was injected into pupae. No label was incorporated into 1,Z3,Z6,Z9-21:H, indicating that the terminal double bond is introduced earlier in the biosynthetic pathway.” Tetraenyl acid intermediate was not detected in our study. Is the step of desaturation linked effectively with decarboxylation? 2) Biosynthesis of even numbered triene in Erannis bajaria (Geometridae) Goller et al., 2007. JCE, 33: 1505 “D4-Z3,Z6,Z9-18:H was detected after treatment of D4-trienyl C19 or C20 acid. The result indicates that the C20 acid is shorted by -oxidation to C19 acid, which can be reduced to an aldehyde and decarbonylated or decarboxylated to the pheromone component.” Biosynthesis of odd numbered triene has not been experimentally confirmed. Where is decarboxylation carried out? Acknowledgments Co-workers Dr. M. Muramatsu The Ayamihabiru Museum, Okinawa, Japan Dr. H. Naka JT Biohistory Research Hall, Osaka, Japan Dr. Y. Kondo Gifu University, Gifu, Japan Chemical Ecology Laboratory of TUAT Dr. M. Yamamoto K. Matsuoka MD. A. Islam R. Yamakawa N. D. Do Y. Adachi A. Yasmine R. Kiyota.