The 3rd International Symposium on Pheromones Sweden, 2003 May

Session III Biosynthesis of Epoxyalkenyl Sex Pheromones: Biosynthetic Pathway, Substrate Specificity, and Endocrine Control

Tetsu ANDO Tokyo University of Agric. & Tech. Graduate School of BASE I. Biosynthetic Pathways for Lepidopteran Pheromones

Tipe I （Acetate of unsaturated fatty alcohol） Anadevidia peponis Acetyl CoA ∆11-desaturation 16:Acyl Z11-16:Acyl

Z9-14:Acyl reduction 12 7 1 O β-oxidation acetylation O Z7-12:Acyl Z7-12:OAc

Type II （Epoxide of unsaturated hydrocabon） A. s. cretacea

Z9,Z12,Z15-18:Acyl decarboxylation epoxidation Z11,Z14,Z17-20:Acyl Z3,Z6,Z9-19:H epo3,Z6,Z9-19:H 1 O 69 19 3 II. In Vivo Experiments with Geometrid ① Ascotis selenaria cretacea Japanese giant looper GC-MS analysis of the Pheromone Gland Extract from the Females Treated Proposed biosynthetic pathway 18 1215 9 1 with D3-Z3,Z6,Z9-19:H CO-SR B A D3-Z3,Z6,Z9-19:H

Acetyl-CoA 20 17 14 11 1 CO-SR

13 6 9 - CO2

19 Z3,Z6,Z9-19:H O epoxidation A) epo3,Z6,Z9-19:H (endogenous) epo3,Z6,Z9-19:H

B) D3-epo3,Z6,Z9-19:H (exogenous) II. In Vivo Experiments with Geometrid Moth ② (A) Specificity of epoxidation in A. s. cretacea Z3,Z6,Z9-19:H Z6,Z9-19:H Z3,Z6,Z9-17:H Z4-19:H no change Z3,Z6,Z9-22:H 3,4-epoxides E3-19:H Z3,Z6-19:H Z2-19:H Z3-19H, Z3-17:H Epoxydase Attacked only (Z)-3-double bond without the substrate specificity on the carbon length and unsaturation degree Strict formation of hydrocarbons Species-specific pheromones (B) Other steps Identification of Z11,Z14,Z17-20:Acid unsuccessful Incorporation of D4-Z11,Z14,Z17-20:Acid Reductase? Z9,Z12,Z15-18:Acyl Z3,Z6,Z9-18:H epo3,Z6,Z9-18:H ? Decarboxylase? Epoxydase Z11,Z14,Z17-20:Acyl Z3,Z6,Z9-19:H epo3,Z6,Z9-19:H III. Analysis of Hemolymph Hydrocarbons

(A) Detection of pheromonal branched-hydrocarbons in hemolymph Homomelina aurantiaca Me2-17:H Schal et al. (1998) (Arctuiidae) Scoliopteryx libatrix Z6,Me13-21:H Subchev & Jurenka (2001) (Noctuidae) Limantria dispar Me2,Z7-18:H Jurenka & Subchev (2000) (Lymantriidae) (precursor of disparlure)

(B) GC-MS analysis of hemolymph from geometird females

A. s. cretacea Z3,Z6,Z9-19:H 25:0 Column: DB-23 19:3 23:0 Temperature: 50 ℃(2 min), 17:0 10 ℃/min (11 min), 16:0 4 ℃/min (15min), (1/4 FE) 18:0 27:0 220 ℃(10 min) 19:0 21:0 15:0 24:0 20:0 14:0 22:0 26:0 29:0 28:0 TIC

10.0 15.0 20.0 25.0 Rt (min) IV. Biosynthetic Sites of Epoxyalkenyl Pheromones

(A) Study with the gypsy moth Jurenka et al. (2003) Proc. Natul. Acad. Sci. USA, 100: 809

Oenocyte Pheromone gland Alkenyl precursor Epoxy pheromone lipophorin

(B) Future works with geometrid Role of Oenocyte and Mechanism of Polyalkene Biosynthesis Analysis of fatty acids and hydrocarbons in oenocytes Incorporation experiments with incubated oenocytes Polyalkene Uptake by Pheromone Glands Analysis of lipophorin In vivo and in vitro experiments for uptake specificity Endocrine Control System V. Endocrine Control of Epoxyalkene Biosynthesis geometrid female Oenocyte Pheromone gland

O CO2H lipophorin

Exp. 1 Occurence in pheromone gland PBAN in vivo Z3,Z6,Z9-19:H epo3,Z6,Z9-19:H Decapitaion ○× Activation of Bom-PBAN injection ○○ epoxidation Head-extract injection ○○ Pheromone gland from decapitated female Exp. 2 Production of in vitro Treatment Z3,Z6,Z9-19:H PBAN epo3,Z6,Z9-19:H Activation of ＋ (topical) ＋○ ＋ (topical) －○ epoxidation Decapitated female Exp. 3 Production of in vivo Treatment Z3,Z6,Z9-19:H PBAN epo3,Z6,Z9-19:H Activation of ＋ (injection) ＋○ ＋ (injection) －× uptake by gland VI. Evolution of Biosynthetic Systems Hybrids of Type I and Type II Pheromones Estigmene acrea (Arctiidae) Achaea janata (Noctuidae) Z3,Z6,epo9-21:H Z3,Z6,Z9-21:H + Z9,Z12,Z15-18:Ald + Z6,Z9-21:H + 21:H + Z9,Z12-18:Ald (27:6:1) + Z9,Z12-18:Ald Neoleucinodes elegantalis (Pyralidae) pulverea (Lymantriidae) E11-16:OH Z11,Z14,Z17-20:OisoBut + Z3,Z6,Z9-23:H (20:1) + Z11,Z14,Z17-20:OMe4-Val (2:1)

Arctiidae Lymantriidae Noctuidae epo9,Z12,Z15-18:Ald Sphingoidea Noctuoidea epo8-12:OH Bombycoidea Papillionoidea Geometroidea Pyraloidea PterophoroideaHesperioidea epo11-16:OAc Zygaenoidea Gelechioidea Tortricoidea Yponomeutoidea Cossoidea Castnioidea Undiscovered Tineoidea Hepialoidea Incurvarioidea Nepticuloidea Ditrysia Epoxydase is a young Eriocranioidea Monotrysia Zeugloptera Phylogenetic Tree enzyme achieved by the Dacnonypha of highly evolved species. Paramecoptera Acknowledgments Chemical Ecology Laboratory of TUAT

1) Study on substrate specificity of epoxidation T. MIYAMOTO, Dr. M. YAMAMOTO, A. ONO, K. OHTANI

2) Study on polyunsaturated hydrocarbons in hemolymph W. WEI, T. MIYAMOTO, M. ENDO, T. MURAKAWA G.-Q. PU

I love experiments. I can find oenocyte.