GC / FT-IR analysis of a novel 2,4,6,9-tetraene occurring in a female pheromone gland of Parasemia plantaginis (Lepidoptera: Arctiidae)

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1 2017 ISCE/APACE Kyoto, Japan (August 24, 2017) GC / FT-IR analysis of a novel 2,4,6,9-tetraene occurring in a female pheromone gland of Parasemia plantaginis (Lepidoptera: Arctiidae) Yuta Muraki, Hideshi Naka, Atsushi Honma, Johanna Mappes, Kaisa Suisto, and Tetsu Ando* * Tokyo University of Agriculture and Technology, JapanJapan JICA, Can Tho University, Vietnam antetsu@cc.tuat.ac.jp (cm -1 )

2 Representative lepidopteran sex pheromones Type I silkworm moth smaller tea tortrix rice stem borer bombykol Sex pheromones have been identified from more than 670 species. Male attractants have been reported for other 1300 species. H Ac A c H H Unsaturated fatty alcohols, acetates and aldehydes with a C 10 C 18 chain Found most commonly (75%) Type II Polyunsaturated hydrocarbons and their epoxides with a C 17 C 23 chain Identified from evolved-insect groups (15%) Z3,Z6,Z9-21:H plum cankerworm moth epo3,z6,z9-19:h Milionia basalis Z3,epo6,Z9-19:H giant geometrid moth Photos from

3 Phylogenetic tree of Lepidoptera plum cankerworm moth Z3,Z6,Z9-21:H Z3,Z6,Z9-23:H Bombycoidea Geometroidea Sesioidea Zygaenoidea Gelechioidea Yponomeutoidea satin moth Z3,epo6,epo9-21:H Noctuoidea yellow peach moth E10-16:Ald Z3,Z6,Z9-23:H Papillionoidea Pyraloidea Hesperioidea Pterophoroidea Tortricoidea Cossoidea Castnioidea Tineoidea Hepialoidea Eriocranioidea Zeugloptera Ditrysia Monotrysia Dacnonypha Paramecoptera Incurvarioidea Nepticuloidea Type I pheromone Type II pheromone

4 Chemical structures of Type II pheromones Linoleic acid H desaturation Z6,Z9-dienes E4,Z6,Z9-trienes Z6,Z9,E11-trienes Z6,Z9,Z12-trienes Δ12 secretion monoepoxides Δ4 Δ11 C n H 2n+1 emerald moth Linolenic acid H desaturation Z3,Z6,Z9-trienes 1,Z3,Z6,Z9-tetraenes Z3,Z6,Z9,E11-tetraenes Z3,Z6,Z9,Z11-tetraenes secretion mono- and di-epoxides Δ1 Δ11 C n H 2n+1 The known chemical diversity is still limited. Novel compounds must be found.

5 Noctuoidea species and their sex pheromones Taxonomy Super-family Family Sub-family Noctuoidea *Erebidae Notodontidae Nolidae Noctuidae Lymantriidae* Arctiidae* <Trifinae> <Quadritinae>* Lithosiinae Syntominae Arctiinae Parasemia plantaqinis, wood tiger moth (Arctiidae; Arctiinae) Four subspecies are recorded in Japan. P. p. macromera P. p. melanissima P. p. jezoensis P. p. melanomera Type of pheromone I II I II, others II, others II, others II II, others Japanese species number 124 [0] 107 [0] 787 [34] 489 [4] 59 [16] 79 [4] 4 [1] 51 [3] [ ] indicate number of species whose sex pheromone has been identified.

6 GC-EAD analysis of the pheromone extract Parasemia plantaqinis, wood tiger moth (Arctiidae; Arctiinae) Four subspecies are recorded in Japan. P. p. macromera P. p. melanissima P. p. jezoensis P. p. melanomera Comp. I Z3,Z6,Z9-21:H Comp. I Comp. III Comp. II Crude pheromone extract (1.0 FE) Comp. IV EAD GC column: DB-23 (0.25 mm X 30 m) Temperature program: 80ºC (1 min) 210ºC (10ºC/min) FID 0.1 mv Mixing ratio I : II : III : IV 30:3:5: Rt (min)

7 GC / MS analysis of the pheromone extract Comp. II E4,Z6,Z9-21:H I TIC Crude pheromone extract (1.0 FE) II III IV Rt (min) GC column: DB-23 (0.25 mm X 30 m) Temperature program: 80ºC (1 min) 210ºC (8ºC/min) 0 Comp. III m/z ,Z3,Z6,Z9-21:H M M m/z Comp. IV 2,4,6,9-tetraene? M GC / FT-IR m/z No information on the configurations

8 Merits of IR analysis -H trans cis H Functional group H Carbon chain length 16 Double bond Number 2 Position 10, 12 Configuration E, Z Determined by the IR spectrum, if a large amount of the pure pheromone is available. IR analysis is not utilized, because the species-specific pheromones are composed of multiple components, which are produced at μg level, at most.

9 GC / FT-IR Capillary GC High separation FT-IR (solid phase) High sensitivity Familiarized spectrum Lq. N 2 Discover IR <Spectra Analysis, Inc.> GC capillary IR beam Detector ZnSe disk cooled at 30ºC

10 IR spectra of four EAG-active components Measured by GC / FT-IR Crude extract (4 FE) Comp. I Disk temperature: 30ºC Disk speed: 2 mm/min Z3,Z6,Z9-21:H Comp. II E4,Z6,Z9-21:H Comp. III ,Z3,Z6,Z9-21:H Comp. IV Conjugated Z,Z-diene Z2,E4,Z6,Z9-21:H (cm -1 ) Comp. IV includes E,Z conjugated systems, and no Z,Z conjugation.

11 Synthesis of 2,4,6,9-tetraene Br Br _ Br Ph 3 + P + P Ph3 Br _ H H Ph 3 P PPh 3 H Poor yield (< 5%), but a very simple route

12 GC / FT-IR analyses of natural and synthetic 2,4,6,9-tetraene [Peak chromatogram] I III II Crude extract (4 FE) IV E2,E4 Crude extract Synthetic Comp. IV good coincident Z2,E4,Z6,Z9-21:H Synthetic Z2,E Rt (min) Column: HP-5 (0.25 mm X 30 m) 2921 Synthetic E2,E4,Z6,Z9-21:H (cm -1 )

13 Summary & discussion 1) Four EAG-active components were found in the pheromone grand of P. p. melanomera and a Finnish subspecies. 2) GC/MS and GC/FT-IR analyses showed the following structures. Comp. I Z3,Z6,Z9-21:H ( linolenic acid) Comp. II E4.Z6,Z9-21:H ( linoleic acid) Comp. III 1,Z3,Z6,Z9-21:H ( linolenic acid) Comp. IV Z2,E4,Z6,Z9-21:H ( linoleic acid) Mixing ratio; I : II : III : IV 30 : 3 : 5 : 1 3) An IR spectrum of the minor Comp. IV measured by a high-sensitive GC/FT-IR instrument indicated its 2Z,4E configuration. 4) The structure of Comp. IV was confirmed by synthesis utilizing the double Wittig reaction. 5) Male attraction by synthetic lures in the field has not succeeded, indicating necessity of additional pheromone component(s).

14 Thank you for attention!! Present address: Can Tho University (CTU) in Can Tho City, Vietnam Chief advisor of JICA s project for Building Capacity for CTU I retired Tokyo University of Agriculture and Technology (TUAT) in I retired Tokyo University of Agriculture and Technology (TUAT) in I am grateful to all member of Chemical Ecology Laboratory of TUAT, I am grateful who studied to all member lepidopteran of Chemical sex pheromones Ecology Laboratory together. of TUAT for study of lepidopteran sex pheromones.