JapaneseJapaneseSociety Society ofAppliedEntomologyof Applied Entomology and Zoology

Appl. Ent, Zool.26 (1): 55-62 (1991)

Sex Pheromone of the Fruit-Piercing , eJtcavata (BuTLER)(: Noctuidae): Isolation and Identification

Yoshihisa OHMAsA,i Sadao WAKAMuRA,2,3 Shtiji KozAi,2 Hajime SuGiE,4 Michio HoRirKE,5 Chisato HiRANo5 and Sukekazu MoRii,6

i Ehime fib'uit TVee Esperiment Station, Shimoiclai, Mketsayama, Ehime 791-Ol, Japan L' Shikoku IVdtional Agriculturag ltweriment Statien, Zentstiji, 1(aga:va 765, Jopan 4 Ibaraki Japan Araiionat institute ofAgro-Environmental Stiences, TIsrkuba, 305, 5 Kbchi JaPan Dopartment ofrigricultural CVicnzistry, Kbchi U}tiversity, IVlrnhoku, 783,

(Reccivcd March 7, 1990; Accepted September 4, 1990)

Two compounds were isolated, as sex phcromone components, from the abdominal tips

.etl..E...1. tL".t". -:.-n:...."An;1.n-nA.:m rlt.pth-hlnrPTt".w.nl TT-e m-;A- s.vulvv"v"Lpnryithn-lntl+ 4.A ura".iuJ Ul UtC ICIIItLIC LIULL-PlelLISi5 111VLn) wlutiasu elleutovubte vJulb-rLl. IllL iuLt]ul was tentatively identified as cis-9,10-epoxy-(Z)-6-heneicosenc. The minor componcnt B characterized as cis-9,10-epoxy-(Z,Z)-3,6-heneicosadiene. They were fbund in a ratio of 86:l4 in t'emale tip extracts. Natural components A and B in thc natural ratio released but they male precopulatory bchaviors including orientation flight and extension of clasper, showed weak pheromone activities when applied separately. In contrast, synthesized racemic component A, with and without synthetic component B (in ratios from 85:15 te IOO;O), elicited a typical male prccopulatory behavior, Synthctic component B alone did not initiate male response, Kay words: fruit piercing moth, Oraesia excavata, sex pheromone, cis-9,10-epoxy-

INTRODUCTION

The fruit-piercing moth, Oraesia exeavata (BuTLER), is one of the most serious pests of various fruits such as peach, orange, grape, pear, and tomato, in Japan. Larvae feed on neither fruit nor fruit tree, but on a weed, Cocculzcs trilobus DC (Ranales: ), grown in hillsides near orchards. Adult migrate nocturnally into orchards, pierce ripe fruits with their acute proboscis, and suck fruit sap. The iajured fruits decompose and soon drop from the trees. Because ofspatial separation offeeding place of adult moths and their main habitat, it has been diMcult to establish the efli ctive contrel system against this particular pest, Application of attractive sex pheromone seems to be one of pessibly successfu1 control methods. This report describes the isolation and identification of the female

3 ?resent address: IVlationai institute of Sericuttural and Entomological Seience, TSukuba, foaraki 3e5} J4Pan 6 Prescnt address: Tlakedu Chemical industties, Ltd,, Dosho-machi, Chuo-ku, Osaka 541, Japan

55

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56 Y. OHMAsA et al.

sex pheromone of O. excavata.

MATERIALS AND METHODS 'Z a r' a a M a t' '3,"" '1#,ffk " '{ cb,l,:':,II'11'igitP' c" fi,G.:･i - ,2 ,tt ,' i.llSiei,t/ ,::,11,k ;Ecg.iii'S/cl2bt,/S.I./f,l btsiC,t,lii.`"'ii? emergenc.e. Aduit //X moths X, were kept in the same rg,Xl,//, cages and provided several slices of apple fruit as food until use. f " Ycl h e S W S C a O y 'e" Z 'e'6,/E' '1'' ` v i /'A' '' /is･ o/j ,:,in ' lil/･2 dii/illl/l l..℃, .･ ,? //X,i //: II//illlli11/i,ltl..iihi,k ,,i. , 4"gX,gi.lrl iarlileil,i.iillli,l/iffifi' e k #n eSci\t:oE, M･li,1:e h S iilk le Wtsa e-,//2,:ii//ifiilil d･:nelc,t,e,rinv//ilg,h 'qZul:ll#-i

?,p,tm,i{PplLeZ,Pe:L!ta.:` t .dS, : .o,pM,ri:altl"" /gl.,lill(Ei, lFg: :aip///". T,he.n,XM.R:r,?f.mskd,Ilil21e7,Ztg,ra,c:dwdLtS,`2O,kZ.::a.n,g3,,fr,?･.mih.e.ww".,":g5gd.u,'l:g lure in each assay, All the field reference assays were conducted 1 to 2 hr after sunset m summer season.

.,,,,.Prf{:::1'gitison.:f,,ehe.'zm.:"g,sstg,acii.･,.Ei./rsta,b,F,2trya,i,`Ms.?o-m,,t-,fo..6,-dgus.ig

a Piece of absorbent cotton･ The extract liquids 72ooc ::Iititlh:geU.gh were stored at Lieuidchromatogropdy. For class separation ofthe lipoidal extract, column chroma- ?o,gr,a,p)Py7ik.Fgzrg'figew7,ZOi2I,";s,ht;6Elog.i/irgC,,ora'l,vt,s,cc:rLeg.z".t,azc,?Edg?,g,;o.C,c",R,o.kL. cessively used as eluting solvents. F ` g P fb Y P io ' i k ' t g /? - ･ ･ ･ ･ - op ig.tl ･ , i3 i3 , li.tii Iag/ie,l,,,, ,, , ,:g/ li. ,,: /!,,/s i,ii･l, ,, iiE. ,,: ,,,tg;iiili£i1:#x,tiee/ec, Gas chromatogrciph-mass I/,il spectrometer Mass spectra were measured ii (GC-MLg). with

aJEOLJMS- eeD300 mass spectrometer combincd with aJEOLJGC-20KP gas chroma- C S a - a a / C2:i,F:' a r a t' 2rii･'tir " ' e /,,,:' 'r ',! '' ' "" c {"T:,oio ,fj ,IX,i /: /i,/..r,Tk .･Xi, ,i,1. 6a :,lgl'S: iPii:32,,:. . /l 1iSii,2i,'1:si/'e. ..gsS] was 1si:

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Sex Pheromone of the Fruit-Piercing Moth 57

Gas chromatogrophic analysis of geometricat isomers. The GC-MS equipped with a SCOT column (Silar-leC, O.28 mmx30 m) and a Shimadzu GC-8A gas chromato- graph equipped with a fused silica capillary column (HR-1701, O.2 mm × 17 m), were used for determination of geometrical isomerism. In certain analyses, eicosane, do- cosane, and tetracosane were used as internal standards for calculation of retention values relative to the straight-chain hydrocarbons (equivalent chain length (ECL) values). Oxidotiwe cleavage of opoxide. Oxidative cleavage with periodic acid was carried out to determine the position ofepoxide (FiEsER and FiEsER, 1967). To 10 pt1 ofhexane solution ofthe sample in a 2 ml-tapered vial was added 10 pt] ofO.1% periodic acid in tetrahydrofuran. The mixture was iajected to the GC-MS within several minutes. Diimide reduction, The monoenyl component was hydrogenated as in YAMAoKA et al. (1976) to determine the geometrical isemerism of the epoxy group, To about 10 paI of hexane solution of the sarnple in a O.5 ml-tapered vial was added 20 pt1 of hy- drazine solution cembined with 20 ul ef hydrogen peroxide solution (N2H4t l.5 rnl of hydrazine hyclrate in 50 ml ethanel, H202: O.2 ml of 31% hydrogen peroxide in 50 ml ethanol). The mixture was maintained at 500C in a water bath to acceierate

the hydrogenation reaction for 4 hr, and then analyzed with the GC-MS equipped with a glass capillary column by applying the mass fragmentographical technique for some characteristic ions, Reductive ozonoC3,sis, Micro-ozonolysis was carried out as in BERozA and BmRL t- tt -t ht t - ny ny ------n- ri A Uxygen

were synthesized by epoxidation of the corresponding alkenes with m-chloroperbenzoic acid. (E)-9-Heneicosene was synthesized by cis-trans isornerization reaction (SGouTAs and KuMMERow, 1969) ofthe corresponding (Z)-isomer obtained from Sigma Chemical Co. Ltd.

RESULTS

isotation of active components The accumulated hexane extracts of about 700 females were fractionated on

Florisil column. Sex stimulant activity for males appeared in the fraction eluted with

5% ether in hexane. GC-EAD analysis of the active fraction indicated that 2 distinct peaks on the FID chromatogram corresponded to potent EAD responses (Fig. 1, Peaks A and B), Com- ponents A and B, corresponding to the GC-peaks A and B, were successfu11y isolated by HPLC with a porous silica bead column. Seventy-eight ug ef component A and 12 ptg ofcompenent B were determined by GC. Thus, the ratio of components A and B in partially purified female extract was estimated to be 86: 14, and the total amount ef them in a single female moth was estimatcd to be 120-130 ng.

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58 Y, OHMAsA et a[.

Solvent

e zlnv e EAD ii l

ABFID

p m O 5 ],O 15 20 25

Rt [inin) 1. Fig, GC-EAD profile of female extracts of Oraesia excavata. One flemale equivalent of 5U/, ether-in-hexane fraction ft'om Florisil eolurnn was used, GC column: Silar-10C SCOT' (O,28 mm l.d,× 30 m)i operated frorn 40 to 1800C at a rate 7.50Cfmin,

Table 1. Male response to isolated components A and B, and synthetic cis-9,10-epoxy-(Z,Z)-3,6-heneicosadiene t tt t-----tt ttttt

Treatmenta Male responseb tt -ttt tt -tt t ...... t.... .t .. t...... -... ComponentA 19% CemponentB 6 Components A+B (86:14) 80 Components A+B (synthetic)c (86:14) 87 Virgin female extract 88

a One female equivalent dosc, b About 40 males tested,

c Syntheticcis-9,10-epoxy-(Z,Z)-3,6-heneicosadiene.

Table 2. Male attraction to isolated components A and B, and synthetic cis-9,IO-epoxy-(Z,Z)-3,6-heneicosadiene

No. of males attracted a'u' it7g" 15 min Treatmenta Test#l Test #2 Test #3

Component A 7

Component B 452'! Components A+B {86 :l4) Components A+B (synthctic)b (86:14) 2455 2916 Virgin female extract

a Five female equivalent dose. b Synthetic cis-9,10-epoxy-(Z,Z)- 3,6-heneicesadiene,

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Sex Phcromone ofthe Fruit-Piercing Moth 59

Biological activities ofthe isolated components are given in Tables 1 and 2. Their activities as male attractant and stimulant were comparable to the crude female extracts

when mixed at the natural ratio 86: 14, and were weak when applied individualiy. These results confirmed that the isolated components A and B were certainly active components of the multicomponent sex pheromone of female O. excavata.

Idlantijication ofcemponent B Mass spectrum of cQmpon ¢ nt B is identical to that of authentic cis-9,IO-epoxy- (Z,Z)-3,6-heneicosadiene (synthetic B) (Fig. 2). Upon GC analysis on a Silar-10C capillary column, component B and synthetic B showed the same retention time and gave a single peak when co-chromatographed. The mixture of component A and synthetic B were as potent in terms of attractant and stimulant activitics as the mixture ofnatural components A and B (Tables 1 and 2). Therefbre, component B was tenta- tively identified as cis-9,10-epoxy-(Z,Z)-3,6-heneicosadiene.

ldentification ofconiponent A Mass spectrum ef component A isquite similar to thatof componentB (Fig. 2).

,oo 55 A l tr ! 1]10 IS3E

/ 2g.o 197･ -308M･-･ I'/eEiFu],a1

o ' 10D 50 leo179 l5e200 250 3ee

b,ut=2.:.y-:-rdset].es B

lF 183151 2ss 30S 197 Mr f I, eIOO 1 5U 1007g. lrJezoe2rJo3oe

108 c

E 183151 288306 ' 197 .1･[' 1 o : 13oe so loe ].se 2.oe 2so ･snl'g

Fig. 2. Mass spectra ofsex pheromone component$ A (A)and B (B), andol' authentic cis-9,IO-epoxy-Z,Z)-3,6-heneicosadiene (C).

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60 Y. OHMAsA et al.

Molecular ion mk S08 suggests a mono-unsatu/rated epoxide with a straight chain of 21 carbon atoms, n-Dodecanal was detected by GC-MS in the oxidative cleavage prod- ucts, indicating that the epoxy group was located at 9,10-position in the carbon chain, An epoxyheneicosane was detected by GC-MS in the diimide reduction products. The calculated ECL value (25.le) is identical to that of authentic cis-9,10-epoxy- the heneicosane (25.10), but diflbrent firom authentic trans-isomer (25.19), indicating geometry of the epoxy guroup is cis-configuration. GC-MS analysis showed presence of 2 preducts, n-hexanal and cis-3,4-epoxypenta- is located decanal, in the ozonolysis preparation. This indicates that the double bond at the 6-position. The ECL fbr component A (23.38) calculated from GC retention time on an HR-1701 fused silica capillary column is comparable with that for synthetic cis-9,IO-epexy-(Z)-6-heneicosene (23.37), but diflerent from that for the (E)-isomer (23.49). Thus component A is identified as cis-9,IO-epoxy-(Z)-6-heneicosene,

Pheromone activity ofsynthetic conipouncts' Sex stimulant activity of the synthetic compounds, cis-9,10-epoxy-(Z)-6-henei- cosene (synthetic A) and cis-9,10-epoxy-(Z,Z)･･3,6-heneicosadiene (synthetic B), was determined at various mixing ratios at 1 ng level (Table 3). Potent activity was obtained at ratios from 95: 5 to 85: 15. Synthetic A alone elicited typical male pre- copulatory behavior. Synthetic B did not initiate any male response. Sex attractant activity was evaluated at the 10 ptg level in a peach orchard (Table 4). Synthetic A alone and binary mixtures of synthetic A and B, at ratios from 95: 5 to 85: 15, attracted more male moths than the virgin female extract. By itse14 syn- thetic B did not attract males.

DISCUSSION

the fruit-piercingrnoth, In the present work, 2 components of the sex pheromone of Oraesia excavata, wcre isolated from abdominal tips of virgin females. The major

Table 3. Male responsc to synthetic sex cornponents A and B ---t pheromone at varlous mlxlng ratlos ' ttt ttt -tt tt-ttt ...-r " ttt- t " Ratio Maleresponseb '--' 'loo,o (A:Eb'i ttt-tttt tt t t .... - .. tttt- 71% gs:s 93 go: lo 88 85: l5 88 80: 20 70 70: 30 62 so: so 31 30: 70 21 O:100 O Virgin female extractC 97 'T'A' 'cis-g;1-)re'bi'y'-(2,z)-s,6-heneicos'adien'e'l : ct's-g,'ib-epoxy-(z)-6-fieneicoscne, i-: £ Dpse: 1 ng as a mixture. b About 40 males tested. e Dose: O.OI female equivalent (about 1 ng as,pheromone components).

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Sex Pheromone of the Fruit-Piercing Moth 61

Table 4. Male attraction to synthetic sex pheromone components A and B at various mixing ratios

Rclativc No, of rnales attracted Ratio (A:B)a #lb#2 #3 #4 #5 #6 #7 #8 Mean

EOO: O95: 131 111 12I1128379

590; 115, 108

IO85: 5342 113 33117 133

1570:

3050: l5 o o 5

50 23 o o 8 o:loe o100 o100(I3) o100

Virgin female extracte 100(13) 100(19) leo(13) 100<14) 100 100 'aA: (8) (6) <9) cis-g,lo-epox"-(z):6-henEi'cosene, B: cis-gllo-epoxy-(z,z)-S,6-heneicosadiene, Dose: 10 ptg as a rnixturc, b Test No･ e Ten femalc equivalent; numerals in parentheses showing actual nurnbers of males attracted to the extract.

compQnent was characterized as cis-9,10-epoxy-(Z)-6-heneicosene. The minor com- penent was tentatively identified as cis-9,IO-epoxy-(Z,Z)-3,6-heneicosadicne, although its positional and geometrical isemerism should be reconfirmed in further studies. The former compound, ais-9,10-epoxy-(Z)-6-hencicesene, was synthesized by RoLuN and PouGNy (1986) as a possible sex pheromone component of an Arctiid species, the ruby tiger moth (Phragmatebiafatiginosa), The latter onc has been reported by HiLL and her co-workers (1981, 1982) as a component of sex pheromenes of twe Arctiid species, the saltmarsh caterpillar rnoth (Estigmene acrea) and the fa11 webworm moth (H)/phantria cunea), In addition to Arctiidae, epoxide compounds have been fbund in Lymantriidae (BiERL et al., 1970) and Geometridae (BEcKER et al., 1985) as sex pheromone com- ponents. Male attractiveness of some epoxides were also reported for Geometrid and Nuctuid moths (WoNG et al., 1985). However, it can be said that epoxide compounds distribute around only limited groups of Lepidoptera as sex pheromone components. Optical isornerism exists in asymmetrical epoxides. In the gypsy moth (lymantria dispar), (7R,8S)-7,8-epoxy-2-methyloctadecane is the natural sex pheromone (IwAKi et al,, 1974). HiLL et al. (I982) demonstrated that the 9S,IOR-isomer of cis-9,10-epoxy- (Z,Z)-3,6-heneicosadiene elicited a response 2 to 3 times greater on male antennae than the 9R,leS-isomer in EAG tests on U>Phantria cunea and Estigmene acrea. In our experiment, the racemic cis-9,IO-epoxy-(Z)-6-heneicesene showed potent pheromone activities by itself (Tables 3 and 4), while the natural component A alone did only weak activities (Tables 1 and 2). Optical isomerism would possibly modify the activity of the synthetic pheromone, and, at least the optical counterpart would not affbct adversely on the pheromone activity. Synthesis of 4 optical isomers of 9,10- epoxy-(Z)-6-heneicosene is now in final steps, and the chilarity of the natural isomer will be determined in near future. Thc synthetic monoenyl epoxy compound, with and without synthetic dienyl epoxy compound, showed a potent attractant activity to wild nales in the field (Table

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62 Y. OHMAsA et ai.

4), suggesting itsusefulness for monitoring andlor controlling this economically lmpor-

tant pest.

ACKNOWLEDGEMENTS

We are gratefuI to Dr. Y. TAMAKi of thc National Institute uf Agro-Environmental Sciences for his hclpful advice throughout this study and for reviews on the rr)anuscripi,, and to Dr, Y. NINoMIyA of the

Nitto Electric (ndustrial Ce, for kindly supplying authentic cis-9,10-epoxy-(Z,Z)-3,6-h eneicosadiene,

REFERENCES

'I'. BEcKER, D., I. MooRE, KiMMEL, ]{. CyJoN, A. CossE ,and M. WysoKI <1985) Sex attractant com- pounds ofthe giant looper, Boarmia selenaTia. Pdytoparasitica 13: l50. BERozA, M. and B. A. BiERL C1967) Rapid determination of olefin position in organic compounds in microgram range by ozonolysis and chromategraphy. Anal. Ckem. 39: 1131-1135. of the moth: BmRL, B, A., "{. BERozA and C. W. CoLLiER (1970) Potent sex attractant gypsy Its ise- lation, identification, and synthesis, S27ience 170: 87-S9. CARRoLL, K. K. (1961) Separation oflipid classes by chrornatograph on blorisil, J. LiPid Res. 2: IB5- 141. FiEsER, L. F. and M, FmsER (1967) Reagentfor Organic S:vnthesis,John Wiley & Sons, Inc., New York, l457 pp, HiLL, A, S. and W. L. RoELoFs (1981) Sex pheromonc or the saltmarsh caterpillar moth, Estigmene acrea, J. Chem. Ecei. 7: 655-668, HiLL, A. S., B, G. KovALEv, L, N, NiKoLAEvA and W. L, RoELoFs (1982> Sex phcromone o{' the fa11 webworm moth, IijtPhantria cunea. J. (:hem. Ecol. 8: 38S-S96. IwAKi, S,, S. MARuMo, T, SAiTo, M, YAMADA and K. KATAc]iRT (1974) Synthesis and activity ofoptically active disparlurc, J, Am, Chem. Soc. 96:7842-7844, OHMAsA, Y. (I991) Fruit-piercing moths, Oraesia excavata, O. emarginata, and Adi'is t7rannus. In Rearing Mlrthods of imects (T. YusmMA, S. KAMANo and Y. TAMA-, eds.) , Japan Plant Protection Association, Tokyo (in press), RoLuN, P, andJ. R, PouGNy (1986) Synthcsis of (6Z)-cis-9S,10R-epoxyheneicosene, a component of the ruby tiger moth pheremonc. 71ftrahedeon 42: 3479-S490. SGouTAs, D. S. and F. A. KuMMERow (1969) Cis-trans isomcrization ofunsaturated fatty acid methyl esters without double bond migration. LiPids 4: 283-287. STRuBLE, D. L. and H. ARN (1984) Combined gas chrornatography and electroantennogram recording ofinsect olfactory responses. In Tlechnieues in Pheromone Research (H, E. HuMMEL and T, A. MiLLER, ecls,). Springer-Verlag, New York, pp. 161-178. WoNG, J. W,, E, W. UNDERmLL, S, L, MAaKENziE and M, D, Cmsfiomf (1985) Sex attractants for geometrid and noctuid moths: Field trapping and electroantennographic responses to triene hydro- carbons and monoepoxydiene derivatives, J. C7iem, Ecot, 11: 727-756. YAMAoKA, R., H. FuKAMi and S. IsHii (1976) Isolation and identlfication ofthe female sex pheromone of the potato tuberworm moth, Rhthorimaea operculella

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