Received: 26 August 2011 /Revised: 3 October 2011 /Accepted: 24 October 2011 /Published online: 24 November 2011# Springer Science+Business Media, LLC 2011

Abstract The pear barkminer moth, Spulerina astaurotaMeyrick (Gracillariidae: Gracillariinae), is a harmful pest ofthe Asian-pear tree. Pheromone components of the femalewere analyzed by gas chromatography (GC) with anelectroantennographic (EAG) detector and GC coupledwith mass spectrometry. The analyses of a crude phero-mone extract and those of a fractionated extract on a Florisilcolumn indicated three EAG-active components,tetradecadien-1-ol, its acetate, and an aldehyde derivative.Characteristic fragment ions in the mass spectra of thedienyl compounds and derivatives with 4-methyl-1,2,4-triazoline-3,5-dione revealed double bonds at the 9- and 11-positions. By comparing the chromatographic behaviors tothose of four authentic geometrical isomers, which weresynthesized by three different routes starting from 1,8-octanediol or 1,9-nonanediol, the configuration of eachnatural component was assigned to be 9Z,11Z; i.e., it was

concluded that the S. astaurota females secreted (9Z,11Z)-9,11-tetradecadien-1-ol (Z9,Z11-14:OH) as a main phero-mone component, and the acetate and aldehyde derivatives(Z9,Z11-14:OAc and Z9,Z11-14:Ald) as minor compo-nents. This identification was confirmed by a fieldevaluation of the synthetic pheromone. While the malemoths could be attracted to a lure baited with Z9,Z11-14:OH alone, Z9,Z11-14:OAc showed a strong synergisticeffect on the attraction. Among the lures tested, the mixtureof alcohol and acetate in a ratio of 7:3 exhibited thestrongest attraction. Addition of Z9,Z11-14:Ald in themixture did not significantly increase the number of malesattracted. Furthermore, the field test indicated that somecontamination of a geometrical isomer of the alcohol didnot impair the activity of the binary mixture with the9Z,11Z configuration.

Key Words Female sex pheromone . Lepidoptera .

Gracillariidae . Gracillariinae . (9Z,11Z)-9,11-Tetradecadien-1-ol . 9,11-Dienyl compounds . Conjugated dienes .Maleattraction . Asian pear . Insect pest monitoring

Introduction

The pear barkminer moth, Spulerina astaurota Meyrick(Gracillariidae: Gracillariinae), is a harmful pest of theAsian-pear tree Pyrus pyrifolia in Japan, and also inhabitsKorea and India (Kuroko, 1982). The larvae mine the barkof stems, and feed within causing damage to the tree. Thetree atrophies and becomes susceptible to infection frominsects and diseases. The mining behavior of the mothprotects it from sprayed insecticide; therefore, the timing ofspraying is important for optimal effectiveness. The sexpheromone facilitates monitoring, and is a potentially

Electronic supplementary material The online version of this article(doi:10.1007/s10886-011-0032-3) contains supplementary material,which is available to authorized users.

N. D. Do : T. Ando (*)Graduate School of Bio-Applications and Systems Engineering,Tokyo University of Agriculture and Technology,Koganei, Tokyo 184-8588, Japane-mail: antetsu@cc.tuat.ac.jp

K. Ohbayashi :H. NakaLaboratory of Applied Entomology, Faculty of Agriculture,Tottori University,4-101, Koyama-minami,Tottori, Tottori 680-8553, Japan

K. NakadaHorticultural Research Center,Tottori Prefectural Agriculture and Forest Research Institute,2048 Yura-shuku,Hokuei, Tottori 689-2221, Japan

J Chem Ecol (2011) 37:1222–1230DOI 10.1007/s10886-011-0032-3

Identification and Field Evaluation of Sex PheromoneComponents of the Pear Barkminer Moth,Spulerina astaurota

Nguyen Duc Do & Kanako Ohbayashi & Hideshi Naka &

Ken Nakada & Tetsu Ando

useful mating disruptant for integrated pest management(IPM) of the moth in pear orchards. In Gracillariidae, sexpheromones have been identified from two Caloptilia, oneConopomorpha, and one Marmara species (Gracillariinae);one Cameraria and 11 Phyllonoryctor species (Lithocolle-tinae) and one Phyllocnistis species (Phyllocnistinae)(Ando, 2011; El-Sayed, 2011).

The components are mono-, di-, and trienyl compoundswith a functional group at the terminus of a C12–C16

straight chain. The positions of the double bonds vary, andseveral compounds with a characteristic polyenyl systemhave been reported as follows: a C14 4,10-diene inPhyllonorycter ringoniella (Sugie et al., 1986; Ujiye etal., 1986), C14 8,10-dienes in Cameraria ohridella (Svatošet al., 1999) and Phyllonorycter emberizaepenella (Mozûr-aitis et al., 2002), a C14 10,12-diene in Phyllonoryctercrataegella (Ferrao et al., 1998), C16 4,6,10-trienes inConopomorpha cramerella (Beevor et al., 1986), and a C16

7,11-diene and a C16 7,11,13-triene in Phyllocnistis citrella(Mafi et al., 2005; Leal et al., 2006; Moreira et al., 2006;Lapointe et al., 2006). Pheromones secreted by theSpulerina species, however, have never been investigated.We became interested in the pheromone structure of S.astaurota, and then found 9,11-dienes from the femalemoths. Results of field evaluation of synthetic pheromonecomponents appear likely to lead to a reliable monitoringmethod.

Methods and Materials

Insects and Pheromone Extract Larvae of S. astaurotaoverwinter and pupate in May. Pupae were collected fromAsian-pear orchards in Tottori Prefecture (Japan) from lateMay to early June 2009, and were reared in plastic boxes inthe laboratory (25±1°C, 16L-8D). After emergence, maleand female moths were sexed and individually placed into5-ml vials. Abdominal tips of fifty 2- to 4-d-old femalemoths were cut and extracted with hexane for 1 hr, filtrated,and kept in a freezer at −30°C until use. A portion of thefemale extract was used for bioassays and chemical analysiswithout purification. Column chromatography of an aliquotof the extract (30 female equivalents, FE) was conducted onFlorisil (500 mg) using 5, 10, 20, and 30% ether in hexane(3 ml each) as an eluant.

Derivatization of Pheromone Components A 25-FE aliquotof crude extract was dissolved in CH2Cl2 (10 μl), andtreated with a CH2Cl2 solution of 4-methyl-1,2,4-triazoline-3,5-dione (MTAD, 10 mg/ml, 40 μl) for 30 min at roomtemperature (Young et al., 1990). After changing the solventto hexane, the MTAD adducts were analyzed by gaschromatography coupled with mass spectrometry (GC-MS).

To confirm the structure determination of the naturalpheromone components, a mixture of authentic 9,11-tetrade-cadienyl compounds (250 ng each) was treated with theMTAD solution in the same manner as for the naturalpheromone, and the products were analyzed by GC-MS.

Analytical Instruments The extracts of females and syn-thetic compounds were analyzed by GC with an electro-antennographic (EAG) detector (GC-EAD). The GC wasequipped with a DB-23 capillary column (0.25 mm×30 m,0.25 μm, J & W Scientific, Folsom, CA, USA), and theeffluent from the column was split into two lines, which ledto a flame ionization detector (FID) and EAD at a ratio of1:1 (Inomata et al., 2005). The oven temperature wasmaintained at 80°C for 1 min, programmed at 8°C/min to210°C, and held for 10 min. Electron ionization (EI, 70 eV)GC-MS was conducted with an HP5975 mass spectrometersystem (quadrupole type, Hewlett Packard) equipped with asplit/splitless injector, and the same DB-23 column as theGC-EAD analysis or an HP-5 column (0.25 mm×30 m,0.25 μm, Hewlett-Packard, Wilmington, DE, USA). Theflow rate of the carrier gas (He) was 1.0 ml/min for all GCanalyses, and the temperature of GC-inlet was 220°C. Theoven temperature for all GC-MS analyses was the same asthat for GC-EAD except for the MTAD derivatizationexperiment, for which the oven temperature of adducts wasmaintained at 100°C for 2 min, programmed at 15°C/min to280°C, and held for 15 min. 1H and 13C NMR spectra wererecorded by a Jeol Delta 2 Fourier transform spectrometer(JEOL Ltd., Tokyo, Japan) at 399.8 and 100.5 MHz,respectively, in CDCl3 solutions containing TMS as aninternal standard.

Synthesis of 9,11-Tetradecadienyl Compounds Four geo-metrical isomers of C14 9,11-dienes were synthesizedstarting from 1,8-octanediol (1) or 1,9-nonanediol (5) asshown in Fig. 1. Experimental details are shown insupplements.

(9Z,11Z)-Isomers (Scheme A): After one hydroxyl groupof the commercially available diol 1 was protected with3,4-dihydro-2H-pyran, another hydroxyl group was iodizedby a treatment with a mixture of I2, triphenylphosphine(PPh3), and imidazole to yield a THP ether of 8-iodooctan-1-ol (2). A coupling reaction of 2 with lithium trimethylsi-lylacetylide in a mixed solvent of THF and HMPA anddesilylation of the product with potassium carbonate inmethanol produced a THP ether of 9-decyn-1-ol (3).Sonogashira-Hagihara coupling (Sonogashira et al., 1975)between 3 and (Z)-1-bromo-1-butene, which was preparedfrom (E)-2-pentenoic acid (Mori and Brevet, 1991; Mori,2009), furnished a THP ether of (Z)-11-tetradecen-9-yn-1-ol(4). 1H NMR analysis of 4 confirmed the Z configuration ofthe double bond at the 11-position; i.e., olefinic proton

J Chem Ecol (2011) 37:1222–1230 1223

resonating at δ 5.40 and 5.80 ppm showed a similarcoupling constant (J=10.5 Hz) as that usually detected for(Z)-isomers of disubstituted alkenes. The triple bond of 4was selectively reduced to a (Z)-double bond byhydroboration-protonolysis using dicyclohexylborane, anddeprotection of the produced THP ether yielded (9Z,11Z)-9,11-tetradecadien-1-ol (Z9,Z11-14:OH). 1H NMR δ ppm:0.99 (3H, t, J=7.5 Hz, CH3CH2), ~1.3 (10H, m), 1.55 (2H,m, CH2CH2OH), 2.17 (4H, m, CH2CH=CHCH=CHCH2),3.61 (2H, t, J=6.5 Hz, CH2OH), 5.44 (2H, m, CH=CHCH=CH), 6.23 (2H, m, CH=CHCH=CH), 13C NMR δ ppm:14.2, 20.8, 25.8, 27.5, 29.2, 29.4, 29.5, 29.6, 32.8, 62.9,123.0, 123.5, 132.1, 133.6. The acetate (Z9,Z11-14:OAc)and aldehyde derivative (Z9,Z11-14:Ald) were synthesizedfrom Z9,Z11-14:OH by treatment with acetic anhydride inpyridine and with pyridinium chlorochromate (PCC) inCH2Cl2, respectively.

(9E,11Z)-Isomers (Scheme B): Half protection of diol(5) with 3,4-dihydro-2H-pyran and Swern oxidation of theremaining hydroxyl group produced a THP ether of 9-hydroxynonanal (6). A bromide derived from (Z)-2-penten-1-ol was converted to (Z)-2-pentenyltriphenylphosphoriumbromide by heating with PPh3. The aldehyde 6 was coupledwith the ylide, which was prepared from the phosphoniumsalt by the treatment with butyllithium (BuLi), to yield aTHP ether of 9,11-tetradecadien-1-ol (7). NMR analysis

revealed that 7 was a mixture of (9E,11Z)- and (9Z,11Z)-isomers in a ratio of about 1:1. After deprotection, Z9,Z11-14:OH and the (9E,11Z)-isomer (E9,Z11-14:OH) wereseparated by column chromatography utilizing silica gelimpregnated with AgNO3. NMR data of E9,Z11-14:OH; 1HNMR δ ppm: 0.99 (3H, t, J=7.5 Hz, CH3CH2), ~1.3 (10H,m), 1.56 (2H, m, CH2CH2OH), 2.08 (2H, m, CH2CH=CH),2.18 (2H, m, CH2CH=CH), 3.63 (2H, t, J=6.5 Hz,CH2OH), 5.30 (1H, dt, J=11, 7.5 Hz, CH=CHCH=CH),5.65 (1H, dt, J=15, 7 Hz, CH=CHCH=CH), 5.91 (1H, dd,J=11, 11 Hz, CH=CHCH=CH), 6.30 (1H, dd, J=15,11 Hz, CH=CHCH=CH); 13C NMR δ ppm: 14.3, 21.0,25.7, 29.2, 29.4 (×2), 29.5, 32.8, 32.9, 63.1, 125.5, 128.0,131.7, 134.7. The acetate (E9,Z11-14:OAc) and aldehydederivative (E9,Z11-14:Ald) were synthesized from E9,Z11-14:OH.

(9Z,11E)- and (9E,11E)-Isomers (Scheme C): In asimilar synthetic procedure from C8 diol 1 to 2, C9 diol 5was converted to a THP ether of 9-iodononan-1-ol (8). Theiodide 8 was heated with PPh3, and a produced phospho-nium salt was treated with BuLi to make an ylide, whichwas coupled with (E)-2-pentenal to yield a THP ether of9,11-tetradecadien-1-ol (9). NMR analysis revealed that 9was a mixture of (9Z,11E)- and (9E,11E)-isomers in a ratioof about 7:3. After deprotection, two alcohols, Z9,E11-14:OH and E9,E11-14:OH, were separated by column chro-

(Scheme A)

OH OTHP OTHPd,cb,aHO I

1 2 32 3

OTHPZ9,Z11-14:OH

hf, ge

ii4

(Scheme B)

OHHO OTHPH OTHPa, j k

O5 6 7O 6 7

g, hg l h

iE9,Z11-14:OH

i

(Scheme C)

OHHO OTHPI OTHPm, nI

5

a, b

8 98h

g, l Z9,E11-14:OHii

E9,E11-14:OAch

E9,E11-14:OH

E9,E11-14:Ald

E9,Z11-14:OAcE9,Z11-14:Ald

Z9,Z11-14:OAc

Z9,Z11-14:Ald

Z9,E11-14:OAcZ9,E11-14:Ald

ii

Fig. 1 Synthetic schemes for 9,11-tetradecadienyl compounds (alco-hols, acetates, and aldehydes); (9Z,11Z)-isomer (Scheme A), (9E,11Z)-isomer (Scheme B), and (9Z,11E)- and (9E,11E)-isomers (Scheme C).Reagents: a, 3,4-dihydro-2H-pyran/p-TsOH/CH2Cl2; b, I2-PPh3/imid-azole/THF; c, TMSC≡CH/BuLi/THF-HMPA; d, K2CO3/MeOH; e,

(Z)-1-bromobutene/Pd(PPh3)4/CuI/PrNH2/benzene; f, (1) BH(C6H11)2/THF, (2) AcOH, (3) NaOH/H2O2 (30%); g, p-TsOH/EtOH; h, Ac2O/pyridine; i, PCC/CH2Cl2; j, (COCl)2/DMSO/Et3N/CH2Cl2; k, (Z)-2-pentenylenetriphenylphosphorane/THF; l, column chromatographywith SiO2-AgNO3; m, Ph3P/Δ; n, (1) BuLi/THF, (2) (E)-2-pentenal

1224 J Chem Ecol (2011) 37:1222–1230

matography utilizing silica gel impregnated with AgNO3.NMR data of Z9,E11-14:OH; 1H NMR δ ppm: 1.01 (3H, t,J=7.5 Hz, CH3CH2), ~1.3 (10H, m), 1.54 (2H, m,CH2CH2OH), 2.12 (4H, m, CH2CH=CHCH=CHCH2),3.58 (2H, t, J=6.5 Hz, CH2OH), 5.28 (1H, dt, J=11,7.5 Hz, CH=CHCH=CH), 5.68 (1H, dt, J=15, 7 Hz, CH=CHCH=CH), 5.93 (1H, dd, J=11, 11 Hz, CH=CHCH=CH), 6.29 (1H, dd, J=15, 11 Hz, CH=CHCH=CH); 13CNMR δ ppm; 13.7, 25.8, 25.9, 27.7, 29.3, 29.5, 29.6, 29.8,32.7, 62.7, 124.8, 128.7, 130.0, 136.1. NMR data of E9,E11-14:OH; 1H NMR δ ppm: 0.99 (3H, t, J=7.5 Hz,CH3CH2), ~1.3 (10H, m), 1.56 (2H, m, CH2CH2OH), 2.06(4H, m, CH2CH=CHCH=CHCH2), 3.62 (2H, t, J=6.5 Hz,CH2OH), 5.59 (2H, m, CH=CHCH=CH), 6.00 (2H, m,CH=CHCH=CH); 13C NMR δ ppm: 13.7, 25.6, 25.7,29.1, 29.39, 29.43, 29.5, 32.6, 32.8, 63.0, 129.4, 130.4,132.4, 133.9. The acetates (Z9,E11-14:OAc and E9,E11-14:OAc) and aldehyde derivatives (Z9,E11-14:Ald and E9,E11-14:Ald) were synthesized from the correspondingalcohols.

Field Trapping of Male Moths The synthetic pheromoneconstituents dissolved in hexane (100 μl) were incorporatedinto rubber septa (white rubber, O.D. 8 mm; Sigma-Aldrich,USA) used as dispensers. Each lure was placed at the centerof a sticky board trap (SE-trap®, 30×27 cm bottom platewith a roof; Sankei Chemical Co., Ltd., Kagoshima, Japan),and hung separately in Asian-pear orchards at intervals of10 and 1.5 m height. Four experiments (A–D) wereconducted in Tottori Prefecture in 2010 and 2011, and thenumber of captured males was counted weekly. In eachfield test, the replicated data were log-transformed before

analysis by Tukey-Kramer’s HSD test (Sokal and Rohlf,1995). The level of significance in all tests was set at 5%.

Results

Structure Elucidation of Pheromone ComponentsGC–EAD analyses of a crude pheromone extract of S.astaurota females showed three EAG-active components(Fig. 2). Of the three, the male antenna responded most toIII. Compounds I and II were detected in the 10% etherfraction of the crude extract on a Florisil column, indicatingan aldehyde and acetate, and III eluted in the 30% etherfraction as would an alcohol. Next, the crude extract wasanalyzed by GC-MS. The total ion chromatogram (TIC)and mass spectra of II and III (Fig. 3), exhibited M+ ionsat m/z 252 and 210 consistent with tetradecadienyl acetateand the parent alcohol, respectively. The chromatographicbehavior with relatively long retention times on a polar GCcolumn suggested conjugated dienyl structures, and char-acteristic fragment ions at m/z 82 and 95 in both spectraindicated two double bonds located at the same 9- and 11-positions (Ando et al., 1988; Ando and Yamakawa, 2011).Since I and an unknown substance were eluted together onthe GC-MS analysis, no clear mass spectrum of I wasrecorded. However, mass chromatograms monitoring somecharacteristic ions for 9,11-tetradecadienal confirmed theoccurrence of this compound in the crude pheromoneextract. The chromatogram at m/z 67 suggested that femalesproduce I–III in a ratio of about 1:2:20, respectively. Thedouble bond positions were confirmed by GC-MS analysis

EAD

Comp. IComp. II

Comp. III

RT (min)16.014.0 18.0

FID

Fig. 2 GC-EAD analysis of the sex pheromone components of Spulerina astaurota females. Analysis of a crude pheromone extract from thefemale (0.5 FE), indicating three EAG-active components (I, RT 15.2 min; II, RT 16.2 min; and III, RT 16.4 min)

J Chem Ecol (2011) 37:1222–1230 1225

of the crude pheromone extract (25 FE) after a treatmentwith MTAD. This analysis showed the formation of anMTAD adduct (RT 15.48 min) derived from III, the majorpheromone component in the gland extract. The massspectrum exhibited an M+ ion at m/z 323, and fragmentions at m/z 294 and 194, characteristic for the adduct of 9,11-tetradecadien-1-ol.

Identification with Authentic Compounds All geometricalisomers of 9,11-tetradecadienyl compounds were synthe-sized by three different schemes, as shown in Fig. 1.(9Z,11Z)-Isomers were stereoselectively synthesized via a(Z)-9-en-11-ynyl intermediate (4), which was partiallyreduced to the conjugated diene by hydroboration (SchemeA). A mixture of (9E,11Z)- and (9Z,11Z)-isomers wasprepared utilizing an ylide derived from (Z)-1-bromo-2-pentene (Scheme B). Another mixture of (9Z,11E)- and(9E,11E)-isomers was synthesized via Wittig coupling with(E)-2-pentenal as a key reaction (Scheme C). The twoisomers in each mixture were separable, and their chemical

structures were confirmed by NMR analyses. The (9E,11Z)-and (9Z,11E)-isomers showed four olefinic protons with adifferent chemical shift. Configuration of the double bondswas revealed by a coupling constant; i.e., 11 Hz for the Zconfiguration and 15 Hz for the E configuration. Althoughthe coupling constant was not clear in the (9Z,11Z)- and(9E,11E)-isomers, allylic carbons resonated with cruciallydifferent chemical shifts reflecting the configuration; i.e.,20.8 and 27.5 ppm for Z9,Z11-14:OH and 25.6 and32.6 ppm for E9,E11-14:OH.

Table 1 shows the RT of each isomer of 9,11-tetradecadienyl compounds (aldehyde, acetate, and alcohol)analyzed by GC-MS on two different columns, a stronglypolar column (DB-23) and a weakly polar column (HP-5).While the RT of the aldehyde was the shortest among thethree compounds in either column, the elution order of theacetate and alcohol changed according to the polarity of thecolumn. Four geometrical isomers of each compoundseparately eluted from both columns in the same order of(9Z,11E)-, (9E,11Z)-, (9Z,11Z)-, and (9E,11E)-isomers. RTs

55

67

82

95

121 135 147

Comp. III

79 95

252 M+

192 [M-60]+

m/z

55

61 121

135 149

Comp. II

0

50

100

0

50

100

Comp. III

RT (min)13.0 14.0 15.0

a

- - - - - - - - - - - - - - - - - - - - - - - -- - - -- - - -

16.012.0

Comp. IIComp. I

67b

c

100 15050 200 250

m/z 100 15050 200 250

192 [M-18]+ 210

M+

Fig. 3 GC-MS analysis of a crude pheromone extract from Spulerinaastaurota females (3.5 FE). a, Total ion chromatogram (TIC); b, massspectrum of II (Z9,Z11-14:OAc, RT 14.68 min); and c, mass spectrumof III (Z9,Z11-14:OH, RT 14.91 min). Mass chromatograms of M+ at

m/z 208 and some characteristic fragment ions of tetradecadienal (ionsat m/z 67, 82, and 95) indicated an occurrence of I (Z9,Z11-14:Ald,RT 13.63 min) in the extract

1226 J Chem Ecol (2011) 37:1222–1230

of three natural pheromone components coincided wellwith those of the (9Z,11Z)-isomers, indicating I, II, and IIIto be Z9,Z11-14:Ald, Z9,Z11-14:OAc, and Z9,Z11-14:OH,respectively. The GC-MS analysis of the crude pheromoneextract indicated that one female possessed about 2 ng ofZ9,Z11-14:OH.

Field Evaluation of the Synthetic Pheromone Since the S.astaurota females produce Z9,Z11-14:OH (III) as themajor pheromone component, the activities of syntheticpheromone lures including Z9,Z11-14:OH alone and mixedwith Z9,Z11-14:OAc (II) in different ratios (totally 0.5 mgfor each septum) were examined first (Experiment A). Theresults from the 2010 tests are shown in Table 2. While Z9,Z11-14:OH without Z9,Z11-14:OAc attracted some males,the activities of the 7:3 and 5:5 mixtures were significantlygreater than the single component lure. No males wereattracted by the lure baited solely with Z9,Z11-14:OAc,indicating that the acetate occurring as a minor pheromonecomponent is an effective synergist. Next, activities of fourgeometrical isomers were compared in order to confirm theconfiguration of the natural pheromone components (Ex-periment B). The 7:3 mixture of Z9,Z11-14:OH and Z9,Z11-14:OAc more effectively attracted male moths than did7:3 mixtures of the dienyl alcohol and acetate with otherconfigurations, as shown in Table 3.

Based on results of the field tests in 2010, the effect ofZ9,Z11-14:Ald (I) on the activity of the 7:3 mixture of Z9,Z11-14:OH (0.35 mg/septum) and Z9,Z11-14:OAc(0.15 mg/septum) was examined in 2011 (Experiment C).Male attraction by the three-component lures including thealdehyde (0.05–0.5 mg/septum) was not significantlydifferent from that by the two-component lures (Table 4).

The number of captured males/trap/night decreased extraor-dinary in 2011, indicating a lower population density thanthat in 2010.

Although no geometrical isomers of Z9,Z11-14:OH werefound in the gland extract, their contamination in thesynthetic pheromone might easily have happened. To applythe synthetic lure as a monitoring tool, it is important tounderstand their effects on male attraction. Each isomer ofZ9,Z11-14:OH was added to the 7:3 mixture of Z9,Z11-14:OH and Z9,Z11-14:OAc, and the lures were evaluated inthe field (Experiment D). Significant differences were notobserved between the lure unmixed and mixed with onegeometrical isomer as a third component even at a ratio of7:3:4 (Table 5).

Discussion

GC-EAD and GC-MS analyses of the pheromone glandextract of Spulerina astaurota females revealed three EAG-active components; 9,11-tetradecadien-1-ol, its acetate, andan aldehyde derivative (Figs. 2 and 3). Comparing theirchromatographic behaviors to those of the synthetic stand-ards of all geometrical isomers confirmed the 9Z,11Zconfiguration of the natural components (Table 1). There-fore, we concluded Z9,Z11-14:OH, Z9,Z11-14:OAc, andZ9,Z11-14:Ald as candidates of the S. astaurota pheromonecomponents. Their biological activities were evaluated in aseries of field-trap experiments using synthetic compounds(Tables 2, 3 and 4). The male moths were attracted to a lurebaited with Z9,Z11-14:OH alone, and Z9,Z11-14:OAcstrongly synergized attraction. Among the lures tested, the

Table 1 Chromatographic behaviour of synthetic 9,11-tetradecadienyl compounds and natural pheromone components of Spulerina astaurotafemales on GC-capillary columns a

Compound Retention time (RT, min)

DB-23 column b HP-5 column c

Aldehyde Acetate Alcohol Aldehyde Acetate Alcohol

(9Z,11E)-Isomer 13.48 14.58 14.79 13.97 16.60 14.86

(9E,11Z)-Isomer 13.60 14.65 14.88 14.09 16.71 14.98

(9Z,11Z)-Isomer 13.63 14.68 14.91 14.20 16.80 15.12

(9E,11E)-Isomer 13.66 14.74 14.94 14.24 16.87 15.16

Natural component d 13.63 14.68 14.91 14.19 16.80 15.12

a Each compound was analyzed by GC-MS equipped with a DB-23 column (0.25 mm ID×30 m) and an HP-5 column (0.25 mm ID×30 m). Thecolumn temperature program was 80°C for 2 min, 8°C/min to 210°C, and 210°C for 10 minb Kováts retention index values of (9Z,11Z)-isomers: Z9,Z11-14:Ald, 2161; Z9,Z11-14:OAc, 2278; Z9,Z11-14:OH, 2297c Kováts retention index values of (9Z,11Z)-isomers: Z9,Z11-14:Ald, 1669; Z9,Z11-14:OAc, 1861; Z9,Z11-14:OH, 1734d Aldehyde (Comp. I, Z9,Z11-14:Ald), acetate (Comp. II, Z9,Z11-14:OAc), and alcohol (Comp. III, Z9,Z11-14:OH). RT of the Comp. I wasdetermined by mass chromatogram monitoring the ion at m/z 67

J Chem Ecol (2011) 37:1222–1230 1227

mixture of Z9,Z11-14:OH and Z9,Z11-14:OAc in a ratio of7:3 exhibited the strongest attraction. On the other hand, theaddition of Z9,Z11-14:Ald to the mixture did not signifi-cantly increase the number of males attracted. The role ofZ9,Z11-14:Ald in this communication system is unclear.

Some other dienyl pheromones are known for thespecies in Gracillariidae, but the 9,11-dienes have not beenidentified from species in this family (Ando, 2011; El-Sayed, 2011). More than 200 Gracillariidae species occur inJapan, including several species in the Spulerina that mineinto specific host plants, such as S. corticicola that attackspine trees. 9,11-Tetradecadienyl compounds, however, arenot novel pheromone components of lepidopteran insects.Interestingly, females of the currant shoot borer, Lampronia

capitella (Incurvarioiidae), also produced the same threecomponents, Z9,Z11-14:OH, Z9,Z11-14:OAc, and Z9,Z11-14:Ald, and the mixture in a ratio of 100:26:13 attracted themales (Löfstedt et al., 2004). The two micro-lepidopteranspecies are classified in different groups even at asuperfamily; i.e., S. astaurota in Gracillarioidea and L.capitella in Incurvarioidea. In addition to these two species,Z9,Z11-14:OAc has been identified from Idaea aversataand I. straminata (Geometridae) as a main pheromonecomponent (Zhu et al., 1996).

Table 2 Attraction of Spulerina astaurota males by lures baited withsynthetic Z9,Z11-14:OH and Z9,Z11-14:OAc mixed in a differentratio (Experiment A) a

Lure (mg/rubber septum) b Captured males

Z9,Z11-14:OH Z9,Z11-14:OAc Ratio /trap/night c Total

0.50 0 10:0 2.43±1.05 b 153

0.45 0.05 9:1 2.51±0.80 b 158

0.35 0.15 7:3 5.97±2.79 a 376

0.25 0.25 5:5 5.40±0.94 a 340

0.05 0.45 1:9 0.75±0.26 c 47

0 0.50 0:10 0.0 0

0 0 0.02±0.02 d 1

a Tested using three traps for each lure from June 22 to July 13, 2010in an Asian-pear orchard of Tottori city and Hokuei town in TottoriPrefectureb Z9,Z11-14:OH (Comp. III), Z9,Z11-14:OAc (Comp. II)c Mean±SE. Values within each test followed by a different letter aresignificantly different at P<0.05 by Tukey-Kramer test

Table 3 Attraction of Spulerina astaurota males by lures baited with9,11-tetradecadienyl alcohol and acetate mixed in a 7:3 ratio(Experiment B) a

Lure (mg/rubber septum) b Captured males

Alcohol (0.35 mg) Acetate (0.15 mg) /trap/night c Total

Z9,Z11-14:OH Z9,Z11-14:OAc 63.05±24.81 a 2,648

Z9,E11-14:OH Z9,E11-14:OAc 3.76±2.41 b 158

E9,Z11-14:OH E9,Z11-14:OAc 1.90±0.89 b 80

E9,E11-14:OH E9,E11-14:OAc 1.98±1.15 b 83

none none 0.10±0.10 c 4

a Tested using three traps for each lure from September 1 toSeptember 15, 2010 in an Asian-pear orchards of Hokuei town inTottori Prefectureb Z9,Z11-14:OH (Comp. III), Z9,Z11-14:OAc (Comp. II)c Mean±SE. Values within each test followed by a different letter aresignificantly different at P<0.05 by Tukey-Kramer test

Table 5 Attraction of Spulerina astaurota males by the syntheticpheromone (Z9,Z11-14:OH and Z9,Z11-14:OAc) mixed with ageometrical isomer of the alcohol component (Experiment D) a

Lure (mg/rubber septum) b Captured males

Z9,Z11-14:OH (0.35)+Z9,Z11-14:OAc (0.15)

Ratio /trap/night c Total

+ none 7:3:0 0.63±0.52 a 53

+ Z9,E11-14:OH (0.05) 7:3:1 0.52±0.38 a 44

+ Z9,E11-14:OH (0.2) 7:3:4 0.80±0.64 a 67

+ E9,Z11-14:OH (0.05) 7:3:1 0.58±0.41 a 49

+ E9,Z11-14:OH (0.2) 7:3:4 0.90±0.72 a 76

+ E9,E11-14:OH (0.05) 7:3:1 1.05±0.89 a 88

+ E9,E11-14:OH (0.2) 7:3:4 0.55±0.31 a 46

control 0±0 0

a Tested using four traps for each lure from June 14 to July 5, 2011 inan Asian pear orchard of Hokuei town in Tottori Prefectureb Z9,Z11-14:OH (Comp. III), Z9,Z11-14:OAc (Comp. II)c Mean±SE. Values within each test followed by a different letter aresignificantly different at P<0.05 by Tukey-Kramer test

Table 4 Attraction of Spulerina astaurota males by lures baited withsynthetic Z9,Z11-14:OH, Z9,Z11-14:OAc, and Z9,Z11-14:Ald mixedin a different ratio (Experiment C) a

Lure (mg/rubber septum) b Captured males

Z9,Z11-14:OH

Z9,Z11-14:OAc

Z9,Z11-14:Ald

Ratio /trap/night c Total

0.35 0.15 0 7:3:0 0.80±0.19 a 28

0.35 0.15 0.05 7:3:1 1.11±0.44 a 39

0.35 0.15 0.20 7:3:4 1.49±0.65 a 52

0.35 0.15 0.50 7:3:10 1.11±0.37 a 39

0 0 0.50 0:0:10 0.03±0.03 b 1

0 0 0 0±0 0

a Tested using five traps for each lure from July 5 to July 12, 2011 inan Asian-pear orchard of Tottori city in Tottori Prefectureb Z9,Z11-14:OH (Comp. III), Z9,Z11-14:OAc (Comp. II), Z9,Z11-14:Ald (Comp. I)c Mean±SE. Values within each test followed by a different letter aresignificantly different at P<0.05 by Tukey-Kramer test

1228 J Chem Ecol (2011) 37:1222–1230

Several lepidopteran sex pheromones including geomet-rical isomers of (9Z,11Z)-9,11-tetradecadienyl compoundsalso have been reported (Ando, 2011; El-Sayed, 2011). Z9,E11-14:OAc is a well-known pheromone component ofNoctuidae species (Spodoptera eridania, S. littoralis, and S.litura), and this compound has been identified from aPyralidae species, Dioryctria abietella. The aldehydederivative with 9Z,11E configuration was produced byanother Pyralidae species (Ectomyelois ceratoniae), and anElachistidae species (Stenoma cecropia). Furthermore, E9,E11-14:OAc is secreted by a Tortricidae species, Epiphyaspostvittana. Thus, taxonomically unrelated species haveevolved to produce the same conjugated dienyl compoundsin the Lepidoptera.

Biosynthesis of the pheromone components have beenstudied in S. littoralis and E. postvittana. In the formerspecies, Z9,E11-14:OAc was biosynthesized by Δ9-desaturation of (E)-11-tetradecenyl intermediate (Dunkelblumand Kehat 1987; Martinez et al., 1990). In contrast, E9,E11-14:OAc was biosynthesized by Δ11-desaturation of (E)-9-tetradecenyl intermediate, which was produced by β-oxidation of (E)-11-hexadecenoate, in the latter species (Fosterand Roelofs, 1990). Two different pathways were proposedfor 9,11-dienes with a different configuration. To date, nobiosynthetic studies have focused on lepidopteran pheromoneswith Z,Z configured conjugated dienyl compounds; therefore,(9Z,11Z)-9,11-dienes of the S. astaurota females might beinteresting in this regard.

The S. astaurota adults appear twice a year; from Junethrough July and in September. In our 2010 field tests, wecaught approximately 4,000 male moths, indicating thatthe synthetic pheromone is powerfully attractive andpotentially useful for accurately detecting mating activityof this pest. In field experiment D, the activity of thesynthetic pheromone was not decreased by admixture ofgeometrical isomers of Z9,Z11-14:OH (Table 5). Thisresult indicates that high purity of the synthetic dienylcompound is not necessary to make a lure for monitoring.Regrettably, some data in Tables 3, 4 and 5 vary with alarge standard error. Each of Experiments B through Dwas carried out in one village, but the traps baited with thesame lure were placed in different orchards located atsome distance apart. It is possible that the populationdensities in the orchards used for the field tests weredifferent, resulting in the variable attraction. These resultsalso suggest that the flight area for each male moth wasnot extensive. The number of captured males in 2011 wasextraordinary small, as shown in Tables 4 and 5,indicating the possibility of a lower population density in2011 than in 2010. Future work utilizing the syntheticpheromone of S. astaurota should clarify the ecology ofthis species, including the extent to which the number oftrapped males correlates with larval damage.

Acknowledgements We thank the Asian-pear growers, especially T.Ikuhashi, Y. Kaigo, Y. Nakahara, H. Ohta, S. Okabe, K. Takeuchi, andI. Tokuyama, who participated in this research and contributed theirfarms and time to the effort. We are grateful to T. Ishiko and S. Toitaof the Tottori Prefectural Agriculture and Forest Research Institute.We also thank Drs. F. Tamura, A. Itai and M. Azuma for offeringimportant information, and Ms. A. Oda and students of the Laboratoryof Applied Entomology in Faculty of Agriculture at Tottori Universityfor their help with field experiments and rearing insects.

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