Appl. Entomol. Zool. 41 (3): 507–513 (2006) http://odokon.org/

Sex pheromone of the soybean pod borer, glycinivorella (: ): Identification and field evaluation

Le Van VANG,1 Masahiro ISHITANI,2 Furumi KOMAI,3 Masanobu YAMAMOTO1 and Tetsu ANDO1,* 1 Graduate School of Bio-Applications and Systems Engineering (BASE), Tokyo University of Agriculture and Technology; Koganei 184–8588, Japan 2 Field Crops and Horticulture Experiment Station, Aomori Prefectural Agriculture and Forestry Research Center; Rokunohe- machi 003–0071, Japan 3 Department of General Education, Osaka University of Arts; Osaka 585–8555, Japan (Received 21 February 2006; Accepted 19 May 2006)

Abstract With reference to the data of synthetic standards, GC-EAD and GC-MS analyses of a pheromone gland extract of the soybean pod borer, Leguminivora glycinivorella (Lepidoptera: Tortricidae), resulted in three acetates [dodecyl acetate, (8E,10E)-8,10-dodecadienyl acetate (E8,E10-12:OAc), and its (8E,10Z)-isomer] at a ratio of 10 : 100 : 2.5 as a candi- date of the pheromone. While the contents in the glands were low and even the titer of the major pheromonal compo- nent was ca. 0.5 ng/female, the positions of the two double bonds were confirmed by a mass spectrum of the adduct with 4-methyl-1,2,4-triazoline-3,5-dione. In a soybean field, synthetic E8,E10-12:OAc successfully attracted male of L. glycinivorella, and highly selective attraction was observed for E8,E10-12:OAc among the geometrical isomers of the 8,10-diene. Neither of the two minor components showed a synergistic effect on field attraction by E8,E10-12:OAc, and their roles were unclear. A binary lure of E8,E10-12:OAc and (E)-8-dodecenyl acetate (E8- 12:OAc) was necessary to attract the male moths of falcana, the soybean pod worm, but E8-12:OAc strongly inhibited the attraction of L. glycinivorella by E8,E10-12:OAc, indicating that E8-12:OAc secreted by M. fal- cana is one of the most important factors in the reproductive isolation of these two species.

Key words: Female sex pheromone; male attractant; ; GC-EAD; GC-MS

tober, overwinters, and pupates in mid-August, INTRODUCTION with the adults appearing from late August to early There are several lepidopteran species that feed September (Ishitani, 1993). Monitoring the adult on soybean pods in Japan. Two olethreutine flight is very important in the control of this pest species, Matsumuraeses falcana Walsingham and since a sprayed pesticide works actively only Leguminivora glycinivorella (Matsumura) in the during a very limited term, when the hatching lar- family Tortricidae, are particularly harmful pests. vae are outside of the pods. While the M. falcana larvae mainly feed on the Lepidopteran sex pheromones have been chemi- hulls of the pods, the L. glycinivorella larvae prefer cally identified from virgin females of more than young beans, and when the beans attacked by this 570 species (Ando et al., 2004; Ando, 2006; El- insect are mixed with healthy beans, the quality of Sayed, 2006). Some of them have been utilized as a the whole crop is reduced. In the Tohoku area of monitoring tool for integrated pest management Japan, soybean cultivation has recently spread as a (Wakamura, 1992). A trap baited with a synthetic replacement for rice; consequently, the loss caused sex pheromone effectively attracts male moths; by L. glycinivorella has increased. In Aomori Pre- thus, the start of the adult stage and the population fecture, L. glycinivorella is a univoltine insect; i.e., density can be successfully monitored. The sex the mature larva makes a cocoon in the soil in Oc- pheromone of M. falcana has been identified

*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2006.507

507 508 L. V. VANG et al.

(Wakamura, 1985; Wakamura and Kegasawa, phy (GC) equipped with an electroantennographic 1986), but that of L. glycinivorella has remained (EAG) detector (GC-EAD, Struble and Arn, 1984) unknown because no method for raising successive and GC-mass spectrometry (GC-MS). To confirm generations has been established and mass collec- the double-bond positions of a dienyl component tion of the pupae is difficult. These two species be- of the pheromone, another crude pheromone long to the same tribe in Olethreutinae extract (40 FE) was treated with a CH2Cl2 solution and co-attack the soybean crop in the same season. of 4-methyl-1,2,4-triazoline-3,5-dione (MTAD, 10 In addition to agricultural applications, we are in- mg/ml, 10 ml) for 10 min at room temperature after terested in whether or not a difference in the sex hexane was removed under a nitrogen stream pheromones of these species is one of the most de- (Young et al., 1990; Vang et al., 2005). The solvent cisive factors in the reproductive isolation between was returned to hexane, and an MTAD adduct was these two species. This report discusses the results analyzed by GC-MS. of a chemical study of the sex pheromone of L. Chemicals. The chemical structures of the glycinivorella and a field evaluation of the syn- pheromone components and related compounds thetic pheromone. are abbreviated as follows: Z(Z)-double bond, E(E)-double bond, number before the hyphen position of the double bond, number after the hy- METHODS AND MATERIALS phencarbon number of the straight chain, OAc and pheromone extracts. In 2003, adult acetate, OHalcohol, and Aldaldehyde. E8,E10- insects of L. glycinivorella (four females and ten 12:OH and E8-12:OH with 98% purity were sup- males) were caught by net sweepings in soybean plied from Shin-Etsu Chemical Co., Ltd. (Tokyo). fields in Aomori Prefecture and sent to the labora- The acetate and aldehyde derivatives were obtained tory of the Tokyo University of Agriculture and by acetylation and PCC oxidation of the corre- Technology for a preliminary experiment. In 2004 sponding alcohols, respectively. Before field exam- and 2005, overwintering larvae were collected ination, each compound was purified by an open- from soybean fields in late July and sent to the uni- column packed silica gel impregnated with 15% versity. To induce pupation, the larvae were then silver nitrate. Three geometrical isomers of placed in humidified soft soil under conditions of a E8,E10-12:OH were newly synthesized following 12D : 12L photo-cycle and 20°C. The male and fe- previously reported procedures (Ando et al., male adults that emerged in late August were indi- 1985b) and were purified by the same method for vidually kept in a small glass tube (1.6 cm ID other dienyl pheromones using HPLC with an ODS 3.8 cm) under the above-mentioned conditions. column (Nishida et al., 2003). Pheromone glands of 2 or 3 d-old virgin females GC-EAD analysis. The EAG-activity of the nat- were excised during the scotophase (2–3 h after ural pheromone was measured by the same GC- lights were turned off) and soaked in distilled EAD instrument as that used in our previous exper- hexane (10 ml/female) for 15 min to extract the iments (Vang et al., 2005). The GC was equipped pheromone. The pheromone was extracted from with a DB-23 capillary column (0.25 mm ID about 150 females and was applied to the following 30 m, 0.25 mm, J & W Scientific, Folsom, CA, analytical experiments. USA), and the oven temperature was maintained at Fractionation and derivatization of the natu- 80°C for 1 min and programmed at a rate of 8°C/ ral pheromone. The crude pheromone extract [50 min up to 210°C, and then maintained at 210°C for female extract (FE)] was chromatographed on a sil- 10 min. An antenna of 2 or 3 d-old male was cut ica gel (200 mg, Kanto Chemical Co., Ltd., Tokyo) near the base and terminal tip and then suspended in a Pasteur pipette. Before charging of the between the two heads of two AgCl-coated silver pheromone extract, the column was washed with electrodes filled with Bombyx mori saline (NaCl distilled hexane (10 ml). The column was succes- 8,620 mg, KCl 100 mg, CaCl2·2H2O 441 mg, sively eluted with 2 ml each of hexane and 1%, 5%, NaH2PO4·12H2O 597 mg, and KH2PO4 227 mg in 20%, and 30% ether in hexane. Each fraction was 1 l H2O; adjusted to pH 6.5). The rate of airflow concentrated by blowing under a gentle nitrogen carrying eluted compounds to the antenna was 3.3 stream and was analyzed using gas chromatogra- cm/s. Three different male antennae were used for Sex Pheromone of the Soybean Pod Borer 509

GC-EAD analyses of the pheromone extract. EAG height of 1 m in a soybean field in Aomori Prefec- activities of synthetic pheromone components and ture where the overwintering larvae of L. glycini- their analogs were also measured by GC-EAD vorella were collected. A randomized complete de- under the same conditions as those used for the sign with two replicates (Test I in 2003) or three gland extract. The process was repeated at least six replicates (Tests II and III in 2005) of each lure times with different male antennae. was examined. Numbers of captured moths were GC-MS analysis. Electron-impact GC-MS was counted everyday, and the positions of the traps carried out on an HP5973 mass spectrometer sys- were rotated after counting to eliminate any posi- tem (Hewlett-Packard) equipped with a capillary tional effect. column operating at a mass range from m/z 30 to 500. The ionization voltage was 70 eV, and the ion RESULTS source temperature was 230°C. For analysis of the natural pheromone, a DB-23 column (0.25 mm EAG active components in the extract ID30 m, 0.25 mm) was used under the same tem- A GC-EAD analysis of the natural pheromone (1 perature program as that used for GC-EAD. For FE), which was extracted from the females caught analysis of the MTAD adduct, an HP-5MS capil- by butterfly nets in 2003, showed two EAG-active lary column (0.32 mm ID30 m, 0.25 mm; Agilent components (Comps. II and III). The analysis of Technologies Inc., Wilmington, DE, USA) was the gland extract (10 FE) from virgin females that used under a column temperature maintained at emerged from the pupae in the laboratory, however, 100°C for 2 min and programmed at a rate of showed three EAG-active components with the fol- 15°C/min up to 280°C. lowing values for the retention time (Rt) and Field examinations. Lures were made from the Kováts relative index (RI); I: 240 mV at 11.14 min synthetic pheromone components impregnated into (RI 1882), II: 580 mV at 13.32 min (RI 2090), and rubber septa (8 mm OD, Aldrich) without any an- III: 150 mV at 13.50 min (RI 2109) (Fig. 1A). The tioxidants or stabilizers. In field tests, each lure was chromatographic behavior of Comp. I was coinci- placed at the center of a sticky trap (3027 cm bot- dent with that of an authentic sample of 12:OAc. tom plate with a roof; Takeda Chemical Ind. Ltd., The RI values of Comps. II and III, which were Osaka), which was hung on a wooden stake at a much larger than those of monoenyl compounds

Fig. 1. GC-EAD analyses with male antennae of L. glycinivorella. [A] The pheromone extract (10 FE), [B] 5% ether fraction chromatographed from the crude extract (25 FE) and [C] a mixture of authentic 12:OAc, E8,E10-12:OAc, and E8,Z10-12:OAc (1 ng of each). 510 L. V. VANG et al. such as E8-12:OAc at 11.35 min (RI 1905) and suggest a conjugated dienyl structure, were equal to those of the synthetic standards of E8,E10- 12:OAc and E8,Z10-12:OAc. Authentic Z8,E10- 12:OAc and Z8,Z10-12:OAc eluted from the GC column at 13.27 min (RI 2085) and 13.48 min (RI 2107), respectively. In the analysis of the extract fractionated with a silica gel column, all three EAG-active components were detected in a fraction eluted with 5% ether in hexane, indicating their acetoxyl functional group (Fig. 1B).

Identification by GC-MS While a GC-MS analysis in 2003 elucidated the chemical structure of the major component (Comp. II) only, further experimentation with the extracts collected in 2004 and 2005 successfully identified Comps. I–III (Fig. 2). In spite of many unknown materials in the crude extract (10 FE) as shown in the total ion chromatogram (TIC, Fig. 2A), mass chromatograms of the ions at m/z 168, 83, and 61 (Fig. 2B) indicated the elution of 12:OAc at 10.36 min (RI 1881, Comp. I), and the mass spectrum at this Rt (Fig. 2C) was almost identical to the au- thentic sample. Mass chromatograms of the other ions at m/z 224, 164, 81, and 61 (Fig. 2B) indicated the elution of C12 dienyl acetates at 12.62 min (RI 2090, Comp. II) and 12.82 min (RI 2109, Comp. III). The mass spectra of Comp. II showed a base Fig. 2. GC-MS analysis of a pheromone extract (10 FE) peak at m/z 81 and an abundant fragment ion at m/z from virgin females of L. glycinivorella. [A] Total ion chro- 68 (Fig. 2D), which were diagnostic for 8,10-di- matogram (TIC), [B] mass chromatograms monitoring ions at enyl compounds (Ando et al., 1985a). The GC-MS m/z 61, 81, 83, 164, 168 and 224, [C] mass spectrum of Comp. I (12:OAc), and [D] mass spectrum of Comp. II (E8,E10- data of Comp. II coincided well with those of syn- 12:OAc). thetic E8,E10-12:OAc. Although the content of Comp. III eluted at the same Rt of authentic tected. Similar GC-MS data were recorded from E8,Z10-12:OAc was very low (Fig. 2A), the mass the 5% ether fraction (25 FE) chromatographed spectrum also represented the characteristic ions with a silica gel column. for the 8,10-dienyl acetate. The calculations from The double-bond positions of the main the TIC indicated that the ratio of these three pheromone component were confirmed by GC-MS acetates, which were estimated to be 12:OAc, analysis of the pheromone extract treated with E8,E10-12:OAc, and E8,Z10-12:OAc, was 10 : MTAD. Separated from many unknown com- 100 : 2.5. The titer of the major pheromonal com- pounds, the MTAD adduct (Rt 14.42 min) derived ponent, Comp. II (E8,E10-12:OAc), in the gland from E8,E10-12:OAc was eluted (Fig. 3A). The extract was ca. 0.5 ng/female. The mass chro- mass spectrum showed a characteristic base peak at matograms of the ions at m/z 166 and 82, which m/z 180 in addition to [M CH3] at m/z 322 (1%) were diagnostic for C12 monoenyl acetates, showed and M at m/z 337 (5%) (Fig. 3B), indicating the no peaks around 11 min; therefore no monoenyl original conjugated 8,10-dienyl structure. Analysis compounds could be detected in the extract. In the of the extract from a limited number of females analysis of the extract fractionated with a silica gel showed no other MTAD adduct derived from column, all three EAG-active components were de- E8,Z10-12:OAc. Sex Pheromone of the Soybean Pod Borer 511

Fig. 3. GC-MS analysis of the pheromone extract (40 FE) treated with MTAD. [A] Total ion chromatogram (TIC), and [B] mass spectrum of an MTAD adduct of Comp. II (E8,E10- 12:OAc). Fig. 4. Electroantennographic (EAG) responses (mean mVSE, n6) of L. glycinivorella males to synthetic E8,E10- EAG responses of the synthetic pheromone and 12:OAc (injection of 0.1, 1, and 10 ng) and its related com- related compounds pounds (injection of 10 ng). The responses were measured by GC-EAD, which delivered about half of the injected com- As shown in Fig. 1C, the synthetic samples of pound to the male antenna. Bars superscripted by a different the three identified acetates (1 ng each) strongly letter indicate a significant difference at p0.05 by the Tukey- stimulated the male antennae of L. glycinivorella. Kramer test. E8,E10-12:OAc is more EAG-active than the other two components. Figure 4 shows the EAG activi- Table1. Attraction of Leguminivora glycinivorella and ties of this main pheromonal component (0.1–10 Matsumuraeses falcana males by lures which were ng) and of other synthetic compounds (10 ng). baited with synthetic E8,E10-12:OAc, or its Among four geometrical isomers of the 8,10-dienyl mixture, with a structure related compound in Aomori Prefecture (Test I)a acetate, E8,E10-12:OAc is the most active com- pound. E8-12:OAc, which was not found in the Total of captured males pheromone extract, as well as E8,E10-12:OAc Lure components L. glycinivorella M. falcana strongly stimulated the antennae. Functional deriv- (mg/rubber septum) atives of E8,E10-12:OAc stimulated the antennae AB AB more weakly than 12:OAc, one of the minor natu- ral components, and the EAG activity of E8,E10- E8,E10-12:OAc (1.0) 12:OH was lower than that of E8,E10-12:Ald. None 22 8 0 0 E8,E10-12:OH (0.2) 13 9 0 1 Field attraction by synthetic lures E8,E10-12:Ald (0.2) 15 9 0 1 Based on the identification of a major E8-12:OAc (0.2) 7 3 6 13 12:OAc (0.2) 16 8 0 0 pheromone component, a field test with synthetic None 6 2 0 0 E8,E10-12:OAc and its binary mixtures with a structure-related compound was carried out in a Tested in two soybean fields (A: 470 m2, and B: 850 m2) 2003 (Test I). Information on the minor compo- separated from each other by 25 m, from August 17 to Sep- nents of the L. glycinivorella pheromone was ex- tember 5, 2003. In each field, one trap was used for each lure. pected, and the results are shown in Table 1. E8,E10-12:OAc obviously attracted the males of L. glycinivorella. Although this test did not reveal the E8,E10-12:OAc. E8-12:OAc is an important pher- structure of a minor component, it revealed that omone component of M. falcana (Wakamura, E8-12:OAc strongly inhibited the activity of 1985), the males of which were specifically caught 512 L. V. VANG et al.

Table2. Attraction of Leguminivora glycinivorella males was recorded on August 25, and the peak of the by rubber septa baited with a synthetic lure in adult detection (65 adults/h) was August 29. a soybean field in Aomori Prefecture (2005, Tests II and III)a DISCUSSION Lure Captured Chemical (mg/rubber septum) males/trapb The virgin females of the soybean pod borer, Leguminivora glycinivorella, produced three EAG- Test II (Aug. 24–Sept. 1) active compounds (Fig. 1). Their chemical struc- E8,E10-12:OAc (0.2) 80.0 32.1 a tures were studied with reference to the GC-MS E8,Z10-12:OAc (0.2) 2.70.5 c Z8,E10-12:OAc (0.2) 2.30.5 c data of synthetic standards, and we concluded that Z8,Z10-12:OAc (0.2) 3.01.4 c the pheromone glands included 12:OAc, E8,E10- Natural typec 14.77.8 b 12:OAc, and E8,Z10-12:OAc at a ratio of None 1.70.5 c 10 : 100 : 2.5 (Fig. 2). While the contents in the glands were low and even the major dienyl compo- Test III (Sept. 2–16) E8,E10-12:OAc (5.0) 8.71.9 a nent occurred below the level of 1 ng/female, the E8,E10-12:OAc (1.0) 8.32.1 a positions of the two double bonds was successfully E8,E10-12:OAc (0.5) 7.00.8 a confirmed by GC-MS analysis of the adduct with E8,E10-12:OAc (0.2) 10.75.4 a MTAD (Fig. 3). The male antennae strongly re- E8,E10-12:OAc (0.01) 7.73.1 a sponded to these three acetates and most effec- None 1.7 1.2 b tively to E8,E10-12:OAc among the compounds a Tested using three traps for each lure. tested (Fig. 4). b MeanSE. Values within each test followed by a different In a soybean field, synthetic E8,E10-12:OAc letter are significantly different at p0.05 by the Tukey- successfully attracted male moths of L. glycini- Kramer test. vorella, and highly selective attraction was ob- c Mixture of E8,E10-12:OAc (0.2 mg), E8,Z10-12:OAc served for this isomer with the 8E,10E configura- (0.025 mg), and 12:OAc (0.1 mg). tion among four geometrical isomers of the 8,10- diene (Tables 1 and 2). Neither of the two minor by the traps baited with a mixture of E8,E10- components showed a synergistic effect on field at- 12:OAc and E8-12:OAc in this study. traction by E8,E10-12:OAc (Table 2), and their Furthermore, after the final chemical identifica- roles were unclear. Synthetic pheromones are usu- tion of the three components, another field evalua- ally evaluated by comparing the attraction ability tion of synthetic compounds was conducted in of females. In this study, traps baited with virgin 2005 (Tests II and III, Table 2). The attraction ac- females of L. glycinivorella were also tested but tivity in a field was specifically observed for failed to attract the males. The reason remained E8,E10-12:OAc among the four geometrical iso- elusive. Since a high population density of L. mers. On the other hand, three component lures glycinivorella was estimated in our test fields from baited with a mixture of 12:OAc, E8,E10-12:OAc, observation of the adults in the daytime and acci- and E8,Z10-12:OAc in a ratio of 10 : 100 : 2.5 at- dental capture of the males by control traps (Tables tracted the targeted males, but the ability was not 1 and 2), attraction by the trap baited with only comparable with that of the single component lure E8,E10-12:OAc seemed to be weak. The term of of E8,E10-12:OAc, indicating that E8,Z10-12:OAc the capture by dienyl acetate, however, corre- might inhibit the activity of E8,E10-12:OAc. Traps sponded to the observations of adult flight, indicat- with lures baited with a different dose of E8,E10- ing the usefulness of the single-component lure as 12:OAc (0.01–5.0 mg) caught a similar number of a monitoring tool. Many ecological aspects of L. males. glycinivorella, including mating behavior, are still The trap of E8,Z10-12:OAc attracted the males unknown. Our preliminary field observation re- from August 17 to September 13, 2005, in a soy- vealed that most males were attracted by the bean field, where the adults could be visually de- pheromone traps during a rather short period, from tected from August 19 to September 10 by a beat- 16:00 to 17:00, a few hours before sunset. Further ing method. The peak of the attraction (15 males/d) study utilizing the pheromone traps will possibly Sex Pheromone of the Soybean Pod Borer 513 reveal the ecology in detail. REFERENCES A binary lure of E8,E10-12:OAc and E8-12:OAc was necessary to attract male moths of Matsumu- Ando, T. (2006) Internet database: http://www.tuat.ac.jp/ raeses falcana (Wakamura, 1985; Wakamura and ~antetsu/LepiPheroList.htm Ando, T., S. Inomata and M. Yamamoto (2004) Lepi- Kegasawa, 1986), but E8-12:OAc strongly inhib- dopteran sex pheromones. Topics Current Chem. 239: ited the field attraction of L. glycinivorella by 51–96. E8,E10-12:OAc. The L. glycinivorella females did Ando, T., Y. Katagiri and M. Uchiyama (1985a) Mass spec- not secrete E8-12:OAc (Fig. 2), but the antennae of tra of dodecadienic compounds with a conjugated double the L. glycinivorella males strongly responded to bond, lepidopterous sex pheromones. Agric. Biol. this monoenyl acetate (Fig. 4). This result indicates Chem. 49: 413–421. Ando, T., Y. Kurotsu, M. Kaiya and M. Uchiyama (1985b) that E8-12:OAc secreted by M. falcana is one of Systematic syntheses and characterization of dodecadien- the most important factors in the reproductive iso- 1-ols with conjugated double bond, lepidopterous sex lation of these two species associated with soy- pheromones. Agric. Biol. Chem. 49: 141–148. bean. Leguminivora and Matsumuraeses are mem- El-Sayed, A. M. (2006) Internet database: http://www. bers of the tribe Grapholitini, whose species in the pherobase.com/ Ishitani, M. (1993) Summer diapause in two univoltine in- Palaearctic region are divided into three sects. In Seasonal Adaptation and Diapause in Insects groups: , , and (M. Takeda and S. Tanaka eds.). Bun-ichi Sogo Shup- genus groups (Komai, 1999). Leguminivora be- pan, Tokyo, pp. 82–91 (in Japanese). longs to the Cydia genus group, and Matsumurae- Komai, F. (1999) A taxonomic review of the genus ses belongs to the Grapholita genus group. This is Grapholita and allied genera (Lepidoptera: Tortricidae) in the first structural determination of the pheromone the Palaearctic region. Entomol. Scand. Suppl. 55: 1– 226. from a species in genus Leguminivora. E8,E10- Nishida, T., L. V. Vang, H. Yamazawa, R. Yoshida, H. Naka, K. 12:OAc has been determined from seven species of Tsuchida and T. Ando (2003) Synthesis and characteri- Cydia (Ando, 2006; El-Sayed, 2006), and records zation of hexadecadienyl compounds with a conjugated on most species of the Cydia genus group, includ- diene system, sex pheromone of persimmon fruit ing L. glycinivorella, indicate the use of this dienyl and related compounds. Biosci. Biotechnol. Biochem. 67: 822–829. acetate. Furthermore, sex attractants of 19 Cydia Struble, D. L. and H. Arn (1984) Combined gas chromatog- species have the acetate as a component (Ando, raphy and electroantennogram recording of insect olfac- 2006; El-Sayed, 2006). tory responses. In Techniques in Pheromone Research (H. In addition to the above-mentioned species, E. Hummel and T. A. Miller eds.). Springer-Verlag, E8,E10-12:OAc has been determined from many New York, pp. 161–178. other olethreutine species: one species of Centro- Vang, L. V., S. Inomata, M. Kinjo, F. Komai and T. Ando (2005) Sex pheromones of five olethreutine species xena whose larvae feed on fruits of a mangrove (Lepidoptera: Tortricidae) associated with the seedlings plant (Vang et al., 2005) (Grapholitini: Grapholita and fruits of mangrove plants in the Ryukyu islands, genus group), one Epiblema and one Rhyacionia Japan: identification and field evaluation. J. Chem. species (Eucosmini), and one Hedya species Ecol. 31: 859–878. (Olethreutini) (Ando, 2006; El-Sayed, 2006). This Wakamura, S. (1985) Identification of sex pheromone com- ponents of the podborer, Matsumuraeses falcana (Wal- dienyl acetate is one of the most common shingham [sic]) (Lepidoptera: Tortricidae). Appl. Ento- pheromone components of species in the subfamily mol. Zool. 20: 189–198. of Olethreutinae and has not been characterized in Wakamura, S. (1992) Development in application of syn- lepidopteran insects in other groups. thetic sex pheromone to pest management. Jpn. Pestic. Inform. 61: 26–31. ACKNOWLEDGEMENTS Wakamura, S. and K. Kegasawa (1986) Sex pheromone of the podborer, Matsumuraeses falcana (Walshingham The authors are grateful to Drs. F. Mochizuki and T. Fuku- [sic]) (Lepidoptera: Tortricidae): activity of the third moto of Shin-etsu Chemical Co., Ltd. for supplying synthetic component, (E,Z)-7,9-dodecadienyl acetate, and 3-com- pheromones for field tests. ponent formulation. Appl. Entomol. Zool. 21: 334–339. Young, D. C., P. Vouros and M. F. Holick (1990) Gas chro- matography-mass spectrometry of conjugated dienes by derivatization with 4-methyl-1,2,4-triazoline-3,5-dione. J. Chromatogr. 522: 295–302.