Biosci. Biotechnol. Biochem., 66 (11), 2461–2464, 2002

Note Identiˆcation of the Sex Pheromone Components Secreted by Female of (: )

Shin-ichi INOMATA,1 Satoshi TSUCHIYA,1 Kazutaka IKEDA,1 Osamu SAITO,2 and Tetsu ANDO1,†

1Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan 2National Agricultural Research Center for Western Region, Fukuyama, Hiroshima 721-8514, Japan

Received March 19, 2002; Accepted June 3, 2002

The variegated cutworm, Peridroma saucia H äubner, that the synthetic pheromone attracted a large num- is a lepidopteran pest to a large number of crops in ber of males in Japan. Canada, the United States, and Europe. It was probably Rearing for successive generations was started naturalized in Japan in the 1970s. The pheromone from eggs of P. saucia whichwerelaidbymatedfe- glands of the female include two components with males caught in the mountainous area around Tsuku- electroantennographic activity in a ratio of 3:1. GC-MS ba in Ibaraki Prefecture. The larvae were reared on a analyses of pheromone extracts untreated and treated semisynthetic diet including powder of mulberry with dimethyl disulˆde revealed the major component leaves [Insecta LF(S), Nippon Nosan Kogyo Co.] at to be (Z )-11-hexadecenyl acetate and the minor compo- 259C under a 16:8 hr light-dark cycle. The terminal nent to be (Z )-9-tetradecenyl acetate. The synthetic abdominal segments, including the pheromone pheromone was used to attract a large number of males glands, were removed from about 100 female moths in a vegetable ˆeld in Tokyo, which suggests that this (2 or 3 days old) in the scotophase and soaked in n- species has already become a harmful pest in Japan. hexane for 15 min to extract the sex pheromone. This crude extract was submitted to GC (HP 6890 GC Sys- Key words: ; pheromone; at- tem), using a capillary column (DB-23, 0.25 mm ID tractant; monoenyl acetate; ˆled attrac- ×30 m, J & W Scientiˆc). The end of the column was tion split into two paths (1:1 split ratio), one being con- nected to a ‰ame ionization detector (FID) and the The variegated cutworm, Peridroma saucia other to an electroantennographic detector (EAD).3) H äubner, is a lepidopteran pest to a large number of AsshowninFig.1andmeasuredwithacolumntem- crops. This noctuid species, which originated in perature program of 809C for 1 min and then 89CW Canada, the United States, and Europe, is now re- minto2109C,themaleantennaeˆxedtotheEAD garded as a cosmopolitan species. The adults were exclusively responded to two components (I and II) discovered in Japan in the 1970s. This species is in the crude extract of one female equivalent (FE). called ``nise-tamanayaga,'' which means false black These components were always detected by the EAD cutworm moth in Japanese, because the appearance in analyses repeated with other injections of the ex- of the adult is similar to that of the black cutworm, tract, and no other materials showed constant elec- Agrotis ipsilon Hufnagel, a harmful noctuid spe- troantennographic (EAG) activity. The peak areas cies.1) P. saucia is presumed to be naturalized in recorded by FID indicate that the pheromone gland Japan, but the distribution areas are still uncon- included I (15 ngWfemale) and II (50 ngWfemale) in a ˆrmed. In order to clarify the ecological aspects and ratio of ca. 1:3, while the EAG response for I was damage to the crops, we were interested in the sex stronger than that for II. The retention times of I and pheromone which can provide a convenient method II coincided well with those of authentic Z9-14:OAc for monitoring the pest. By ˆeld screening with a (13.72 min) and Z11-16:OAc (15.69 min), respec- large number of synthetic chemicals in Canada, Stru- tively. Elution of their geometrical isomers was faster ble et al. have found that the sex attractant for the than that of the natural components, i.e., (E )-9- male moth was composed of (Z )-9-tetradecenyl tetradecenyl acetate (13.54 min) and (E )-11-hex- acetate (Z9-14:OAc) and (Z )-11-hexadecenyl acetate adecenyl acetate (15.51 min). (Z11-16:OAc).2) We report here that these two com- The pheromone extract was analyzed by GC-MS pounds were actually produced by the females and (HP 5973 MS System, electron impact ionization at

† To whom correspondence should be addressed. TelWFax:+81-42-388-7278; E-mail: antetsu＠cc.tuat.ac.jp 2462 S. INOMATA et al.

Fig. 1. GC Analysis of the Pheromone Extract of Peridroma saucia (1 FE) with a Flame Ionization Detector (FID) [A] and an Electroantennographic Detector (EAD) [B]. Fig. 2. GC-MS Analysis of the Pheromone Extract of Peridroma saucia (1 FE) Treated with Dimethyl Disulˆde (DMDS), Total 70 eV) operated under the same GC conditions as Ion Chromatogram (TIC) [A], and Mass Spectra of DMDS Ad- those for the above-mentioned analysis. Measure- ducts of Component I (Z9-14:OAc) [B] and Component II (Z11- ment with the crude extract of one FE successfully 16:OAc) [C]. showed the mass spectra of components I and II. Their characteristic ions at m Wz 61 ([CH3 CO2H+ + + H] )andmWz M-60 ([M-CH3CO2 H] , m Wz 194 for I and mWz 222 for II) conˆrmed their chemical struc- tures, i.e., a mono-unsaturated C14 chain with a ter- minal acetoxy group for I and the C16 analog for II. The other spectral patterns of I and II were also simi- lar to those of the authentic monoenyl acetates with a

C14 or C16 straight chain. Another GC-MS measure- ment of the extract (10 FEs) was carried out, but no other pheromone-like substance that could be consi- dered as a minor component was detected. The double-bond positions of the natural components were determined by a GC-MS analysis of their ad- ducts with dimethyl disulˆde (DMDS), according to the method of Buser et al.4) The crude extract (10 Fig. 3. Electroantennographic (EAG) Response (Mean mV±SE, n＝5) of Peridroma saucia Males to the Synthetic Pheromone FEs) was dissolved in a mixture of DMDS (50 ml) and Components, (Z )-9-Tetradecenyl Acetate (Z9-14:OAc) []and diethyl ether (100 ml), including iodine (500 mg), and (Z )-11-Hexadecenyl Acetate (Z11-16:OAc) []. warmed overnight at 409C.Afteraddinga5z sodi- um thiosulfate solution (0.5 ml), the crude products were extracted with n-hexane and injected for GC- the double bond at the 11-position of the C16 chain by MS operated under the same conditions as those for M+ at m Wz 376 and fragment ions at mWz 259, 199, the analysis of the extract that had not been treated and 117 (Fig. 2C). with DMDS. Figure 2A shows a total ion chromato- The dose responses of the two synthetic compo- gram of this measurement. The DMDS adduct der- nents were measured by the GC-EAD method. The ived from I was eluted from the GC column overlap- EAG activity of the minor component, Z9-14:OAc, ping with an unknown compound, but its mass was interestingly about 100 times stronger than that spectrum at 23.80 min included M+ at m Wz 348 and of the major component, Z11-16:OAc, as shown in fragment ions at m Wz 231, 171, and 117, which indi- Fig. 3. Next, their activity to attract male moths was cated the double bond at the 9-position of the C14 evaluated in a vegetable ˆeld in Tokyo (Fuchu-shi). chain (Fig. 2B). The DMDS adduct of II was eluted The chemicals were mixed in several diŠerent ratios at 26.84 min, and its spectrum evidently conˆrmed and applied to natural rubber septa (8 mm OD, white Sex Pheromone of the Noctuid Moth 2463

Table 1. Attraction of Male Noctuid Moths, Peridroma saucia and Mamestra brassicae, by Synthetic Lures Baited with (Z )-9-Tetradecenyl Acetate (Z9-14:OAc) and (Z )-11-Hexadecenyl Acetate (Z11-16:OAc) in 2001a

Captured males trapb Components of lure W (mgWrubber septum) April 24–May 16 June 15–July 6 Aug. 4–25 Sept. 20–Oct. 11

Z9-14:OAc Z11-16:OAc P.s. M.b. P.s. M.b. P.s. M.b. P.s. M.b.

0 1.00 0.0 63.7 0.0d 8.0 0.0c 0.0 0.0d 2.0 0.05 0.95 — — 4.0c 0.0 0.3c 0.0 0.0d 0.0 0.15 0.85 — — 15.3ab 0.0 16.7ab 0.0 2.3c 0.0 0.25 0.75 70.3 0.0 23.7a 0.0 21.3a 0.0 6.0b 0.0 0.40 0.60 — — 6.0bc 0.0 16.0ab 0.0 7.3b 0.0 0.60 0.40 — — 10.0abc 0.0 8.0b 0.0 14.7a 0.0 1.00 0 0.0 0.0 0.0d 0.0 0.0c 0.0 0.0d 0.0 0 0 0.0 0.0 0.0d 0.0 0.0c 0.0 0.0d 0.0

a Tested with three traps for each lure in a ˆeld cultivating several vegetable species in Tokyo (Fuchu-shi). b Values within each column followed by a diŠerent letter are signiˆcantly diŠerent at Pº0.05 by the Tukey-Kramer test. rubber, Aldrich) that were used as dispensers set at sect pest in Japan,7) this result suggests the possibility the center of sticky-type traps (30 cm×27 cm, Take- that P. saucia has already become a harmful pest da Chemical Ind.). The traps were ˆxed at a height of with an unlimited ‰ight period, at least in the vegeta- 1.5 m in the ˆeld in November 2000, and the lures bleˆeldusedinthisexperiment,andthatdamageto were renewed every two months. The species, sex, crops caused by this species has been confused with and number of every insect captured by each trap that by other noctuid species, especially A. ipsilon. were recorded once a week for one year. Table 1 Our future studies with pheromone traps will focus shows the total number of attracted males during on the habitat of P. saucia in Japan. four diŠerent periods. Strong attraction of P. saucia We conclude that the sex pheromone of the males was constantly observed by the mixture of Z9- Japanese strain of P. saucia consists of two compo- 14:OAc and Z11-16:OAc in a ratio of 1:3, the natural nents, Z9-14:OAc (minor) and Z11-16:OAc (major). ratio in the gland extract, although another 3:2 mix- These monoenyl compounds, which might be biosyn- ture was the most successful in attracting males thesized via the D11-desaturation of palmitic acid,8) among the lures tested from September 20 to October are common pheromone components of many 11. A number of males were caught in May and June. lepidopteran species, particularly the noctuid species. After a decrease in the attraction during July, Lepidopteran sex pheromones have been identiˆed another clear population increase was recorded in for more than 500 species, and one-ˆfth of them be- August. However, male moths were captured long to the family of Noctuidae.9,10) Even though the throughout the year, even during the winter, indicat- survey of pheromones is limited in this group, we ing the possibility of the adults overwintering in the know that as many as 18 species have Z11-16:OAc as Tokyo area. their main pheromone component. Among them, 12 In the ˆeld tests, other male noctuid pests, Telorta species secrete a minor component(s) diŠering from divergens Butler and Mamestra brassicae L., were Z9-14:OAc to attract partners. The sex pheromones captured by a single component lure of Z11-16:OAc, of the remaining 6 species include not only Z9- as expected from our previous ˆeld screening tests.5) 14:OAc but also an extra component(s) to devise an Larvae of T. divergens eat ‰owers of the peach tree, original communication system. Surprisingly, the and 45 males in total were captured from December same simple pheromone system that has been identi- to February by this test. M. brassicae is well known ˆed in this study for P. saucia has not been reported as a harmful cosmopolitan pest to many crops. In elsewhere. total, 322 males of this species were captured from April to October. The main component of M. brassi- Acknowledgments cae pheromone is Z11-16:OAc. The pheromone gland of M. brassicae includes other minor compo- The authors are grateful to Dr. S. Sugi of the nents whose synergistic activity has not been de- Japan Heterocerists' Society for identifying the in- ˆned.6) Presumably, trapping by this single- compo- sects attracted in the ˆeld test, and to Associate Prof. nent lure, as shown in Table 1, did not directly re‰ect S. Matsumura of Field Science Center (Tokyo Uni- the population density of M. brassicae in the ˆeld. versity of Agriculture and Technology) for assistance Comparing this attraction of M. brassicae, however, in the ˆeld screening test. quite a large number of males of P. saucia (more than 1,500 in total) were captured by the traps. Although P. saucia is not currently regarded as an in- 2464 S. INOMATA et al. References Bestmann, H. J., Technique for injecting intact glands for analysis of sex pheromones of Lepidoptera 1) Inoue, H., Sugi, S., Kuroko, H., Moriuti, S., and by capillary gas chromatography: reinvestigation of Kawabe, A., ``Moths of Japan'', Kodansha Co., pheromone complex of Mamestra brassicae. J. 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