Agric. Biol. Chem., 44 (4), 765 775, 1980 765

Sex Pheromone of the Rice Green Caterpillar: Chemical Identification of Three Components and Field Tests with Synthetic Pheromones

Tetsu ANDO, Ken-ichi KISHINO,* Sadahiro TATSUKI,**

and Nobutaka TAKAHASHI•õ

Department of Agricultural Chemistry, Faculty of Agriculture,

The University of Tokyo, Tokyo 113, Japan *Tohoku National Agricultural Experiment Station , Omagari, Akita 014-01, Japan **The Institute of Physical and Chemical Research , Wako, Saitama 351, Japan

Received August 30, 1979

The sex pheromone of the rice green caterpillar, Naranga aenescens Moor, was presumed

to be a mixture of some unsaturated acetates using electroantennogram technique. Abdominal

tips of virgin females were shown to contain (Z)-9-tetradecenyl acetate (compound I), (Z) - 9-hexadecenyl acetate (compound ‡U) and (Z)-11-hexadecenyl acetate (compound ‡V) in a

ratio of 1:1:4, respectively. These synthetic three compounds were individually active on the

male antenna but only the mixture of the three compounds attracted the male in a field. Field traps containing the three compounds in a ratio of 1: 1: 4 (1 mg/rubber septum) con

sistently attracted more males than traps baited with two unmated females.

The rice green caterpillar, Naranga aene crowded conditions of three or more indivi scens Moor (: , Eustro duals per tube, even though they are reared tiinae), is one of the most harmful pest under a long day photoperiod. Then, in this of rice plant in eastern Asia. The larvae of study we have used efficiently electroanten this species feed on the leaves, causing foliar nogram (EAG) technique as a bioassay instead damage which often results in a great loss in of the customary laboratory bioassay which is rice yield. In the Tohoku district this pest based on the observation of the male sexual usually occurs three times every year and over responses using the many strictly pre-condi winters as a pupa.'' Since the adults which tioned male moths. EAG shows the electro emerge from overwintering pupae late in spring physiological response at the antennal level eli mate in the daytime, population monitering by cited by the olfactory stimuli, such as phero light traps is unsuccessful at the first flight." mone, odor of food and so on. EAG techni Developments of effective synthetic sex phero que had been developed by Schneider2) and mone of this are required for a monitor Roelofs and his co-workers identified sex ing tool to know the timing of insecticide ap pheromones of many species in Lepidoptera by plication, and also for a selective suppression of the application of this technique." Every the pest. antenna is available for EAG measurement of The population density during the larval ten or more test materials in succession and the stage of this species is known to be one of the repetition with several antennae can give trust factors responsible for inducing larval dia worthy results.'' It is expected that EAG pause.1) The constant supply of the many technique is good for the identification of the moths for the laboratory bioassay of the sex sex pheromone of the rice green caterpillar. pheromone is very difficult, because diapause We had already reported the identification of is induced when larvae are reared under this sex pheromone preliminarily.') In this t To whom future correspondence should be paper we will describe minutely the process of addressed. the structure presumption of the sex phero- 766 T. ANDO, K. KISHINO, S. TATSUKI, and N. TAKAHASHI

mone at first, which includes not only chemical reactions with the crude extract of virgin females but also EAG assay to a series of syn thetic mono-unsaturated compounds. On the bases of this estimate we have succeeded in isolating and characterizing three EAG-active components from the extract, and then it was confirmed that these three components are necessary to attract the male moths in the field.

STRUCTURE PRESUMPTION

In the EAG analysis of the fractions obtained by thin-layer chromatography (TLC) of 50 female extract on a silica gel plate using n hexane-benzene (1: 3) as developing solvent the strong activity was observed at acetate zone FIG. 2. EAG Assay of the Fractions Obtained by Preparative TLC (AgNO3 Impregnated Silica Gel) of (Rf 0.40.5), while no activity at alcohol zone 50 Female Extract. (Rf 0-0.1) (Fig. 1). Another TLC of the Developing solvent; benzene. Standard material; crude extract on 20% AgNO3/silica gel plate A: Tetradecyl acetate, B: (E)-9-Tetradecenyl acetate, using benzene as developing solvent and sub C: (Z)-9-Tetradecenyl acetate, D: (Z)-9, (E)-12 sequent EAG assay showed the good activity - Tetradecadienyl acetate. at (Z)-monoene acetate zone (Rf 0,4-0.5) as shown in Fig. 2 (saturated acetate; Rf ca. 0.8, hydrogenation or by the treatment with me (E)-monoene acetate ; Rf ca. 0.6, (E), (Z)-homo thanolic potassium hydroxide, but the treat conjugated diene acetate; Rf ca. 0.3). The ment of crude alkaline hydrolyzed mixture with EAG activity of the crude extract was lost by acetic anhydride-pyridine restored the original activity. EAG activity on TLC, and combina tion of chemical reactions and EAG tests in dicated that the sex pheromone of this species was acetate of unsaturated alcohol. Further more above conclusion was confirmed by the field tests. In our random field attraction tests using about one hundred mono-unsaturated straight chain (C,,-C,5) alcohols or acetates it was observed that male insects of ninety-three species were attracted specifically,' ~but none of these synthetic compounds could attract the moths of the rice green caterpillar when tested in a single source. This result suggested that the sex pheromone of the rice green caterpil lar might have multi-component and/or

FIG. 1. EAG Assay of the Fractions Obtained by polyene nature. Preparative TLC (Silica Gel) of 50 Female Extract. To get the information on the carbone number of the pheromone, the extract from 100 Developing solvent; n-hexane-benzene (1:3). Stand ard material; A: (Z)-9-Tetradecenyl acetate, B: (Z)-9 females was injected onto the nonpolar gas - Tetradecen-l-ol. liquid chromatography (GC) column (OV-1, Sex Pheromone of the Rice Green Caterpillar 767

(a) Tetradecenyl Acetate (C14) (b) Hexadecenyl Acetate (C16)

FiG. 3. EAG Activities of 10 jig Monoene Acetates.

The response to each test chemical is expressed as percent of the response to the standard compound, 10ƒÊg (Z)-9-tetradecenyl acetate. Sensitivity of the male antenna was checked by the standard

compound prior to appling each test chemical. Each EAG test was replicated by the use of at least seven different antennae.

2 %, 0.3 x 200 cm). In EAG analysis of the for thirty minutes to get the extract without excessive impurities. The crude extract ob preliminary collected fractions activities were detected at the retention times of 14-carbon tained by evaporation of the solvent was only

acetate and 16-carbon acetate. Then the EAG responses to a series of mono-unsaturated 14

- and 16-carbon acetates were measured using 10ƒÊg synthetic pure samples. The result of

this measurement is shown in Fig. 3. This assay revealed that some 14 and 16-carbon

monoene acetates in tested chemicals have

good activities. (Z)-9-Tetradecenyl acetate elicited the greatest antennal response (5.4 mV) in the tested 14-carbon acetates and (Z)-11

hexadecenyl acetate (1.7 mV) in the 16-carbon

acetates.

The above experiments strongly suggested

that the sex pheromone of the rice green cater

pillar was the mixture of several unsaturated acetates which included (Z)-9-tetradecenyl

acetate and (Z)-11-hexadecenyl acetate as

main components.

Mass Chromatography Mass Chromatography

IDENTIFICATION Fin. 4. Procedure of the Identification of EAG - Active Substances. The abdominal tips excised from about 1200 * shows that EAG analysis was carried out at this virgin female moths were soaked in n-hexane step. 768 T. ANDO, K. KISHINO, S. TATSUKIand N. TAKAHASHI

46.2 mg and the most EAG-active material sessed the following fragment ions; m/e 194 could be extracted with the nonpolar solvent (M+-CH3COOH), 166 (C12H+22), 152 (C11H+20), during this short period of time. The extract 138 (C10H+18), 124 (C9H+6), 110 (C8H+14), 61 and was subjected to the treatment shown in Fig. 43. The retention time and mass spectrum was 4 for the identification of the EAG-active com identical with those of a standard material, ponents. By the preparative TLC (silica gel, (Z)-9-tetradecenyl acetate as shown in Fig. 5. developing solvent; n-hexane-benzene (1: 3)), As double bond positional isomers have been 3.8 mg of EAG-active fraction was obtained known to show similar mass spectra, the posi from the acetate zone (Rf 0.4 0.5). This par tion of unsaturation could not be decided from tially purified sex pheromone was fractionated this GC-MS. Similarly the mass spectrum by preparative GC with a nonpolar column of the major peak of fraction B at 9.0 min was (OV-1, 2 %, 0.3 x 120 cm, 140°C) to give two shown to be a hexadecenyl acetate as shown in EAG-active fractions, fractions A and B, Fig. 6. Comparison of the GC peak area which were collected at 3.6 5.6 min and at between fractions A and B indicated the ratio 9.9 13.2 min, respectively. In this GC con of tetradecenyl acetate to hexadecenyl acetate dition, retention times of tetradecyl acetate to be 1 to 5. and hexadecyl acetate were 4.2 min and 10.4 To determine the location of the double min, respectively. bound in these compounds, ozonolysis was Gas-liquid chromatography combined with conducted after further purification of both mass spectroscopy (GC-MS) of fraction A on a fractions A and B by the preparative GC with PEG 20M column (5 %, 0.3 x 200 cm, 190°C) a polar column (PEG 20M, 15 %, 0.3 x 120 cm, showed one major peak at 4.8 min which pos 190•KC). GC-MS of the ozonolysis product of

FIG. 5. Result of the GC-MS of Standard (Z)-9-Tetradecenyl Acetate (a) and Fraction A of the Female Extract (b). Sex Pheromone of the Rice Green Caterpillar 769

FIG. 6. Result of the GC-MS of Standard (Z)-11-Hexadecenyl Acetate (a) and Fraction B of the Female Extract (b).

the purified fraction A on a PEG 20M column scanning every six seconds. Fig. 7-a shows the

(5%, 0.3 x 200 cm, 100-190•KC, 10•KC/min) mass chromatography of the ozonolysis pro indicated that one major component was 9 ducts of the mixture of two standard com - acetoxynonanal in the respect of thr retention pounds, (Z)-9-tetradecenyl acetate and (Z) time (ca. 14 min) and the mass spectrum which 11-hexadecenyl acetate. It shows the ap

possessed following fragment ions; m/e 157 pearance of 9-acetoxynonanal and 11-acetoxy

(M+-CH3CO), 112 (C8H16+), 97 (C7H13+), 83 undecanal at 4.8 min and 9.3 min, respectively. (CBH11+), 69 (C5H9+), 55 (C,H,+), 61 and 43. Mass chromatography of the ozonolysis pro Fraction B gave two peaks, which were indi ducts of fraction A revealed the presence of 9 cated to be due to 9-acetoxynonanal (minor - acetoxynonanal as shown in Fig. 7-b, and that component) and 11-acetoxyundecanal (major of fraction B the presence of 9-acetoxynonanal component). Retention time of 11-acetoxy and ll-acetoxyundecanal as shown in Fig. undecanal is ca. 19 min and this compound 7-c. Therefore it has been clearly concluded shows the following mass fragment ions; m/e that fraction A consists of 9-tetradecenyl ace 185 (M+-CH3CO), 140 (C10H20), 125 (C9H17+), tate and fraction B consists of 9-hexadecenyl 111(C8H15+), 97(C,H13+), 83 (C6H11+), 61 and 43. acetate and 11-hexadecenyl acetate. In confirmation of the above GC-MS measure The behavior of the EAG-active material on ment, mass chromatographic analyses of the the AgNO3 impregnated silica gel TLC indi ozonolysis products were conducted on eight cated that all the sex pheromone components selected fragment ions (m/e 185, 157, 140, 125, possessed (Z)-configuration as described al 112, 97, 61 and 43) under the following condi ready. Further, stronger EAG activities of tion; PEG 20M, 5%, 0.3 x 200 cm, 190°C, (Z)-isomers than (E)-isomers as shown in 770 T. ANDO, K. KISHINO, S. TATSUKI and N. TAKAHASHI

FIG. 7. Mass Chromatogram of the Ozonolysis Product on Eight Selected Fragment Ions; (a) Mixture of Two Standard Compounds, (Z)-9-Tetradecenyl Acetate and (Z)-11-Hexadecenyl Acetate, (b) Fraction A and (c) Fraction B.

Fig. 3 are in good agreement with this presump FIELD TESTS tion of geometry of double bond. In con Field tests of the synthetic pheromones were clusion the EAG-active components in the conducted in a paddy field of the Tohoku virgin female extract of the rice green caterpillar National Agricultural Experiment Station, were characterized as (Z)-9-tetradecenyl acetate Omagari, Akita Pref. in 1977 and 1978. In this (compound ‡T), (Z)-9-hexadecenyl acetate (com field three flights of the rice green caterpillar pound ‡U) and (Z)-11-hexadecenyl acetate (corn are usually observed from late in April to nound ‡V). early in August. The third flight is always

quantitatively more exceeding than others. 1) In the field tests with compounds ‡T-‡V using

rubber septa attractancy of single-component

and two-component lures were found to be The quantitative ratio of compound ‡U to negligible, while the mixture of compounds

compound ‡V could not be estimated directly, ‡T-‡Vin the ratio observed in the female extract

because they were not detected as the separated (1: 1: 4) could strongly attract the male moths. two peaks on GC (PEG 20M, 5 %, 0.3 x 200cm). Thus three compounds identified from the Fragment ion at m/e 61 is characteristic of female extract were shown to be indispensable

acetates and has been observed with similar for the field attraction of the males. This

intensity in several ƒÖ-acetoxyalkanals.6) The result confirms that the sex pheromone of the

comparison of the peak area between 9 rice green caterpillar is composed of com

- acetoxynonanal and 11-acetoxyundecanal on pounds ‡T, ‡U and ‡V. m/e 61 ion shown in Fig. 7-c indicates that For getting the optimum ratio of the syn

compounds ‡U and ‡V are included in the female thetic pheromone, three-component mixtures

extract in the ratio of 1: 4. Since the quanti in many different ratios were tested. Table ‡T

tative ratio of compound I to the sum of com shows the result of field test of nine lures

pounds ‡U and ‡V is I to 5 as described before, holding compound ‡V as major component at the virgin female must contain compounds ‡T, the third flight. The trap baited with the

‡U and ‡V in the ratio of 1: 1: 4, respectively. rubber septum which holds a mixture of 0.125, Sex Pheromone of the Rice Green Caterpillar 771

TABLE I. FIELD ATTRACTION OF MALE MOTHS OF RICE GREEN CATERPILLAR To THREE-COMPONENT LURES¢

a Data obtained from 2 traps in each treatment during July 18 -August 4, 1977.

b In rubber septa .

e. It is expressed as percent of the activity of the mixture in the natural ratio (‡T: ‡U: ‡V =1: 1: 4).

FIG. 8. Summary of Field Attractancy of 26 Lures for Male Moths of Rice Green Caterpillar.

Activity are expressed as percent of the flights to the mixture of compounds ‡T-‡V in a ratio of

1: 1: 4. Ratios shown by the same letter (a d) were tested in one block, using the traps of same type.

0.125 and 0.5 mg of compounds I, ‡U and ‡V, that the lures loading compound I or ‡U as a is most attractive in all the traps. This at major component showed less activity than tractancy decreases when the content of com 20% of the original mixture. Results of the pound I increases, the mixture containing above tests are summarized in Fig. 8. This 0.5 mg of compound I showing only about 25 figure intelligibly shows that the optimum activity of the original mixture. The rise of ratio of compounds ‡T-‡V for the attraction the ratio of compound ‡U also weakens the of the rice green caterpillar is about 1: 1: 4 attractancy exceedingly. Another test revealed in good coincidence with the natural ratio, 772 T. ANDO, K. KISHINO, S. TATSUKI and N. TAKAHASHI and that the more the mixing ratio of lures mixtures of compounds ‡U and ‡V. The traps keeps apart from 1: 1: 4 the more their at baited with the two compounds in a ratio of tractancy diminishes. 1: 4 caught more males (seventeen moths) Further, attractancy of the mixture in the than others in July. Specific attraction of optimum ratio (1: 1: 4) at the total amounts of another species was not noticed at every trap

0.1, 0.5, 1 and 5 mg was examined also using tested from April to August. rubber septa as dispensers. In this test total catches of male moths increased according to the amount of dosage, but the difference be DISCUSSION

tween the 1-mg and 5-mg traps was little. It is concluded that the rice green caterpillar,

This result indicates that the 5-mg dosage was Naranga aenescens, utilizes (Z)-9-tetradecenyl not necessary for the attraction of the males acetate (compound ‡T), (Z)-9-hexadecenyl ace and 1 mg/rubber septum was satisfactory like tate (compound ‡U) and (Z)-11-hexadecenyl many other species. Then comparison of the acetate (compound ‡V) as the components of

attractiveness of the rubber septum loading the sex pheromone for male attraction and that

1 mg of the synthetic pheromone and virgin the unmated female moth may release com females was conducted. Field traps contain pounds ‡T, ‡U and ‡V in a ratio of 1: 1: 4, respec ing 1 mg pheromone attracted males about tively. Compounds ‡T and ‡V have been known

two times as many as two virgin females at the to be the sex pheromone components of first and second flights as shown in Table II. some other noctuid species, but compound ‡U

The data obtained at the third flight is coinci has not been identified hitherto as the phero dent with this result, although we missed the mone component. This is the first case. timing of the test due to the unusual weather Recently the multi-component nature of lepido and could not examined throughout this pterous sex pheromone have been claimed period. The strong activity of this synthetic by the finding of several two-component sex pheromone suggests a possibility of use of the pheromones,7,8) and we conducted the random synthetic pheromone as a substitute for the screening tests of two-component lures in a virgin females to monitor the population of this field.9) But the three-component sex phero

species. mone whose every components are indispen

In this field tests it was interestingly observed sable for the field attraction of male moths that the male moths of Scoparia sp. (Pyrali is known very seldom. Roelofs and Carde dae: Scopariinae) were attracted only to the distinguished between the primary sex phero mone components, which elicit long-distance TABLE II. THE. NUMBER OF RICE GREEN upwind anemotaxis in the responding insect, CATERPILLAR MALE MOTHS CAPTURED BY PHEROMONE AND LIGHT TRAPS and the secondary components, which evoke close-range responses, such as landing, wing

fanning, hair pencilling and copulatory at tempts.8) According to their definition all

compounds I III found in the female extract

are primary pheromone components. Field traps containing the three compounds were

consistently more attractive than traps baited

with virgin females. This result indicates a Data obtained from three traps . that secondary pheromone components may a Data obtained from one trap . Similar number of not be effluted in particular by the female moth female moths was also attracted. of the rice green caterpillar other than three May 5-.29, 1978. d June 15•`July 3 , 1978. primary pheromone components. The role July 18•`August 4, 1977. of each of three components on the seriated Sex Pheromone of the Rice Green Caterpillar 773

sexual behavior of the male moth, i. e. orienta extract of the pheromone; (2) the preparation tion, precopulatory behavior and mating, of the EAG profile of male antennal responses should be clarified by the observation of the to a series of mono-unsaturated compounds sexual behavior of this species in a field and which agree with the chemical nature defined laboratory. in step (1); (3) certification of the presence of Noctuidae, one of the biggest families in the candidate(s) selected in step (2), in the Lepidoptera, is grouped into sixteen subfami virgin female extract purified with application lies. In this family sex pheromones have been of EAG assay; and (4) comfirmation of at studied on more than twenty species from the tractive activity of the identified component(s) following five subfamilies; Amphipyrinae, in the field tests. If the synthetic candidate(s) Heliothidinae, Noctuinae, Hadeninae and can be available sufficiently before the (3), it is Plusiinae, and chemically characterized as a short way for the elucidation of the phero unsaturated straight chain alcohols and/or mone to conduct the field test with the candi their derivatives (acetates and aldehydes). date(s) directly after step (2). The sex pheromone of the rice green caterpillar The field test shown in Table II proved that is the first one identified from the subfamily the synthetic sex pheromone possessed strong . Together with the fact that one er attractancy than virgin females. And our species in Eustrotiinae, Micardea argentata preliminary test for the utilization of the syn Butler, was attracted to (E)-7-dodecenylacetate thetic pheromone revealed that a rubber septum or its (Z)-isomer in our random screening which was applied with I mg of the pheromone tests,5) this identification suggests that the before the first flight (late in April) consistently sex pheromones of species in the subfamily showed the same attractive activity as a new Eustrotiinae are generally unsaturated straight dispenser till the end of the last flight (early in chain compounds. It is interesting to reveal August). These results show that it is possible the chemical nature of pheromones in the other to utilize the synthetic pheromone as a simple subfamilys; Catocalinae, Apatelinae, Hermini tool monitoring the insect population. Parti inae, etc. cularly to get the timing of insecticide applica The success of the identification of this sex tion this synthetic sex pheromone must be pheromone with such a small number of the indispensable in place of the virgin females at virgin females (ca. 2000 in total) may be due to the first flight when this species mates in the appropriate application of EAG technique as daytime and a light trap is impotent. But the monitoring tool of the pheromone and the pheromone trap was rather inferior to the light selection of pheromone candidates by EAG trap in attraction at the third flight (Table II). survey with synthetic compounds prior to It seems that the pheromone trapping does not chemical identification. Especially it must be always reflect the popuration density of the pointed out that EAG technique is most useful insect. Hence the information of relation for the identification of the multi-component between the number of trapped moths and the pheromones. Each component separated real insect population in a field will be needed. from multi-component pheromone possesses some EAG-activities, while it may not be able to excite the male moth. Now based on EXPERIMENTAL our experience we wish to propose the general EAG analyses. The proceduresof our experiments identification method which is applicable to a were modifiedfrom that describedby Roelofs.3) The lepidopterous sex pheromone composed of head of the male moth of the rice green caterpillarwas unsaturated primary alcohols and/or their removedand placedonto the surfaceof the tissuepaper whichwas wettedwith insectRinger's solution.') The derivatives, as follows: (1) the determination ground electrode was submerged in the above saline of the functional group, carbon number of the solutionto make contact with the antennal base. The chain and double bond number, using the crude input electrode was fixed into a glass capillary con- 774 T. Armo, K. KISHINO, S. TATSUKI and N. TAKAHASHI

taining the same saline solution. The antennal end was evapolation of the solvent was treated with 2 ml of 2 N introduced into this capillary. For the assay of the methanolic potassium hydroxide for 1 hr at room natural pheromone and synthetic compounds, 50 female temperature. After the addition of 4 ml of 1 N hydro extract and 10 ƒÊg of chemicals were used, respectively. chloric acid and a trace amount of NaHCO3, the The test material dissolved in organic solvent (usually hydrolyzed product derived from the pheromone was n-hexane) was soaked to a folded filter paper (I x extracted with 100 ml of n-hexane. Half of this extract 2.5 cm, Toyo Roshi No. 2) placed in a glass cartridge was appropriated to assay its activity in EAG and (1.2 (O. D.) x 2.5 cm) and the solvent was allowed to field tests. The remaining extract was treated with 0.5 evaporate. The cartridge was then put into a glass ml of pyridine and 0.5 ml of acetic anhydride succes syringe (5 ml) and 1 ml of air in this syringe was puffed sively for re-acetylation after the removal of n-hexane. by hand into the purified air-flow passing over the This mixture was left at room temperature for 1 hr and antenna constantly (1 cm (I. D.) at the outlet, 3 1/min). added to 5 ml of water. The crude product extracted As a control, the filter paper soaked with the solvent with 100 ml of n-hexane was used for EAG and field alone was prepared. EAG responses were displayed test. on a cathode ray oscilloscope and photographed. Ac tivity of each material was expressed by the EAG Hydrogenation of the pheromone. Crude extract of amplitude (mV) subtracted that of a control. 100 females dissolved in 2 ml of n-hexane was hydro genated over 2 mg of 10 % Pd-C for 10 min at room Field trapping. Tub-type traps10) and sticky type temperature. After the filtration of the catalyst, the traps (Takeda pheromone trap*') were set on the road crude hydrogenated product was used for EAG and side above the ground and at a height of 0.4 m, respec field tests. tively. For the test of the natural pheromone, test material derived from 5 females was soaked into a filter Ozonolysis of the pheromone. Fractions A and B paper (9 cm in diameter) which was placed at the center purified from 600 female extract were dissolved in of the trap and renewed every other day. Attractivity 0.2 ml of n-hexane and treated with ozone for 1 min at of the female for the male moths was examined using -77°C . Since the reductive decomposition of the the trap baited with two virgin females in a plastic vial ozonide was achieved by a high temperature in gas with wire-screen ends (6 x 3 cm). The females were chromatograph, the above solution was directly injected also renewed every other day. Synthetic chemicals into gas chromatograph-mass spectrometer to analyze were applied to rubber septa*1 as dispensers, which were the (o-acetoxyalkanals decomposed from the pheromone used without renewal during the experiment. The components. number of the attracted moths was counted every morning. Acknowledgment. We thank Dr. Y. Tamaki of National Institute of Agricultural Sciences for his Chemicals. All of the monoene compounds were help in measuring GC-MS and useful advice, and Ms. synthesized in our laboratory in the method described T. Sato of Tohoku National Agricultural Experiment before, using the stereo-selective reduction of acetylene Station for rearing of the insect and examination of the compounds.') For the EAG test, all the chemicals field traps. were purified by preparative TLC with AgNO3 impre

gnated silica gel plates. Although this purification was not conducted for the chemicals used in field tests,

the contamination of a trace amount (less than 3 %) of REFERENCES

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Shorey and J. J. McKelvey, Jr, John Wiely & Sons Oku and N. Takahashi, Agric. Biol. Chem., 42, Inc., N. Y., 1977, pp. 253-285. 1081 (1978). 8) W. L. Roelofs and R. T. Carde, Ann. Rev. Ent., 10) S. Tatsuki, M. Kurihara, K. Uchiumi, J. Fukami, 22, 377 (1977). Y. Fujimoto, T. Tatsuno and K. Kishino, Appl. 9) T. Ando, H. Kuroko, S. Nakagaki, O. Saito, T. Ent. Zool., 14, 95 (1979).