Appl. Entomol. Zool. 38 (1): 73–78 (2003)

Sex pheromone of the flax budworm, maritima adaucta Butler (: )

Masashi KAKIZAKI1,*,† and Hajime SUGIE2 1 Hokkaido Ornamental Plants and Vegetables Research Center; Takikawa, Hokkaido 073–0026, 2 National Institute of Agro-Environmental Sciences; Tsukuba, Ibaraki 305–8604, Japan (Received 4 January 2002; Accepted 11 November 2002)

Abstract Sex pheromone of the flax budworm, Heliothis maritima adaucta, was extracted from abdominal tips of virgin fe- males using hexane. Three EAD-active peaks were detected in the extract by the GC-EAD method. These peaks were identified to be (Z)-11-hexadecenal (Z11-16:Ald), (Z)-11-hexadecen-1-ol (Z11-16:OH), and n-hexadecanal (n-16:Ald) by GC-MS analysis. The ratio of Z11-16:Ald, Z11-16:OH, and n-16:Ald was 100 : 24.5 : 2.4, and the amount of Z11- 16:Ald was about 18.1 ng per female. Mixtures of Z11-16:Ald and Z11-16:OH in ratios of 100 : 1–6 attracted males in the field, but the each compound alone did not. White rubber septa impregnated with 1.01 mg and 3.03 mg of a mix- ture of Z11-16:Ald and Z11-16:OH in a ratio of 100 : 1 could capture more males than those with smaller doses. Ad- dition of n-16:Ald to binary mixtures did not increase their attraction activity. Therefore, Z11-16:Ald and Z11-16:OH were concluded to be essential components of the sex pheromone of H. m. adaucta for male attraction. Also, addition of (Z)-9-hexadecenal, a pheromone component in a subspecies in Hungary, to the above binary mixture did not in- crease the attraction activity.

Key words: Sex pheromone; Heliothis maritima adaucta; (Z)-11-hexadecenal; (Z)-11-hexadecen-1-ol; n-hexadecanal

1993). Further, Szocs et al. (1993) reported that INTRODUCTION mixtures of Z11-16:Ald, Z11-16:OH, and n-16:Ald Heliothis maritima has been reported to have the showed attraction activity for male H. maritima in following subspecies: the flax budworm, H. m. the field, and that the addition of Z9-16:Ald en- adaucta Butler distributed in Japan, the fulvous hanced male response in a flight tunnel test, but clover , H. m. maritima Grasl in France, H. m. had no effect on the attraction activity in the field. angarensis and H. m. centralasiae in , H. m. The sex pheromone for H. m. adaucta has not been bulgarica in Bulgaria, H. m. warneckei (septentri- previously analyzed. Therefore, this report de- onalis) in Jutland, and H. m. hungarica in Hungary scribes the identification of female sex pheromone (Pool, 1989). H. m. adaucta is a pest of the legumi- of this subspecies. nous crops (soy bean, adzuki bean, kidney bean), leguminous forage plants, pimento, lettuce, and MATERIALS AND METHODS some ornamental plants (babies’-breath, carnation, etc.). Larvae feed on the leaves and buds of these . Larvae of H. m. adaucta were collected plants. Traps using sex pheromone would be useful from a soybean field in August 1995 (Naganuma, for population monitoring of this species. Four sex Hokkaido), and June 2001 (Takikawa, Hokkaido). pheromone components—(Z)-11-hexadecenal (Z11- They were reared on soybean leaves at 22°C under 16:Ald), (Z)-11-hexadecen-1-ol (Z11-16:OH), n- a 16L:8D photoregime, and the adults obtained hexadecanal (n-16:Ald), and (Z)-9-hexadecenal were used. (Z9-16:Ald)—were identified in a ratio of 74:7: Pheromone extracts. Since calling behavior of 4 : 1 in the virgin female extracts of H. maritima female H. m. adaucta , 2–3 d after emer- collected in Ukraine (Kovalev et al., 1991) and gence, is observed from 30 min to 4 h after lights 88.0 : 7.2 : 4.8 : trace in Hungary (Szocs et al., off, abdominal tips of females were cut 2 to 3 h

* To whom correspondence should be addressed at: E-mail: [email protected] † Present address: Hokkaido Dohnan Agricultural Experiment Station; Ohono, Hokkaido 041–1201, Japan

73 74 M. KAKIZAKI and H. SUGIE after lights off. Twenty-six females were used for disulfide (DMDS) (Buser et al., 1983). Fifty micro- analyses of sex pheromone compounds. The tips liter DMDS and 5 ml iodine solution (60 mg I2 in were immediately dipped into 1 ml hexane, and ex- 1 ml diethyl ether) were added to 50 ml hexane so- tracted for 3 min at room temperature. The extract lution containing each peak compound (sample I), was condensed to a 100 ml solution using a N2 gas which was collected by preparative GC, and the stream and stored at room temperature during a mixture was stored overnight at 40°C. Then, 200 ml few days. The extract (sample I) was used for of 5% Na2S2O3 solution in distilled water was measurement of antennal receptor responses and added to the mixture, which was extracted with identification of active compounds. Three female 100 ml hexane. abdominal tips in 30 ml hexane were used to deter- Geometric isomerism of each mono-unsaturated mine the amount and ratio of pheromone compo- compound was determined by comparing the GC nents without condensing treatment (sample II), to retention time (Rt) with those of synthetic com- avoid the influence of this treatment. pounds using the polar column (HP-INNOWAX). Column chromatography. The crude extracts Chemicals. Z11-16:Ald, Z9-16:Ald, n-16:Ald, of sample I were also separated by column chro- and Z11-16:OH were supplied from Shin-Etsu matography on Florisil containing 7% H2O (Car- Chem. Co. Ltd. The synthetic chemicals were puri- roll, 1961). The column was successively eluted fied by column chromatography on Silica-gel con- with hexane and 5%, 15%, 25% and 50% ether in taining 16.7% silver-nitrate. The purity of Z11- hexane. 16:Ald was 99.9% with less than 0.04% E isomer Bioassay by gas chromatography-electroan- and that of Z11-16:OH was 99.9% with less than tennographic detector (GC-EAD). The responses 0.01% E isomer. The purity of n-16:Ald was of antennae of H. m. adaucta males to the crude 99.8%. extract and each fraction from the Florisil column Lures. A white rubber septum (8 mm OD, Cat. chromatography were recorded with a GC-EAD No. Z10072-2, Aldrich Co. Ltd.) impregnated with system (Struble and Arn, 1984). A gas chromato- 100 ml hexane solution of synthetic compounds graph (Hewlett-Packard HP5980) with an HP-IN- was used as a lure. NOWAX capillary column (30 mϫ0.53 mm ID, Field experiments. Field experiments were car- 1.0 mm film thickness) coupled to a thermal con- ried out in a 22,500 m2 field of leguminous forage ductivity detector (TCD) was used. The oven tem- plants (red clover, white clover, and alfalfa) in Au- perature was programmed at 5°C/min from 60°C gust, 1997 (tests I–IV, VI–VII, Naganuma), and in (1.5 min) to 220°C (30 min). Preparative GC was a 12,000 m2 field of the same plants in June, 2001 performed by collection of the effluent containing (test V, Takikawa). The population densities of H. each EAD active peak by glass capillary tubes m. adaucta in these two years were at the middle from the vent tube of TCD. level. Sticky board traps (Takeda Chem. Co. Ltd.) Chemical analysis. Gas chromatograph-mass were placed at intervals of about 15 m and set at 40 spectrometer (GC-MS) analyses (EI mode) were cm above ground. performed on a Hewlett-Packard HP5971A with an Numbers of males captured by each trap were HP-INNOWAX capillary column (60 mϫ0.32 mm counted at intervals of 2 to 3 d, and the trap loca- ID, 0.25 mm film thickness) for analysis of sample tions were rotated at that time. Total numbers of I. The oven temperature was programmed in the males captured by each trap (X) were transformed same manner as above. The interface temperature to square root (Xϩ0.5) before analysis of variance, was 250°C. Also, JEOL JMS-SX102A with a TC- followed by Tukey’s test. FFAP capillary column (GL Science; 30 mϫ0.25 mm ID, 0.25 mm film thickness) was used for RESULTS analysis of 2 ml of sample II. The oven temperature was programmed at 7°C/min from 80°C (1 min) to Antennal responses in female extracts 210°C (30 min). The interface temperature was Three EAD active peaks (peaks A, B, and C) by 210°C. male antennae were detected in the female extract Double bond positions in unsaturated com- (Fig. 1). The large EAD amplitudes of peaks A and pounds were determined by adduction of dimethyl B produced by the crude extracts of 0.52 female Sex Pheromone of Heliothis maritima adaucta 75

Fig. 2. Mass spectra of EAD active peaks A, B, and C in Fig. 1. GC-EAD analysis from a male Heliothis maritima the GC-MS analyses of the female extract of Heliothis mar- adaucta antenna to the crude extracts of abdominal tips of itima adaucta. 0.52 female equivalents. Peaks A, B, and C indicate repro- ducible EAD active peaks (upper: TCD trace; lower: EAD trace). ϭ the synthetic Z11-16:Ald (Rt 36.15 min; Rt of E isomerϭ35.87 min). The characteristic fragment equivalents were 1.68–1.73 mV, 0.62–0.66mV, re- ions of peak B showed at m/z 96 (100%), 55 spectively, and the small EAD amplitude of peak C (84.0%), and 222 (32.0%, MϩϪ18), and these sug- was 0.064–0.048 mV. The EAD amplitude of peak gested that peak B is a mono-unsaturated C16 alco- C was not easily distinguished from other noisy hol. The characteristic fragment ions of DMDS ϭ ϩ peaks, however, it was also reproducible on the adduct of peak B showed m/z 117 (C5H10 SCH3 ), ϩϭ ϩ GC-EAD tests for other extracts. Rt of peaks A, B, 217 (H3CS C11H21OH), and 334 (M : C4H9CH- and C were 30.78 min, 34.85 min, and 29.95 min, (SCH3)CH(SCH3)C10H20OH), which were identical respectively. Peaks A and C were found in the frac- to those of 11-16:OH. Rt of peak B was 42.78 min tion of 5% ether in hexane, and peak B was found and almost the same as that of the synthetic Z11- ϭ ϭ in the fraction of 25% ether in hexane with the 16:OH (Rt 42.77 min; Rt of E isomer 42.35 min). Florisil column chromatography. Although some The characteristic fragment ions of peak C showed peaks after peak B are visible in the EAD chart of at m/z 57 (100%), 55 (76.7%), and 222 (42.0%, Fig. 1, they were not reproducible in other extracts. MϩϪ18), and these suggested that peak C is a sat-

Thus, these peaks were not analyzed as pheromone urated C16 aldehyde. Rt of peak C was 35.12 min, candidates. almost the same as that of the synthetic n-16:Ald ϭ (Rt 35.09 min). Therefore, the compounds of Identification of active compounds peaks A, B, and C were identified to be Z11- Mass spectra of peaks A, B, and C are shown in 16:Ald, Z11-16:OH, and n-16:Ald, respectively. Fig. 2. The characteristic fragment ions of peak A The ratios of Z11-16:Ald, Z11-16:OH, and n- showed at m/z 55 [relative intensity: 100% (base 16:Ald were calculated to be 100 : 24.5 : 2.4 in the peak)], 194 (7.2%, MϩϪ44), 220 (52.0%, MϩϪ female extract (sample II), and the average amount 18), and 238 (24.8%, Mϩ), and these suggested of Z11-16:Ald was about 18.1 ng per female. that peak A is a mono-unsaturated C16 aldehyde. Meanwhile, Z9-16:Ald was not detected in the fe- The characteristic fragment (diagnostic) ions of male extract of H. m. adaucta. ϭ DMDS adduct of peak A showed m/z 117 (C5H10 ϩ ϩϭ ϩ SCH3 ), 215 (H3CS C11H20O), and 332 (M : Field attraction tests using the synthetic com- C4H9CH(SCH3)CH(SCH3)C10H19O), which were pounds identical to those of 11-16:Ald. Rt of peak A on Mixtures of Z11-16:Ald and Z11-16:OH in ra- GC-MS was 36.17 min, almost the same as that of tios of 100 : 1–3 showed significant attraction activ- 76 M. KAKIZAKI and H. SUGIE

Table 1. Field attraction tests using 2-compound mixtures in others (Table 3). Heliothis maritima adaucta (Tests I–III)

Components (mg)a No. of males DISCUSSION captured per trap Z11-16:Ald Z11-16:OH (meanϮSE)b Three compounds, Z11-16:Ald, Z11-16:OH, and n-16:Ald, found in the abdominal tip extract of fe- Test I, 5–13 Aug. 1997, Naganuma male H. m. adaucta showed EAD activity. The an- 100 — 0Ϯ0a tennae of H. m. adaucta males also showed EAD Ϯ 100 1 2.0 1.0 b activity to the synthetic compounds of Z11-16:Ald, 100 5 0Ϯ0a 100 25 0Ϯ0a Z11-16:OH, and n-16:Ald (Kakizaki, unpublished 100 50 0Ϯ0a data), the EAD amplitude by the synthetic n- 100 75 0Ϯ0a 16:Ald showed about 1/3–1/4 of those to the same 100 100 0Ϯ0a amount of the synthetic Z11-16:Ald. Thus, these Ϯ 75 100 0 0a compounds were considered the pheromone candi- 50 100 0Ϯ0a 25 100 0Ϯ0a dates. However, a mixture of two compounds, Z11- 5 100 0Ϯ0a 16:Ald and Z11-16:OH, was responsible for male 1 100 0Ϯ0a attraction, but the individual compounds did not at- — 100 0Ϯ0a tract males. Further, addition of n-16:Ald to the bi- Blank 0Ϯ0a nary mixture did not increase the attraction activ- ity. Therefore, Z11-16:Ald and Z11-16:OH are Test II, 15–25 Aug. 1997, Naganuma 1,000 10 3.2Ϯ0.9 b concluded to be the sex pheromone components of 10 1,000 0Ϯ0a H. m. adaucta. The amount of Z11-16:Ald in H. m. Blank 0Ϯ0a adaucta was estimated to be about 18.1 ng from 3- female sample, however, the variation of amount in Test III, 26–30 Aug. 1997, Naganuma population should be certified by further analysis 1,000 30 1.7Ϯ0.3 ab 1,000 10 3.7Ϯ1.7 a using many individuals. Szocs et al. (1993) re- 1,000 3 0.7Ϯ0.3 b ported that a mixture of Z11-16:Ald, Z11-16:OH, 1,000 1 0.7Ϯ0.3 b and n-16:Ald in a ratio of 100:5:6 attracted male Blank 0Ϯ0b H. maritima in Hungary, whereas the mixtures of

a Z11-16:Ald and Z11-16:OH did not attract males. These compounds were impregnated onto white rubber These results indicated that the essential compo- septa. b Two traps (test I), five traps (test II), or three traps (test III) nents of sex pheromone in H. m. adaucta are dif- were used. Means followed by the same letter in each test ferent from those in H. maritima in Hungary. Addi- are not significantly different at the 5% level by Tukey’s tion of Z9-16:Ald, which was reported as the sex test. pheromone component based on a flight tunnel test in H. maritima in Hungary (Szocs et al., 1993), ity (tests I, II, and III in Table 1). In contrast, Z11- also did not increase attraction activity. 16:Ald or Z11-16:OH alone, and mixtures of Z11- In H. maritima in Ukraine and Hungary, the ra- 16:Ald and Z11-16:OH in ratios of 100 : 0.1, 100 : tios of Z11-16:Ald, Z11-16:OH, n-16:Ald, and Z9- 0.3, 100 : 5–100, and 0.1–75 : 100 did not show at- 16:Ald were 100 : 9.5 : 5.4 : 1.4 (Kovalev et al., traction activity (tests I–III). Sometimes, mixtures 1991) and 100 : 8.2 : 5.5 : trace (Szocs et al., 1993), of Z11-16:Ald and Z11-16:OH in ratios of 100 : respectively. On the other hand, in H. m. adaucta, 5–6 showed the same level of attraction activity as the ratio of Z11-16:Ald, Z11-16:OH, and n-16:Ald those of 100 : 1 (tests IV and V in Table 2). The ad- was 100 : 24.5 : 2.4. The relative ratio of Z11- ditions of n-16:Ald and/or Z9-16:Ald to the mix- 16:OH to Z11-16:Ald in H. m. adaucta was higher ture of Z11-16:Ald and Z11-16:OH did not in- than those in H. maritima in Ukraine and Hungary. crease attraction activity of the binary mixture Differences in sex pheromone components and (tests IV and V in Table 2). Among the doses rang- their ratios may exist among local populations of ing from 0.101 mg to 3.03 mg, 1.01 mg and 3.03 H. maritima. mg of the binary mixture attracted more males than The optimum ratio of Z11-16:Ald and Z11- Sex Pheromone of Heliothis maritima adaucta 77

Table 2. Field attraction tests using 2-, 3-, and 4-compound mixtures in Heliothis maritima adaucta (Tests IV and V)

Components (mg)a No. of males captured per trap Z11-16:Ald Z11-16:OH n-16:Ald Z9-16:Ald (meanϮSE)b

Test IV, 22–31 Aug. 1997, Naganuma 1,000 60 30 3.0Ϯ1.3 a 1,000 10 30 2.3Ϯ0.9 a 1,000 10 2.7Ϯ1.2 a Blank 0Ϯ0b

Test V, 1–11 June 2001, Takikawa 3,000 150 150 30 4.5Ϯ1.4 a 3,000 30 30 30 7.0Ϯ1.8 a 3,000 30 30 4.8Ϯ1.1 a 3,000 30 30 7.0Ϯ2.5 a 3,000 30 9.5Ϯ0.6 a Blank 0Ϯ0b

a These compounds were impregnated onto white rubber septa. b Three traps (test IV) or four traps (test V) were used. Means followed by the same letter in each test are not significantly differ- ent at the 5% level by Tukey’s test.

Table 3. Field attraction test using various amounts and the influences of the purity of Z11-16:Ald and/ of a 2-compound mixture in Heliothis or Z11-16:OH on the attractiveness remain un- maritima adaucta (Test VI) known. Therefore, further studies on a lure would be necessary for effective monitoring. Components (mg)a No. of males captured per trap ACKNOWLEDGEMENTS Z11-16:Ald Z11-16:OH (meanϮSE)b We thank Dr. Shin-ichi Yoshimatsu of the National Institute Test VI, 26–30 Aug. 1997, Naganuma of Agro-Environmental Sciences for kind advice on Heliothid- 3,000 30 2.3Ϯ0.3 ab inae species, Dr. Fumiaki Mochizuki of Shin-Etsu Chem. Co. 1,000 10 3.7Ϯ1.2 a Ltd. for supplements of synthetic chemicals, Dr. Kenjiro 300 3 0.3Ϯ0.3 bc Kawasaki of the National Institute of Sericultural and Entomo- 100 1 0.7Ϯ0.3 bc logical Sciences for his advice about a GC-EAD method, and Blank 0Ϯ0c Dr. Hiroshi Noguchi of the National Institute of Agro-Environ- mental Sciences for his advice. a These compounds were impregnated onto white rubber septa. REFERENCES b Three traps were used. Means followed by the same letter Buser, H. R., H. Arn, P. Guerin and S. Rausher (1983) Deter- are not significantly different at the 5% level by Tukey’s mination of double bond position in mono-unsaturated test. acetates by mass spectrometry of dimethyl disulfide adducts. Anal. Chem. 55: 818–822. 16:OH for the field attraction of H. m. adaucta was Carroll, K. K. (1961) Separation of lipid classes by chro- different from the ratio found in the female extract matography on Florisil. J. Lipid Res. 2: 135–141. (sample II). This may be related to differences be- Kovalev, B. G., K. Löftstedt, S. F. Nedopekina, E. A. Nesterov and E. Viik (1991) Isolation and identification of the tween the release rates or release patterns of the sex pheromone of females of Heliothis maritima (Lepi- compounds from dispensers and from live females. doptera, Noctuidae). Chem. Natur. Comp. 27: 503–506 Although rubber septa impregnated with 1.01 mg [Translated from Kh. Pri. Soy. (1991) 4: 574–577 (in and 3.03 mg of a mixture of Z11-16:Ald and Z11- Russian)]. 16:OH could capture more H. m. adaucta males Pool, R. W. (1989) Heliothis, Noctuidae Part 1. In Lepi- dopterorum Catalogus (New Series) (J. B. Heppner ed.). than those with 0.101 mg and 0.303 mg, attraction E. J. Brill/Flora & Fauna Publication, Leiden, pp. activity of lures with higher doses has not yet been 492–494. tested. Moreover, the effective periods of this lure Struble, D. L. and H. Arn (1984) Combined gas chromatog- 78 M. KAKIZAKI and H. SUGIE

raphy and electroantennographic recording in ol- Szocs, G., A. Raina, M. Tóth and B. A. Leonhardt (1993) factory responses. In Techniques in Pheromone Research Sex pheromone components of Heliothis maritima: (H. E. Hummel and T. A. Miller eds.). Springer-Verlag, Chemical identification, flight tunnel and field tests. En- Tokyo, pp. 161–178. tomol. Exp. Appl. 66: 247–253.