Appl. Entomol. Zool. 38 (1): 73–78 (2003) Sex pheromone of the flax budworm, Heliothis maritima adaucta Butler (Lepidoptera: Noctuidae) Masashi KAKIZAKI1,*,† and Hajime SUGIE2 1 Hokkaido Ornamental Plants and Vegetables Research Center; Takikawa, Hokkaido 073–0026, Japan 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 moth, H. m. maritima Grasl in France, H. m. had no effect on the attraction activity in the field. angarensis and H. m. centralasiae in China, 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 Insects. 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 moths, 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 m30.53 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 m30.32 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 (X10.5) 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 m30.25 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). 5 the synthetic Z11-16:Ald (Rt 36.15 min; Rt of E isomer535.87 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%, M1218), and these sug- was 0.064–0.048 mV.