Journal of Chemical Ecology, Vol. 18, No. 3, 1992

FEMALE SEX PHEROMONE OF ORIENTAL TOBACCO BUDWORM, Helicoverpa assulta (GUENEE) (LEPIDOPTERA: NOCTUIDAE): IDENTIFICATION AND FIELD TESTING

A. C O R K , 1"* K . S . B O O , 2 E. D U N K E L B L U M , 3 D . R . H A L L , 1 K. J E E - R A J U N G A , 4 M. K E H A T , 3 E. K O N G J I E , 5 K . C . P A R K , 2 P. T E P G I D A G A R N , 4 and L I U X U N 5 ~Natural Resources Institute Central Avenue, Chatham Maritime Chatham, Kent, ME4 4TB, UK 2Department of Agricultural Biology College of Agriculture Seoul National University Suwon 441-744, Republic of Korea 3Institute of Plant Protection, ARO The Volcani Center Bet Dagan 50 250, Israel 4Division of Entomology and Zoology Department of Agriculture Bangkhen, Bangkok 10900, Thailand 51nstitute of Zoology Academia Sinica Beijing, China (Received June 19, 1991; accepted October 31, 1991) Abstract--Analysis of ovipositor washings from virgin female Helicoverpa assulta (Guen~e) (Lepidoptera: Noctuidae) from Korea by gas chromatography (GC) linked to electroantennography and GC linked to mass spectrometry resulted in the identification of nine compounds, hexadecanal, (Z)-9-hexadecenal, (Z)-ll-hexadecenal, hexadecyl acetate, (Z)-9-hexadecenyl acetate, (Z)-ll-hexadecenyl acetate, hexadecan-l-ol, (Z)-9-hexadecen-l-ol, and (Z)11-hexadecen-l-ol. However, ovipositor washings from females from Thailand contained mainly the 16-carbon aldehydes with very small amounts of (Z)-9-hexadecenyl acetate. Field tests conducted in Korea, China, and Thai*To whom correspondence should be addressed. 403 0098-0331192/0300-0403506.50/0 9 1992PlenumPublishingCorporation

404

CORK ET AL. land indicated that a binary blend of (Z)-9-hexadecenaland (Z)-I 1-hexadecenal was sufficient for attraction, although the most attractive ratio of compounds varied with location. In Korea a 20" 1 blend of compounds was the most attractive, while in Thailand a 7.5 : 1 blend was most attractive. In China both blends of hexadecenal isomers were equally attractive. Addition of the hexadecenylacetates to the 20: 1 blend of hexadecenalsin the ratio of 1: 3.3 increasedthe trap catch of male H. assulta comparedto lurescontaining the aldehydes alone in Korea but reduced trap catch in China. Additionof the hexadecenyl acetates to the 7.5:1 blend of hexadecenals had no significant effect on trap catch in Thailand or China compared to the aldehydes alone. The addition of the 16-carbon alcohols to the aldehydes had a significantly inhibitory effect in all three countries, suggesting they are not pheromone components. Taken together these results indicate that H. assulta is polymorphic with at least two populationsrespondingto differentsex pheromones. Key Words--Helicoverpa assulta, Heliothis, Lepidoptera, Noctuidae, hexadecanal, (Z)-9-hexadecenal,(Z)-ll-hexadecenal, hexadecyl acetate, (Z)-9hexadecenyl acetate, (Z)-ll-hexadecenyl acetate, hexadecan-l-ol, (Z)-9-hexadecen-l-ol, (Z)-ll-hexadecen-l-ol, sex pheromone.

INTRODUCTION The Oriental tobacco budworm, Helicoverpa assulta (Guenre) is found throughout the Far East, Africa, and Australasia, where it is a pest of such crops as peppers (Cho and Boo, 1988) and tobacco and onions (Hill, 1983). Control of this pest in the larval stages with insecticides is particularly difficult because of its habit of feeding within the fruits of host plants, and these difficulties have been further aggravated by the increasing resistance of larvae to the insecticides currently used for its control (Lee and Boo, 1985). In order to achieve effective control strategies, there is a need for a sensitive, species-specific monitoring system. A n attractive bait based on the sex pheromone of the species would fit this requirement and have the added advantage that traps based on such a lure are highly mobile and cheap to operate, requirements that are particularly important to pest monitoring systems when utilized in developing countries (Beevor et al., 1983). The present study describes our efforts to identify components of the female sex pheromone and develop a species-specific lure for monitoring H. assulta by conducting field trials of the compounds identified in Korea, China, and Thailand.

METHODS AND MATERIALS

Insect Material. Larvae were collected in the Suwon area of Korea and reared on either tobacco leaves or an artificial diet (Kim et al., 1984). Pupae were sexed and dispatched by air to England. On arrival they were separated

Helicoverpa assulta PHEROMONE

405

into single sex groups of 10-15 individuals and placed in Perspex containers (30 • 15 • 15 cm) with access to 10% sucrose solution and tissue paper in vertical strips to allow them to expand their wings on emergence (Cho and Boo, 1988). The containers were placed in an environmental cabinet maintained at 25~ and 60% humidity during the reversed 12-hr light period and 22~ and 60% humidity in the 12-hr dark period. In Thailand, insects were reared on artificial diet (Kim et al., 1984) and maintained on a 12-hr: 12-hr light-dark cycle. Pheromone Collection. Pheromone gland washings were prepared in England from Korean insects using hexane and 0- to 2-day-old females 4-6 hr and 10 hr into the scotophase as described by Sower et al. (1973). In Thailand gland extracts were prepared 10 hr into the scotophase (10 glands/sample) and dispatched to Israel for chemical analysis. Gas Chromatography (GC). GC analyses of pheromone gland extracts made from Korean insects were conducted on a Carlo Erba Mega series 5300 instrument fitted with two Grob split/splitless injectors (200~ and flame ionization detector (FID) (220~ Fused silica capillary columns (25 m • 0.32 mm ID) were used throughout the study, coated with either nonpolar CP Sil 5CB (chemically bonded methylsilicone; Chrompack) or polar CP Wax 52CB (chemically bonded Carbowax 20 M equivalent; Chrompack). The carrier gas was helium with an inlet pressure of 0.4 kg/cm 2. All injections were made with the split valve closed for 40 sec onto a column held at 70~ for 2 min then temperature programmed at 20~ to 120~ and then at 4~ to 210~ GC analyses of Thai pheromone gland extracts were conducted on a Varian 3700 instrument equipped with a FID detector, splitless injector system, and polar fused silica capillary column (30 m • 0.25 mm ID) coated with DB-225 (chemically bonded cyanosilicone; J & W). Samples were injected onto the analytical column, held at 60~ for 2 min, and then temperature programmed to 150~ at 10~ The purge valve was opened 1 min after injection, and the inlet pressure of the helium carrier gas was 1.0 kg/cm 2. Retention times of compounds identified in the ovipositor washings are summarized in Table 1 and quoted as equivalent chain lengths (ECLs) relative to the retention times of straight-chain acetates; thus, for example, tetradecyl acetate = 14.00 (Christie, 1988; Harris and Habgood, 1966). Electroantennography (EAG). Linked GC-EAG analyses were carried Out essentially as described by Cork et al. (1990) by splitting half of the analytical column effluent to the FID detector and the other half to a silanized glass reservoir in the GC oven. Column effluent was expelled from the reservoir at 17-sec intervals for 3-sec with a stream of nitrogen (250 cm3/min) into a constant stream of nitrogen (250 cmS/min) over the EAG preparation. The series of EAG responses generated were then compared to identify those that were significantly above background responses in order to determine the presence of electrophysiologically active compounds.

406

CORK ET AL.

TABLE 1. G C RETENTION TIMES AND RELATIVE COMPOSITION OF LAG-ACTIVE COMPOUNDS IDENTIFIED IN PHEROMONE GLAND WASHINGS OF H. assulta FROM KOREA

Retention times (ECL)"

Nonpolar column~

Polar columnd

Compositionb normalised to Z9-16 : Aid

Compound

Synthetic

Natural

Synthetic

Natural

4-6 hr dark

10 hr dark

Z9-16 : Aid Z11-16 : Aid 16 : Aid

13.77 13.86 14.02

13.78 13.86 14.02

14.65 14.76 14.39

14.65 14.76 14.39

100 7.5 26.5

100 4.4 17.8

Z9-16 : OH Z1 t-16 : OH 16 : OH

14.49 14.59 14.73

14.50 ND ND

17.00 17.09 16.71

16.97 17.09 16.70

1.7 0.1 0.2

55 6.5 2.5

Z9-16 : Ac Z11-16: Ac 16:Ac

15.75 15.86 16.00

15.75 15.85 t6.00

16.23 16.36 16.00

16.23 16.36 16.00

31.6 1.4 4.0

254 2.3 25

~Retention times in equivalent chain length units relative to the retention times of straight chain acetates. bND = not detected. CNonpolar column, chemically bonded methylsilicone liquid phase, CP Sil 5CB. '/Polar column, chemically bonded Carbowax 20 M, CP Wax 52CB.

Mass Spectrometry (MS). Electron impact (EI) and chemical ionization (CI) mass spectra with isobutane as reagent gas were obtained on a Finnigan MAT Ion Trap Detector, model 700, under continuous ion monitoring between m/z 30-250 at 220~ for EI-MS and m/z 60-300 at 200~ for CI-MS as described by Cork et al. (1991). EI mass spectra of dimethyl disulfide (DMDS) adducts were obtained in the multiple ion detection (MID) mode on a Finnigan 5100 EI machine at 70 eV, coupled to a Super Incos data system and interfaced with a Finnigan MAT 9610 GC. Samples were injected onto a fused silica capillary column (30 m • 0.25 mm ID) with low-polarity SE 54 (chemically bonded phenylmethylsilicone; J & W) held at 60~ for 4 min and then temperature prograrmned at 20~ to 230~ The injector was operated in the splitless mode with the purge valve opened 0.5 min after injection. The inlet pressure of the helium carder gas was maintained at 0.7 kg/cm 2. Synthetic Chemicals. Monounsaturated acetates, alcohols,and aldehydes were obtained by standard Wittig and acetylenic coupling reactions (Henrick, 1977) or purchased from Shin Etsu Chemical Co. (Tokyo, Japan). Compounds used for field testing were further purified by argentation chromatography (Houx

Helicoverpa assulta PHEROMONE

407

et al., 1974) to give material at least 99.9% isomerically and chemically pure by GC analysis. Microreactions. Dimethyl disulfide derivatives were prepared essentially as described by Buser et al. (1983) and Dunkelblum et al. (1985). Field Trials. Field trials in China were conducted in Hunan province in tobacco and hot-pepper fields using water traps (Campion et al., 1974). Related trials in Korea were conducted in hot-pepper fields using cone traps (Hartstack et al., 1979), while those in Thailand were conducted in tobacco fields with sleeve traps (Dunkelblum and Kehat, 1989; Neumark and Teich, 1980). In Korea and China treatments were placed out in randomized complete blocks with at least 20 m between treatments in a block and 100 m between blocks. The position of treatments within a block was rotated in a clockwise direction daily when trap catches were recorded and insects discarded. Female moths tested in Korea were provided with sugar solution and changed daily. In Thailand traps were rotated and trap catches counted once or twice a week. Pheromone dispensers were white rubber septa (Aldrich, catalog No. Z10,072-2) impregnated with 0.1 ml of hexane solution containing the synthetic pheromone blend and an equivalent weight of 2,6-di-tert-butyl-4-methylphenol as antioxidant. The results were statistically analyzed by analysis of variance (ANOVA) after combining catches for each treatment and transforming them to log (x + 1). If differences in treatment means were significant at the 5 % level or lower, then they were ranked by Duncan's multiple-range test (DMRT) (Duncan, 1955).

RESULTS Structure Determination. GC-EAG analyses of extracts prepared from insects collected in Korea using typically 0.1 pheromone gland equivalents on a nonpolar CP Sil 5CB column under temperature programmed conditions indicated three regions of EAG activity significantly above background. GC retention times of compounds eluting in these regions were consistent with those of saturated and monounsaturated 16-carbon, straight-chain aldehydes, alcohols, and acetates, respectively (Table 1). Analysis of the 16-carbon aldehyde region by EI-MS indicated the presence of three compounds (Figure 1). The compounds had MS consistent with synthetic hexadecanal and two hexadecenal isomers. CI-MS of the compounds confirmed these results, with significant M + 1 ions at m/z 241 and 239 for hexadecanal and the two hexadecenal isomers, respectively, and significant M - 1 and M + 1 - 18 ions associated with the hexadecenal isomers at m/z 237 and m/z 221, respectively. Comparison of GC retention times of the 16-carbon aldehydes with synthetic analogues on polar and nonpolar phases under iso-

4O8

CORK ET AL.

I1

58~

U

U~n~n

TIC HC

~

III

"1

2[ 011

....

I ....

2t. 5a

IO t ....

I ....

23. ell

I ....

OlI~ I ....

I ....

I . . . . . . . .

24.56 26. ell ti,e (,in)

Retention

I ....

I ....

27.58

FIc. 1. Total ion chromatogram in EI mode, m/z 30-250, of 0.2 equivalents of H. assulta ovipositor extract prepared from insects reared in Korea and chromatographed on a polar GC column. The mass spectra and GC retention times of the labeled compounds corresponded with I, 16 : Aid; II, Z9-16: Aid; III, Z 11-16 : Aid; IV, 16: Ac; V, Z9-16:Ac; VI, Zll-16:Ac; VII, 16:OH; VIII, Z9-16:OH; IX, Zll-16:OH; Unknown, unidentified compound; and HC, saturated straight-chain hydrocarbon.

thermal conditions (Cork et al., 1988) confirmed the presence of hexadecanal (16:Aid), (Z)-9-hexadecenal (Z9-16:Ald) and (Z)-ll-hexadecenal ( Z l l 16: Aid) in the approximate ratio of 3.5:13.3:1 for pooled extracts prepared between 4 and 6 hr into the scotophase. In a related study of 10 individual female ovipositor extracts prepared 10 hr into the scotophase, the average ratio of 16: Aid, Z9-16: Aid, and Z11-16 : Aid was 4:23 : 1. Extracts prepared 4-6 hr into the scotophase contained on average 150 ng/female equivalent of Z 9 16:Ald, whereas ovipositor extracts prepared 10 hr into the scotophase contained on average 60 ng of Z9-16:Ald, but this varied from 4 to 180 ng. Similarly three 16-carbon alcohols were observed by EI-MS (Figure 1) that had the same GC retention times on a polar column (Table 1) and mass spectra as hexadecan-l-ol (16 : OH), (Z)-9-hexadecen-l-ol (Z9-16: OH), and (Z)-I 1hexadecen-l-ol ( Z l l - 1 6 : O H ) . The alcohols were present in amounts approximately 2 % of the corresponding aldehydes in extracts prepared 4-6 hr into the scotophase, but this increased to an average of 55 % in extracts prepared 10 hr into the scotophase. Only the most abundant alcohol, Z9-16: OH, could be confirmed on both GC phases and by CI-MS with M + 1 ions at m/z 241.

Helicoverpa assulta PHEROMONE

409

EAG activity elicited by compounds eluting in the 16-carbon acetate region was shown by EI-MS to be associated with three compounds that had characteristic ions at m/z 61 (CH3COOH2) + and M - 6 0 ions at m/z 224 and 222, indicative of hexadecyl acetate (16:Ac) and two hexadecenyl acetate isomers, respectively. CI-MS was only able to confirm the major hexadecenyl acetate with M + I ions at 283 and M - 6 0 ions at m/z 222. The GC retention times of these compounds on polar and nonpolar GC columns were consistent with those of 16:Ac, (Z)-9-hexadecenyl acetate (Z9-16:Ac), and (Z)-I 1-hexadecenyl acetate (Z 11-16 : Ac) (Table 1). The amount of Z9-16 : Ac relative to Z9-16 : Aid was found to be approximately 26 % in extracts prepared 4-6 hr into the scotophase, but this increased dramatically to an average of 232 % in extracts prepared 10 hr into the scotophase, although there was considerable variation between ovipositor extracts prepared from individual female moths of from 20 % to 600%. There was no evidence from GC-EAG or GC-MS analyses for the presence of any tetradecenal isomers that are observed in the sex pheromones of some Heliothidinae (Dunkelblum and Kehat, 1989; Klun et al., 1980a). Ovipositor washings from insects collected in Thailand were analyzed by capillary GC and by GC-MS after conversion to the dimethyl disulfide adducts (Buser et al., 1983; Dunkelblum et al., 1985). The latter analyses confirmed the presence of Z9-16 : Aid and Z 11-16: Aid with characteristic retention times and ions at m/z 145, 187, and 332 (M +) for Z 9 - 1 6 : A l d , and 117, 215, and 332 (M +) forZ11-16 :Ald. The average ratio of 16 : Aid, Z9-16 : Aid, and Z 1 1 16:Ald was 1.5:5:1, respectively, and pheromone yields were 1-5 ng per female. Small amounts of Z 9 - 1 6 : A c were detected in some extracts, but the alcohols were never observed. FieM Tests. Preliminary field trials were conducted in China, Korea, and Thailand with different ratios of Z9-16 : Aid and Z 11-16: Aid. Different results were obtained at each location. In Korea the most attractive blend tested was the 20:1 ratio of components, in Thailand the 7.5:1 ratio of compounds was the most attractive, and in China there was no significant difference in catches with lures containing either the 50:1 or 5:1 ratio of hexadecenal isomers, although the 1 : 1 ratio of hexadecenal isomers was significantly less attractive (Table 2). In Korea, the 20 : 1 blend of hexadecenal isomers was shown to be significantly more attractive to male moths than two virgin females in field trials (Table 2). The 20:1 blend of Z 9 - 1 6 : A l d and Z11-16:Ald was then tested in China and Korea at different loadings and in both instances the highest catch was obtained with the highest loading tested, 5000/zg, although differences between catches for each treatment were not significant in the trial conducted in China (Table 3). A 7.5 : 1 blend of compounds was tested in China and Thailand with

410

CORK ET AL. TABLE 2. CATCHES OF MALE H. assulta IN CHINA, THAILAND, AND KOREA WITH DIFFERENT BLENDS OF Z 9 - 1 6 " ALD AND Z 1 1 - 1 6 : ALD

Ratio of compounds (1000/~g loading)

Total catch/treatmen# Thailandd

Z9-16: Aid

Z11-16 : Aid

Chinab

Koreac

Test I

Test II

50 25 20 10 7.5 5 I 2femMes

1 1 1 1 1 1 1

279a 367a NT 205a 317a 239a lib NT

219b 393a 450a 213b NT 249b NT 180b

0c 9bc NT 16b 54a 22b lbc NT

0c llbc NT 21b 46a 20b 6bc NT

~

followed by the same letter are not significantly different P > 0.05 by DMRT using log (x + 1)-transformed data. NT = not tested. b Four replicates, 30 nights. CThree Replicates, eight nights. dTest I, two replicates, 14 nights; test II, two replicates, 49 nights.

TABLE 3. CATCHES OF MALE n. assulta IN CHINA AND KOREA WITH DIFFERENT LOADINCS OF 20 : 1 BLEND OF Z 9 - 1 6 : ALD AND Z 1 1 - 1 6 : ALD

Total catch/treatment~ Loading of dispenser (#g)

Chinab

Koreac

5000 1003 500 100 2females Blank

284a 169a 181a 103a NT 0b

107a 51b 29bc ld 13cd NT

aTotals followed by the same letter are not significantly different P > 0.05 by DMRT using log (x + 1)-transformed data. NT = not tested. 9Four replicates, 57 nights. CThree replicates, seven nights.

Helicoverpa assulta PHEROMONE

411

the same result (Table 4). Lures with 1000 /xg or more o f the 2 0 : 1 ratio o f hexadecenal isomers were found to be significantly more attractive than two female moths in Korea (Table 3). The effect on attractiveness o f adding Z 9 - 1 6 : OH, Z 9 - 1 6 : Ac, and Z 1 1 1 6 : A c to a 2 0 : 1 blend o f Z 9 - 1 6 : A l d and Z l l - 1 6 : A I d was tested in China and Korea (Table 5). The compounds were added at levels comparable to those found in ovipositor extracts prepared 4 - 6 hr into the scotophase, when the highest levels o f mating were observed in Korea (Kim and Boo, 1986). At both locations Z 9 - 1 6 : O H was found to reduce trap catch significantly when added at 2 % o f Z 9 - 1 6 : Aid. However, the effect o f adding a 20: 1 blend o f Z 9 - 1 6 : Ac and Z 1 1 - 1 6 : Ac to an equivalent blend o f Z 9 - 1 6 : Aid and Z 1 1 - 1 6 : Aid in the ratio o f 1 : 3.3 was to increase trap catch significantly in Korea, but reduce trap catch significantly in China. In a related experiment in China and Thailand with a 7 . 5 : 1 ratio o f Z 9 - 1 6 : A I d and Z 1 1 - 1 6 : A i d , the addition o f the related alcohols in the same isomeric ratio reduced trap catch significantly, while the addition of the related acetates had no statistically significant effect on trap catch (Table 6).

DISCUSSION The female sex pheromones o f Heliothidinae moths characterized so far are blends o f saturated and monounsaturated 14-carbon aldehydes, 16-carbon aldehydes, 16-carbon acetates, and 16-carbon alcohols (Table 7). The E A G TABLE 4. CATCHES OF MALE H. assulta IN CHINA AND THAILAND WITH DIFFERENT LOADINGS OF 7.5 : 1 BLEND OF Z 9 - 1 6 : ALD AND Z 1 1 - 1 6 : ALD

Total catch/treatment~ Thailandc Loading of dispenser (#g)

Chinab

Test I

Test II

5000 2000 1000 500 100 50

141a NT NT t0b 7b NT

55a NT 3lab 19b 4b 13b

133a NT 23b llbc 0c 12bc

~Totals followed by the same letter are not significantly different P > 0.05 by DMRT using log (x + 1)-transformeddata. NT = not tested. bFour replicates, 25 nights. CTest 1, two replicates, 14 nights; test II, two replicates, 49 nights.

412

CORK ET AL.

TABLE 5. CATCHES OF MALE H. assulta WITH 16-CARBON ACETATES AND ALCOHOLS ADDED TO BLEND OF Z 9 - 1 6 : ALD AND Z 1 1 - 1 6 : ALD IN 20 : 1 RATIO

Composition (/zg)

16: Aid

16 : Ac

Total catch/ treatmenff

16: OH

Sat

Z9-

ZI 1-

Sat

Z9-

Zl 1-

Sat

Z9-

China b

Korea"

0 0 0 0 250

1000 1000 100O 1000 1000

50 50 50 50 50

0 0 0 0 85

0 300 300 0 300

0 15 15 0 15

0 0 0 0 5

0 0 20 20 20

503a 238b 20c 23c 32c

48b 129a 0c 3c lc

Blank

0d

NT

~Totals followed by the same letter are not significantly different P > 0.05 by DMRT using log (x + 1)-transformed data. NT = not tested. bThree replicates, 57 nights. ~Three replicates, four nights.

TABLE 6. CATCHES OF MALE n. assulta WITH DIFFERENT LOADINGS OF 7.5 : 1 BLEND OF Z 9 - 1 6 : ALD AND Z 1 1 - 1 6 : ALD AND RELATED ACETATES AND ALCOHOLS

Composition (#g) 16 : Ald

16: Ac

16 : OH

Total catch/treatmenff

Z9-

Z11-

Z9-

Z11-

Z9-

Z11-

Chinab

Thailand c

880 880 880 880

120 120 120 120

9 265 265 0

0 35 35 0

0 0 17.6 17.6

0 0 2.4 2.4

l16a l15a 2b 0b

392a 668a 44b 3c

Blank

0c

NT

~Totals followed by the same letter are not significantly different P > 0.05 by DMRT using log (x + 1)-transformed data. NT = not tested. ~ replicates, 32 nights, CFive replicates, 64 nights.

Helicoverpa assulta PHEROMONE

413

TABLE 7. CHEMICALS IDENTIFIED IN OVIPOSITOR WASHINGS OF Helicoverpa AND

Heliothis SPECIES Speciesa

Compund

a

b

c

d

e

f

g

h

14:Ald Z9-14:AId Z9-14 : OH Z9-14 : Ac 16:Ald Z7-16:Ald Z9-16 :Ald Zll-16:AId Z9-16:OH Z11-16: OH 16 : Ac Z7-16: Ac Z9-16:Ac Zll-16:Ac

0 0 0 0 49 0 7 100 0 21 0 0 0 0

0 0 0 0 353 0 1333 100 22.7 tr 53.7 0 421.3 18.4

0.8 14.6 6.5 2 3.7 1.1 1.5 100 0 24.3 0 0 0 4.8

0 0 0 0 8.2 0 0.5 100 0 7.2 0 0 0 0

0 0 0 0 9 tr tr 100 0 75 0 0 0 50

0 0 0 0 18 0 66 100 48 40.6 0 5.5 14.3 41

2 3.9 0 0 11.7 1.6 1.2 100 0 3.9 0 0 0 0

0 0 0 0 4.8 1.2 18.4 100 0 0 0 0 0 0

Species a b c d e f g h

H. H. H. H. H. H. H. H.

armigera assulta peltigera phloxiphaga punetiger subflexa virescens zea

Origin

Reference

Malawi Korea Israel United States Australia United States United States United States

Nesbitt et al., 1980. This study. Dunkelblum and Kehat, 1989. Raina et al., 1986. Rothschild et al., 1982. Teal et al., 1981. Klun et al., 1980a. Klun et al., 1980b.

active c o m p o u n d s identified in o v i p o s i t o r washings o f H. a s s u l t a moths collected in K o r e a and T h a i l a n d w e r e similar 16-carbon aldehydes, acetates, and alcohols, but the relative abundances o f the c o m p o u n d s differed both in c o m p a r i s o n with o t h e r species and b e t w e e n the populations studied. In all other H e l i c o v e r p a and H e l i o t h i s p h e r o m o n e s characterized, the m a j o r c o m p o n e n t is Z 1 1 - 1 6 : A i d , but Z 9 - 1 6 : A i d was the m o s t abundant hexadecenal i s o m e r found in o v i p o s i t o r washings f r o m H. a s s u l t a obtained f r o m Korea and Thailand with Z 1 1 - 1 6 : A i d detected as a m i n o r c o m p o n e n t (Table 1). Significant differences in the ratio o f Z 9 - 1 6 : A l d to Z 1 1 - 1 6 : A l d w e r e found in ovipositor w a s h i n g s prepared f r o m insects obtained f r o m K o r e a and Thailand, and this could not be attributed to differences in sampling protocols as samples were prepared by the s a m e m e t h o d ( S o w e r et al., 1973) and in the s a m e t i m e period

414

CORKET AL.

in file scotophase. Extracts were prepared at times when calling behavior or mating had been observed, notably 4-6 hr into the scotophase for Korean insects (Kim and Boo, 1986) and 10 hr for Thai insects. Extracts prepared from Thai insects at earlier periods in the scotophase contained no detectable quantities of pheromone. Ovipositor washings made from Korean insects also contained significant amounts of hexadecenyl acetates and hexadecen-1-ols, and again the (Z)-9 isomer predominated over the (Z)-I 1 isomer. The relative abundance of Z9-16: Aid, Z9-16: Ac, and Z9-16 : OH changed from 1 : 0.3 : 0.02 in extracts prepared from females 4-6 hr into the scotophase to 1:2.5:0.55 in extracts prepared 10 hr into the scotophase (Table 1). This is in line with similar observations by Heath et al. (1991) working with the (Z)-I 1 isomers from H. subflexa ovipositor washings. In contrast, ovipositor washings prepared from Thai insects 10 hr into the scotophase contained only trace amounts of Z 9 - 1 6 : A c and the hexadecen-l-ols were not detected. In view of the large differences in ratios of EAG-active compounds found in the ovipositor washings of female H. assulta, it was not possible to define the natural blend of EAG-active compounds that would elicit the optimal matefinding response from receptive male moths without either collecting volatiles released from calling female moths or field testing different blends of the compounds identified. In this instance the latter course of action was taken. Field tests in Korea, China, and Thailand showed male H. assulta moths were attracted by blends of the two hexadecenal isomers alone in which Z916:Aid predominated, but the optimum ratio varied with the location and reflected differences in the ratios found in the ovipositor washings. The most attractive blend of Z9-16:Ald and Z11-16:Ald tested in Thailand was 7.5:1, while 20:1 was significantly more attractive in Korea. In China there was no difference in attractiveness with blends containing between a 50: 1 and 5 : 1 ratio of Z9-16:Ald and Z11-16:Aid although the 1:1 blend was significantly less attractive. Taken together the laboratory and field data suggest there may be two populations of H. assulta and that both were present in the Hunan province of China when the field tests were conducted. Addition of corresponding blends of the two hexadecenyl acetates to the optimum blend of aldehydes increased attractiveness in Korea (Table 5) but did not significantly affect attractiveness in Thailand (Table 6). Addition of the hexadecenyl acetates to a 20:1 blend of Z9-16:Ald and Z11-16:Ald significantly decreased attractiveness in China (Table 5), but addition of the acetates to a 7.5 : 1 blend had no significant effect on trap catches (Table 6). These results provide further evidence for the existence of different populations of H. assulta. However, addition of Z9-16 : OH to blends of the hexadecenal isomers with or without the hexadecenyl acetate isomers at a level of only 2 % of the Z9-16: Aid dramatically reduced trap catches in China, Korea, and Thailand (Tables 5 and

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6), suggesting that the alcohol found in ovipositor washings might have been a pheromone precursor (Teal and Tumlinson, 1986). This study has provided another example of the way in which related species achieve species specificity by varying the relative amounts of a limited range of related chemicals in their sex pheromones (Kaae et al., 1973). In India H. assulta coexists with both H. armigera and H. peltigera. H. armigera is attracted by mixtures of Z l l - 1 6 : A l d with 1-10% Z9-16:Ald (Kehat et al., 1980), in agreement with relative amounts of pheromone components found in analyses of ovipositor washings (Nesbitt et al., 1979, 1980). Ten potential pheromone components were detected in female ovipositor washings from H. peltigera by Dunkelblum and Kehat (1989), with Z11-16 :Aid, Z11-16: OH, and Z9-14:Ald the most abundant components. Laboratory and field tests showed Z9-16:AId was not necessary for attraction of this species, and a 2:1 mixture of Z 11-16 : Aid and Z9-14 :Ald attracted male H. peltigera moths and only a few male H. armigera. Addition of Z l l - 1 6 : O H further reduced the attractiveness to 1-1. armigera without affecting attraction of H. peltigera. Presumably male H. assulta moths would not be attracted to H. armigera female moths because of the relative amounts of Z l l - 1 6 : A l d and Z9-16:Ald, nor would they be attracted to H. peltigera female moths because of the virtual absence of Z9-16:Ald and the presence of Z l l - 1 6 : O H . The Z9-14:Ald is vital for attraction of 11. peltigera and reduces attractiveness to H. armigera, but it was not detected in ovipositor washings from H. assulta and its effect on the behavior of this species was not tested. In Australia, H. assulta is sympatric with both H. armigera and the native budworm, H. punctiger. Rothschild et al. (1982) showed that a 20: 20 : 1 mixture of Z l l - 1 6 : A I d , Z l l - 1 6 : A c , and Z9-14:Ald provided an attractive lure for male H. punctiger moths, again in line with analyses of ovipositor washings from virgin female moths. Cross-attraction with H. armigera would not be expected because of the absence of Z9-16:AId and the presence of Z9-14:Ald in the 1t. punctiger pheromone, while male H. assulta moths would not be attracted because of the absence of Z9-16:Ald. From this work, the best blends recommended for monitoring H. assulta are a 20: 1 mixture of Z9-16: Aid and Z 11-16: Aid with 30 % of a corresponding mixture of Z9-16: Ac and Z 11-16 : Ac in Korea and a 7.5 : 1 mixture of the two aldehydes with 30% of the corresponding acetates in Thailand. In China the situation is complicated by the apparent mixing of these populations and, until insect material is analyzed from China, only the blend recommended for Thailand should be used. Pheromone polymorphism involving different ratios of pheromone components has been observed previously in a number of lepidopterous species (e.g., Guerin et al., 1984; Bailey et al., 1986; L6fstedt et al., 1986; L6fstedt, 1990), but whether the different populations can be described as different species will depend, in part, on the degree of homogeneity of the

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pheromone blends produced by each population and the degree of cross-attractiveness of the pheromones (Foster and Roelofs, 1987). Although this study provides evidence for the possible existence of at least two separate populations of H. assulta, confirmation will have to await further analyses of the pheromone produced by insects collected in different regions and further field testing of the different blends. Obviously, this is particularly important if pheromone lures are to be used for reliable monitoring of H. assuha in different areas. Acknowledgments--Research conducted in Israel and Thailand was supported by grant C5020 from the U.S~-Israel Co-operative Development Research (CDR) Program. In Korea the research was supported by grants from the Korea Science and Engineering Foundation (862-1503-007-2).

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