Journal of Chemical Ecology, Vol. 7, No. 6, 1981

IDENTIFICATION OF A SEX PHEROMONE OF Heliothis subflexa (GN.) (LEPIDOPTERA: NOCTUIDAE) AND FIELD TRAPPING STUDIES USING DIFFERENT BLENDS OF COMPONENTS ~

P.E.A. TEAL, 2 R.R. HEATH, J.H. TUMLINSON, and J.R. McLAUGHLIN

Insect Attractants, Behavior, and Basic Biology Research Laboratory Agricultural Research, Science and Education Administration, USDA Gainesville, Florida 32604 (Received November 24, 1980; revised January 27, 198 I) Abstract Eight compounds were isolated from the sex pheromone gland of Heliothis subflexa (Gn.) and identified as hexadecanal, (Z)-9-hexadecenal, (Z)-I l -hexadecenal, (Z)-7-hexadecen- 1-01 acetate, (Z)-9-hexadecen-l-ol acetate, (Z)-I 1-hexadecen-l-ol acetate, (Z)-9-hexadecen-l-ol, and (Z)-I I-hexadecen-l-ol. Although the whole blend was found to be an effective male attractant, the deletion of alcohols from the blend increased trap captures considerably. Further, although the binary mixture of (Z)-9hexadecenal and (Z)-ll-hexadecenal caught some male H. sut~lTexa, significant increases in captures were noted when the three acetate components were included in the blend.

Key Words--Sex pheromone, Heliothis subflexa, Lepidoptera, Noctuidae, reproductive behavior, field trapping, capillary chromatography.

INTRODUCTION

Heliothis subflexa ( G n . ) is a n i n n o x i o u s n o c t u i d m o t h w h i c h feeds e x c l u s i v e l y o n g r o u n d c h e r r y (Physallis spp.) ( B r a z z e l et al., 1953). A l t h o u g h n o t a pest, it has r e c e n t l y b e c o m e t h e s u b j e c t o f n u m e r o u s p e s t c o n t r o l s t u d i e s d u e to tMention of a commercial or proprietary product in this paper does not constitute an endorsement of that product by the USDA or the State of Florida. 2Department of Entomology and Nematology, University of Florida under a cooperative agreement with the Insect Attractants, Behavior, and Basic Biology Research Laboratory, Gainesville, Florida 32604. 1011 0098-0331/81 / 1100-1011503.00/0 9 1981 Plenum Publishing Corporation

1012

TEAc ETAL.

success in laboratory hybridization between itand the tobacco budworm, H. vireseens (F.), a broadly sympatric sibling species of great economic importance (Laster, 1972). The basis of these control studies lies in the production of sterile male and fertile female hybrid and backcross progeny which, when released into a natural 1t. virescens population, should reduce the numbers of H. virescens via the induction of male sterility. However, the considerable difficulties in obtaining viable hybrid progeny or even interspecific mating under laboratory conditions (Brazzel et al., 1953; Easter, 1972; Proshold and Lachance, 1974) indicate that the pair have developed highly effective mechanisms ofpremating reproductive isolation. Due to the broadly sympatric distributions and intersecting reproductive periods of these species (Tingle et al., 1977), barriers to interspecific gene flow are most likely the result of differences in their respective sex pheromone communication systems (Roelofs and Card6, 1974). Recent chemical studies on several HeIiothis species have indicated the existence of a common trend in the types of compounds forming the pheromone blend, with (Z)-ll-hexadecenal and (Z)-9-hexadecenal being components common to all species studied thus far (Klun et al., 1980a,b; Nesbitt et al., 1979, 1980). Hence, it appears that pheromonal mechanisms of isolation between species result from different blends and ratios of components. We report the identification of a highly effective species-specific sex pheromone blend produced by female H. subflexa. METHODS AND MATERIALS

General. 11. subflexa used for both the collection of pheromone and bioassay studies were reared from laboratory stocks maintained at either Gainesville, Florida, or Stoneville, Mississippi. After pupation the insects were sexed and allowed to emerge in isolation from members of the opposite sex under test conditions. Newly emerged adults, collected daily, were placed in 30-cm s Plexiglas cages and provided with a 10% sucrose solution for nutrient. All laboratory studies were carried out on moths maintained under a 16: 8-hr light-dark cycle at a temperature of 20~C and a relative humidity of 60%. Insects used for both the collection of pheromone and bioassay studies were selected on the basis of good physical appearance at between 2 and 5 days of age. Pheromone extracts were prepared from groups of five actively calling females which had entered into a protracted bout of calling as indicated by ovipositor extension for a minimum of 2 rain (Teal et al., 1981). Individual calling females were removed from the holding cage and the ovipositors removed as described by Klun et al. (1980a). Immediately after removal, the ovipositors were placed (5/microvial) in 250 #1 of ethyl ether (Mallinckrodt|

PHEROMONEOF Heliothis subflexa

1013

anhydrous reagent grade) and allowed to soak for a period of 2-3 min. The ethyl ether extract was pipetted into another vial which contained 15/~1 of isooctane (Fisher, 99 mole %) and the ether allowed to evaporate under a fine nitrogen stream. Extracts were then stored at - 6 0 ~ until use. Chemical Analysis. Gas chromatographic (GC) analysis of 3- to 5-#1 samples [1-2 female equivalents (FE)] of the ovipositor extracts was done with a Hewlett-Packard model 5710A| GC equipped with a splitless injector system. The output of the flame ionization detector was interfaced to a Nicolett 1180 | data system capable of storing 32,000 real time data points. Nitrogen (linear flow velocity of 9.8 cm/sec) was used as a carrier gas. Initial GC studies were performed with a 66-m • 0.25-mm (ID) glass capillary column coated with SP 2340 (Supelco, Belefonte, Pennsylvania) capable of separating most of the geometrical and positional isomers of C14 and C16 alcohols, aldehydes, and acetates (Heath et al., 1980). Samples were injected at an initial column temperature of 60 ~ C (injector temperature = 250 ~ C) with a 60-sec delay prior to injector purging. The column temperature was programed after 2 min at 32 ~ C/min to a final temperature of 150~ C. Extracts were also chromatographed on a 3 l-m • 0.25-mm (ID) glass column coated with cholesteryl cinnamate (Heath et al., 1979). Splitless injections were made at the mesophase transition temperature (159 ~C) using decane as the solvent. The retention times of the compounds eluting during GC analysis of the ovipositor extracts as well as those of the synthetic standards were reduced to equivalent chain length (ECL) units with slight modification (Jamison and Reid, 1969). For the purpose of this study, the acetates of primary saturated alcohols varying in chain length from 12 to 20 carbons were used as the functional retention index (Swoboda, 1962), regardless of the compound's functionality. In subsequent studies, ovipositor extracts were cochromatographed with isomerically pure (+98%) synthetic standards. Further chemical characterization was accomplished by GC-mass spectrometry with a Finigan model 3200| chemical ionization mass spectrometer equipped with a GC inlet. The combined extract from 50 calling females was injected onto a 2-m • 2-mm (ID) glass column packed with 3% OV-17| on Gas Chrom Q| (100/120 mesh) and the total effluent was introduced directly into the ionization source. Methane was used as both a carrier and reagent gas. Spectra of the natural products were compared with those of candidate synthetic compounds. All synthetic standards used (Figure la), with the exception of (E)-7hexadecenal, (E)-9-hexadecenal, (E)- 11-hexadecenal, (E)-9-hexadecen- ! -ol, (E)-ll-hexadecen-l-ol, and (Z)-9-hexadecen-l-ol acetate, were obtained from Chemical Samples Co. (Columbus, Ohio), as were the starting materials used in preparation of the above standards. (E)-9-Hexadecen-l-ol was obtained from a lithium bronze reduction of 9-hexadecyn-l-ol (Mueller and

1014

TEAL ET AL.

Gillick, 1978) and a portion of the product was oxidized to (E)-9-hexadecenal with pyridinium chlorochromate (Corey and Suggs, 1975). (E)-7-Hexadecenal a.nd (E)-I l-hexadecenal were similarly prepared by oxidation of their corresponding alcohols. The (E)-I l-hexadecen-l-ol was obtained by saponification of (E)-I l-hexadecen-l-ol acetate, while the (Z)-9-hexadecen-l-ol acetate was prepared by acetylation of( Z)-9-hexadecen-! -ol. Isomeric purity was assessed by GC analysis with the SP2340 column and, when necessary, geometric isomers were separated by high-performance liquid chromatography on a 25 X 2.5-cm (OD) AgNO3 column eluted with toluene (Heath and Sonnet, 1980). All standards were assesssed as being at least 98% pure. Bioassays and Field Testing. Laboratory-reared males were used to assess the ability of gland extracts to elicit male reproductive behaviors. In these tests, I-FE samples of the extracts were placed on ! • 3-cm filter papers and suspended in the upwind end of a 1.5 X 0.5 X0.5-m Plexiglas wind tunnel (Teal et al., 1981a). Individual males were then released into the center of the downwind end and behaviors monitored over a 5-min period. The data were recorded on audio cassette tape and later transcribed (Teal et al., 198 la). The ability of each extract to induce upwind taxis, landing on the dispenser, and genital segment exposure was assessed. Field studies were conducted during July and August of 1980 in a fallow field near Gainesville, Florida, containing ground cherry, Physalis spp., and supporting a high larval H. subflexa population. Cone traps (Hartstack et al., 1979) spaced 10 m apart were set in two lines at 90 ~ to one another in the field. The traps were randomly baited with three calling-age females, a 2-ml polyethylene vial containing 30 mg of the synthetic mixture plus 5/~g of B HT [2,6-bis(l,l-dimethylethyl)-4-methylphenol, as an antioxidant], or a blank vial. Both the females and vials were rebaited every 2 days. In a second series of tests, traps were baited with 3 females, a blank 8.5-cm-diam. filter paper, or filter papers baited at 30-min intervals during the calling period with either 5 FE of the ovipositor extract or an equivalent amount (ca. 75 ng as indicated by GC analysis) of the synthetic blend. All baits were rerandomized daily. The species specificity of the H. subflexa synthetic blend was assessed during field trapping studies in tobacco and corn fields having populations of either H. virescens or H. zea (Boddie) and in the original test field. All sites had fruiting ground cherry present. Cone traps, spaced 10-12 m apart and located in the vicinity of Physalis plants, were baited with 3 female H. subflexa, 30 mg of the H. subflexa synthetics dispensed from polyethylene vials, 3 female H. zea or H. virescens, or 30 mg of either the 4-component H. zea or the 7-component H. virescens synthetic blend reported by Klun et al. (1980a,b) depending upon the field. Tests in the original test field included females and synthetic blends of all three species. Two series of tests were conducted to assess the effects of groups of

PHEROMONEOFHeliothis subflexa

1015

components on trap capture. In the first series the effect of deleting the alcohol, acetate, or aldehyde components from the blend was assessed with rubber septa (A. H. Thomas Co.) (Flint et al., 1979) impregnated with the same concentration of blend components as in the 25-mg complete blend. The second test was designed to assess the effectiveness of the aldehyde, alcohol, and acetate component groups independently, and to assess combinations of the aldehyde and acetate fractions. Pherocon| IC sticky traps spaced 10 m apart were positioned in two lines in the original field and randomized daily during the test periods. Daily trap captures were recorded and the data transformed to log,0 (n + 1) prior to statistical analysis.

RESULTS AND DISCUSSION Components contained within the ovipositor extracts were tentatively identified by comparison of their ECL values with those of a standard mixture of synthetic compounds. This mixture contained the A7, A9, and A 11 isomers of hexadecenal, hexadecen-l-ol, and hexadecen-l-ol acetate. The saturated analogs of the above compounds were also added to the mixture. Although the SP2340 column provided adequate resolution of most of the compounds (Figure la), the cholesteryl cinnamate column was also used to further define the assignments of the components. Table 1 lists the ECL units for the synthetic compounds used in this study on both the SP2340 and cholesteryl cinnamate columns. Bioassays of I-FE concentrations of the whole ovipositor extracts gave consistent results, indicating that the complete range of reproductive behaviors assessed was elicited with glandular extracts while only minimal random flight was observed when solvent blanks were presented. Capillary chromatography of these extracts revealed several maj or peaks having retention times coinciding with hexadecanal, (Z)-9-hexadecenal, (Z)I I -hexadecenal, (Z)-7-hexadecen- 1-ol acetate, (Z)-9-hexadecen- !-ol acetate, ( Z)- I I -hexadecen- 1-ol acetate, ( Z)-9-hexadecen- 1-ol, and ( Z)- l 1-hexadecen1-ol (Figures l and 2). Several other peaks were also variably present but, when present, each composed less than 1% of the total mixture. Mass spectra obtained from peaks eluting from the OV-17 column when ovipositor extracts were injected had identical fragmentation patterns with the monounsaturated aldehyde, acetate, and alcohol standards. Points of unsaturation were further confirmed by cochromatography of the ovipositor extracts and synthetic compounds on the SP2340 capillary and cholesteryl cinnamate columns. The relative proportion of each of these compounds within the natural blend is indicated in Table 2. Field tests of the synthetic blend formulated either in polyethylene vials

1016

TEAL ET AL.

1A 3a 5 6

II 12 la

10 41

~5

I

2N BHT

1B

r--

6

2o2, FIG. I. (A) Chromatogram of standard compounds eluting from the SP2340 column. 1 = Hexadecanal, 2 = (E)-7-hexadecenal, 3 = (E)-9-hexadecenal, 4 = (E)-I l-hexadecenal, 5 = (Z)-7-hexadecenal, 6 = (Z)-9-hexadecenal, 7 = (Z)-I 1-hexadecenal, 8 = hexadecan- 1-ol acetate, 9 = (E)-7-hexadecen- 1-ol acetate, 10 = ( E)-9-hexadecen- 1-ol acetate, 11 = (E)- I 1-hexadecen- I-ol acetate, 12 = ( Z)-7-hexadecen- 1-ol acetate, 13 = (Z)-9-hexadecen- I-ol acetate, 14 = (Z)- I l-hexadecen- I-ol acetate, 15 = hexadecanI-ol, 16 = (E)-7-hexadecen-l-ol, 17 = (E)-9-hexadecen-I-ol, 18 = (E)-ll-hexadecen1-ol, 19 = (Z)-7-hexadecen-l-ol, 20 = (Z)-9-hexadecen-l-ol, 21 = (Z)-I l-hexadecen1-ol. (B) Chromatogram of components in the ovipositor extracts eluting from the SP2340 column. 1 = Hexadecanal, 6 = (Z)-9-hexadecenal, 7 = (Z)-I l-hexadecenal, 12 = (Z)-7-hexadecen-l-ol acetate, 13 = (Z)-9-hexadecen-l-ol acetate, 14 = (Z)-I 1hexadecen-I -ol acetate, 20 = ( Z)-9-hexadecen-1 -ol, 21 = ( Z)- 11-hexadecen- 1-ol. or o n filter paper indicated that the synthetics were as effective in c a p t u r i n g males as either females or the crude ovipositor extracts in cone traps (Table 3). Field observations indicated that males generally u n d e r w e n t u p w i n d taxis toward the traps baited with the synthetic blend, e x t e n d i n g their genitalia a n d hovering at 10-15 cm from the dispenser prior to flying up a n d into the trap. However, close-range courtship behaviors (Teal et al., 1981 a) such as l a n d i n g on the dispenser, a b d o m i n a l curving, a n d c o p u l a t o r y a t t e m p t s were not observed when the synthetic blend was used, which indicates that cues responsible for eliciting courtship behaviors were absent, masked, or inhibited in these tests. Such stimuli may result from several different features absent in

PHEROMONE OF Heliothis

subflexa

1017

our tests such as the precise blend of components released by calling females and visual and tactile stimuli (Baker and Card~, 1979a,b). Further, the absence of stimuli responsible for landing and close-range copulatory behaviors are presumed to have little effect upon cone trap efficiency since males commonly move up and out of the pheromone plume when incomplete stimuli are presented (Teal et al., 1981b) and are therefore captured in cone traps. Neither the synthetic blends nor caged females of the three species captured males of other Heliothis species, indicating that the pheromone blends are species specific. However, while both the H. subflexa and H. virescens synthetic blends caught as effectively as caged females (15 c~ to H. subflexa synthetics/21 c~ to H. subflexa 9 , and 6 d to H. virescens synthetics/10 d to H. virescens ? ) the blend described for H. zea by Klun et al. (1980a) was considerably less effective than females (3 c5 to synthetics/56 d to 9 ). This may indicate a disparity in the actual blend released during calling and that maintained within the pheromone gland or cuticle overlying it TABLE I. EQUIVALENT CHAIN LENGTH UNITS OF G C STANDARDS ON S P 2 3 4 0 AND CHOLESTERYL CINNAMATE CAPILLARY COLUMNS AS CALCULATED USING SATURATED Cj4-C16 ACETATES AS THE FUNCTIONAL RETENTION iNDEX

Equivalent chain length Standard

SP2340

Cholesteryl cinnamate

Hexadecanal (E)-7-Hexadecenal (Z)-7-Hexadecenal (E)-9-Hexadecenal (Z)-9-Hexadecenal (E)-I l-Hexadecenal (Z)-I I-Hexadecenal Hexadecen- 1-ol acetate (E)-7-Hexad ecen-1 -ol acetate (Z)-7-Hexadecen-l-ol acetate (E)-9-Hexadecen- I-ol acetate (Z)-9-Hexadecen-l-ol acetate (E)-I I-Hexadecen-l-ol acetate (Z)-I 1-Hexadecen- I -ol acetate Hexadeca n- 1-ol (E)-7-Hexadeeen-I-ol (Z)-7-Hexadecen-!-ol (E)-9-Hexadecen-l-ol (Z)-9-Hexadecen-l-ol (E)-I 1-Hexadecen~ I-ol (Z)-I I-Hexadecen- 1-ol

1513 1555 1577 1559 1582 1567 1584 1600 1628 1645 1634 1653 1643 1665 1693 1734 1752 1737 1756 1744 1768

1434 1412 1402 1415 1404 1419 1410 1600 1578 1566 1583 157 I 1585 1575 1561 152I 1508 1526 151 I 1541 1518

1018

TEAL ET AL.

2A 234

BH~

t491011

21~I l l j

1

8

7

2B 14

L_

1

FIG. 2. (A) C h r o m a t o g r a m of standard compounds eluting from the cholesteryl cinnamate column. C o m p o u n d labels as in Figure 1A. (B) C h r o m a t o g r a m of components in the ovipositor extracts eluting from the cholesteryl cinnamate column. Component labels as in Figure 1B.

TABLE 2. CHEMICAL COMPONENTS ISOLATED FROM H. subflexa OVIPOSITOR WASHES

Compound

Mean % composition (15 9 )

Composition by weight (ng/~ )

Hexadecanal (Z)-9-Hexadecenal (Z)-I l-Hexadecenal (Z)-7-Hexadecen- 1-ol acetate (Z)-9-Hexadecen-l-ol acetate (Z)-I I-Hexadecen-l-ol acetate (Z)-9-Hexadecen-l-ol (Z)-I l-Hexadecen-l-ol

5.4 19.8 30.0 1.6 4.3 12.3 14.4 12.2

0.82 3.01 4.56 0.25 0.65 1.87 2.19 1.85

PHEROMONEOF Heliothis subflexa

1019

TABLE3. COMPARISONOF CONETRAP CAPTURESOF MALE1-1.subflexa US~N~ FEMALES,SYNTHETICCHEMICALS,AND CRUDEEXTRACTSa Trap bait 3 females 20 mg synthetics in vials 5 FE synthetics on filter papers 5 FE ovipositor extracts on filter papers Blank

Mean trap capture/night h 7.6 8.0 7.3 3.0 0.0

a a a a b

~Means followed by the same letter are not significantly different at a 0.05 level in a Duncan's multiple-range test. hSix replicates.

(cf. Weatherston and Maclean, 1974). Although the obvious differences in the blends of the three species suggest several avenues by which reproductive isolation could be effected, we have not assessed the behavioral effects of differences in the blend ratios or presence or absence of components on any of the three species and are unable to define the basis for chemical isolation at present. Laboratory interspecific communication studies between H. virescens and H. subflexa (Teal et al., 1981 b) indicate that long-distance semiochemical isolation occurs between female 11. virescens and male H. subflexa. However, it is the close-range orientation that is disrupted when the male H. virescens are released downwind from calling H. subflexa females. Studies conducted to assess the effects of deleting the alcohol, aldehyde, or acetate components from the blend indicated that the removal of any one of these groups did not completely stop trap capture (Table 4, experiment 1). While the whole synthetic blend was as effective as virgin females in cone trap studies, a significant decrease in captures relative to females was noted when the whole blend was employed in sticky traps. Further, considerably more males were captured in the sticky traps when the alcohols were deleted, suggesting that the alcohols may act as an inhibitor to landing, a prerequisite for both mating (Teal et al., 1981a) and being caught in sticky traps. This is further supported by field observations of moths orienting to cone traps which indicated that, although males did approach dispensers containing the whole blend, none landed or exhibited any close-range copulatory behaviors. Tests employing baits composed of various blends of components in sticky traps indicated that those containing either the acetates or alcohols alone were ineffective in trapping males, while the binary mixture of (Z)-9hexadecenal and (Z)-I l-hexadecenal was considerably more effective than the whole blend of alcohols, aldehydes, and acetates (Table 4, experiment 2). The acetates alone are ineffective trap baits, but their addition to the binary mixture of monounsaturated aldehydes causes a pronounced increase in the

16:AI

+ + + + +

+ + +

Z9-16:AI

+ + + + +

+ + +

Z II-16:AI

+ +

+ +

+ +

+ +

+ + + +

+

Z9-16:Ac

+

2'7-16:Ac

subflexa

+ +

+ +

+ +

+

ZII-16:Ac

+ +

+

+ + +

ZII-16:OH

+

+ + +

Z9-16:OH

2.899 1.000 0.941 0.803 0.578 0.148 0.148 0.000

2.742 1.912 0.774 0.706 0.045

(a) (b) (b) (b,c) (c,d) (c,d) (c,d) (d)

a a b b c

Mean (males/night) ~

MALES USING DIFFERENT BLENDS OF COMPONENTS a

a R a w d a t a t r a n s f o r m e d to log~0 ( X + I) p r i o r to a n a l y s i s (16 replicates over 8 nights, e x p e r i m e n t 1; 10 replicates o v e r 5 nights, e x p e r i m e n t 2). M e a n s followed by the s a m e letter are not s i g n i f i c a n t l y different in a D u n c a n ' s m u l t i p l e - r a n g e test at a P = 0.05 level, t 6 : A I = H e x a d e c a n a l ; Z 9 16:AI = ( Z ) - 9 - h e x a d e c e n a l ; Z I 1 - 1 6 " A l = ( Z ) - I I - h e x a d e c e n a l ; Z 7 - 1 6 : A c = ( Z ) - 7 - h e x a d e c e n - l - o l acetate; Z 9 - 1 6 : A c = (Z)-9-hexadecen-l-ol acetate; Z 1 1 - t 6 : A c = ( Z ) - I 1 - h e x a d e c e n - l - o l acetate; Z 9 - 1 6 : O H = ( Z ) - 9 - h e x a d e c e n - l - o l ; Z 11-16: O H = ( Z ) - I 1 - h e x a d e c e n - l - o l . M e a n s f r o m the t w o e x p e r i m e n t s are n o t c o m p a r e d w i t h one a n o t h e r . The presence of a c o m p o u n d in a tes~/blend is i n d i c a t e d by a +.

+ +

+

Experiment 2

Experiment 1 + + + +

3(2

TABLE 4. COMPARISON OF STICKY TRAP CAPTURES OF tl.

t~

H

PHEROMONE OF Heliothis subflexa

1021

number of males captured, indicating their necessity for effective sexual communication. The decrease in captures recorded on addition of hexadecanal to either the monounsaturated aldehyde or monounsaturated aldehyde + acetate blends tends to suggest a slight inhibitory function. However, insufficient behavioral analysis has been conducted to determine the validity of this hypothesis at present. Although the blend reported here is highly complex and may contain chemicals having no behavioral significance, a definite biochemical theme based on the use of C16 compounds is quite obvious. The use of these compounds is common among heliothids throughout the world, with (Z)-l lhexadecenal being the major pheromone component in all cases reported (Klun et al., 1980a,b; Nesbitt et al., 1979, 1980). The distinction appears to be the presence of acetates within the H. subflexa blend. However, Rothschild (1978) has recently reported that a mixture of (Z)-I l-hexadecenal, (Z)-9tetradecenal, and (Z)-l l-hexadecen-l-ol acetate caught considerably more male H. punctigera (Wllgn.) than did the aldehyde blend alone. Hence, while the aldehydes are of obvious major importance to sexual signaling, the acetates may indeed form an integral part of the pheromone blend of many Heliothis species. Acknowledgments--We wish to thank M.L. Laster, MAFES Delta Branch, for providing some of the 11. subflexa used in this study, R.E. Doolittle for help in the preparation of the synthetic compounds used, and R. Rush for assistance with the field studies.

REFERENCES

BAKER, T.C., and CARDE, R.T. 1979a. Analysis of pheromone-mediated behaviors in male Grapholitha molesta, the Oriental fruit moth (Lepid optera: Tortricidae). Environ. Entomol. 8: 956-968. BAKER, T.C., and CARDE, R.T. 1979b. Courtship behavior of the Oriental fruit moth (Grapholitha molesta): Experimental analysis and consideration of the role of sexual selection in the evoluation of courtship pheromones in Lepidoptera. Ann. Entomol. Soc. Am. 72:173-188. BRAZZEL, J.R., NEWSOME, L.D., ROUSEL,T.S., LINCOLN, C., WILHAMS, F.J., and BARNES,G. 1953. Bollworm and tobacco budworm as cotton pests in Louisiana and Arkansas. L. Agric. Exp. Stn. Tech. Bull. 482, 47 pp. COREr, E.J., and SUGGS, J.W. 1975. Pyridinium chlorochromate. An efficient reagent for oxidation of primary and secondary alcohols to carbonyl compounds. Tetrahedron Lett. 31:2647-2650. FLINT, H.M., McDoNouGH, L.M., SALTER,S.S., and WALTERS,S. 1979. Rubber septa: A long lasting substrate for (Z)-I 1-hexadecenal and ( Z)-9-tetradecenal, the primary components of the sex pheromone of the tobacco budworm. J. Econ. EntomoL 72:798-800. HARTSTACK,A.W., WITZ, J.A., and BUCK,D.R. 1979. Moth traps for the tobacco budworm. J. Econ. EntomoL 72:519-522. HEATh, R.R., and SONNET, P.E. 1980. Techniques for in situ coating of Ag § onto silica gel in H PLC columns for the separation of geometrical isomers. J. Liq. Chromatogr. 3:1129-1135.

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