Journal of Chemical Ecology, Vol. 18, No. 3, 1992
IDENTIFICATION OF MALE CABBAGE LOOPER SEX PHEROMONE ATTRACTIVE TO FEMALES
R.R. HEATH,*
P.J. L A N D O L T , B . D . D U E B E N , R.E. SCHNEIDER
R.E. MURPHY,
and
Insect Attractants, Behavior, and Basic Biology Research Laboratory Agricultural Research Service, U.S. Department of Agriculture Gainesville, Florida 32604 (Received May 24, 1991; accepted November 4, 1991) Abstract--A multicomponent pheromone produced by male cabbage looper moths that is attractive to female moths in a flight tunnel bioassay was isolated and identified. Based on analyses of hairpencil extracts of male cabbage loopers and volatiles emitted by males, the pheromone has been identified as a blend consisting of (S)-(+)-linalool, p-cresol, and m-cresol. The chirality of the major component, (S)-(+)-linalool, is important for behavioral response of females. These pheromonal compounds were also identified as volatiles released by males when males were exposed to the principal pheromone component of female cabbage loopers, (Z)-7-dodecen-l-ol acetate. The amount of male pheromone released was increased significantly when males were exposed to a combination of (Z)-7-dodecen-l-ol acetate and the odor from cabbage. Neither linalool nor the cresols were detected in volatiles from cabbage or from males exposed to cabbage odor. Key Words--Male-produced pheromone, cabbage looper, Trichoplusia ni, Lepidoptera, Noctuidae, female attractant, linalool, p-cresol, m-cresol, enantiomers.
INTRODUCTION Considerable research has been focused on f e m a l e - p r o d u c e d sex p h e r o m o n e s o f lepidopteran insects. The n u m b e r o f female sex attractants reported in the literature is in excess o f 1700 ( M a y e r and M c L a u g h l i n , 1990). M a l e - p r o d u c e d sex attractants are less c o m m o n in insects ( T h o m h i l l and A l c o c k , 1983) and unusual in Lepidoptera. In a r e v i e w o f m a l e p h e r o m o n e s in Lepidoptera, Birch * To whom correspondence should be addressed. 441
442
HEATH ET AL.
and Hefetz (1987) listed six species in which female attraction to male pheromones is known. There are now four reports of female flight attraction responses to males: the aretiids Estigmene acrea (Drury) (Willis and Birch, 1982) and possibly Creatonotos transiens (Walker) and C. ganges (L.) (Wunderer et al., 1986) and the cabbage looper Trichoplusia ni (Hfibner) (Landolt and Heath, 1990). Female behavior often associated with the release of male-produced pheromones includes mate acceptance and occurs only at close range (Birch 1974). Male cabbage loopers produce volatile compounds that may influence female behavior in attraction and courtship interactions. An electrophysiological response of female cabbage loopers to material extracted from male hairpencils was reported by Grant (1970). In subsequent investigations Grant (1971) and Grant and Brady (1973) speculated on the possible function of chemicals associated with hairpencils and brushes of the male cabbage looper. Jacobson et al. (1976) reported the identification of 2-phenylethanol in male cabbage looper hairpencils and a wing flutter response by females to hairpencil extracts and to 2-phenylethanol. These findings were contradicted by Hagan and Brady (1981), who were unable to find 2-phenylethanol in male cabbage loopers. Gothilf and $horey (1976), however, were unable to demonstrate a male scent brush role in mating success of this species. Based on reports of female catches in traps baited with female sex pheromone (Mitchell et al., 1972; Creighton et al., 1973; Birch, 1977), Birch (1977) suggested that females may have responded to chemicals released by males at the trap rather than to the female pheromone used as the trap lure. Recently, Landolt and Heath (1989, 1990) demonstrated attraction of unmated females to live males, extracts of males, and extracts of male hairpencils in flight-tunnel experiments. We report here the isolation and identification of a multicomponent male cabbage looper pheromone attractive to females in a flight-tunnel bioassay. The release of these identified pheromone components by males exposed to female sex pheromone also was demonstrated, and the release rate and ratio of pheromone components from stimulated males were determined.
MATERIALS AND METHODS
Insects and Bioassays. All moths used were reared as larvae on artificial diet according to the procedures reported by Guy et al. (1985). Pupae were sorted by sex and placed in 22 x 22 • 22-cm aluminum and fiberglass screen cages. Cages of pupae and emerged moths were held in environmental chambers on a 14:10 light-dark cycle, at 25 + 1 ~ and 60 +_ 10% relative humidity. Pupae were transferred daily to new cages, providing emerged moths of discrete
M A L E CABBAGE LOOPER A T T R A C T A N T
443
age groups. Moths were held with cups of a sugar-honey solution on cotton as food. Bioassays were conducted during the fifth to seventh hour of the 10-hr scotophase in a Plexiglas flight tunnel constructed as a 1 × 1 × 2-m box, with air drawn through by a fan at 0.22 m/sec. Lures were hung from a wire loop near the center of the upwind end of the tunnel and moths were released singly from a 20-ml polystyrene vial with a screen bottom and open top hung horizontally from a clamp on a ring stand near the center of the downwind end of the flight tunnel. Observations were aided by overhead red lights at < 1 footcandles. Virgin female moths 4-6 days old were placed in the flight tunnel chamber 1-3 hr before bioassays to allow adjustment to the conditions in the chamber. All bioassays consisted of a 2-min observation period following the release of a single moth. Test material was applied to 5.5-cm-diam. filter papers and was air dried in the tunnel for 60 sec before testing. Generally, five moths were tested sequentially per test sample. Responses scored included flight, anemotactic (upwind) flight, plume tracking (zig-zagging upwind flight within the apparent odor plume), and contact. Five females were also released sequentially to solvent treated filter paper at the upwind end and observed for 2 min each, as controls. Hairpencil Extracts and Collection of Folatiles. Extracts of male hairpencils were prepared by removing the terminal large paired abdominal hairpencils with forceps and placing them in a vial of hexane. Hairpencils from 50 males were accumulated in 2 ml of hexane. The hexane extract was pipetted to a clean vial after 20 min and concentrated when necessary under a stream of N2. A modified push-pull system (Landolt and Heath, 1987) was used to collect volatiles emitted by 3- to 6-day-old males. A schematic of the system is shown
FIG. 1. Schematic of system used to Collect volatiles from male cabbage loopers, suspected male stimulants, and combination of males and stimulants.
444
HEATH ET AL.
in Figure t. Briefly, compressed air, purified by passing through two charcoal filters, was separated into two airstreams. One of these streams was saturated with water by passage through a water-filled gas dispersion cylinder. The two airstreams were recombined to provide an adjusted relative humidity Jof 6065 %. This purified and humidified air was introduced into a glass chamber(s) that contained a frit near the upwind end of the chamber and provided a laminar airflow. Volatiles were collected on one of three collector traps positioned at the downwind end of the chambers, any one of which could be selected for volatile collection. Vent flow was monitored with a meter from a vent in the chamber, and the vent flow was maintained at 2 % of the airflow through the filter. Airflow was maintained at 1 liter/min with a slight positive pressure ( + 0.2 cm H20) established in the glass chamber(s). Pressure drop in the chamber(s) due to the collector trap was reduced to near zero by the use of vacuum downwind of the collector filter. This system was used to collect volatiles from males exposed to airflow passed over a cabbage plant, from males exposed to airflow containing (Z)-7 dodecen-l-ol acetate (Z7-12:Ac), from males exposed to airflow passed over a cabbage plant and Z7-12:Ac, from airflow over cabbage plants, and from airflow containing Z 7 - 1 2 : A c released from a robber septum loaded with 10 #g of the acetate. Collections were made using a 10-cm-OD • 30-cm-long glass chamber, which held the cabbage, or a 5-cm-OD • 30-cm-long chamber, which contained five moths. The smaller chamber contained a port that permitted the introduction of Z 7 - 1 2 : A c upwind of the males, via ground glass joints, and the chamber that housed the cabbage could be coupled in series with the chamber containing the insects. Volatiles were collected on traps using Super-Q as the adsorbent. Super-Q traps were prepared by packing ca. 20 mg of the adsorbent in 4-cm-long • 4.0mm-ID glass tubes resulting in a bed length of 5 mm. Two stainless-steel frits were used to contain the adsorbent. The Super-Q traps were cleaned by Soxhlet extraction with methylene chloride for 24 hr prior to use. Volatiles collected on the traps were eluted with 200 /~1 of methylene chloride and then 50 ng of tridecan-l-ol acetate was added as internal standard for subsequent analyses. Isolation and Analysis of Pheromone. Capillary gas chromatography was done using a retention gap column prior to the capillary column. This system permitted the injection of samples without concentration in 5-100 #1 of solvent. A combination of three fused silica columns connected in series using Glassseal connectors (Supelco Inc., Bellefonte, Pennsylvania) were used. The primary deactivated fused silica column, 8 cm • 0.5 mm ID, was connected between the injector and the retention gap column. This primary column permitted the use of 0.4-mm-OD stainless-steel needles with a septum injector. Selection of retention gap columns for use with the analytical column was determined by the
MALE CABBAGE LOOPER ATTRACTANT
445
phase ratio of both columns (Grob, 1982; Murphy, 1989). For the purpose of this investigation the retention gap columns were 10-m • 0.25-mm-ID silane deactivated fused silica (Quadrex, New Haven, Connecticut). Analytical columns used for analyses were a 50-m • 0.25-mm-ID Supelcowax 10 (bonded Carbowax), a 50-m • 0.25-mm-ID bonded BP-1 and a 25-m • 0.25-mm Chirasil-val (Chrompak, Raritan, New Jersey). A 15-m x 0.53-mm-ID, 1.0/~m film Carbowax fused silica column (Quadrex) was used for micropreparative purification of hairpencil extracts, extracts of coriander seeds, and candidate synthetics. Gas chromatographic analyses were conducted with a Hewlett-Packard model 5890 gas chromatograph, equipped with cool-on column capillary injector (septum injector) and flame ionization detector. Helium was used as the carder gas at a linear flow of 18 cm/sec. The temperature program was isothermal at 60~ for 2 min, and then programmed at 20~ to 160~ The chromatographic data were stored and analyzed in a Nelson 4000 data system. Initial micropreparative purification of hairpencil extracts on the 0.53-mmID capillary column resulted in the collection of three large fractions. Fraction 1 included the collection of compounds having an equivalent chain length unit (ECLU) (Swaboda, 1962) of heptan-l-ol acetate (ECLU = 700) to compounds eluting before nonan-l-ol acetate (ECLU = 900) (material collected from 6 to 14 min during GC run). Fraction 2 included compounds having an ECLU of 900-1400 (material collected from 18.2 to 20 min during GC run), and fraction 3 included compounds within the range of 1400-1800 (material collected from 20.2 to 30 min during GC run). Effluent from the column was split, with 2 % of the effluent routed to the FID detector and 98% of the effluent collected in a cooled, 30-cm glass capillary tube. The capillary was contained in a temperature gradient collector similar to that described by Brownlee and Silverstein (1968) and liquid nitrogen was used to cool the capillary. Collected fractions were washed from the capillary using 200 #1 of 1 : 1 hexane-ether. Fractions that were used in identifications were analyzed on the analytical columns described to ensure that the peak contained only one component. This same analytical method was used to further purify fractions of natural extracts, synthetics, and linalool from coriander seeds. Identification of Male Pheromone. Mass spectra of active natural compounds and candidate compounds were obtained using the BP-1 column, operated as described above, coupled to a Nermag model R1010 mass spectrometer in either electron impact (EI-MS) or chemical ionization (CI-MS) mode. The reagent gas used for CI was either methane or isobutane. Vapor-phase infrared spectra were obtained with a Nicolet 20SXC GC-FTIR spectrometer. Samples were introduced to the FTIR spectrometer via a BP-1 column connected to the Hewlett-Packard model 5890 gas chromatograph equipped with cool-on column
446
HEATH ET AL.
capillary injector. Nuclear magnetic resonance spectra (1H) were obtained with a Nicolet NT-300 (300 MHz) Fourier transform spectrometer using either CDCI3 o r C6D6 a8 the solvent and tetramethyl silane as an internal reference. Reagents and Derivatization. The chirality of linalool (3,7-dimethyl-l,6octadien-3-ol) was determined by formation of the diastereomeric 1-phenylethylurethanes with 1-phenylethyl-isocyanate (PEI) (Fluka Chemical Corp., Ronkonkoma, New York) diastereomers according to the method of Gaydou and Randriamiharisda (1987). The (R)-(+)- and (S)-(-)-PEI were used without purification. Approximately 200 ng of linalool isolated from male hairpencils, coriander, or synthetic racemic or (R)-(-)-linalool was dissolved in 20 #1 of methylene chloride, to which approximately 1 t~g of the (R)-(+)-PEI was added. After heating at 100~ for 1 hr, the sample was concentrated with dry N2. The sample was redissolved in HPLC grade methanol (J.T. Baker) containing 100 ng of n-tetradecanol as the internal standard for GC analysis. Racemic (Aldrich Chemical Corp., Milwaukee, Wisconsin) and (R)-(-)linalool (K&K Laboratories, Cleveland, Ohio) were purified using preparative GC. (S)-(+)-linalool was obtained from coriander seeds (Harris Seeds, Rochester, New York). Seeds were crushed and then soaked in GC-grade methylene chloride over night. The concentrated residue was filtered and purified by preparative GC. Ortho-, meta-, and paracresol were purchased from Aldrich Chemicals and purified by preparative GC prior to use. Linalool from coriander seed and synthetic compounds used for bioassays were > 98 % purity based on GC analyses. Synthetic pheromone components and blends were tested, using flight tunnel bioassays, for attractiveness to unmated female cabbage loopers and were compared to hairpencil extracts and solvent blanks. All materials tested were applied to 5.5-cm-diam. filter paper at dosages of 5 male equivalents (ca. 122.5 ng of linalool, 25.5 ng of p-cresol, and 3.5 ng of m-cresol) in hexane. A comparison (at amounts equal to 5 male equivalents) was conducted of attraction to: (1) racemic linalool, p-cresol, and m-cresol; (2) (R)-(-)-linalool, p-cresol, and m-cresol; (3) (S)-(+)-linalool, p-cresol, and m-cresol; and (4) hairpencil extracts from five males. Fifty females were tested per treatment, as 10 sets of five. Using a randomized complete block design, a complete treatment comparison of five females tested per treatment, covering all five treatments, was conducted per day for 10 days to provide the data set. In a second test, the following treatments were similarly compared: hexane (200 /zl); p-cresol and m-cresol at 25.5 and 3.5 ng per aliquot; (R)-(-)-linalool at 122.5 ng per aliquot; the complete identified blend of (R)-( -)-linalool, p-cresol, and m-cresol at 122.5, 25.5, and 3.5 ng, respectively; and hairpencil extracts from five males. Odors from cabbage plants, Brassica oleraceae (L), and Z7-12 : Ac loaded at 10/zg on rubber septa were tested as stimulants to elicit pheromone release from male cabbage loopers. Cabbage plants were grown in a greenhouse and
MALE CABBAGE LOOPER ATTRACTANT
447
were 8-12 weeks old, 20-25 cm wide and 15-20 cm tall when used, with no head development. The Z 7 - 1 2 : A c was purchased from Aldrich Chemicals and purified ( > 98 % purity) prior to use by AgNO3 high-performance liquid chromatography (Heath and Sonnet, 1980). Volatiles from males without stimuli, males exposed to cabbage odor, males exposed to Z 7 - 1 2 : A c released from a rubber septum, and a combination of Z 7 - 1 2 : A c and cabbage odor were collected and analyzed. Additionally, volatiles from the septum containing Z712 : Ac and cabbage were analyzed. Data on female bioassay response to fractions, isolates, synthetic, and linalool from coriander were subjected to ANOVA after arcsin transformation, with significant differences between treatment response means determined by Student's t test (Steel and Torrie, 1960) or Duncan's new multiple-range test (Duncan, 1955).
RESULTS
Response of female cabbage loopers in the flight tunnel to fraction 1 (material collected from 6 to 14 min during GC run), fraction 2 (material collected from 18.2 to 20 min during GC run), fraction 3 (material collected from 20.2 to 30 min during GC run), and combined fractions collected from the GC of male hairpencil extracts indicated that no loss in activity had occurred during chromatography (Table 1). Further testing of combinations of fractions indicated
TABLE 1. PERCENTAGES OF VIRGIN FEMALE CABBAGE LOOPERS RESPONDING IN FLIGHT TUNNEL BIOASSAYS TO G C FRACTIONS OR HAIRPENCIL EXTRACTS (A, N = 45) OR COMBINATIONS OF G C FRACTIONS (B, N = 50) a
Gas chromatographic fractions b Behavior
1
2
3
Combined
Extract
0a 0a
0a 0a
13b 9b
44d 31 c
31c 16b
1+2
1+3
2+3
2a 0a
26c 20c
6a 4ab
A Attraction Contact
3
1+2+3
B
Attraction Contact
12b 6b
24c 18c
aAmounts bioassayed were equal to that found in extracts of five males. bNumbers in rows followed by the same letter are not significantly different by Duncan's (1955) new multiple-range test at P < 0.05.
448
HEATH ET AL.
that all activity was accounted for by fractions 1 and 3 (Table 1), containing three peaks eluting with equivalent chain lengths (Swaboda, 1962) of 838.50, 1434.77, and 1439.61 (designated A, B, and C in Figure 2). Chemical ionization mass spectra of peak A, in which methane was used as the reagent gas, established that the molecular weight of this compound was 154 with diagnostic ions at m/e 153 (M - 1) and 155 (M + 1). Diagnostic ions at m/e 135 (M + 1 18) suggested the loss of water from the parent molecule. Electron impact did not provide an ion indicative of the molecular weight of the compound. Based on this information, peak A was proposed to be a 10-carbon alcohol containing two degrees of unsaturation. Further analysis by infrared spectroscopy indicated an alcohol stretch at 3636 cm -], and a terminal methylene characterized by C=CH2 stretch at 3090 cm -~. Proton NMR spectra with decoupling experiments on ca. 4 fzg of material provided the information for the identification of the compound as linalool based on the following spectral data: [~H]NMR (C6D6) ~1.28 (s, 3 H), 1.51-1.65 (m, 3H), 1.68 (s, 3H), 1.96-2.11 (m, 2H), 5.07 (dd, 1H J = 1.9, 11.2 Hz), 5.08-5.17 (m, 1H), 5.21 (dd, 1H, J = 1.9, 16.9 Hz),
A
CHROMATOGRAM 1 . . . . . . . .
|
B
1----1
CHROMATOGRAM 2
1~5
'
25
30
MINUTES FIG. 2. Chromatograms of volatiles collected from males in the presence of Z7-I2:Ac and cabbage odor (chromatogram 1) and volatiles from males in the presence of cabbage (chromatogram 2). Peak identification is A = linalool, B = p-cresol, and C = m-cresol. Gas chromatograph analysis was done on a 50-m x 0.25-mm-ID Supelcowax 10 column.
M A L E C A B B AG E L O O P E R A T T R A C T A N T
449
5.92 (dd, 1H, J = 11.2, 16.9 Hz). These data are in agreement with that reported for linalool by Ohwa et al. (1986). Preliminary experiments using (R)-(+)- and (S)-(-)-l-phenylethyl isocyanate as the derivitizing agent indicated that the (R)-(+)-urethane derivative was enantiomerically more pure (97%) and provided greater separation factor then the ( S ) - ( - ) derivative. Thus, the chirality of isolated linalool was determined by formation of the (R)-(+)-l-phenylethyl urethane diastereomer of the natural linalool and comparison of its retention time with the linalool diastercomers of known configuration. The products were chromatographed on a Chiralsil-val column and coeluted with the diastereomer formed with the linalool obtained from coriander seeds, previously reported as (S)-(+)-linalool (Cornforth et al., 1962; Ohloff and Klein, 1962). The elution time of the diastereomer formed with the linalool from male hairpencils was 14.2 min compared with 14.2 rain for the (S,R) diastereomer and 16.3 rain for the (R,R) diastereomer. Electron impact mass spectra of the smaller active peaks (B and C in Figure 2) found in the hairpencil extracts gave identical spectra. The molecular weight of these compounds were 108 with diagnostic ions at m/e 108 (M +) and 107 (M + - 1). Diagnostic ions at m/e 90 (M + - 18) suggested the loss of water from the parent molecule. A computer search of the National Institute of Standards and Technology mass spectral library resulted in a significant match with the spectra of cresol. Confirmation of the identity of these two compounds was obtained by comparison of vapor-phase IR spectra of the natural product with synthetic material and GC retention time comparisons. Based on these analyses, compounds B and C were identified as para- and meta-cresol, respectively. The average amount of (S)-(+)-linalool, p-cresol, and m-cresol found in male hairpencils was 24.5, 5.1, and 0.7 ng for each compound, respectively (Table 2). Wind-tunnel bioassays of the synthetic cresols and (S)-(+)-linalool obtained from coriander seeds resulted in comparable female response when compared to the hairpencil extracts (Table 3). Synthetic blends prepared using (R)-(-)-linalool and racemic linalool in combination with p- and m-cresols were significantly less attractive. In a comparison of a blend of the three components-(S)-(+)-linalool, p-cresol, and m-cresol--and hairpencil extracts, (S)-(+)-linalool alone and p- and m-cresol were significantly less attractive than the complete blend or extracts. Having identified pheromonal components from male hairpencils that elicited positive behavioral responses of females, we investigated the emission of these compounds from males. Results of the GC analysis of the volatiles collected are shown in Table 2. The identified male-produced pheromonal components were found only in volatiles collected from males exposed to Z712:Ac. In a previous study (Landolt and Heath, 1990) we showed that males exposed to cabbage odor were much more attractive to females than were males alone or cabbage alone. Similar results were obtained with males exposed to
450
HEATH ET AL.
TABLE 2. AMOUNT IN OF LINALOOL, p-CRESOL, AND m-CRESOL IN 15-MIN COLLECTIONS FROM MALE CABBAGE LOOPERS OR POTENTIAL MALE STIMULANTSa
Source (S)-(+)-Linalool (ng/male)
p-Cresol (ng/male)
m-Cresol (ng/male)
24.5 + 5.1 0.0a 5.0 _ 2.3b 0.0a 9.0 +__4.3c 0.0a
5.1 + 2.4 0.0 0.3 _+ 0.2 0.0 0.6 + 0.6 0.0
0.7 + 0.2 0.0 0.1 _ 0.1 0.0 0.2 + 0.1 0.0
Male hairpencil extracts (N = 12) Males (N = 5) Males + Z7-12:Ac (N = 5) Males + cabbage (N = 5) Males + cabbage + Z 7 - t 2 : A c (N = 5) Cabbage (N = 5)
~Mean amounts of linalool collected followed by the same letter are not significantly different by Duncan's (1955) new multiple-range test at P < 0.05. Values are •
TABLE 3. PERCENTAGESa OF VIRGIN FEMALE CABBAGE LOOPERS RESPONDING IN FLIGHT TUNNEL BIOASSAYS TO NATURALLY DERIVED OR SYNTHETIC MALE PHEROMONE CHEMICALS, BLENDS, AND HAIRPENCIL EXTRACTS ( g = 50 FOR PART 1, 35 FOR PART 2)
Behavior Part 1 Plume tracking Contact
Part 2 Plume tracking Contact
Racemic linalool (R)-(-)-Linalool (S)-(+)-Linalool 2 x racemic Hairpencil + cresols + cresols + cresols + cresols extract
34b 16a
20a 10a
52c 42b
24a 16a
54c 44b
p and m cresols
Hexane
(S)-(+)-Linalool
Synthetic blend
Hairpencil extract
14b 0a
3a 0a
23b 9b
60c 37c
54c 34c
aNumbers in rows followed by the same letter are not significantly different by Duncan's (1955) new multiple-range test at P < 0.05.
Z7-12:Ac, which were much more attractive to females than were unexposed males. We were unable to detect the presence of linalool or cresols in volatiles from males, cabbage, or males placed in the odor of cabbage (Table 2 and Figure 2). There was, however, an increase in the amount of pheromonal components released by males exposed to both cabbage and Z7-12:Ac compared
451
MALE CABBAGE LOOPER ATTRACTANT
tO males exposed to Z 7 - 1 2 : A c alone (Table 2). It was also noted that less pheromonal material was obtained from volatiles collected from these stimulated males than was found in analyzed hairpencil extracts. This difference in amount is due to the experimental design. It is important to note however, that the active materials isolated from male hairpencils were emitted from stimulated males. DISCUSSION Our results demonstrate that unmated female cabbage looper moths are attracted to a blend of three components identified in the terminal genital hairpencils of conspecific males. Linalool, the major component of the pheromone blend, is ubiquitous in plants and is often the major component of essential oils (Masada, 1976). It occurs in both the dextrorotatory [(S)-(+)-linalool or coriandrol] and levorotatory [(R)-(-)-linalool or licerol] forms. Additionally, linalool has been reported as a chemical component involved in the attraction of many other insects. For example, it is reported as a component of the attractant of the carrot fly larva, Psila rosae (Ryan and Guerin, 1982), a component of a female attractant of the spined soldier bug, Podisus maculiventris (Aldrich et al., 1984), and a component of a synthetic blend that attracts eastern yellowjackets, Vespula maculifrons (Aldrich et al., 1986). The effect of the chirality of linalool on the response of the carrot fly larvae, spined soldier bug, and eastern yellowjackets has not been reported. The chirality of linalool is of significance to the behavioral response of female cabbage loopers to the male pheromone. In addition to linalool, two cresols were found as components in the pheromonal blend attractive to female cabbage loopers, p-Cresol has been reported as a component of female pheromone of two hard ticks, Rhipicephalus appendicalatus and R. pulchellus (Wood et al., 1975) and as a component of an oviposition attractant of the tree-hole mosquito, Aedes triseriatus (Bentley et al., 1981). Bioassays conducted with the cresols were based on the ratio found either in hairpencil extracts or as collected volatiles. Studies are currently being conducted to determine the importance of the ratio of these components to the response of females to blends containing altered ratios. We have also demonstrated that, in addition to the identification of (S)(+)-linalool, p-cresol, and m-cresol in male hairpencils, these compounds are released by males when males are exposed to Z7-12:Ac, and the amount of pheromone released was increased when males were exposed to Z7-12 : Ac and the odor from cabbage. Neither linalool nor the cresols were evident in volatiles from cabbage or unstimulated males in the presence of cabbage odor. This suggests that either these compounds are not responsible for previously reported female attraction to cabbage and increased attraction of females to males on cabbage or that they occur in amounts not detectable with the analytical methods employed in these experiments.
452
HEATH ET AL.
Although male pheromone in the cabbage looper may function as an aphrodisiac or have some other courtship role, under the conditions of our studies the hairpencil blend of (S)-(+)-linalool and p- and m-cresols attracts unmated females. Field studies wilt be required to fully determine the potential use of these compounds as a female attractant. Captures of female cabbage loopers in traps baited with Z7-12 : Ac reported by Mitchell et al. (1972), Creighton et al. (1973), and Birch (1977) may have been due to female attraction to (S)-(+)linalool, p-cresol, and m-cresol in male hairpencils displayed at or in the traps. REFERENCES ALDRICH, J.R., LUSBY, W.R., KOCHANSKY, J.P., and ABRAMS, C.B. 1984. Volatile compounds from the predatory insect Podisus maculiventris: Male and female metathoracic scent gland and female dorsal abdominal gland secretions. J. Chem. Ecol. 10:561-568. ALDRICH, J.R., LUSBY, W.R., and KOCHANSKY,J.P. 1986. Identification of a new predaceous stink bug pheromone and its attractiveness to the Eastern Yellowjacket. Experientia 42:583-585. BENTLEY, M.D., MCDANIEL, I.N., YATAGAP, M., LEE, H., and MAYNARD, R. 1981. Oviposition attraction and stimulants of Aedes triseriatus (Say) (Diptera: Culicidae). Entomol. Soc. Am. 10:186-189. BIRCH, M.C. 1974. Aphrodisiac pheromone in insects, pp. 115-134, in M.C. Birch (ed.). Pheromones. North Holland, Amsterdam. BIRCH, M.C. 1977. Response of both sexes of Trichoplusia ni (Lepidoptera: Noctuidae) to virgin females and to synthetic pheromone. Ecol. Entomol. 2:99-104. BIRCH, M.C., and HEFETZ, A. 1987. Extmsible organs in male moths and their role in courtship behavior. Bull Entomol. Soc. Am. 33:222-229. BROWNLEE,R.G., and SILVERSTEIN,R.M. 1968. A micro-preparative gas chromatograph and a modified carbon skeleton determinator. Anal. Chem. 40:2077-2079. CORNFORTH,R.H., CORNFORTH, J.W., and PRELOG,V. 1962. Uben die configuration yon linalool, eine berichtigung. Justus Liebigs Ann. Chem. 634:197-198. CREIGHTON,C.S., McFADDEN, T.L., and CUTHBERT,E.R. 1973. Supplementary data on phenylacetaldehyde: an attractant for Lepidoptera. J. Econ. Entomol. 66: 114-115. DUNCAN, D.B. 1955. Multiple range and multiple F. Tests. Biometrics 11:1-41. GAYDOU, E.M., and RANDRIAMIHARISDA,R.P.J. 1987. Gas chromatographic separation of linalool derivatives. Chromatography 396:378-381. GOTHILF,S., and SHOREY,H.H. 1976. Sex pheromones for Lepidoptera: Examination of the role of male scent brushes in courtship behaviour of Trichoplusia hi. Environ. Entomol. 5:115119. GRANT, G.G. 1970. Evidence for a male sex pheromone in the noctuid, Trichoplusia hi. Nature 227:1345-1346. GRANT, G.G. 1971. Scent apparatus of the male cabbage looper, Trichoplusia hi. Ann. Entomol. Soc. Am. 64:347-352. GRANT,G.G., and BRADY,U.E. 1973. Topography of the scent scales of the male cabbage looper, Trichoplusia hi. J. Ga. Entomol. Soc. 8:99-106. GROB, J.J. 1982. Band broadening in space and the retention gap in capillary gas chromatography. J. Chromtagr. 237:15-23. GuY, R.H., LEPPLA, N.C., RYE, J.R., GREEN, C.W., BARETTE, S.L., and HOLLIEN, K.A. 1985. Trichoplusia hi, pp. 487-494, in P. Singh and R.F. Moore (eds.). Handbook of Insect Rearing, Vol. 2. Elsevier, Amsterdam.
MALE CABBAGELOOPER ATTRACTANT
453
HAGAN, D.V., and BRADY, U.E. 1981. Absence of detectable 2-phenylethanol in Trichoplusia ni, a reported pheromone of males. J. Ga. Entomol. Soc. 16:192-196. HEATH, R.R., and SONNET, P.E. 1980. Technique for in situ coating of Ag + onto silica gel in HPLC columns for the separation of geometrical isomers. J. Liquid Chromatogr. 3(8): 11291135. JACOBSON,M., ADLER,V.E., KIStlABA,A.N., and PRIESNER,E. 1976.2-Phenylethanol, a presumed sexual stimulant produced by the male cabbage looper moth, Trichoplusia Hi. Experientia 32:964-966. LANDOLT, P.J., and HEATH, R.R. 1987. Role of female-produced sex pheromone in behavioral reproductive isolation between Trichoplusia ni (Htibner) and Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae: Plusiinae). J. Chem. Ecol. 13:1005-1018. LANDOLT, P.J., and HEATH, R.R. 1989. Attraction of female cabbage looper moths (Lepidoptera: Noctuidae) to male-produced sex pheromone. Ann. Entomol. Soc. Am. 82(4):520-525. LANDOLT, P.J., and HEATH, R.R. 1990. Sexual role reversal in mate-finding strategies of the cabbage looper moth. Science 219:1026-1028. MASADA, Y. 1976. Analysis of Essential Oil by Gas Chromatography and Mass Spectroscopy. Wiley, New York. MAYER, M.S., and MCLAUGHLIN,J.R. 1990. Handbook of Insect Pheromones and Sex Attractants. CRC Press, Boca Raton, Florida. MITCHELL, E.R., WEBB, J.C., and HINES, R.W. 1972. Capture of male and female cabbage loopers in field traps baited with synthetic sex pheromone. Environ. Entomol. l:525-526. MURPHY, R.E. 1989. The fractionation gap: An optimized coupling of fused silica columns in open tubular gas chromatography. Master's thesis. University of Florida, _ _ . OHLOFF, G., and KLEIN, E. 1962. Determination of configuration of linalool. Tetrahedron 18:3739. OHWA, M., KOGURE, T., and ELIEL, E.L. 1986. An asymmetric synthesis of enantiomerically pure (S)-(+)-linalool (3,7-dimethyl-l,6-octadien-3-ol) and a formal synthesis of (R)-(-)-linalool. J. Org. Chem. 51:2599-2601. RYAN, M.F., and GUERIN, P.M. 1982. Behavioral responses of the carrot fly, Psila rosa, to carrot volatiles. Physiol. Entomol. 7:314-324. STEEL,R.G.D., and TORRIE,J.H. 1960. Principles and Procedures of Statistics. McGraw-Hill, New York. SWABODA,P.A.T. 1962. Qualitative and quantitative analysis of flavour volatiles from edible fats, pp. 273-291, in M. van Swacy (ed.). Gas Chromatography. Butterworth, London. THORNHILL, R., and ALCOCK, J. 1983. The Evolution of Insect Mating Systems. Harvard University Press, Cambridge, Massachusetts. WILLIS, M.A., and BIRCH, M.C. 1982. Male lek formation and female calling in a population of the aretiid moth, Estigmene acrea. Science. 218:168-170. WOOD, W.F., LEAHY, M.G., GALUN, R., PRESTWITHC,G.D., ME1NWALD,J., PURNELL,R.E., and PAYNE,R.C. 1975. Phenols as pheromones of ixodid ticks: A general phenomenon? J. Chem. Ecol. 1:501-509. WUNDERER, H., HANSEN, K., BELL, T.W., SCHNEIDER, D., and MEINWALD, M. 1986. Sex pheromones of two Asian moths (Creatonotos transiens, C. ganges; Lepidoptera: Arctiidae): Behavior, morphology, chemistry, and electrophysiology. Exp. Biol. 46:11-27.
IDENTIFICATION OF MALE CABBAGE LOOPER SEX PHEROMONE ATTRACTIVE TO FEMALES
R.R. HEATH,*
P.J. L A N D O L T , B . D . D U E B E N , R.E. SCHNEIDER
R.E. MURPHY,
and
Insect Attractants, Behavior, and Basic Biology Research Laboratory Agricultural Research Service, U.S. Department of Agriculture Gainesville, Florida 32604 (Received May 24, 1991; accepted November 4, 1991) Abstract--A multicomponent pheromone produced by male cabbage looper moths that is attractive to female moths in a flight tunnel bioassay was isolated and identified. Based on analyses of hairpencil extracts of male cabbage loopers and volatiles emitted by males, the pheromone has been identified as a blend consisting of (S)-(+)-linalool, p-cresol, and m-cresol. The chirality of the major component, (S)-(+)-linalool, is important for behavioral response of females. These pheromonal compounds were also identified as volatiles released by males when males were exposed to the principal pheromone component of female cabbage loopers, (Z)-7-dodecen-l-ol acetate. The amount of male pheromone released was increased significantly when males were exposed to a combination of (Z)-7-dodecen-l-ol acetate and the odor from cabbage. Neither linalool nor the cresols were detected in volatiles from cabbage or from males exposed to cabbage odor. Key Words--Male-produced pheromone, cabbage looper, Trichoplusia ni, Lepidoptera, Noctuidae, female attractant, linalool, p-cresol, m-cresol, enantiomers.
INTRODUCTION Considerable research has been focused on f e m a l e - p r o d u c e d sex p h e r o m o n e s o f lepidopteran insects. The n u m b e r o f female sex attractants reported in the literature is in excess o f 1700 ( M a y e r and M c L a u g h l i n , 1990). M a l e - p r o d u c e d sex attractants are less c o m m o n in insects ( T h o m h i l l and A l c o c k , 1983) and unusual in Lepidoptera. In a r e v i e w o f m a l e p h e r o m o n e s in Lepidoptera, Birch * To whom correspondence should be addressed. 441
442
HEATH ET AL.
and Hefetz (1987) listed six species in which female attraction to male pheromones is known. There are now four reports of female flight attraction responses to males: the aretiids Estigmene acrea (Drury) (Willis and Birch, 1982) and possibly Creatonotos transiens (Walker) and C. ganges (L.) (Wunderer et al., 1986) and the cabbage looper Trichoplusia ni (Hfibner) (Landolt and Heath, 1990). Female behavior often associated with the release of male-produced pheromones includes mate acceptance and occurs only at close range (Birch 1974). Male cabbage loopers produce volatile compounds that may influence female behavior in attraction and courtship interactions. An electrophysiological response of female cabbage loopers to material extracted from male hairpencils was reported by Grant (1970). In subsequent investigations Grant (1971) and Grant and Brady (1973) speculated on the possible function of chemicals associated with hairpencils and brushes of the male cabbage looper. Jacobson et al. (1976) reported the identification of 2-phenylethanol in male cabbage looper hairpencils and a wing flutter response by females to hairpencil extracts and to 2-phenylethanol. These findings were contradicted by Hagan and Brady (1981), who were unable to find 2-phenylethanol in male cabbage loopers. Gothilf and $horey (1976), however, were unable to demonstrate a male scent brush role in mating success of this species. Based on reports of female catches in traps baited with female sex pheromone (Mitchell et al., 1972; Creighton et al., 1973; Birch, 1977), Birch (1977) suggested that females may have responded to chemicals released by males at the trap rather than to the female pheromone used as the trap lure. Recently, Landolt and Heath (1989, 1990) demonstrated attraction of unmated females to live males, extracts of males, and extracts of male hairpencils in flight-tunnel experiments. We report here the isolation and identification of a multicomponent male cabbage looper pheromone attractive to females in a flight-tunnel bioassay. The release of these identified pheromone components by males exposed to female sex pheromone also was demonstrated, and the release rate and ratio of pheromone components from stimulated males were determined.
MATERIALS AND METHODS
Insects and Bioassays. All moths used were reared as larvae on artificial diet according to the procedures reported by Guy et al. (1985). Pupae were sorted by sex and placed in 22 x 22 • 22-cm aluminum and fiberglass screen cages. Cages of pupae and emerged moths were held in environmental chambers on a 14:10 light-dark cycle, at 25 + 1 ~ and 60 +_ 10% relative humidity. Pupae were transferred daily to new cages, providing emerged moths of discrete
M A L E CABBAGE LOOPER A T T R A C T A N T
443
age groups. Moths were held with cups of a sugar-honey solution on cotton as food. Bioassays were conducted during the fifth to seventh hour of the 10-hr scotophase in a Plexiglas flight tunnel constructed as a 1 × 1 × 2-m box, with air drawn through by a fan at 0.22 m/sec. Lures were hung from a wire loop near the center of the upwind end of the tunnel and moths were released singly from a 20-ml polystyrene vial with a screen bottom and open top hung horizontally from a clamp on a ring stand near the center of the downwind end of the flight tunnel. Observations were aided by overhead red lights at < 1 footcandles. Virgin female moths 4-6 days old were placed in the flight tunnel chamber 1-3 hr before bioassays to allow adjustment to the conditions in the chamber. All bioassays consisted of a 2-min observation period following the release of a single moth. Test material was applied to 5.5-cm-diam. filter papers and was air dried in the tunnel for 60 sec before testing. Generally, five moths were tested sequentially per test sample. Responses scored included flight, anemotactic (upwind) flight, plume tracking (zig-zagging upwind flight within the apparent odor plume), and contact. Five females were also released sequentially to solvent treated filter paper at the upwind end and observed for 2 min each, as controls. Hairpencil Extracts and Collection of Folatiles. Extracts of male hairpencils were prepared by removing the terminal large paired abdominal hairpencils with forceps and placing them in a vial of hexane. Hairpencils from 50 males were accumulated in 2 ml of hexane. The hexane extract was pipetted to a clean vial after 20 min and concentrated when necessary under a stream of N2. A modified push-pull system (Landolt and Heath, 1987) was used to collect volatiles emitted by 3- to 6-day-old males. A schematic of the system is shown
FIG. 1. Schematic of system used to Collect volatiles from male cabbage loopers, suspected male stimulants, and combination of males and stimulants.
444
HEATH ET AL.
in Figure t. Briefly, compressed air, purified by passing through two charcoal filters, was separated into two airstreams. One of these streams was saturated with water by passage through a water-filled gas dispersion cylinder. The two airstreams were recombined to provide an adjusted relative humidity Jof 6065 %. This purified and humidified air was introduced into a glass chamber(s) that contained a frit near the upwind end of the chamber and provided a laminar airflow. Volatiles were collected on one of three collector traps positioned at the downwind end of the chambers, any one of which could be selected for volatile collection. Vent flow was monitored with a meter from a vent in the chamber, and the vent flow was maintained at 2 % of the airflow through the filter. Airflow was maintained at 1 liter/min with a slight positive pressure ( + 0.2 cm H20) established in the glass chamber(s). Pressure drop in the chamber(s) due to the collector trap was reduced to near zero by the use of vacuum downwind of the collector filter. This system was used to collect volatiles from males exposed to airflow passed over a cabbage plant, from males exposed to airflow containing (Z)-7 dodecen-l-ol acetate (Z7-12:Ac), from males exposed to airflow passed over a cabbage plant and Z7-12:Ac, from airflow over cabbage plants, and from airflow containing Z 7 - 1 2 : A c released from a robber septum loaded with 10 #g of the acetate. Collections were made using a 10-cm-OD • 30-cm-long glass chamber, which held the cabbage, or a 5-cm-OD • 30-cm-long chamber, which contained five moths. The smaller chamber contained a port that permitted the introduction of Z 7 - 1 2 : A c upwind of the males, via ground glass joints, and the chamber that housed the cabbage could be coupled in series with the chamber containing the insects. Volatiles were collected on traps using Super-Q as the adsorbent. Super-Q traps were prepared by packing ca. 20 mg of the adsorbent in 4-cm-long • 4.0mm-ID glass tubes resulting in a bed length of 5 mm. Two stainless-steel frits were used to contain the adsorbent. The Super-Q traps were cleaned by Soxhlet extraction with methylene chloride for 24 hr prior to use. Volatiles collected on the traps were eluted with 200 /~1 of methylene chloride and then 50 ng of tridecan-l-ol acetate was added as internal standard for subsequent analyses. Isolation and Analysis of Pheromone. Capillary gas chromatography was done using a retention gap column prior to the capillary column. This system permitted the injection of samples without concentration in 5-100 #1 of solvent. A combination of three fused silica columns connected in series using Glassseal connectors (Supelco Inc., Bellefonte, Pennsylvania) were used. The primary deactivated fused silica column, 8 cm • 0.5 mm ID, was connected between the injector and the retention gap column. This primary column permitted the use of 0.4-mm-OD stainless-steel needles with a septum injector. Selection of retention gap columns for use with the analytical column was determined by the
MALE CABBAGE LOOPER ATTRACTANT
445
phase ratio of both columns (Grob, 1982; Murphy, 1989). For the purpose of this investigation the retention gap columns were 10-m • 0.25-mm-ID silane deactivated fused silica (Quadrex, New Haven, Connecticut). Analytical columns used for analyses were a 50-m • 0.25-mm-ID Supelcowax 10 (bonded Carbowax), a 50-m • 0.25-mm-ID bonded BP-1 and a 25-m • 0.25-mm Chirasil-val (Chrompak, Raritan, New Jersey). A 15-m x 0.53-mm-ID, 1.0/~m film Carbowax fused silica column (Quadrex) was used for micropreparative purification of hairpencil extracts, extracts of coriander seeds, and candidate synthetics. Gas chromatographic analyses were conducted with a Hewlett-Packard model 5890 gas chromatograph, equipped with cool-on column capillary injector (septum injector) and flame ionization detector. Helium was used as the carder gas at a linear flow of 18 cm/sec. The temperature program was isothermal at 60~ for 2 min, and then programmed at 20~ to 160~ The chromatographic data were stored and analyzed in a Nelson 4000 data system. Initial micropreparative purification of hairpencil extracts on the 0.53-mmID capillary column resulted in the collection of three large fractions. Fraction 1 included the collection of compounds having an equivalent chain length unit (ECLU) (Swaboda, 1962) of heptan-l-ol acetate (ECLU = 700) to compounds eluting before nonan-l-ol acetate (ECLU = 900) (material collected from 6 to 14 min during GC run). Fraction 2 included compounds having an ECLU of 900-1400 (material collected from 18.2 to 20 min during GC run), and fraction 3 included compounds within the range of 1400-1800 (material collected from 20.2 to 30 min during GC run). Effluent from the column was split, with 2 % of the effluent routed to the FID detector and 98% of the effluent collected in a cooled, 30-cm glass capillary tube. The capillary was contained in a temperature gradient collector similar to that described by Brownlee and Silverstein (1968) and liquid nitrogen was used to cool the capillary. Collected fractions were washed from the capillary using 200 #1 of 1 : 1 hexane-ether. Fractions that were used in identifications were analyzed on the analytical columns described to ensure that the peak contained only one component. This same analytical method was used to further purify fractions of natural extracts, synthetics, and linalool from coriander seeds. Identification of Male Pheromone. Mass spectra of active natural compounds and candidate compounds were obtained using the BP-1 column, operated as described above, coupled to a Nermag model R1010 mass spectrometer in either electron impact (EI-MS) or chemical ionization (CI-MS) mode. The reagent gas used for CI was either methane or isobutane. Vapor-phase infrared spectra were obtained with a Nicolet 20SXC GC-FTIR spectrometer. Samples were introduced to the FTIR spectrometer via a BP-1 column connected to the Hewlett-Packard model 5890 gas chromatograph equipped with cool-on column
446
HEATH ET AL.
capillary injector. Nuclear magnetic resonance spectra (1H) were obtained with a Nicolet NT-300 (300 MHz) Fourier transform spectrometer using either CDCI3 o r C6D6 a8 the solvent and tetramethyl silane as an internal reference. Reagents and Derivatization. The chirality of linalool (3,7-dimethyl-l,6octadien-3-ol) was determined by formation of the diastereomeric 1-phenylethylurethanes with 1-phenylethyl-isocyanate (PEI) (Fluka Chemical Corp., Ronkonkoma, New York) diastereomers according to the method of Gaydou and Randriamiharisda (1987). The (R)-(+)- and (S)-(-)-PEI were used without purification. Approximately 200 ng of linalool isolated from male hairpencils, coriander, or synthetic racemic or (R)-(-)-linalool was dissolved in 20 #1 of methylene chloride, to which approximately 1 t~g of the (R)-(+)-PEI was added. After heating at 100~ for 1 hr, the sample was concentrated with dry N2. The sample was redissolved in HPLC grade methanol (J.T. Baker) containing 100 ng of n-tetradecanol as the internal standard for GC analysis. Racemic (Aldrich Chemical Corp., Milwaukee, Wisconsin) and (R)-(-)linalool (K&K Laboratories, Cleveland, Ohio) were purified using preparative GC. (S)-(+)-linalool was obtained from coriander seeds (Harris Seeds, Rochester, New York). Seeds were crushed and then soaked in GC-grade methylene chloride over night. The concentrated residue was filtered and purified by preparative GC. Ortho-, meta-, and paracresol were purchased from Aldrich Chemicals and purified by preparative GC prior to use. Linalool from coriander seed and synthetic compounds used for bioassays were > 98 % purity based on GC analyses. Synthetic pheromone components and blends were tested, using flight tunnel bioassays, for attractiveness to unmated female cabbage loopers and were compared to hairpencil extracts and solvent blanks. All materials tested were applied to 5.5-cm-diam. filter paper at dosages of 5 male equivalents (ca. 122.5 ng of linalool, 25.5 ng of p-cresol, and 3.5 ng of m-cresol) in hexane. A comparison (at amounts equal to 5 male equivalents) was conducted of attraction to: (1) racemic linalool, p-cresol, and m-cresol; (2) (R)-(-)-linalool, p-cresol, and m-cresol; (3) (S)-(+)-linalool, p-cresol, and m-cresol; and (4) hairpencil extracts from five males. Fifty females were tested per treatment, as 10 sets of five. Using a randomized complete block design, a complete treatment comparison of five females tested per treatment, covering all five treatments, was conducted per day for 10 days to provide the data set. In a second test, the following treatments were similarly compared: hexane (200 /zl); p-cresol and m-cresol at 25.5 and 3.5 ng per aliquot; (R)-(-)-linalool at 122.5 ng per aliquot; the complete identified blend of (R)-( -)-linalool, p-cresol, and m-cresol at 122.5, 25.5, and 3.5 ng, respectively; and hairpencil extracts from five males. Odors from cabbage plants, Brassica oleraceae (L), and Z7-12 : Ac loaded at 10/zg on rubber septa were tested as stimulants to elicit pheromone release from male cabbage loopers. Cabbage plants were grown in a greenhouse and
MALE CABBAGE LOOPER ATTRACTANT
447
were 8-12 weeks old, 20-25 cm wide and 15-20 cm tall when used, with no head development. The Z 7 - 1 2 : A c was purchased from Aldrich Chemicals and purified ( > 98 % purity) prior to use by AgNO3 high-performance liquid chromatography (Heath and Sonnet, 1980). Volatiles from males without stimuli, males exposed to cabbage odor, males exposed to Z 7 - 1 2 : A c released from a rubber septum, and a combination of Z 7 - 1 2 : A c and cabbage odor were collected and analyzed. Additionally, volatiles from the septum containing Z712 : Ac and cabbage were analyzed. Data on female bioassay response to fractions, isolates, synthetic, and linalool from coriander were subjected to ANOVA after arcsin transformation, with significant differences between treatment response means determined by Student's t test (Steel and Torrie, 1960) or Duncan's new multiple-range test (Duncan, 1955).
RESULTS
Response of female cabbage loopers in the flight tunnel to fraction 1 (material collected from 6 to 14 min during GC run), fraction 2 (material collected from 18.2 to 20 min during GC run), fraction 3 (material collected from 20.2 to 30 min during GC run), and combined fractions collected from the GC of male hairpencil extracts indicated that no loss in activity had occurred during chromatography (Table 1). Further testing of combinations of fractions indicated
TABLE 1. PERCENTAGES OF VIRGIN FEMALE CABBAGE LOOPERS RESPONDING IN FLIGHT TUNNEL BIOASSAYS TO G C FRACTIONS OR HAIRPENCIL EXTRACTS (A, N = 45) OR COMBINATIONS OF G C FRACTIONS (B, N = 50) a
Gas chromatographic fractions b Behavior
1
2
3
Combined
Extract
0a 0a
0a 0a
13b 9b
44d 31 c
31c 16b
1+2
1+3
2+3
2a 0a
26c 20c
6a 4ab
A Attraction Contact
3
1+2+3
B
Attraction Contact
12b 6b
24c 18c
aAmounts bioassayed were equal to that found in extracts of five males. bNumbers in rows followed by the same letter are not significantly different by Duncan's (1955) new multiple-range test at P < 0.05.
448
HEATH ET AL.
that all activity was accounted for by fractions 1 and 3 (Table 1), containing three peaks eluting with equivalent chain lengths (Swaboda, 1962) of 838.50, 1434.77, and 1439.61 (designated A, B, and C in Figure 2). Chemical ionization mass spectra of peak A, in which methane was used as the reagent gas, established that the molecular weight of this compound was 154 with diagnostic ions at m/e 153 (M - 1) and 155 (M + 1). Diagnostic ions at m/e 135 (M + 1 18) suggested the loss of water from the parent molecule. Electron impact did not provide an ion indicative of the molecular weight of the compound. Based on this information, peak A was proposed to be a 10-carbon alcohol containing two degrees of unsaturation. Further analysis by infrared spectroscopy indicated an alcohol stretch at 3636 cm -], and a terminal methylene characterized by C=CH2 stretch at 3090 cm -~. Proton NMR spectra with decoupling experiments on ca. 4 fzg of material provided the information for the identification of the compound as linalool based on the following spectral data: [~H]NMR (C6D6) ~1.28 (s, 3 H), 1.51-1.65 (m, 3H), 1.68 (s, 3H), 1.96-2.11 (m, 2H), 5.07 (dd, 1H J = 1.9, 11.2 Hz), 5.08-5.17 (m, 1H), 5.21 (dd, 1H, J = 1.9, 16.9 Hz),
A
CHROMATOGRAM 1 . . . . . . . .
|
B
1----1
CHROMATOGRAM 2
1~5
'
25
30
MINUTES FIG. 2. Chromatograms of volatiles collected from males in the presence of Z7-I2:Ac and cabbage odor (chromatogram 1) and volatiles from males in the presence of cabbage (chromatogram 2). Peak identification is A = linalool, B = p-cresol, and C = m-cresol. Gas chromatograph analysis was done on a 50-m x 0.25-mm-ID Supelcowax 10 column.
M A L E C A B B AG E L O O P E R A T T R A C T A N T
449
5.92 (dd, 1H, J = 11.2, 16.9 Hz). These data are in agreement with that reported for linalool by Ohwa et al. (1986). Preliminary experiments using (R)-(+)- and (S)-(-)-l-phenylethyl isocyanate as the derivitizing agent indicated that the (R)-(+)-urethane derivative was enantiomerically more pure (97%) and provided greater separation factor then the ( S ) - ( - ) derivative. Thus, the chirality of isolated linalool was determined by formation of the (R)-(+)-l-phenylethyl urethane diastereomer of the natural linalool and comparison of its retention time with the linalool diastercomers of known configuration. The products were chromatographed on a Chiralsil-val column and coeluted with the diastereomer formed with the linalool obtained from coriander seeds, previously reported as (S)-(+)-linalool (Cornforth et al., 1962; Ohloff and Klein, 1962). The elution time of the diastereomer formed with the linalool from male hairpencils was 14.2 min compared with 14.2 rain for the (S,R) diastereomer and 16.3 rain for the (R,R) diastereomer. Electron impact mass spectra of the smaller active peaks (B and C in Figure 2) found in the hairpencil extracts gave identical spectra. The molecular weight of these compounds were 108 with diagnostic ions at m/e 108 (M +) and 107 (M + - 1). Diagnostic ions at m/e 90 (M + - 18) suggested the loss of water from the parent molecule. A computer search of the National Institute of Standards and Technology mass spectral library resulted in a significant match with the spectra of cresol. Confirmation of the identity of these two compounds was obtained by comparison of vapor-phase IR spectra of the natural product with synthetic material and GC retention time comparisons. Based on these analyses, compounds B and C were identified as para- and meta-cresol, respectively. The average amount of (S)-(+)-linalool, p-cresol, and m-cresol found in male hairpencils was 24.5, 5.1, and 0.7 ng for each compound, respectively (Table 2). Wind-tunnel bioassays of the synthetic cresols and (S)-(+)-linalool obtained from coriander seeds resulted in comparable female response when compared to the hairpencil extracts (Table 3). Synthetic blends prepared using (R)-(-)-linalool and racemic linalool in combination with p- and m-cresols were significantly less attractive. In a comparison of a blend of the three components-(S)-(+)-linalool, p-cresol, and m-cresol--and hairpencil extracts, (S)-(+)-linalool alone and p- and m-cresol were significantly less attractive than the complete blend or extracts. Having identified pheromonal components from male hairpencils that elicited positive behavioral responses of females, we investigated the emission of these compounds from males. Results of the GC analysis of the volatiles collected are shown in Table 2. The identified male-produced pheromonal components were found only in volatiles collected from males exposed to Z712:Ac. In a previous study (Landolt and Heath, 1990) we showed that males exposed to cabbage odor were much more attractive to females than were males alone or cabbage alone. Similar results were obtained with males exposed to
450
HEATH ET AL.
TABLE 2. AMOUNT IN OF LINALOOL, p-CRESOL, AND m-CRESOL IN 15-MIN COLLECTIONS FROM MALE CABBAGE LOOPERS OR POTENTIAL MALE STIMULANTSa
Source (S)-(+)-Linalool (ng/male)
p-Cresol (ng/male)
m-Cresol (ng/male)
24.5 + 5.1 0.0a 5.0 _ 2.3b 0.0a 9.0 +__4.3c 0.0a
5.1 + 2.4 0.0 0.3 _+ 0.2 0.0 0.6 + 0.6 0.0
0.7 + 0.2 0.0 0.1 _ 0.1 0.0 0.2 + 0.1 0.0
Male hairpencil extracts (N = 12) Males (N = 5) Males + Z7-12:Ac (N = 5) Males + cabbage (N = 5) Males + cabbage + Z 7 - t 2 : A c (N = 5) Cabbage (N = 5)
~Mean amounts of linalool collected followed by the same letter are not significantly different by Duncan's (1955) new multiple-range test at P < 0.05. Values are •
TABLE 3. PERCENTAGESa OF VIRGIN FEMALE CABBAGE LOOPERS RESPONDING IN FLIGHT TUNNEL BIOASSAYS TO NATURALLY DERIVED OR SYNTHETIC MALE PHEROMONE CHEMICALS, BLENDS, AND HAIRPENCIL EXTRACTS ( g = 50 FOR PART 1, 35 FOR PART 2)
Behavior Part 1 Plume tracking Contact
Part 2 Plume tracking Contact
Racemic linalool (R)-(-)-Linalool (S)-(+)-Linalool 2 x racemic Hairpencil + cresols + cresols + cresols + cresols extract
34b 16a
20a 10a
52c 42b
24a 16a
54c 44b
p and m cresols
Hexane
(S)-(+)-Linalool
Synthetic blend
Hairpencil extract
14b 0a
3a 0a
23b 9b
60c 37c
54c 34c
aNumbers in rows followed by the same letter are not significantly different by Duncan's (1955) new multiple-range test at P < 0.05.
Z7-12:Ac, which were much more attractive to females than were unexposed males. We were unable to detect the presence of linalool or cresols in volatiles from males, cabbage, or males placed in the odor of cabbage (Table 2 and Figure 2). There was, however, an increase in the amount of pheromonal components released by males exposed to both cabbage and Z7-12:Ac compared
451
MALE CABBAGE LOOPER ATTRACTANT
tO males exposed to Z 7 - 1 2 : A c alone (Table 2). It was also noted that less pheromonal material was obtained from volatiles collected from these stimulated males than was found in analyzed hairpencil extracts. This difference in amount is due to the experimental design. It is important to note however, that the active materials isolated from male hairpencils were emitted from stimulated males. DISCUSSION Our results demonstrate that unmated female cabbage looper moths are attracted to a blend of three components identified in the terminal genital hairpencils of conspecific males. Linalool, the major component of the pheromone blend, is ubiquitous in plants and is often the major component of essential oils (Masada, 1976). It occurs in both the dextrorotatory [(S)-(+)-linalool or coriandrol] and levorotatory [(R)-(-)-linalool or licerol] forms. Additionally, linalool has been reported as a chemical component involved in the attraction of many other insects. For example, it is reported as a component of the attractant of the carrot fly larva, Psila rosae (Ryan and Guerin, 1982), a component of a female attractant of the spined soldier bug, Podisus maculiventris (Aldrich et al., 1984), and a component of a synthetic blend that attracts eastern yellowjackets, Vespula maculifrons (Aldrich et al., 1986). The effect of the chirality of linalool on the response of the carrot fly larvae, spined soldier bug, and eastern yellowjackets has not been reported. The chirality of linalool is of significance to the behavioral response of female cabbage loopers to the male pheromone. In addition to linalool, two cresols were found as components in the pheromonal blend attractive to female cabbage loopers, p-Cresol has been reported as a component of female pheromone of two hard ticks, Rhipicephalus appendicalatus and R. pulchellus (Wood et al., 1975) and as a component of an oviposition attractant of the tree-hole mosquito, Aedes triseriatus (Bentley et al., 1981). Bioassays conducted with the cresols were based on the ratio found either in hairpencil extracts or as collected volatiles. Studies are currently being conducted to determine the importance of the ratio of these components to the response of females to blends containing altered ratios. We have also demonstrated that, in addition to the identification of (S)(+)-linalool, p-cresol, and m-cresol in male hairpencils, these compounds are released by males when males are exposed to Z7-12:Ac, and the amount of pheromone released was increased when males were exposed to Z7-12 : Ac and the odor from cabbage. Neither linalool nor the cresols were evident in volatiles from cabbage or unstimulated males in the presence of cabbage odor. This suggests that either these compounds are not responsible for previously reported female attraction to cabbage and increased attraction of females to males on cabbage or that they occur in amounts not detectable with the analytical methods employed in these experiments.
452
HEATH ET AL.
Although male pheromone in the cabbage looper may function as an aphrodisiac or have some other courtship role, under the conditions of our studies the hairpencil blend of (S)-(+)-linalool and p- and m-cresols attracts unmated females. Field studies wilt be required to fully determine the potential use of these compounds as a female attractant. Captures of female cabbage loopers in traps baited with Z7-12 : Ac reported by Mitchell et al. (1972), Creighton et al. (1973), and Birch (1977) may have been due to female attraction to (S)-(+)linalool, p-cresol, and m-cresol in male hairpencils displayed at or in the traps. REFERENCES ALDRICH, J.R., LUSBY, W.R., KOCHANSKY, J.P., and ABRAMS, C.B. 1984. Volatile compounds from the predatory insect Podisus maculiventris: Male and female metathoracic scent gland and female dorsal abdominal gland secretions. J. Chem. Ecol. 10:561-568. ALDRICH, J.R., LUSBY, W.R., and KOCHANSKY,J.P. 1986. Identification of a new predaceous stink bug pheromone and its attractiveness to the Eastern Yellowjacket. Experientia 42:583-585. BENTLEY, M.D., MCDANIEL, I.N., YATAGAP, M., LEE, H., and MAYNARD, R. 1981. Oviposition attraction and stimulants of Aedes triseriatus (Say) (Diptera: Culicidae). Entomol. Soc. Am. 10:186-189. BIRCH, M.C. 1974. Aphrodisiac pheromone in insects, pp. 115-134, in M.C. Birch (ed.). Pheromones. North Holland, Amsterdam. BIRCH, M.C. 1977. Response of both sexes of Trichoplusia ni (Lepidoptera: Noctuidae) to virgin females and to synthetic pheromone. Ecol. Entomol. 2:99-104. BIRCH, M.C., and HEFETZ, A. 1987. Extmsible organs in male moths and their role in courtship behavior. Bull Entomol. Soc. Am. 33:222-229. BROWNLEE,R.G., and SILVERSTEIN,R.M. 1968. A micro-preparative gas chromatograph and a modified carbon skeleton determinator. Anal. Chem. 40:2077-2079. CORNFORTH,R.H., CORNFORTH, J.W., and PRELOG,V. 1962. Uben die configuration yon linalool, eine berichtigung. Justus Liebigs Ann. Chem. 634:197-198. CREIGHTON,C.S., McFADDEN, T.L., and CUTHBERT,E.R. 1973. Supplementary data on phenylacetaldehyde: an attractant for Lepidoptera. J. Econ. Entomol. 66: 114-115. DUNCAN, D.B. 1955. Multiple range and multiple F. Tests. Biometrics 11:1-41. GAYDOU, E.M., and RANDRIAMIHARISDA,R.P.J. 1987. Gas chromatographic separation of linalool derivatives. Chromatography 396:378-381. GOTHILF,S., and SHOREY,H.H. 1976. Sex pheromones for Lepidoptera: Examination of the role of male scent brushes in courtship behaviour of Trichoplusia hi. Environ. Entomol. 5:115119. GRANT, G.G. 1970. Evidence for a male sex pheromone in the noctuid, Trichoplusia hi. Nature 227:1345-1346. GRANT, G.G. 1971. Scent apparatus of the male cabbage looper, Trichoplusia hi. Ann. Entomol. Soc. Am. 64:347-352. GRANT,G.G., and BRADY,U.E. 1973. Topography of the scent scales of the male cabbage looper, Trichoplusia hi. J. Ga. Entomol. Soc. 8:99-106. GROB, J.J. 1982. Band broadening in space and the retention gap in capillary gas chromatography. J. Chromtagr. 237:15-23. GuY, R.H., LEPPLA, N.C., RYE, J.R., GREEN, C.W., BARETTE, S.L., and HOLLIEN, K.A. 1985. Trichoplusia hi, pp. 487-494, in P. Singh and R.F. Moore (eds.). Handbook of Insect Rearing, Vol. 2. Elsevier, Amsterdam.
MALE CABBAGELOOPER ATTRACTANT
453
HAGAN, D.V., and BRADY, U.E. 1981. Absence of detectable 2-phenylethanol in Trichoplusia ni, a reported pheromone of males. J. Ga. Entomol. Soc. 16:192-196. HEATH, R.R., and SONNET, P.E. 1980. Technique for in situ coating of Ag + onto silica gel in HPLC columns for the separation of geometrical isomers. J. Liquid Chromatogr. 3(8): 11291135. JACOBSON,M., ADLER,V.E., KIStlABA,A.N., and PRIESNER,E. 1976.2-Phenylethanol, a presumed sexual stimulant produced by the male cabbage looper moth, Trichoplusia Hi. Experientia 32:964-966. LANDOLT, P.J., and HEATH, R.R. 1987. Role of female-produced sex pheromone in behavioral reproductive isolation between Trichoplusia ni (Htibner) and Pseudoplusia includens (Walker) (Lepidoptera: Noctuidae: Plusiinae). J. Chem. Ecol. 13:1005-1018. LANDOLT, P.J., and HEATH, R.R. 1989. Attraction of female cabbage looper moths (Lepidoptera: Noctuidae) to male-produced sex pheromone. Ann. Entomol. Soc. Am. 82(4):520-525. LANDOLT, P.J., and HEATH, R.R. 1990. Sexual role reversal in mate-finding strategies of the cabbage looper moth. Science 219:1026-1028. MASADA, Y. 1976. Analysis of Essential Oil by Gas Chromatography and Mass Spectroscopy. Wiley, New York. MAYER, M.S., and MCLAUGHLIN,J.R. 1990. Handbook of Insect Pheromones and Sex Attractants. CRC Press, Boca Raton, Florida. MITCHELL, E.R., WEBB, J.C., and HINES, R.W. 1972. Capture of male and female cabbage loopers in field traps baited with synthetic sex pheromone. Environ. Entomol. l:525-526. MURPHY, R.E. 1989. The fractionation gap: An optimized coupling of fused silica columns in open tubular gas chromatography. Master's thesis. University of Florida, _ _ . OHLOFF, G., and KLEIN, E. 1962. Determination of configuration of linalool. Tetrahedron 18:3739. OHWA, M., KOGURE, T., and ELIEL, E.L. 1986. An asymmetric synthesis of enantiomerically pure (S)-(+)-linalool (3,7-dimethyl-l,6-octadien-3-ol) and a formal synthesis of (R)-(-)-linalool. J. Org. Chem. 51:2599-2601. RYAN, M.F., and GUERIN, P.M. 1982. Behavioral responses of the carrot fly, Psila rosa, to carrot volatiles. Physiol. Entomol. 7:314-324. STEEL,R.G.D., and TORRIE,J.H. 1960. Principles and Procedures of Statistics. McGraw-Hill, New York. SWABODA,P.A.T. 1962. Qualitative and quantitative analysis of flavour volatiles from edible fats, pp. 273-291, in M. van Swacy (ed.). Gas Chromatography. Butterworth, London. THORNHILL, R., and ALCOCK, J. 1983. The Evolution of Insect Mating Systems. Harvard University Press, Cambridge, Massachusetts. WILLIS, M.A., and BIRCH, M.C. 1982. Male lek formation and female calling in a population of the aretiid moth, Estigmene acrea. Science. 218:168-170. WOOD, W.F., LEAHY, M.G., GALUN, R., PRESTWITHC,G.D., ME1NWALD,J., PURNELL,R.E., and PAYNE,R.C. 1975. Phenols as pheromones of ixodid ticks: A general phenomenon? J. Chem. Ecol. 1:501-509. WUNDERER, H., HANSEN, K., BELL, T.W., SCHNEIDER, D., and MEINWALD, M. 1986. Sex pheromones of two Asian moths (Creatonotos transiens, C. ganges; Lepidoptera: Arctiidae): Behavior, morphology, chemistry, and electrophysiology. Exp. Biol. 46:11-27.
