Identification of Floral Compounds FromAbelia Grandiflora That Stimulate Upwind Flight In Cabbage...

Journal of Chemical Ecology, Vol. 17, No. 3, 1991

IDENTIFICATION OF FLORAL COMPOUNDS FROM Abelia grandiflora THAT STIMULATE UPWIND

FLIGHT IN CABBAGE LOOPER MOTHS

K E N N E T H F. H A Y N E S , * J I N G Z H A N G Z H A O , and A B D U L L A T I F

Department of Entomology University of Kentucky

Lexington, Kentucky 40546

(Received September 18, 1990, accepted November 19, 1990)

Abstract--Four major volatile components emitted from flowers of Abelia grandiflora were identified based on retention time using two capillary columns of different polarities and electron impact mass spectrometry. These are phenylacetaldehyde, benzaldehyde, 2-phenylethanol, and benzyl alcohol. A blend of these compounds was as effective as a cluster of flowers in stimulating upwind flight by male Trichoplusia ni to the source in a wind-tunnel test. Phenylacetaldehyde or 2-phenylethanol were each as effective as the complete blend in stimulating source location by male moths. Attraction to a source of the synthetic blend was demonstrated in virgin males and females and mated males and females, but virgin moths of both sexes were more likely than mated moths to complete the sequence of behavioral responses necessary to locate the odor source.

Key Words--Lepidoptera, Noctuidae, Trichopiusia hi, floral attractants, anemotaxis, wind tunnel.

INTRODUCTION

Vola t i le chemica l s emi t t ed f rom plants faci l i tate host- f inding in a wide range o f

insects. In moths , such vola t i le chemica l s of ten enable females to locate suitable

ov ipos i t ion sites (Phelan and Baker , 1987; L iu et a l . , 1988; R a m a s w a m y , 1988;

Haynes and Baker , 1989; Landol t , 1989; T ing le et a l . , 1989) but may also

a l low male and female adults to find their o w n food resources (Wiesenborn and

Baker , 1990). Feed ing by adult f emale moths m a y improve reproduct ive suc-

* To whom correspondence should be addressed.

637

0098-0331/91/0300-0637506.50/0 © 1991 Plenum Publishing Corporation

638 HAYNES ET AL,

cess by increasing egg production, extending the ovipositional period, improving ovipositional host-finding success, and/or increasing resources allocated to each egg. Feeding by adult males may improve mate-finding success, increase longevity and the associated chances for multiple mating, or increase "nutri- t ive" spermatophore size, thereby indirectly improving reproductive success. Adult cabbage looper moths, Trichoplusia ni (Hfibner) and other Plusiinae feed on nectar from the ornamental shrub Abelia grandiflora (Andrr), (honeysuckle family, Caprifoliaceae); both male and female moths are attracted to these flowers from sunset to 1 AM (Grant, 1971a). Olfactory stimuli play an important role in mediating nectar-finding by T. ni, but the chemical composition of these cues has not been defined.

METHODS AND MATERIALS

Abelia grandiflora plants were maintained in a greenhouse (maximum day- time temperature 35~ minimum nighttime temperature 21~ Volatile collections were conducted for 24 hr from flower clusters of six A. grandiflora plants. Volatile floral compounds were collected on 70 mg of Tenax-TA (All- tech, Deerfield, Illinois), which had been loaded into a glass tube (0.5 cm diam. x 5.5 cm long) plugged at both ends with glass wool. The large open ends of two glass funnels (10 cm diam.) were butted against one another to enclose a flower cluster containing 6-10 open flowers. The funnels were sealed together with Teflon tape and supported in position with a ringstand to avoid stress to the plant stem. A Tenax trap was attached to the 3.0-cm stems of both funnels with a 2-cm section of Tygon tubing. Air was drawn through the system at 175- 200 ml/min via a vacuum pump attached to one of the traps. Approximately 400/~1 of HPLC- grade hexane was added to the Tenax and was allowed to drip into a graduated vial. Additional hexane was added to the Tenax until exactly 600/zl was recovered in the vial. One hundred microliters of this solution was combined with 100 /zl of a 100 ng/tzl solution of (R)-(+)-l-phenylethanol (internal standard). One microliter of this solution was injected into a Hewlett- Packard (Avondale, Pennsylvania) 5890A gas chromatograph linked to a Hew- lett-Packard 5970B mass selective detector (MSD). The gas chromatograph was equipped with a 30-m DB-Wax capillary column (J&W Scientific, Folsom, Cal- ifornia, 0.25 /~m film thickness, 0.25 mm diam.). The oven temperature was held at 40~ for 2 min and then increased by 10~ to a final temperature of 210~ The MSD scanned from 40 to 400 m/z. Analyses were also conducted using 30-m DB-5 capillary column (J&W Scientific, 0.25 /zm film-thickness, 0.25 mm diam.) with the same temperature program. Identifications were based on comparisons of retention times and mass spectra of the floral volatiles to authentic phenylacetaldehyde (Aldrich Chem. Co., Milwaukee, Wisconsin),

ATTRACTANTS FOR Trichoplusia ni 639

2-phenylethanol (Sigma Chem. Co., St. Louis, Missouri), benzaldehyde (Sigma) and benzyl alcohol (Sigma).

Cabbage looper larvae were reared on an artificial diet following the pro- cedures described by Shorey and Hale (1965). Pupae were separated according to sex. Pupae and adults were held in 3.8-liter paper cartons with nylon screen lids. Males and females were maintained in separate environmental chambers under a photoperiod of 16:8 (light-dark) with temperature cycling from 25 ~ during the photophase to 22~ during the scotophase. Adults had access to a cotton pad saturated with 10% sucrose-water. In most of the bioassays virgin males were used, but when mated adults were required, 2- to 3-day-old virgin male and female moths were placed together 4 hr into the scotophase in a 40 x 4 6 x 40-cm (1 x w x h) observation box with a clear Plexiglas top that allowed observations to be made under dim red light. Pairs in copula were transferred from the box to 0.5-liter cartons. At the initiation of the photophase, mated males and females were sorted into separate containers by observing the dorsal long abdominal scales found only on males (Grant, 1971b).

To conduct wind-tunnel tests for behavioral responses of T. ni to floral volatiles, 3- to 4-day-old adult moths were placed into hardware cloth cylinders (8 x 4.5 cm diam.) with aluminum lids before the start of the scotophase. A wind tunnel similar to that described by Miller and Roelofs (1978) was used. These behavioral assays were conducted under dim red light 0-2 hr after the initiation of a scotophase. Moths were released individually 1.5 m downwind of the stimulus source. Behavioral responses to the test stimulus were noted in the following categories: wing fanning in the release cage (WF), upwind flight in the odor plume (UWF, consisting of repeated counterturns directly downwind of the stimulus source), the distance of the moth's closest approach to the test stimulus, and source contact (SC).

A randomized complete block design was used for four experiments. An arcsine-square root transformation was used on percentage data before analyses of variance were performed. The first experiment included the following test stimuli: a cluster of 10 A. grandiflora flowers whose stem was placed in a 4-dram vial with water, and 0.1, 1.0, and 10.0 mg of phenylacetatdehyde in 100/zl of hexane. In all experiments, this volume was delivered onto ca. 35 mg of cotton sealed in the hollow interior of a polyethylene vial cap that had a removable lid (vial closure, 1.9 cm diam. x 1.2 cm high). A 3-mm hole was cut in the cap to increase the rate of volatilization from this source. This experiment consisted of nine blocks with five moths per treatment per block. In the second experiment, a blend of synthetic chemicals was prepared to approximate the blend ratios of compounds identified in volatile emissions from A. grandiflora flowers (1.0 mg phenylacetaldehyde, 0.77 mg 2- phenylethanol, 0.80 mg benzaldehyde, 0.13 mg benzyl alcohol in 100 tzl of hexane). This synthetic blend and 100/~1 of hexane (blank) were delivered to the plastic caps as above.

6 4 0 H A Y N E S ET AL.

Again, 10 A. grandiflora flowers served as a positive control. Twelve blocks with five moths per treatment per block were used. In the third experiment, an evaluation of male moths' responses to the blend of synthetic compounds was compared to each compound individually (1 mg) (eight blocks, five moths per treatment per block). In the last experiment, an evaluation of the responses of virgin males and females and mated males and females to the complete synthetic blend vs. hexane blank was conducted (eight blocks, five moths per treatment for each sex and mating status per block).

RESULTS

Compounds with retention times (RT) identical to authentic phenylacetaldehyde, 2-phenylethanol, benzaldehyde, and benzyl alcohol were found in volatile collections from flower clusters using relatively polar (DB-Wax, Figure 1) and relatively nonpolar (DB-5) capillary columns. These identifications were supported by mass spectra. Authentic benzaldehyde and the first unknown were characterized by ions at m/z 51, 77 (base peak), 105, and 106 (molecular ion). Synthetic phenylacetaldehyde and an unknown peak with RT identical to this

5q

o 01

~9

Synthetic Standards

3 2

IS Volatile Collection

lO l l

T i m e

I I I

8 9 12 13 15 16

( m i n . )

I

14

FIG. 1. Total ion chromatograms of synthetic standards and volatile collection from Abelia grandiflora flowers. The internal standard (IS) was (R)-(+)-l-phenylethanol.1 = benzaldehyde, 2 = phenylacetaldehyde, 3 = benzyl alcohol, 4 = 2- phenylethanol.


compound were characterized by ions at m/z 65, 91 (base peak) and 120 (molecular ion). A third floral compound and authentic benzyl alcohol were characterized by ions at m/z 79 (base peak), 107, and 108 (molecular ion). Synthetic 2-phenylethanol and a fourth floral compound were characterized by ions at m/z 65, 91 (base peak), and 122 (molecular ion).

The quantity of phenylacetaldehyde, 2-phenytethanol, benzaldehyde, and benzyl alcohol emitted from A. grand• flowers in a 24-hr period was deter- mined using two capillary columns (Table 1). Results using the DB-Wax column and MS detector indicated that phenylacetaldehyde was the most abundant compound recovered from these collections (37.4%), followed by benzaldehyde (33.3%), 2-phenylethanol (24.2%), and benzyl alcohol (5.1%). Results were similar using a DB-5 column and a flame ionization detector. The results from the DB-Wax column were used to formulate a blend of synthetic compounds that mimics the proportions of compounds found in volatile collections from flowers.

In the first behavioral experiment, 38 % of male moths flew upwind and contacted a plastic cap containing 0.1 mg of phenylacetaldehyde, which was significantly lower than the response stimulated by a cluster of flowers (76%, overall F = 7.7, P < 0.05, analysis of variance, Scheffr's multiple comparison procedure). The percentage of males contacting sources containing 1.0 mg (60%) and 10.0 mg (67%) was not significantly different from the response to the flower cluster (P > 0.05). One milligram of phenylacetaldehyde was selected as the standard dose for additional experiments.

The synthetic blend of floral compounds stimulated wing fanning, upwind flight, flight to within 0.5 m, and source contact at levels not significantly different from levels stimulated by a cluster of Abelia flowers (Table 2). Of those

TABLE 1. QUANTITIES OF VOLATILE CHEMICALS EMITTED FROM Abelia grandiflora FLOWERS AND TRAPPED ON TENAx-TA '~.

Quantity per flower • SEM (~g)~'

Compound DB-Wax' DB-4 d

Phenylacetaldehyde 4.04 • 0.63 4.84 • 0.65 2-Phenylethanol 2.62 • 0.22 1.75 • 0.45 Benzaldehyde 3.60 • 0.78 4.27 • 0.88 Benzyl alcohol 0.55 • 0.17 0.64 _+ 0.36

~Collections were run for 24 hr (N = 6) and were made from flower clusters on six different potted Abelia grand• plants.

1,Flower clusters contained 6-10 flowers, but quantity per flower was calculated. "30-m DB-Wax capillary column. d30-m DB-5 capillary column.

642 HAYNES ET AL.

TABLE 2. BEHAVIORAL RESPONSES OF MALE Trichoplusia ni TO HEXANE CONTROL (BLANK), CLUSTER OF Abelia grandiflora FLOWERS, and BLEND OF SYNTHETIC

COMPOVNDS IDENTIFIED FROM THESE FLOWERS (N = 12) a

Behavioral response ___ SEM (%)b

Treatment WF UPW < 0.5 m SC

Blank 73.3 4- 4.5b c 1.7 _+ 1.7b 0 b 0 b Flowers 86.7 4- 3.8ab 56.7 4- 7.3a 53.3 + 7.5a 53.3 4- 7.5a Synthetic Blend a 91.7 4- 3.0a 65.0 4- 6.1a 56.7 + 6.0a 56.7 4- 6.0a

a Twelve experimental blocks, each block consisted of five moths released individually to a specific test stimulus.

bWF = wing fanning in the release cage; UPW = upwind flight in odor plume; <0.5 m = moth progressed to within 0.5 m of the test stimulus source; SC = source contact.

CMeans in the same column are significantly different if they do not share a letter in common (P < 0.05, ANOVA and Scheff6 multiple comparison procedure).

dThe synthetic blend consisted of 1 mg phenylacetaldehyde, 0.77 mg 2-phenylethanol, 0.80 mg benzaldehyde, and 0.13 mg benzyl alcohol applied to plastic cap.

moths that initiated upwind flight to flowers or the synthetic blend, most com-

pleted this flight by contacting the source (94.0 and 87.2%, respectively). Wing

fanning in the release cage was not a good assay for activity of test stimuli, because the percentage of moths exhibiting this behavior was high even for the

hexane (blank) treatment.

The synthetic blend was not significantly better in stimulating source con-

tact than two of its components, phenylacetaldehyde or 2-phenylethanol (Table 3). While moderate numbers of male moths initiated upwind flight to benzal-

dehyde or benzyl alcohol, very few of these were able to maintain their upwind

progress and contact the source.

The synthetic blend stimulated upwind flight to the source in virgin males

and females and in mated males and females (Figure 2). There was a significant

difference between the blank and synthetic blend regardless of mating status or sex of the moths in terms of the percentage of moths that fanned their wings, initiated upwind flight, progressed to within 0.5 m of the source, and contacted the source (P < 0.05, F = 25, 103, 140, 196, respectively). Interestingly, the mating status of the moth also had a significant effect on upwind flight, flying

to within 0.5 m, and contacting the source (P < 0.05, F = 11, 7, 7, respec-

tively). Mated moths were less likely to complete this sequence of behaviors

than were virgins. Males and females were equally likely to exhibit each of the

behaviors recorded (P > 0.05, F = 0, 0.1, 0.1, 0.3, in sequence).


TABLE 3. BEHAVIORAL RESPONSES OF MALE Trichoplusia ni To FOUR VOLATILE CHEMICALS IDENTIFIED FROM Abelia grandiflora FLOWERS AND TO

A BLEND OF THESE COMPOUNDS (N = 8) a

Behavioral Response • SEM (%)b

Treatment WF UPW _<0.5 m SC

Synthetic blend c 95.0 + 3.3a d 85.0 + 5.0a 62.5 • 4.5a 55.0 • 5.0a Phenylacetaldehyde

(1 mg) 92.5 + 3.6a 75.0 + 6.3ab 52.5 + 6.5ab 47.5 • 6.5a 2-Phenylethanol

(1 mg) 85.0 + 6.3a 60.0 • ll.0ab 42.5 _ 8.8abc 40.0 + 8.4ab Benzaldehyde

(1 mg) 85.0 + 7.3a 42.5 • ll.0b 12.5 + 5.3c 2.5 • 2.5c Benzyl alcohol

(1 mg) 82.5 _+ 8.0a 62.0 _+ 14.lab 25.5 +_ 8.2bc 15.0 _+ 8.2bc

~Eight experimental blocks, each block consisted of five moths released individually to a specific test stimulus.

b See Table 2 for behavioral categories. c See Table 2 for chemical composition of this blend. dMeans in the same column are significantly different if they do not share a letter in common (P < 0.05, ANOVA and Scheff6 multiple comparison procedure).

DISCUSSION

Phenylacetaldehyde is reported to be an attractant for Plusiinae moths

(including T. ni) and other Noctuidae (Smith et al., 1943; Creighton et al.,

1973). It has been identified as a volatile compound emitted by the bladder

flower, Araujia sericofera Brothero, that attracts and traps many species of moths (Cantelo and Jacobson, 1979). From our study it appears to be a major

constituent of the blend of volatile compounds emitted from flowers of A. grandiflora, an ornamental shrub that is frequently visited by T. ni moths foraging

for nectar (Grant, 1971a). Another component of the fragrant blend of compounds from these flowers

is 2-phenylethanol. 2-Phenylethanol has been identified from male scent brushes of several species of moths (Aplin and Birch, 1970; Birch et al., 1990). Jacob- son et al. (1976) isolated this compound from the genital scent brushes of male

T. ni, but others have been unable to detect it (Hagan and Brady, 1981; Landolt and Heath, 1990). It is possible that the overt behavioral response of female T. ni to 2-phenylethanol documented by Jacobson et al. (1976) was due to its proposed role in stimulating nectar-finding behavior, rather than to their pro- posal that it mediated precopulatory behavior. Benzaldehyde and benzyl alco-

644 H A Y N E S ET AL.

o

oj

(J (0.Sin

100

80

80

4o

zo

11 HI VtrB. 0" Mat. d Vlrg. 9 Mat.

N Virg. cr Mat, d Virll. 9 Mat.

FIG. 2. Behavioral responses of virgin and mated, male and female Trichoplusia ni to hexane blank (cross-hatched bars) and a blend of phenylacetaldehyde, 2-phenylethanol, benzaldehyde, and benzyl alcohol (open bars). WF = wing fanning in the release cage, UPW = upwind flight at the level of the odor plume, <0.5 m = flying to within 0.5 m of the source, SC = source contact. Bars indicate •

hol, two additional components of Abelia's volatile blend, have been identified from scent brushes of other noctuid moths (see Birch et al., 1990). While our wind tunnel assays have not identified an important role for these two compounds in mediating upwind flight to Abelia flowers, it is possible that their presence increases the specificity of the response. Grant (1971a) indicated that mainly plusiine moths were observed feeding on A. grandiflora flowers. The response to phenylacetaldehyde includes species from other subfamilies in Noc- tuidae (Cantelo and Jacobson, 1979). Benzaldehyde has been reported to have a repellent effect on several species of moths (Hirai, 1982). We did not observe any decrease in source location as a consequence of including benzaldehyde in the synthetic blend, and in fact benzaldehyde by itself did stimulate a few moths to fly upwind and contact the source.

The demonstration that a synthetic blend of floral chemicals stimulates upwind flight and source-finding in virgin and mated individuals of both sexes suggests the importance of nectar-finding for reproductive success in all adult T. ni. Our finding that mated adults were less successful than virgins in locating these odor sources may reflect a refractory period for foraging behavior after mating. In females, this change could indicate a shift towards ovipositional host-finding activity during the hours in which nectar-feeding usually occurs.

Acknowledgments--We thank J.D. Clark and J.T. Collins for technical assistance. D.M Campero, R.E. Hunt, P.J. Landolt, Y. Liu, D.A. Potter, and K.V. Yeargan reviewed and improved an earlier draft of this manuscript. We acknowledge support of the University of Kentucky Major Research Instrumentation Bond Program in the purchase of equipment used in this study (ID#'s


7E-8164-11, L- 250-8A080, L-251-8A080, and L-252-8A080). This investigation (paper No. 90- 7-174) was conducted in connection with a project of the Kentucky Agricultural Experiment Station and is published with the approval of the director.

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BIRCH, M.C., POPPY, G.M., and BAKER, T.C. 1990. Scents and eversible scent structures of male moths. Annu. Rev. Entomol. 35:25-58.

CANTELO, W.W. and JACOBSON, M. 1979. Phenylacetaldehyde attracts moths to bladder flower and to blacklight traps. Environ. Entomol. 8:444-447.

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.

GRANT, G.G. 1971a. Feeding activity of adult cabbage loopers on flowers with strong olfactory stimuli. J. Econ. Entomol. 64:315-316.

GRANT, G.G. 1971b. Scent apparatus of the male cabbage looper, Trichoplusia ni. Ann. Entomol. Soc. Am. 64:347-352.

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.

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JACOBSON, M., ADLER, V.E., KtSHABA, A.N. , and PRIESNER, E. 1976. 2-Phenytethanol, a pre- sumed sexual stimulant produced by the male cabbage looper moth, Trichoplusia ni. Exper- ientia 32:964-966.

LANDOLT, P.J. 1989. Attraction of the cabbage looper to host plants and host plant odor in the laboratory. Entomol. Exp. Appl. 53:117-124.

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LIu, S.-H., NORRIS, D.M., and MARTI, E. 1988. Behavioral responses of female adult Trichoplusia ni to volatiles from soybeans versus a preferred host, lima bean. Entomol. Exp. Appl. 49:99- 109.

MILLER, J.R., and ROELOFS, W.L. 1978. Sustained-flight tunnel for measuring insect responses to wind-borne sex pheromones. J. Chem. Ecol. 4:187-198.

PHELAN, P.L., and BAKER, T.C. 1987. An attracticide for control of Amyelois transitella (Lepi- doptera: Pyralidae) in almonds. J. Econ. Entomol. 80:779-783.

RAMASWAMY, S.B. 1988. Host finding by moths: Sensory modalities and behaviours. J. Insect Physiol. 34:235-249.

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Identification of Floral Compounds FromAbelia Grandiflora That Stimulate Upwind Flight In Cabbage...

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