Journal of Chemical Ecology, Vol. 10, No.

9, 1984

I D E N T I F I C A T I O N OF NEW SEX P H E R O M O N E
C O M P O N E N T S IN Trichoplusia ni, P R E D I C T E D
FROM BIOSYNTHETIC PRECURSORS

L.B. B J O S T A D , l C . E . L I N N , 1 J . - W . D U , 2, a n d W . L . R O E L O F S I

Department of Entomology
New York State Agricultural Experiment Station
Geneva, New York 14456
2Shanghai Institute of Entomology
Academia Siniea
Chungkin Road (S.) 225
Shanghai, P.R.C.

(Received August 16, 1983; revised January 9, 1984)

Abstract--In addition to the previously identified components (Z)-7-

dodecenyl acetate and dodecyl acetate, sex pheromone glands of Tri-
choplusia ni release (Z)-5-dodecenyl acetate, 1l-dodecenyl acetate, (Z)-7-
tetradeeenyl acetate, and (Z)-9-tetradecenyl acetate. Bioassays in a flight
tunnel showed that a synthetic blend of these six compounds elicited
complete flights to the source from 95% of the males tested and elicited
hairpenciling responses at the end of the flights from 88% of the males
tested. This blend was not significantly different from intact pheromone
glands, which elicited complete flights to the source from 98% of the males
tested and hairpenciling responses from 91% of the males tested. In contrast,
the previously identified two-component blend elicited significantly fewer
complete flights to the source (33%) and did not elicit hairpenciling
responses from any of the males tested. The search for additional sex
pheromone components was prompted by our previous identification of
unusual fatty acyl moieties in the gland that seemed to be possible bio-
synthetic intermediates.

Key Words--Lepidoptera, Noctuidae, Trichoplusia ni, pheromone, (Z)-

7-dodecenyl acetate, (Z)-5-dodecenyl acetate, 1 l-dodecenyl acetate, (Z)-7-
tetradecenyl acetate, (Z)-9-tetradecenyl acetate, biosynthesis.

INTRODUCTION

T h e m a i n s e x p h e r o m o n e c o m p o n e n t o f t h e c a b b a g e l o o p e r m o t h , Tri-
c h o p l u s i a ni ( N o c t u i d a e ) , w a s i d e n t i f i e d as ( Z ) - 7 - d o d e c e n y l a c e t a t e ( B e r g e r ,

1309
0098-0331/84/0900-1309503.50/0 9 1984 Plenum Publishing Corporation
1310 BJOSTAD ET AL.

1966). Dodecyl acetate was later identified as a second component, important

mainly in close-range courtship behavior (Bjostad et al., 1980a). An extensive
series of flight tunnel tests by Linn and Gaston (1981a,b) provided a detailed
comparison of the behavioral effects of a large number of blends of these two
components.
We recently demonstrated that the main pheromone component, (Z)-7-
dodecenyl acetate, arises by chain shortening of the fatty acyl moiety (Z)-I 1-
hexadecenoate to (Z)-9-tetradecenoate and then to (Z)-7-dodecenoate, which
is reduced and acetylated (Bjostad and Roelofs, 1983). Appreciable amounts
of (Z)-l 1-octadecenoate and (Z)-9-hexadecenoate were also observed in the
gland, as were smaller amounts of (Z)-7-tetradecenoate and (Z)-5-dodecen-
oate. It was apparent that (Z)-7-tetradecenoate could arise from ( Z ) - l l -
octadecenoate by chain shortening and that (Z)-5-dodecenoate could be
produced by further chain shortening. Small peaks with the retention times of
methyl (Z)-7-tetradecenoate and methyl (Z)-5-dodecenoate were in fact
observed in eapillary gas-liquid chromatograph (GLC) analyses of methan-
olyzed pheromone gland extracts, and small peaks with the retention times of
(Z)-7-dodecenyl acetate and (Z)-5-dodecenyl acetate were also observed.
Because the (Z)- 11-hexadecenoate and (Z)- 11-octadecenoate moieties in
the gland are apparently produced by a ( Z ) - l l desaturase acting on the
saturated moieties hexadecanoate and octadecanoate, respectively, we sus-
pected that tiny amounts of (Z)-I 1-tetradecenoate and 11-dodecenoate might
also be produced by the desaturase. Small peaks with the correct retention
times were observed in gas chromatograph traces, as were small peaks with the
retention times of (Z)-I 1-tetradecenyl acetate and 11-dodecenyl acetate. A
summary of the proposed biosynthetic routes is shown in Figure 1. We now
report the chemical identifications of these compounds, and a behavioral
evaluation of their status as pheromone components.

METHODS AND MATERIALS

Extracts. The insects were reared on a semisynthetic medium (Shorey

and Hale, 1965), and females were separated from males as pupae. Adults were
maintained on a light cycle (14:10 light-dark). Pheromone glands were
dissected from the ovipositors of 3 to 4-day-old females with fine forceps and
allowed to stand overnight in 1 ml glass-distilled dichloromethane in a 4-ml
vial with a Teflon-lined screw cap.
Volatile extracts were made from individual females essentially as
described by Baker et al. (1981) and Pope et al. (1982) by trapping volatiles
from the gland on glass wool, but we found that larger quantities of
pheromone components were obtained by introducing a stream of nitrogen
through a capillary tube whose outlet was about 1 mm from the gland. After a
Trichoplusia ni PHEROMONE 131 1

FATTY ACYL PRECURSORS

~~ ~: >CHAIN SHORTENN
,,,~(z)-11 DESATURATION
IG

REDUCTO
1I N

F ~ . 1. Proposed biosynthetic pathways leading to compounds observed in the sex

pheromone gland of Trichoplusia ni. Height of each block is proportional to amount
of compound in gland. Names of compounds are abbreviated. Z7-12:fatty acyl
precursor = (Z)-7-dodecenoate, Z7-12 : acetate = (Z)-7-dodecenyl acetate, etc.

volatile e x t r a c t h a d been m a d e f r o m a n i n d i v i d u a l g l a n d for 10 min, the g l a n d

was excised f r o m the o v i p o s i t o r a n d e x t r a c t e d with 10 ~1 of d i c h l o r o m e t h a n e .
Both the volatile e x t r a c t a n d the g l a n d e x t r a c t f r o m the s a m e female were then
a n a l y z e d by c a p i l l a r y G L C .
Chemical Identifications. C a p i l l a r y G L C was c o n d u c t e d with a 45-m
C a r b o w a x 20 M c o l u m n a n d with a 25-m cross-linked methyl silicone c o l u m n ,
used with splitless injection and p r o g r a m e d f r o m 80~ to 200~ at 10~
1312 BJOSTAD ET AL.

after an inital delay of 2 min. Quantification of relative abundances of

compounds was performed by electronic integration of peak areas (flame
ionization detection). Packed G L C columns were 3% OV-101 (methyl
silicone) on 100- to 120-mesh Gas-Chrom Q in a 2-m glass column (inside
diameter, 4 mm) at 180~ , and 10% XF- 1150 (50% cyanoethyl, methyl silicone)
on 100- to 120-mesh C h r o m o s o r b W - A W - D M C S in a 2-m glass column
(inside diameter, 2 ram) at 140~ The resolution of the XF-1150 column
deteriorated rapidly if used at temperatures higher than 140~ Fractions
were collected from packed GLC columns with 30-cm glass capillary tubes.
Crude extracts of pheromone glands in dichloromethane were injected
onto an OV-101 packed column, and the fractions containing 12-carbon
acetates and 14-carbon acetates were recovered by rinsing through each
collection tube with 0.5 ml dichloromethane. Positional isomers were resolved
by collection from the XF-1150 packed column. Each isomer was identified
on the basis of ozonolysis, base methanolysis, and acetylation.
For determination of double-bond positions by ozonolysis (Beroza and
Bierl, 1967), a solution of ozone was prepared in 20/~1 of CS2 in a small test
tube (5 m m diameter) in a bath of acetone and dry ice ( - 7 8 ~ C). A carbon
disulfide solution of a given isomer to be ozonized was injected into this
solution with a syringe, a stream of nitrogen was applied to dispel excess
ozone, and a carbon disulfide solution containing 1 ~g triphenylphosphine
was injected to decompose the ozonide. The solution was analyzed by
capillary GLC, and the retention time of the acetoxy aldehyde was compared
with the retention times of acetoxy aldehydes of synthetic positional isomers.
Base methanolysis was performed to verify the conversion of acetates to
the corresponding alcohols. A dichloromethane solution of a given isomer in a
4-ml vial with a Teflon-lined screw cap was evaporated to apparent dryness
with a stream of nitrogen, and 0.5 ml of a solution of K O H in methanol (0.5
M) was added. After 1 hr at 25 ~ C, 1 ml water and 1 ml hexane were added to
the vial, and the mixture was shaken. The hexane portion was reduced in
volume by evaporation with a nitrogen stream, and the retention time of the
methanolysis product was determined by capillary GEC.
Acetylation was performed to verify the conversion of alcohols to the
corresponding acetates. Acetyl chloride (0.1 ml) was added to a given isomer
in a 4-ml vial, was allowed to stand at 25 ~ C for 1 hr, and was then evaporated
to apparent dryness with a stream of nitrogen. Hexane (0.5 ml) was added to
recover the acetylation product, and after concentration with a stream of
nitrogen, the solution was analyzed by capillary GEC.
Behavioral Tests. Males 2-3 days old were used for behavioral tests,
using procedures described by Linn and Gaston (1981a,b) and Linn and
Roelofs (1981). The design and application of the flight tunnel have been
described by Miller and Roelofs (1978). Males were tested individually in the
fight tunnel, at the midpoint of the scotophase at a light intensity of 0.3 lux,
Trichoplusia ni PHEROMONE 1313

with a windspeed of 50-55 cm/sec at 21 ~ C. As emphasized in an earlier report

(Linn and Gaston, 1981a), care in handling the male moths was critical in
achieving high proportions of successful male flights. Males were allowed to
acclimate quietly at 0.3 lux in the room housing the flight tunnel for 1 hr
before testing, and each male was moved slowly to the tunnel for testing.
Synthetic compounds for behavioral testing were prepared in our
laboratory, and isomeric purity greater than 99.9% was verified by capillary
GLC analysis. Polyethylene caps were prepared by adding dichloromethane
solutions of synthetic compounds to the insides of the caps with Pasteur
pipets. After 1 hr the caps were closed and left in the hood for 36 hr. Caps were
held in glass vials at - t 0 ~ when not in use. Sex pheromone glands were
bioassayed by excising the glands from five females and placing them on the
head of an insect pin mounted on a cork. In addition, individual pheromone
glands were bioassayed using the same gland extrusion tube (described in
Baker et al., 1981) used in the volatile extraction apparatus.

RESULTS

Chemical Identifications. Analysis of crude gland extracts by capillary

GLC indicated the presence of a number of compounds in small amounts in
addition to the main pheromone component (Z)-7-dodecenyl acetate.
Volatile extracts from the glands of individual females contained only a few of
the compounds observed in gland extracts (Figure 2). The compounds in the
gland extracts were separated by GLC (Tables 1 and 2). Most of these
compounds were acetates. The acetate functional group was verified in each
case by methanolysis to form the corresponding alcohol (and comparison of
the retention time with that of the same synthetic compound) and by
reacetylation of the alcohol to assure that the retention times of the parent
compound and of its reacetylation product were identical. The chain length of
the parent acetate was verified by comparison of its retention time with that of
synthetic acetates. Double-bond positions were determined by comparison of
the retention times of the acetoxy aldehydes from ozonolysis with the
retention times of the acetoxy aldehydes from ozonolysis with the retention
times of acetoxy aldehydes from ozonolysis of synthetic unsaturated acetates.
All retention times were determined with the Carbowax 20M GLC
capillary column. This column was able to resolve most positional isomers, as
indicated by tests with synthetic compounds. Synthetic (Z)-5, (Z)-7, and
(Z)-9-dodeceuyl acetates were separated, as were synthetic (Z)-5, (Z)-7,
(Z)-9, and (Z)-I 1-tetradecenyl acetates. The retention times of synthetic
(Z)-9-dodecenyl acetate and l l-dodecenyl acetate were identical on the
Carbowax 20 M column (within 0.01 min, according to cochromatography),
but these compounds could be separated on the methyl silicone column.
1314 BJOSTAD ET AL.

*~'- Z7-12:OAc
j. ~o
Z5-12:OAc --'~1
12:OAc-~.~ |1 T: n~ GLAND EXTRACT

III 16:~
I I I Z7 14OAc I / / Zll-16:OAc

'~
Z7-12:OH
~'Zl1-14:OAc

T. ni VOLATILE EXTRACT

FIG. 2. Capillary GLC traces of gland extract and volatile extract of Trichoplusia ni.
Names of compounds are abbreviated. Z7-12 : OAc = (Z)-7-dodecenyl acetate, Z7-
12: OH = (Z)-7-dodecenol, etc. The Z7-12: OH peak was added for completeness
in the gland extract trace, but three of four extracts lacked this compound (see text).

Ozonolysis indicated that (Z)-5, (Z)-7, and 11-dodecenyl acetates all occurred
in the gland, but (Z)-9-dodecenyl acetate was apparently completely absent
(acetoxy aldehyde as little as 1% the amount that was observed from
ozonolysis of 11-dodecenyl acetate would have been detected). Moreover,
(Z)-7, (Z)-9, and (Z)-I l-tetradecenyl acetates all occurred in the gland, but
(Z)-5-tetradecenyl acetate was apparently completely absent [less than 0.01%
the amount of (Z)-7-dodecenyl acetate].
An anomaly became apparent with respect to the occurrence of
dodecenyl alcohols in the gland extracts and volatile extracts. Capillary GLC
analyses of volatile extracts from individual glands indicated the presence of a
small peak with the retention time of (Z)-7-dodecenol, and this peak was also
apparent in analyses of extracts of the individual excised glands (Table 2).
Four extracts of 50 glands each were also prepared, and the dodecenyl alcohol
peak was apparently absent from three of the extracts (less than 0.05% of the
Trichoplusia ni PHEROMONE 1315

TABLE 1. RELATIVE PROPORTIONS OF COMPOUNDS IDENTIFIED FROM SEX PHEROMONE

GLAND EXTRACTS OF Trichoplusia ni (50 GLANDS EACH) a

Extract

C o m p o u n d ~' 1 2 3 4 X SD

1. 12: OAc 4.55 5.43 4.93 4.17 4.77 0.54

2. Z5-12 : OAc 7.83 4.85 8.38 7.72 7.20 1.59
3. Z7-12: OAc 100.00 100.00 100.00 I00.00 100.00
4. I 1-12 : OAc 2.79 3.14 2.98 2.75 2.92 0.18
5. 14: OAc c 0.04
6. Z7-14 : OAc 0.45 0.31 0.29 0.40 0.36 0.075
7. Z9-14 : OAc 0.26 0.26 0.28 0.31 0.28 0.024
8. ZI 1-14 : OAc" 0.03
9. 16: OAc 0.17 0.11 0.12 0.08 0.12 0.039
10. Z11-16: OAc 0.17 0.10 0.11 0.06 0.11 0.046
11. 12:OH a 0.03
12. Z5-12 : OH J 0.09
13. Z7-12: OH a 0.81
14. 11-12:OH d 0.01

~Relative proportions were calculated with respect to the main component Z7-12: OAc.
bNames of c o m p o u n d s are abbreviated. Z 7 - 1 2 : O A c = (Z)-7-dodecenyl acetate, Z7-
12 : OH = ( Z)-7-dodecenol, etc.
"Detected by capillary Carbowax 20 M analysis of C14 acetate fraction from OV-101 packed
column; direct analysis of gland extract would have required overloading capillary column
with (Z)-7-dodecenyl acetate.
aOnly present in one extract of four extracts prepared; relative proportions determined by
analysis of acetate derivatives.

a m o u n t of (Z)-7-dodecenyl acetate), but was present in the fourth [0.8% the

amount of (Z)-7-dodecenyl acetate]. The fourth extract was fractionated by
G L C on OV-101, and the dodecenyl alcohol fraction was acetylated with
acetyl chloride and analyzed by G L C on the Carbowax 20 M capillary
column. Peaks with the retention times of the acetates of dodecanol, (Z)-5-
dodecenol, (Z)-7-dodecenol, and 1 l-dodecenol were observed in essentially
the same proportions as the native acetates in the gland (Table 1).
Males Responses to Pheromone Glands. Males were tested individually
in a flight tunnel to make a comparison of pheromone glands with synthetic
blends (Table 3). Single pheromone glands were displayed from the same
extrusion tube used to make volatile extracts, assuring that the handling
procedures for bioassays and for volatile extractions were the same. In
addition, five excised pheromone glands were placed on the heacl of an insect
pin mounted on a cork, and male responses to this treatment were compared
with their responses to polyethylene caps containing synthetic blends. The
estimated release rate of (Z)-7-dodecenyl acetate from the five glands was
 1316 BJOSTAD ET AL.

TABLE 2. RELATIVE PROPORTIONS OF COMPOUNDS FROM VOLATILE EXTRACTS AND

GLAND EXTRACTS OF INDIVIDUAL SEX PHEROMONE GLANDS OF T. n i a

Female

Compound b I 2 3 4 5 6 Mean SD

I. 12: OAc
Volatiles 8.19 6.83 6.49 7.54 5.71 8.38 7.19 1.03
Gland 10.80 7.61 6.49 8.32 7.71 7.94 8.15 1.43
2. Z5-12 : OAc
Volatiles 10.83 10.68 8.26 4.32 10.69 11.13 9.35 2.59
Gland 10.80 10.15 8.27 4.46 10.82 10.05 9.09 2.45
3. Z7-12: OAc
Volatiles 100.00 100.00 100.00 100.00 100.00 100.00 100.00
Gland 100.00 100.00 100.00 100.00 100.00 100.00 100.00
4. ll-12:OAc
Volatiles 2.51 2.36 2.13 3.48 2.80 3.38 2.78 0.55
Gland 2.90 2.28 2.13 3.40 3.23 3.10 2.84 0.52
5. Z7-14 : OAc
Volatiles 1.89 1.74 0.71 0.23 0.49 1.25 1.05 0.68
Gland 1.98 3.17 0.71 0.47 1.62 1.12 1.51 0.99
6. Z9-14 : OAc
Volatiles 0.76 0.62 0.24 0.12 0.12 0.88 0.46 0.34
Gland 0.79 2.92 0.24 0.35 0.50 0.62 0.90 1.01
7. Z7-12: OH
Volatiles 1.51 2.11 0.59 0.35 1.70 1.13 1.23 0.67
Gland 1.58 0.89 0.59 0.35 0.62 1.12 0.86 0.44

~ proportions were calculated with respected to the main component Z7-12: OAc.
~Names of compounds are abbreviated. Z7-12:OAc = (Z)-7-dodecenyl acetate, ZT-
12:OH = (Z)-7-dodecenol, etc.

1 0 0 n g / m i n (Bjostad et al., 1980b), c o m p a r a b l e to the release rate of d o d e c y l

a c e t at e f r o m p o l y e t h y l e n e caps (6.1 ~ g / h r ) m e a s u r e d by K u h r et ai. (1972).
Single glands elicited c o m p l e t e flights to the source f r o m 98% of the males
tested, and the t r e a t m e n t with five excised glands elicited c o m p l e t e flights
f r o m 95% of the males tested.
Male Responses to Synthetic Blends. Only the c o m p o u n d s that a p p e a r e d
in the volatile extracts were tested in synthetic blends in the flight tunnel
bioassays (Table 3). Because it was u n c l e a r w h e t h e r or not the ( Z ) - 7 -
d o d e c e n o l in the volatile extracts was an artifact, a blend lacking it and a blend
i n c l u d i n g it were b o t h tested. The blend lacking ( Z ) - 7 - d o d e c e n o l (blend
A) elicited c o m p l e t e flights f r o m 95% o f the males tested and was n o t
significantly different f r o m intact p h e r o m o n e glands, which elicited c o m p l e t e
flights f r o m 98% of the males tested. The blend including ( Z ) - 7 - d o d e c e n o l
(blend B) elicited c o m p l e t e flights f o r m only 33% of the males tested. The
Trichoplusia ni PHEROMONE 1317

TABLE 3. RESPONSES OF MALE Trichoplusia ni It," FLIGHT TUNNEL TO COMPOUNDS

IDENTIFIED FROM FEMALE SEX PHEROMONE GLAND EXTRACTSa'b

Number Taking Plume Upwind Source Hairpencil

Treatment tested flight orientation flight contact display

Five excised glands 40 39 a 38a 38 a 38 a 38a

Single gland
(intact female in
holder used
for volatile
extractions) 40 40 a 39 a 39 a 39a 37 a
Blend A:
12:OAc
(0.16 mg)
Z5-12:OAc
(0.17 mg)
Z7-12 : OAc
(3.00 rag)
ll-12:OAc
(0.10 mg)
Z7-14 : OAc
(0.0093 mg)
Z9-14 : OAc
(0.0078 mg) 40 40 a 39a 38 a 38a 35 a
Blend B:
blend A

Z7-12:OH
(0.024 mg) 40 40 a 31 b 26 b 13b 2b
Blend C:
12 : OAc
(0.16 mg)
Z7-12 : OAc
(3.00 mg) 40 36 a 20c 14c 13b 0b

aNames of compounds are abbreviated. Z7-12 : OAc = (Z)-7-dodecenyl acetate, etc. Values in
each column with different letters are significantly different ( P ~< 0.05) according to method of
adjusted significance levels for proportions (Ryan, 1960).

t w o - c o m p o n e n t b l e n d i d e n t i f i e d p r e v i o u s l y ( B j o s t a d et al., 1980a) e l i c i t e d
c o m p l e t e f l i g h t s f r o m 3 3 % o f t h e m a l e s t e s t e d ( b l e n d C), b u t n o n e o f t h e s e
males exhibited the characteristic hairpenciling behavior that usually occurs
a f t e r c o m p l e t e d f l i g h t s t o f e m a l e s ( G o t h i l f a n d S h o r e y , 1975). T h e six-
component synthetic blend reported here (blend A) elicited hairpenciling
f r o m 8 8 % o f t h e m a l e s t e s t e d . T h e b l e n d i n c l u d i n g ( Z ) - 7 - d o d e c e n o l ( b l e n d B)
elicited hairpenciling from 5% of the males tested.
 1318 BJOSTAD ET AL.

The low number of male responses to the synthetic blend containing

(Z)-7-dodecenol (blend B) is inconsistent with our observation that this
compound occurs in volatile extracts. The intact pheromone glands used for
bioassay were displayed from the same glass holder used in the apparatus for
volatile extraction, and this seems to rule out the possiblity of a difference in
handling procedures. In order to check the possibility of hydrolysis during the
volatile extraction, a small piece of filter paper bearing 10 ~g of (Z)-7-
dodecenyl acetate was tested in the volatile extraction apparatus, but no
(Z)-7-dodecenol was found in the volatile extract [less than 0.05%the amount
of (Z)-7-dodecenyl acetate recovered]. Because gland extracts typically lacked
(Z)-7-diodecenol, and because males responded poorly to a blend containing
it, the weight of evidence seems to suggest that its presence in volatile extracts
was an artifact, but we have not yet been able to identify the source.

DISCUSSION

After the identification of (Z)-7-dodecenyl acetate from the gland

extracts of T. ni by Berger (1966), a number of studies b y Shorey and co-
workers demonstrated the importance of this single compound in attracting
males for mating. These included trapping experiments with synthetic (Z)-7-
dodecenyl acetate (Saario et al., 1970; Gaston et al., 1971; Kaae and Shorey,
1972; Kaae et al., 1973a,b) and experiments demonstrating disruption of
communication by the release of synthetic (Z)-7-dodecenyl acetate from
many point sources (Shorey et al., 1967, 1972; Gaston et al., 1967; Kaae et al.,
1974; Farkas et al., 1974).
A second sex pheromone component, dodecyl acetate, was later isolated
from T. ni pheromone gland extracts and volatile extracts (Bjostad et al.,
1980a), and its behavioral role was rigorously investigated with flight tunnel
studies (Linn and Gaston, 1981a,b). This compound was initially detected
using packed columns for quantitative analyses of (Z)-7-dodecenyl acetate in
individual T. ni sex pheromone glands (Bjostad et al., 1980b). The columns
used were not able to resolve positional isomers, however, and compounds
present in small amounts were difficult to detect because the peaks from
packed GLC columns were relatively broad. A capillary GLC became
available for subsequent work on the pathway of (Z)-7-dodecenyl acetate
biosynthesis in T. ni (Bjostad and Roelofs, 1983), and the much greater
resolving power and sensitivity of capillary GLC allowed the detection of a
number of additional compounds in gland extracts of T. ni.
A variety of empirical studies have been conducted to determine the
behavioral effects of synthetic compounds that are structurally related to
(Z)-7-dodecenyl acetate. The geometric isomer (E)-7-dodecenyl acetate was
behaviorally neutral in trapping experiments (McLaughlin et al., 1975), but
Trichoplusia ni enE~OMOYE 1319

was marginally effective in disruption experiments (Kaae et al., 1974). The

functional group analog (Z)-7-dodecenol was tested in trapping experiments
in which it was dispensed along with (Z)-7-dodecenyl acetate, and dramatic
reductions in trap catch were observed (Tumlinson et al., 1972). In flight
tunnel experiments in which (Z)-7-dodecenyl acetate and (Z)-7-dodecenol
were dispensed simultaneously, males made copulatory attempts at a point
considerably downwind from the source, although (Z)-7-dodecenyl acetate
dispensed alone elicited copulatory attempts at the source (McLaughlin et al.,
1974). Attempted disruption with (Z)-7-dodecenol was somewhat successful
(Kaae et al., 1974). Steck et al. (1982a,b) tested 30 synthetic compounds for
trace coattractant activity in T. ni and other noctuid moths with a two-stage
screening procedure. In the first stage, (Z)-7-dodecenyl acetate was mixed with
10% of each of the 30 compounds and tested by trapping in the field.
Compounds that greatly reduced trap catches relative to those obtained with
(Z)-7-dodecenyl acetate alone were retested at 1% and 0.1% levels, with the
rationale that these compounds might be trace coattractants causing inhibi-
tion only at superoptimal levels. A significant increase in trap catches of T. ni
was observed when 0.5% (Z)-7-tetradecenyl acetate was added to (Z)-7-
dodecenyl acetate. This complements our observation that (Z)-7-tetradecenyl
acetate is released from sex pheromone glands of T. ni, and is present at
0.5-1% the amount of (Z)-7-dodecenyl acetate.
The biosynthesis of the main pheromone component (Z)-7-dodecenyl
acetate involves chain shortening of (Z)-I 1-hexadecenoate to (Z)-9-tetra-
decenoate and then to the immediate fatty acyl precursor (Z)-7-dodecenoate
(Bjostad and Roelofs, 1983). We suspected that the corresponding acetates
(Z)-I 1-hexadecenoate and (Z)-9-tetradecenoate might be produced in ad-
dition to (Z)-7-dodecenoate. This proved to be true. The gland also contains
(Z)-I 1-octadecenoate in abundance, and we reasoned that chain shortening
of this compound would produce (Z)-9-hexadecenoate, (Z)-7-tetradecenoate,
and (Z)-5-dodecenoate and that the corresponding acetates might also be
expected. (Z)-7-Tetradecenyl acetate and (Z)-5-dodecenyl acetate were
observed, but (Z)-9-hexadecenyl acetate was not; this was not surprising,
because (Z)-I l-hexadecenoate is much more abundant in the gland than
(Z)-9-hexadecenoate, and (Z)-ll-hexadecenyl acetate was only slightly
above the limit of detection. Finally, because the (Z)-l 1-hexadecenoate and
(Z)-I l-octadecenoate in the gland were apparently produced by a (Z)-I 1
desaturase, it seemed likely that (Z)-I 1-tetradecenoate and 11-dodecenoate
might also occur in small amounts and that the corresponding acetates might
be produced as well. This also proved to be true.
Although biosynthetic considerations led us to propose the presence of a
number of additional compounds that were in fact observed in the gland, we
were equally interested in isomers that were not observed in the gland. For
example, (Z)-5-dodecenyl acetate, (Z)-7-dodecenyl acetate, and 1 l-dodecenyl
1320 BJOSTAD ET AL.

acetate were observed, but (Z)-9-dodecenyl acetate was not. In the bio-
synthesis of (Z)-7-dodecenyl acetate, a relatively small proportion of the large
(Z)-I 1-hexadecenoate pool in the gland is chain shortened. The biosynthesis
of (Z)-5-dodecenyl acetate similarly appears to involve chain shortening of a
relatively small proportion of the large (Z)-ll-octadecenoate pool in the
gland. The biosynthesis of (Z)-9-dodecenyl acetate would therefore be
expected to involve chain shortening of only a small proportion of the
(Z)-I 1-tetradecenoate in the gland, but so little of this compound is present
that chain shortening apparently produces a negligible amount of the
precursor (Z)-9-dodecenoate. (In the biosynthesis of 11-dodecenyl acetate,
the precursor ll-dodecenoate is apparently produced directly by the de-
saturase and does not involve chain shortening). As another example, (Z)-7-
tetradecenyl acetate, (Z)-9-tetradecenyl acetate, and (Z)-ll-tetradecenyl
acetate were present in the gland, but (Z)-5-tetradecenyl acetate was not.
Presumably this is because the precursor (Z)-5-tetradecenoate would arise
from chain shortening of (Z )-11-eicosenoate, which was not detected in these
pheromone glands.
In light of these interpretations, we were particularly interested in a
recent paper by Klun et al. (1983) reporting that the sex pheromone of the
noctuid moth Loxagrotis albicosta comprises the components dodecyl
acetate, (Z)-5-dodecenyl acetate, (Z)-7-dodecenyl acetate, and 11-dodecenyl
acetate, in the respective proportions 5 : 5 : 1 : 5. These are the same C12 acetates
that we found in Trichoplusia ni (albeit in different proportions), and the
isomer (Z)-9-dodecenyl acetate is evidently absent from pheromone gland
extracts of L. albicosta just as it is absent from gland extracts of T. ni. We
suggest that the biosynthetic features discussed above with reference to T. ni
may explain the isomeric array of L. albicosta, including a (Z)-11 desaturase
that produces large amounts of (Z)-ll-hexadecenoate and ( Z ) - l l - o c t a -
decenoate (and produces much smaller amounts of (Z)-11-tetradecenoate and
1 l-dodecenoate), and also including a chain-shortening system involved in
making (Z)-5-dodecenyl acetate from (Z)-I l-octadecenoate, in making (Z)-
7-dodecenyl acetate from (Z )-l l-hexadecenoate, and in making dodecyl
acetate from hexadecanoate.
Volatile extracts of T. ni contained fewer compounds than gland extracts
and in much smaller amounts. Some compounds that were observed in very
tiny amounts in gland extracts, such as (Z)-I 1-hexadecenoate and (Z)-I 1-
tetradecenoate, were below the limit of detection in volatile extracts. Only the
six compounds dodecyl acetate, (Z)-5-dodecenyl acetate, (Z)-7-dodecenyl
acetate, 11-dodecenyl acetate, (Z)-7-tetradecenyl acetate, and (Z)-9-tetra-
decenyl acetate were observed in all gland extracts and in all volatile extracts.
The male response to this blend (95% completed flights) was not significantly
different from the response to intact pheromone glands (98% completed
flights) in flight tunnel bioassays. It is conceivable that additional components
Trichoplusia ni PHEROMONE 1321

i n v o l v e d in sex p h e r o m o n e c o m m u n i c a t i o n in T. n i m a y be released in trace

a m o u n t s , as o b s e r v e d in o t h e r m o t h s (Steck et al., 1982a,b, a n d references
therein), but male responses to the synthetic blend of six c o m p o u n d s r e p o r t e d
here were so high that it would have been i m p o s s i b l e to detect a n y further
c o n t r i b u t i o n of o t h e r c o m p u n d s .
Volatile e x t r a c t s also c o n t a i n e d a small a m o u n t of ( Z ) - 7 - d o d e c e n o l
(Table 2), b u t o f f o u r g l a n d e x t r a c t s a n a l y z e d , three lacked ( Z ) - 7 - d o d e c e n o l
a l t o g e t h e r , a n d s y n t h e t i c blends c o n t a i n i n g ( Z ) - 7 - d o d e c e n o l greatly r e d u c e d
male responses (Table 3), an effect o b s e r v e d by o t h e r w o r k e r s ( T u m l i n s o n et
al., 1972; M c L a u g h l i n et al., 1974). We have not ruled out the possibility that
the ( Z ) - 7 - d o d e c e n o l in the volatile extracts m a y be an artifact.
We c o n c l u d e f r o m these e x p e r i m e n t s t h a t the c o m p l e m e n t o f six acetates
o b s e r v e d in volatile e x t r a c t s of T. n i p h e r o m o n e g l a n d s elicits the c o m p l e t e
c o u r t s h i p sequence f r o m significantly m o r e males t h a n does the previously
identified blend, ( Z ) - 7 - d o d e c e n y l acetate plus d o d e c y l acetate. A l t h o u g h all
six acetates are released by females, it m a y be t h a t the significant increase in
m a l e r e s p o n s e is only due to s o m e of them. A c o m p r e h e n s i v e set o f tests is
u n d e r w a y to d e t e r m i n e if each of the six c o m p o u n d s has a significant effect on
male behavior.

Acknowledgments--We are grateful to Dr. Abner Hammond and Dr. Thomas Baker for
providing us with cultures of Trichoplusia ni. We thank Frances Wadhams, Kathy Poole, and
Michael Christopher Poole for rearing the insects. This work was supported by National
Science Foundation grant PCM-8109850.

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