Journal of Chemical Ecology, VoL 7, No.

4, 1981

SEX PHEROMONE
OF THE SALTMARSH
CATERPILLAR
M O T H , E s t i g m e n e a c r e a 1'2

A D A S. H I L L a n d W E N D E L L

L. R O E L O F S

Department of Entomology
New York State Agricultural Experiment Station
Geneva, New York 14456
(Received August 4, 1980; revised September 22, 1980)
Abstract--Three compounds have been identified as components of the sex
pheromone emitted by females of the saltmarsh caterpillar moth, Estigmene
acrea (Drury). These are (Z,Z)-9,12-octadecadienal (I), (Z,Z,Z)-9,12,15octadecatrienal (II), and (Z,Z)-3,6-cis-9,10-epoxyheneicosadiene
(III). In
female tip extract they were found in a ratio of 1 : 6: 25, respectively, and in
trapped female effluvia the ratio was 1 : 6: 27, respectively. Combinations of
1II with either I or II elicited sustained upwind flight in a wind tunnel, but
none of these compounds by themselves did so. There is evidence that the
antennal acceptor site for II1 is chiral.
Key Words--Saltmarsh caterpillar moth, Estigmene acrea, (Z,Z)-9,12octadecadienal, ( Z,Z,Z)-9,12,15-octadecatrienal, ( Z,Z)-3,6-cis-9,10-epoxyheneicosadiene, fall webworm moth, Hyphantria cunea, insect sex pheromone, cis-9,10-epoxyheneicosane, trans-9,10-epo xyheneicosane, linolealdehyde, linolenaldehyde, Lepidoptera, Arctiidae.

INTRODUCTION

The saltmarsh caterpillar moth, Estigmene acrea (Drury), occurs throughout

the North American continent and appears to be unknown elsewhere. The
l a r v a e f e e d o n t h e f o l i a g e o f a w i d e v a r i e t y o f g a r d e n a n d field c r o p s a n d a r e
u s u a l l y a pest o f m i n o r e c o n o m i c i m p o r t a n c e ( M e t c a l f et al., 1962).
T h e sex p h e r o m o n e s o f o n l y a f e w species in t h e A r c t i i d a e f a m i l y h a v e
b e e n r e p o r t e d to date. T h e s e i n c l u d e m e m b e r s o f t h e H o l o m e l i n a a u r a n t i a c a
c o m p l e x , H o l o m e l i n a laeta ( G u e r i n ) , a n d Isia isabella (J. E. S m i t h ) ( G u e r i n )
Lepidoptera: Arctiidae.
2Supported in part by the Rockefeller Foundation and by National Science Foundation Grants
GB-38020 and PCM 78-13241.
655
0098-0331 / 81 / 0700-0655503.00/ 0 9 1981 Plenum Publishing Corporation

656

HILL AND ROELOFS

(formerly Pyrrhactia isabella), for all of which 2-methylheptadecane has been

reported as a pheromone component (Roelofs and Carde, 1971). We initiated
an investigation of the sex pheromone system of E. acrea because it
represented another arctiid species. Initial tests involving electroantennogram
(EAG) studies of materials obtained from female tip extracts indicated that
2-methylheptadecane probably was not involved, so a full-scale investigation
of the Eo acrea sex pheromone system was undertaken.
METHODS AND MATERIALS

Solvents were distilled through a 10-plate Oldershaw column. Temperatures are given in ~ C. Gas chromatography (GC) columns were glass, either 2
mm 1.8 m, 2 mm X 3.6 m, or 4 mm 1.8 m, and were packed with one of
the following: OV-1 or OV-101 (methyl silicone, 3% on 100-120 mesh GasChrom Q); Hi-Eft (Hi-Eft 8BP, cyclohexanedimethanol succinate, 3% on
100-120 mesh Gas-Chrom Q); XF-1150 (GE XF-1150, 50% cyanoethyl,
methyl silicone~ 10% on 100-120 mesh Chromosorb W-AW-DMCS), Porapak Q ( 100-120 mesh) or Tenax (Tenax GC, 60-80 mesh). The carrier gas for
GC was nitrogen and hydrogen flame ionization was used. Mass spectra (MS)
were determined with either a Perkin-Elmer Hitachi model RMU6 interfaced
with an OV-I GC column, or with a Finnigan 3300 dual quadrupole mass
spectometer interfaced with an OV-101 GC column (Cornell University Mass
Spectrometry Center). The infrared spectra were recorded using either a
Perkin-Elmer model 257 grating infrared spectrophotometer, a Nicolet 7199
Fourier Transform Interferometer| located at the USDA Laboratories in
Gainesville, Florida, or a Nicolet Fourier Transform Interferometer| located
at the USDA Laboratories in Beltsville, Maryland. Proton magnetic resonance
(PMR) spectra were either from a Varian HA 100 or a Varian XL 100
equipped with FT located at the School of Forestry, Syracuse, New York.
Ultraviolet (UV) spectra were determined with a Carey model 15 recording
spectrophotometer.
Electroantennograms (EAGs) were determined as described by Roelofs
(1977, and references therein). The sustained flight tunnel, or wind tunnel, has
been described previously (Miller and Roelofs, 1978) and was modeled after
those described by Kennedy (1977) and Farkas et al. (1974).
Insects were reared on a pinto bean diet (Shorey and Hale, 1965). These
insects originated from two different sources. One was from a culture
previously maintained in Texas (Texas culture), which was used during the
first phase of this investigation, and the other was from a single female
collected from a blacklight trap in Geneva, New York (Geneva culture); this
culture was supplemented occasionally with additional field-collected specimens, and was the one used during the later phase of this investigation,
including the wind tunnel bioassays.

657

PHEROMONE OF THE SALTMARSH C A T E R P I L L A R

Pupae were segregated according to sex and were allowed to emerge at

ca. 20-25 ~ C under a 16:8 light-dark cycle. Females were collected during the
first part of their scotophase at 1-3 days posteclosion; each abominal tip was
extruded manually, snipped off, and extracted with methylene chloride.
Alternately, the females were collected at any time during their light phase,
and the glands were pulled out from the insects' abdomens with dissecting
forceps. The glands occur as a pair of tubes about 3 mm long by about 0.2 mm
OD, with separate openings at either side of the mid-dorsal line on the last
abdominal segment. These also were extracted with methylene chloride.
Extracts were stored in a freezer. Males were held 1-10 days at 20-25 ~ C under
a 16:8 light-dark cycle. They were removed any time during the last half of
their scotophase, brought into light (~> 1 lux), and then bioassayed in the flight
tunnel starting about 15 min after removal from scotophase conditions. The
bioassay period lasted 30-60 rain.
The linoleyl and linolenyl alcohols used for synthesis of the corresponding
aldehydes were purchased from NuCheck Corp. (Elysian, Minnesota).
Acetaldehyde, propanal, nonanal, and dodecanal were purchased from
Aldrich Chemical Co. Pentadecanal and octadecanal were prepared from the
corresponding alcohols, which were purchased from Aldrich Chemical Co.
Aldehydes were prepared from the corresponding alcohols by treatment with
pyridinium chlorochromate (PCC) by the method of Corey and Suggs (I 975).
The (Z)-9-tetradecenyl acetate was purchased from Farchan Chemical Co.
Compound IIl [Figure l, (Z,Z)-3,6-cis-9,10-epoxyheneicosadiene] was
prepared by B. Kovalev via a route described elsewhere (Hill et al., 1981).

I.

(Z,Z)-9,12-octadecadienal

0
~

II.

(Z,Z,Z)-9,12,15-octadecatrienal

III.

\H

- -

(Z, Z) -3,6-cis-9,

lO-epoxyheneicosadiene

FIG. 1. Structures of I, I1, and Ill.

658

HILL AND ROELOFS

The two enantiomers of c/s-7,8-epoxy-2-methyloctadecane (disparlure)

were prepared by Dr. K. Mori (Mori et al., 1979).
Microchemical reactions were usually carried out in 1-dram vials having
Teflon-lined screw.-caps and routinely involved the use of a substantial excess
of the reagents involved. Some of these reactions have been described in detail
elsewhere, such as ozonolysis (Beroza and Bierl, 1967) and acetylation with
acetyl chloride (Hill et al., 1977). Catalytic hydrogenation was with Pd/CaCO3
in methanol or ethanol at I atmosphere of H2 and room temperature.
Epoxides were converted to diols by treatment with 0.5% H2804 in 50%
aqueous tetrahydrofuran [THF: freshly distilled form lithium aluminum
hydride (LAH)] for 4 hr at room temperature; the product was recovered by
dilution with H20 and extraction with Skellysolve-B (petroleum ether 60-68 ~ )
(Skelly B). Reductions with LAH were carried out either in dry Et20 or in dry,
purified TH F for 10 min to 1 hr at room temperature; products were recovered
by addition of dilute, aqueous NaOH (1-5%) at ice-bath temperature under
N2 and extraction with SkeUy B. Hydroxy compounds were converted to the
corresponding bromides by reaction for 1 hr or longer at room temperature
with triphenylphosphine dibromide (TPPDB), freshly prepared from triphenyl
phosphine and bromine in CH2C12 (Sonnet and Oliver, 1976); products were
recovered by evaporation of the solvent in a stream of N2 and extraction of the
product with Skelly B. Hydroxyl groups were trimethylsilated with hexamethyldisilazane (HMDS) and trichlorosilane (TCS) in pyridine (Sweeley
et al., 1963); the products were either analyzed directly by GC or were
recovered by evaporation in a stream of Nz, addition of water, and extraction with
Skelly B.
Synthesis of cis- and trans-9,10-Epoxyheneicosanes. 1-Decyne (1.7 g,
12.3 raM; Farchan Research Laboratories) in dry dioxane (50 ml) was treated
with lithium amide (1.1 g, 48 mM; Alpha Inorganics) for 3.5 hr at reflux, after
which it was cooled to room temperature and l-bromoundecane (6.4 g, 27
mM; Chemical Samples Co.) was added dropwise over a period of ca. 30 min.
The mixture was kept at reflux overnight. After cooling, the mixture w a s
diluted with water and extracted twice with Skelly B; the Skelly B layers were
combined and washed successively with dilute nitric acid (ca. 1%), water,
saturated sodium bicarbonate, and water. Filtration through a magnesium
sulfate bed was followed by evaporation of the solvent. The recovered crude
product, 9-heneicosyne (6.1 g), was filtered through Florisil (petroleum ether),
and the recovered materials were converted to the alkenes without further
purification. Reduction with sodium-liquid ammonia (Warthen and Jacobson, 1973) yielded (E)-9-heneicosene, which still contained large amounts of
the alkyne. The E isomers required were purified by GC collection from
Hi-Eft. Catalytic reduction with Pd/CaCO3 and quinoline (Green et al., 1967)
yielded (Z)-9-heneicosene. The epoxides were formed from alkenes using
m-chloroperbenzoic acid.

PHEROMONE OF THE S A L T M A R S H C A T E R P I L L A R

659

RESULTS

Initially, an aliquot (ca. 10 FE) of a crude female tip extract from E. acrea
was collected in timed fractions from a GC column and the fractions were
assayed for E A G activity. Using an OV- 101 column, one distinct area of EAG
activity was always evident, with a second, earlier, active area sometimes
present. This early area was more distinct and reproducibly detected when
gland extracts were used instead of whole tip extracts. Female effluvium
recovered from P o r a p a k Q also reproducibly showed two distinct areas of
activity when collected from OV-101. When each of the two EAG-active
materials was recovered and re-collected from a Hi-Eft column, the early
OV-101 area of activity resolved into two EAG-active materials, whereas the
late OV-101 fraction showed only o n e EAG-active component that had a
longer retention time on Hi-Eft than either of the other two components.
These components were designated I, II, and III, in their order ofelution from
Hi-Eft. Prominent GC peaks coincident with each of these areas of EAG
activity were evident in GC tracings of these extracts. Their equivalent carbon
numbers were calculated to be the following (using a series of n-hydrocarbons
as reference standards): on OV-101,200 ~ , 19.73 for I and II, and 22.35 for III;
on Hi-Eft, 200 ~ , 23.6 for I, 24.1 for II, and 25.3 for II1. The approximate ratios
between the three components were calculated t o ~ e as follows: for a whole tip
extract 1 : 6:25, for a gland extract 1 : 6 : 10, and for recovered female effluvium
1:6:27.
Identification of I and H. C o m p o u n d I was identified as (Z,Z)-9,12octadecadienal (linolealdehyde, CAS registry No. 2541-61-9), and II was
identified as (Z,Z,Z)-9,12,15-octadecatrienal (linolenaldehyde, CAS registry
No. 2423-13-4), as described below. These structures are presented in Figure 1.
Samples of I and II were purified by collection of the crude pheromone extract
from an OV-1 or an OV-101 column followed by re-collection of the EAGactive materials from a Hi-Eft column, which completely resolved the two
components.
Catalytic hydrogenation of I and of II yielded indistinguishable products.
In both eases the product had the same GC retention time as n-octadecanal on
OV-101 and on Hi-Eft. The C I - M S (methane) of hydrogenated I, hydrogenated II, and n-octadecanal were identical (m/e 269, M H +, as the one
prominent ion).
C I - M S (isobutane) of I and II showed M H ions at 265 and 263,
respectively, indicating that I is diunsaturated and II is triunsaturated. These
spectra were very similar to those obtained for linolealdehyde and linolenaldehyde, respectively. The GC retention times observed for I and II on OV-101,
Hi-Eft, and XF-1150 were within 0.1 rain or less of those for linolealdehyde
and linolenaldehyde, respectively.

660

HILL AND ROELOFS

Samples of I and II were purified and each was subjected to the following
sequence of reactions: (1) treatment with LAH in dry Et20 to produce the
alcohols, (2) acetylation with acetyl chloride to produce the acetates, and (3)
microozonolysis to produce a compound identified as 9-acetoxynonanal.
After each reaction, the products were examined by GC on a nonpolar column
and a polar column (6.7 min at 170~ and 4.0 min at t70 ~ respectively) and
matched those of the expected products within 0.1 min or less. Prior to the
ozonolyses, the acetates from I and II were collected from OV-101. Authentic
9-acetoxynonanal was prepared by ozonolysis of (Z)-9-tetradecen-l-yl acetate.
The CI-MS (isobutane) of all three ozonolysis products were, for all practical
purposes, the same (m/e 201 M H with other ions all less than 20% of this).
These data support a 9-position double bond in I and II.
Ozonolysis of purified I produced n-hexanal, as determined by comparison of the GC retention times of the product with synthetic hexanal on
XF-1150, 100 ~ (5.2 and 5.3 min, respectively). These data indicate a 9,12
double-bond system in compound I.
Purified II was also ozonized to produce n'propanal, which was
identified by its GC retention time on XF-1150 (4.4 min at 50 ~ ) compared to
that for synthetic n-propanal (4.35 rain). These data indicate that the end
double bonds of the three in compound II are in positions 9 and 15.
A UV scan of II (cyctohexane) showed no evidence of conjugation. The
GC retention times of II on the various columns also confirmed the absence of
conjugation in II. This leaves only positions 9,12, and 15 for the double-bond
system in compound II.
An FT-IR spectrum of II revealed that the double bonds in II are all Z
double bonds (Figure 2). There was strong carbonyl absorption at 1731 c m -1
Identification of llI as (Z,Z)-3,6-cis-9,10-Epoxyheneicosadiene. Samples of III were purified for the various analyses by collection of the crude
female tip extract from OV-1 or OV-101 followed by re-collection from
Hi-Eft. In some instances, the collections were preceded by chromatography
on Florisil, from which III eluted with ca. 5-10% Et20 in Skelly B (the
effective mixture was dependent on the activity of the Florisil). This was
consistent with an epoxide structure.
A mass spectrum (El, 70 eV) of III showed a molecular ion at role 306,
with an ion at role 288 (M-18), consistent with an epoxide structure.
Hydrogenation of III over Pd/CaCO3 produced a compound with a shorter
retention time than III on OV-101 and Hi-Eft (equivalent carbon number of
21.2). The mass spectrum (El, 70 eV) of hydrogenated III had a molecular ion
at 310, and an ion at m / e 292 (M-I 8). This was consistent with the presence of
two clouble bonds in III.
To establish that III is unbranched, hydrogenated III was treated
successively with LAH in Et20, triphenylphosphine dibromide (TPPDB) in
CH2C12, and then again with LAH. This sequence reductively removed the

661

P H E R O M O N E OF T H E S A L T M A R S H C A T E R P I L L A R

3000

2000
WAVENUMBERS

1000

800 PPM

1II

3000

2000

1000

800

WAVENUMBERS

FIC. 2. FT-IR of II and III (as films).

epoxide group and produced a compound with the same GLC retention times
on OV-101,181 ~ (20.7 min), and Hi-Eff, 181 ~ (9.1 min) as n-heneicosane. In
addition, the mass spectra (E 1, 70 eV) ofn-heneicosane and of treated II1 were
the same (rn/e 296, M*, and sequence ions characteristic of an unbranched
saturated hydrocarbon chain).
The presence of the epoxide group in III was confirmed, and its location
on the 21-carbon chain was established, by the following route: treatment of
catalytically hydrogenated III with 0.5% sulfuric acid in 1 : 1 aqueous T H F
followed by preparation of the trimethylsilyl derivative using H M D S - T M C S pyridine. The mass spectrum (El, 70 eV) of this trimethylsilated diol showed
two prominent peaks at m/e 215 and 257, corresponding to the fragments
produced by cleavage between carbons 9 and 10. This type of fragmentation is
well-documented (Capella and Zorzut, 1968; Eglinton et al., 1968).
A sample of catalytically hydrogenated Ill had the same retention time
on Hi-Eft, 170~ (25.2 min) as a synthetic sample ofcis-9,10-epoxyheneicosane.

6.52

HILL

AND

ROELOFS

Synthetic trans-9,10-epoxyheneicosane has a different retention time (24.0

min) on this column than the corresponding cis isomer (25.2 min).
The location of the d o u b l e bond closest to the epoxide in III was
established by reductive removal of the epoxide group and subsequent
ozonolysis. The epoxide group was removed, without alteration of the double
bonds, by the following sequence of reactions: (1) treatment of III with L A H
in dry Et20 to produce a secondary alcohol, (2) reaction of this alcohol with
freshly prepared T P P D B in benzene to produce the corresponding bromide,
and (3) treatment with L A H in dry Et20 to remove the bromide group. The
product was collected from Hi-Eft, 190 ~ , at 3.2-4.25 min (with n-heneicosane
at 2.9 min). This material had an equivalent carbon number of 20.9 on
OV-101,210 ~ . This purified hydrocarbon was ozonized in CS2 to produce as
the major product a material with retention times on OV-101,170 ~ (6.5 min),
and on XF- 1150, 170~ (4.25 min), similar to those of synthetic n-pentadecanal
(6.4 min and 4.35 rain on the two columns, respectively).
The position of the other double bond was determined by ozonolysis of
untreated III in CS2, which yielded a product with a similar retention time on
XF-1150 (4.4 min at 50 ~ as propionaldehyde.
An F T - I R of III showed that the two double bonds are of the Z
configuration, since there was no peak in the 960-980 cm -~ region (Figure 2).

100 MHz

%a aa

0
L'k

I~

~:"~'e

f
f

L
g

(1

"-T--

0 PPM

FIG. 3. FT-PMR of II1 (C6D6), with chemical shift assignments.

PHEROMONE

OF

THE

SALTMARSH

663

CATERPILLAR

A [1H]NMR spectrum (FT) of III is shown in Figure 3 with the chemical

shift assignments, which are all consistent with the nonconjugated dieneepoxide structure assigned to III.
All the above data lead to the unequivocal structure assignment of
(Z,Z)-3,6-cis-9,10-epoxyheneicosadiene for III, which can exist in two
enantiomeric forms, 9S, 10R and 9R, 10S. Tests have, as yet, not established
the enantiomeric composition of III produced by female E. acrea. It also is
possible that minor quantities of the various geometric isomers of I, II, and III
are present in the gland extract.
EA G Responses o f Male Antennae to (
(-)-Disparlures. Typical
EAG responses given by a male E. aerea antennae to (+)-disparlure, ( - ) disparlure and III are presented in Figure 4. Structures of these compounds
are given in Figure 5. The two disparlure samples were evaporated into the
stream of air going over the test antenna from filter paper cartridges having
100 g of each material; the sample o f III was from a capillary tube (GC
collected). The highest response was to III (1.8-2.9 mV), followed by that to
(-)-disparlure (1.0-1.4 mV), and then by a distinctively lower response to the
(+)-disparlure (0.2-0.6 mV). In addition, the response to (-)-disparlure
showed the same slow recovery as the response of III, indicative of a binding
interaction with the acceptor site.
Observations o f Male Flights in a Wind Tunnel. Table 1 presents the
results obtained when male E. aerea moths were exposed in a flight tunnel to I,
II, III, and various combinations of these materials; this includes the samples
isolated from female E. acrea as well as synthetic samples of the three
compounds (III was racemic).

Male E. a c r e a
EAG responses to Disparlure enantiomers
and to female-produced epoxide (1TO

--4+)
-4-4-b~

,,,

(_')
,,,,

....

~L~t

,ll ....
/1I

FIG. 4. ElectroantennogramresponsesofmaleE.acreaantennatolII,(+)-disparlure,
and (-)-disparlure.

664

HILL

AND

ROELOFS

0
/\

--

--

--

Estigmene

acrea

Utetheisa

ornatrix

0
Orgyia

pseudotsugata

FIG. 5. Comparison of III with other female-produced sex pheromone components

known for Lepidoptera.
It is clear t h a t m a l e s will n o t fly to III a l o n e , b u t t h a t III in c o m b i n a t i o n
w i t h e i t h e r I o r II will c a u s e the m a l e s to fly u p to the s o u r c e . T h e y will also
h o v e r n e a r it, e x t e n d their c l a s p e r s while w a l k i n g o n the s o u r c e with v i g o r o u s
w i n g f a n n i n g , a n d e v e n a t t e m p t to m a t e w i t h o t h e r i n d i v i d u a l s either at the
s o u r c e or at the site o f flight i n i t i a t i o n . A f t e r f l y i n g to the source, i n d i v i d u a l s
c o u l d be p l a c e d d o w n w i n d r e p e a t e d l y a n d a g a i n w o u l d fly to the souree. T h e y
c o u l d also be k e p t in flight for several m i n u t e s ( u p to 10-15 m i n ) b y m o v i n g
the s t r i p e d f l o o r u n d e r n e a t h t h e m b a c k w a r d s ( M i l l e r a n d R o e l o f s , 1978). T h e
p e r i o d o f this flight a c t i v i t y s t a r t e d a b o u t 15 m i n after i n i t i a t i o n of the
p h o t o p h a s e a n d lasted a b o u t 1 hr.
TABLE 1. RESPONSESOF MALE E . a c r e a MOTHS IN A FLIGHT TUNNEL TO I, II, I11, AND
COMBINATIONSTHEREOF
No. of male respondingb
(% of total)
Samplea
E. acrea,
E. acrea,

effluvium
tip extract

I!
IH(
HI (
II1 (
Ili(
111(
III +

)
)+ 1
) + II
) + I + II
)+Z9-18:ALD
I + I1

No. of
observations

Fanning

Flying
at source

3
2

3 (100)a
2 (100)a

3 (100)a
2 (100)a

2(50) b

2
10
14
20
13
5
6

O(O) b
8(80) b
13 ( 93)ab
20 (100)a
13 (100)a
1(20) b
6 (100)a

0(0)

Average flight
time to
source c

O(O) b
0( 0) b
8 ( 57)a
14 ( 70)a
10( 77)a
0(0) b
5 ( 83)a

226 sec
659 sec
204 sec
300 sec

aSamples were synthetic unless indicated to be from females. Samples from moths were purified
by GC collection. Z 9-18:ALD is (Z)-9-octadecenal. Synthetic III was racemic.
b 9
.
.
.
.
.
Figures followed by the same letter in
each column are not significantly
different,
according to
the method of Ryan (1960), at the 5% experimental error rate.
CTotal numbers of flights timed were 7, 9, 10, and 1, respectively (for samples 6, 7, 8, and 10).

PHEROMONE OF THE SALTMARSH C A T E R P I L L A R

665

Observation o f Calling E. acrea Females and Location of Female Sex

Pheromone Gland. Females were kept routinely on a 16 : 8 light-dark cycle.
Under these conditions, females were seen calling just before scotophase and
also just after scotophase. Calling could be initiated in the females by removal
of the females from scotophase at any time during the last 5 hr of scotophase;
when this was done, the females would call starting about 15 min after
initiation of the photophase.
The calling stance is one in which the female elevates her wings slightly,
sometimes flutters them while walking around, and her abdominal tip is
pushed out slightly and retracted in a pulsing motion. The rate of pulsing at
room temperature was timed at about 80 pulses per min (three individuals
were observed).
Various crude dissections of female abdominal tips were carried out and
each portion was extracted and analyzed for the pheromone components.
Only a pair of tubes, existing dorsally at the base of the penultimate
abdominal segment, were found (using GC tracings) to contain any appreciable amounts of the sex pheromone components, I, II, and III. These tubes are
visible under the cuticular abdominal covering under slight magnification
(25 Each is about 3 mm in length and about 0.2 mm OD, translucent, and
almost colorless (light cream color). They appear to be similar to the tubular
glands of the geometrid moth, Rheumaptera hastata (L.) (Werner, 1977),
except the pair of tubes in E. acrea exist separately, whereas the tubes of R.
hastata join and have a common funnel-shaped opening.

DISCUSSION

The three compounds (I, I1, and III; Figure 1) identified as sex
pheromone components for E. acrea and Hyphantria cunea (Drury) (Hill
et al., 1981) have not been reported previously as components of other sex
pheromone systems. Compounds I and II are the aldehydes corresponding to
the widely distributed polyunsaturated fatty acids linoleic and linolenic acids,
which are known to be essential fatty acids for insects (Downer, 1978). The
epoxide component, III, although hitherto unknown, is similar to the
compounds shown in Figure 5, which have been reported as sex pheromone
components for other lepidoptera: one is from another arctiid, Utetheisa
ornatrix bella (L.) (Conner et al., 1980); the other two are from lymantriids,
the Douglas-fir tussock moth, Orgyiapseudotsugata (McDunnough) (Smith
et al., 1975) and the gypsy moth, Lymantria dispar (L.) (Bierl et al., 1970). The
top two pheromones in Figure 5 and compound III are all unbranched
21-carbon chain structures with a Z double bond at the 6 position. The U.
ornatrix component also has a (Z)-3 double bond in common with III, and its
other double bond, at position 9, occurs at the same locus on the carbon chain

666

HILL AND ROELOFS

and has the same Z configuration as the epoxy group of III. The points of
similarity between III and the O. pseudotsugata pheromone component are
fewer. Although the oxygen functionalities of these two compounds are not in
exactly the same place along the carbon chain, they do occur at vicinal
positions. The only other epoxide so far reported as an insect sex pheromone,
cis-7,8-epoxy-2-methytoctadecane (dispar!ure), is that from L. dispar. Field
data (Miller et al., 1977; Carde'et al., 1977) strongly support assignment of the
7R,8S structure [(+)-disparlure] as that of the natural pheromone. No other
sex pheromone components for lymantriids have been reported, although
males of one other species in this family, the nun moth, Lymantria monacha
(L.), has been captured in traps baited with disparlure.
The only other known arctiid sex pheromone structure is 2-methylheptadecane, which has been reported for the banded woolly bear moth, lsia
isabella (J. E. Smith; formerly Pyrrarctia isabella), and for a number of tiger
moths in the Holomelina genus (Roelofs and Carde', 1971). This hydrocarbon
has the 2-methyl branch feature in common with the disparlure structure.
Male E. acrea antennae can discriminate between the levo- and
dextrorotatory isomers of cis-disparlure, with the (-)-isomer consistently
producing EAG responses that are 2-5 times greater in amplitude than those
produced with the (+)-isomer. The acceptor site is, presumably, that for III, so
this acceptor site appears to be chiral. If the structures of the two isomers of II1
and the two enantiomers of cis-disparlure can be correlated as shown in
Figure 6, then a prediction of the absolute configuration of III seems possible.
The correspondence of the two longer-chain portions of each compound,
shown in Figure 6, to the right of each epoxide group, is made on the
assumption that the 10-carbon chain portion of the cis-disparlure molecule is
too bulky to be accomodated at the locus on the acceptor site that ordinarily

7R,8S (+)

7S,8R (-)

FIG. 6. Stereochemical relationships between 11I and the disparlure enantiomers.

PHEROMONE OF THE SALTMARSHCATERPILLAR

667

fits the 8 - c a r b o n u n s a t u r a t e d p o r t i o n of I l l . Based o n these a s s u m p t i o n s , a

9S, 10R c o n f i g u r a t i o n at the epoxide g r o u p of III is predicted. A d d i t i o n a l l y ,
because of the m a r k e d difference in the E A G responses to (+) a n d ( - )
disparlures, it seems reasonable to expect that III p r o d u c e d by female E . a c r e a
will be f o u n d to be exclusively or p r e d o m i n a n t l y the 9S,10R e n a n t i o m e r .
W i n d t u n n e l o b s e r v a t i o n s of male E . a c r e a flights have d e m o n s t r a t e d
that u p w i n d flight by males is initiated a n d sustained in the presence of III in
c o m b i n a t i o n with either I or II a n d that racemic, synthetic III is effective.
Since all three c o m p o n e n t s are emitted by the females, b u t only two of the
three a p p e a r to be required for sustained u p w i n d a n e m o t a x i s , the f u n c t i o n a l
reason for emission of both aldehydes by the female is not clear at present.
At this stage it is evident that all three c o m p o u n d s , I, II, a n d III, are
emitted by female E . a c r e a a n d that all three can be perceived by the males a n d
mediate the specific b e h a v i o r of u p w i n d flight ( a m e n o t a x i s ) by them. F u r t h e r
testing of the biological significance of these c o m p o n e n t s , i n c l u d i n g field
t r a p p i n g tests a n d investigation of the biosynthesis of these c o m p o u n d s , will
be carried out.
A c k n o w l e d g m e n t s - - W e are very grateful to the following for providing specialized
services: Dr. J.H. Tumlinson and R.R. Heath for the FT-1R spectrum of III; Dr. J. Kochansky for
the FT-IR spectrum of II; Dr. R.M. Silversteinfor the 100-MHz FT-NMR of III; Dr. T. Wachs
and D. Angell for the mass spectra. We are grateful also to F. Wadhams and K. Poole for
maintaining the insect cultures and for preparation of most of the female tip extracts, to Drs. R.T.
Carde'and T.C. Baker for some of the early EAG determinations, to Dr. J. Barnard for the
statistical analyses, to C. Anway for assistance with the flight tunnel bioassays, and to Drs. P.J.
Chapman and S.E. Lienk for providing the gravid E. acrea female from the blacklight trap.
We are also extremely grateful to Dr. B. Kovalevfor providing the synthetic sample of III,
and to Dr. K. Mori for the enantiomers of cis-disparlure.

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