(Z,Z)-4,7-tridecadien-(S)-2-YL acetate: Sex pheromone of Douglas-fir cone gall midge, Contarinia oregonensis

Article abstract of DOI:10.1023/A:1021005517389

Our objectives were to identify and field test the sex pheromone of female Douglas-fir cone gall midge, Contarinia oregonensis (Diptera: Cecidomyiidae). Coupled gas chromatographic-electroantennographic detection (GC-EAD) analyses of pheromone extract revealed a single compound (A) that elicited responses from male antennae. Hydrogenation of pheromone extract, followed by renewed GC-EAD analysis, revealed a new EAD-active compound with chromatographic characteristics identical to those of tridecan-2-yl acetate on five fused silica columns (DB-5, DB-210, DB-23, SP-1000, and Cyclodex-B). Syntheses, chromatography, and retention index calculations of all possible tridecen-2-yl acetates suggested that the candidate pheromone A was a tridecadien-2-yl acetate with nonconjugated double bonds. Synthetic candidate pheromone component (Z,Z)-4,7-tridecadien-2-yl acetate (Z4Z7) cochromatographed with A on all analytical columns and elicited comparable antennal activity. In GC-EAD analyses that separated the enantiomers (Z,Z)-4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7) and (Z,Z)-4,7-tridecadien-(R)-2-yl acetate (2R-Z4Z7) with baseline resolution, only 2S-Z4Z7 as a component in a racemic standard or in pheromone extract elicited antennal responses. In Douglas-fir seed orchards, sticky traps baited with 2S-Z4Z7 captured male C. oregonensis, whereas 2R-Z4Z7 was behaviorally benign. Comparable catches of males in traps baited with racemic Z4Z7 (50μg) or virgin female C. oregonensis suggested that synthetic pheromone baits could be developed for monitoring C. oregonensis populations in commercial Douglas-fir seed orchards.

Full text of DOI:10.1023/A:1021005517389

C
Journal of Chemical Ecology, Vol. 28, No. 11, November 2002 ( 2002)
(Z,Z)-4,7-TRIDECADIEN-(S)-2-YL ACETATE: SEX
PHEROMONE OF DOUGLAS-FIR CONE GALL MIDGE,
Contarinia oregonensis
REGINE GRIES,1 GRIGORI KHASKIN,1 GERHARD GRIES,1,
ROBB G. BENNETT,2 G. G. SKIP KING,1 PETRA MOREWOOD,1
KEITH N. SLESSOR,3 and W. DEAN MOREWOOD1
1Centre for Environmental Biology
Department of Biological Sciences, Simon Fraser University
Burnaby, British Columbia, V5A 1S6 Canada
2Ministry of Forests
Tree Improvement Branch
Saanichton, British Columbia, V8M 1W4 Canada
3Department of Chemistry
Simon Fraser University
Burnaby, British Columbia, V5A 1S6 Canada
(Received March 29, 2002; accepted July 5, 2002)
Abstract—Our objectives were to identify and field test the sex pheromone
of female Douglas-fir cone gall midge, Contarinia oregonensis (Diptera: Ce-
cidomyiidae). Coupled gas chromatographic–electroantennographic detection
(GC-EAD) analyses of pheromone extract revealed a single compound (A) that
elicited responses from male antennae. Hydrogenation of pheromone extract,
followed by renewed GC-EAD analysis, revealed a new EAD-active compound
with chromatographic characteristics identical to those of tridecan-2-yl acetate
on five fused silica columns (DB-5, DB-210, DB-23, SP-1000, and Cyclodex-
B). Syntheses, chromatography, and retention index calculations of all pos-
sible tridecen-2-yl acetates suggested that the candidate pheromone A was a
tridecadien-2-yl acetate with nonconjugated double bonds. Synthetic candi-
date pheromone component (Z,Z)-4,7-tridecadien-2-yl acetate (Z4Z7) cochro-
matographed with A on all analytical columns and elicited comparable
antennal activity. In GC-EAD analyses that separated the enantiomers (Z,Z)-
4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7) and (Z,Z)-4,7-tridecadien-(R)-2-yl
acetate (2R-Z4Z7) with baseline resolution, only 2S-Z4Z7 as a component
in a racemic standard or in pheromone extract elicited antennal responses.
In Douglas-fir seed orchards, sticky traps baited with 2S-Z4Z7 captured
To whom correspondence should be addressed. E-mail: gries@sfu.ca
2283
C
0098-0331/02/1100-2283/0 2002 Plenum Publishing Corporation

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GRIES ET AL.
male C. oregonensis, whereas 2R-Z4Z7 was behaviorally benign. Compara-
ble catches of males in traps baited with racemic Z4Z7 (50 g) or virgin fe-
male C. oregonensis suggested that synthetic pheromone baits could be devel-
oped for monitoring C. oregonensis populations in commercial Douglas-fir seed
orchards.
Key Words—Douglas-fir cone gall midge, Contarinia oregonensis, Cecidomyi-
idae, sex pheromone, enantiomers, (Z,Z)-4,7-tridecadien-(S)-2-yl acetate,
(Z,Z)-4,7-tridecadien-(R)-2-yl acetate.
INTRODUCTION
The life history of the Douglas-fir cone gall midge, Contarinia oregonensis Foote
(Diptera: Cecidomyiidae) is closely associated with the phenology of its host plant
Douglas-fir, Pseudotsugamenziesii. Inearlyspringasconeletsopenforpollination,
female C. oregonensis eclose from their pupae in the duff, mate, and seek open
conelets for oviposition. Females place their eggs singly or in clusters near the
base of cone scales by extending their flexible ovipositor. Neonate larvae tunnel
into cone scales and induce formation of, and develop within, galls near ovules. In
the fall, larvae leave mature cones and overwinter in the duff as prepupae or pupae
(Hedlin, 1961).
In natural forests, C. oregonensis is one of many insects that feed and develop
on seeds. However, in coastal seed orchards of British Columbia, in Canada and
Washington and Oregon, in the United States, C. oregonensis has become the sin-
gle most important insect pest of Douglas-fir seeds. Larvae-caused gall formation
results in seed fusion to the scale, preventing seed extraction at harvest or destruc-
tion of seeds at high populations. Severe infestations destroy the entire seed crop
(Hedlin, 1961, 1974; Ruth, 1980).
C. oregonensis is the only coastal Douglas-fir seed orchard pest in British
Columbia monitored annually. Monitoring entails tedious and time-consuming
counts of egg-infested scales in samples of conelets. When a threshold number
of infested scales per conelet is reached, insecticidal control of C. oregonensis is
implemented.
Miller and Borden (1981, 1984) presented evidence for a sex attractant
pheromone produced by female C. oregonensis. Traps baited with synthetic
C. oregonensis pheromone would allow investigation of relationships between
captures of male C. oregonensis and numbers of both egg-infested scales at pol-
lination and galled scales at harvest. If reliable correlations could be established,
decisions about C. oregonensis control could be based solely on captures of males
in pheromone-baited traps. Monitoring of C. oregonensis would become simple
and effective.
We report identification and field testing of the pheromone.

SEX PHEROMONE OF C. oregonensis
METHODS AND MATERIALS
2285
Experimental Insects. In late August and early September 1995–1996, ma-
ture, dry Douglas-fir cones infested with larvae of C. oregonensis were col-
lected into loosely woven burlap sacks. These sacks were then partially sub-
merged in water-filled pails for 2–3 days to induce the mature larvae to leave
the cones. After removing the sacks from the pails, the larvae that had exited
the cones and sacks and accumulated in the bottom of pails (apparently un-
affected by their temporary submergence) were obtained by pouring the water
through a No. 30 standard sieve (600 m). Sieve-restrained larvae were then
transferred to 500-ml glass jars half filled with clean, damp sand and closed with
fine mesh screening. To break larval developmental diapause, jars were placed in
environmental chambers (18 C; 70% relative humidity.) with temperature low-
ered by 2 C every 5 d to 4 C. After 3.5 months of cold exposure (4 C), jars
were subjected to a temperature increase from 4 to 18 C in increments of 2 C
every 5 d. Emergent adults were collected several times a day and kept individu-
ally in Petri dishes. Ovipositors (with pheromone glands) of calling females and
abdominal tips of males were removed and separately extracted in hexane for
5–50 min.
Laboratory Analyses of Pheromone Extract and General Instrumentation.
Aliquots of 5–10 female and male equivalents (FE, ME) of abdominal tip
extract were analyzed by gas chromatography (GC) and coupled gas
chromatographic–electroantennographic detection (GC-EAD) (Arn et al., 1975),
employing an Hewlett-Packard (HP) 5890 gas chromatograph fitted with a fused
silica column (30 m 0.25 or 0.32 mm ID) coated with DB-210, DB-5, DB-23,
Cyclodex-B (J&W Scientific, Folsom, California, USA) or SP-1000 (Supelco,
Bellefonte, Pennsylvania, USA). For GC-EAD recordings, a severed insect head
(with both antennae intact) was placed into the opening of a glass capillary elec-
trode (1.0 0.58 100 mm) (A-M Systems, Inc., Carlsborg, Washington, USA)
filled with saline solution (Staddon and Everton, 1980). One antenna with its tip re-
moved by spring microscissors (Fine Science Tools Inc., North Vancouver, British
Columbia, Canada) was placed into the recording capillary electrode. Coupled GC-
mass spectrometric (MS) analyses of pheromone extract (300 FE) and of synthetic
standards employed a Varian II Ion Trap GC-MS fitted with the above-referenced
DB-5 column.
High-performance liquid chromatography (HPLC) employed a Waters LC
626 high-performance liquid chromatograph equipped with a Waters 486 vari-
able wavelength UV visible detector set to 210 nm, HP Chemstation software
˚
(Rev.A.07.01), and a normal phase Nova-Pak silica column (60A, 4 m;
3.9 150 mm) with 1 ml/min of hexane–ether (97:3) flow. Nuclear magnetic
resonance (NMR) spectroscopy of synthetic compounds was conducted on a

2286
GRIES ET AL.
Bruker AMX-400 spectrometer at 400.13 MHz for 1H NMR spectra. 1H chemical
shifts are reported as parts per million (ppm; ) relative to TMS. Elemental analy-
ses were performed using a Carlo Erba model 1106 elemental analyzer. Chemicals
obtained from commercial sources were used without further purification unless
otherwiseindicated. Allmoisture-andair-sensitivereactionswereconductedunder
argon. Column chromatography refers to flash chromatography, using silica gel
60 (230–400 mesh, E. Merck, Darmstadt) (Still et al., 1978).
Syntheses
(Z,Z)-4,7-Tridecadien-2-ol (5) (Scheme 1). To a THF solution of the benzyl
ether of 4-pentyn-2-ol (1) (4.35 g, 25 mmol), 11 ml of 2.5 M butyllithium in hexane
(10% excess) was added at 70 C. The mixture was stirred under argon and then
warmed to 25 C before addition of CuI (475 mg, 0.1 equiv.). After stirring for 1 hr
at 25 C, a THF solution of freshly prepared 2-octyn-1-yl methanesulfonate (2)
(30 mmol) (from 2-octyn-1-ol, methanesulfonyl chloride, triethylamine, 0 C) was
added dropwise. The reaction mixture was allowed to warm to room temperature,
quenched with aq. conc. NH4Cl, and after 2 hr of stirring was extracted with
hexane–ether (1:1). The desired benzyl ether of 4,7-tridecadiyn-2-ol (3) (40%
yield) was purified (85% based on GC) by flash chromatography and reduced to
the benzyl ether of (Z,Z)-4,7-tridecadien-2-ol (4) by catalytic hydrogenation with
P-2 Ni in ethanol. Flash chromatography on AgNO3-impregnated (20%) SiO2,
with a gradient of hexane–ether (99:1 to 95:5) eluent afforded (Z,Z)-diene (4)
(76% yield, 94 % pure). Removal of the benzyl group of 4 by exposure to excess
calcium (15 equiv.) in liq. ammonia (Hwu et al., 1986) for 15 min at 70 C
yielded (Z,Z)-4,7-tridecadien-2-ol (5) (71%) and corresponding monoenes (7%),
as determined by GC-MS. Therefore, 5 was repurified by flash chromatography as
above to give 1.05 g (5.40 mmol) of pure 5. Anal. calcd. for C13H24O: C 79.13; H
12.22. Found: C 79.53; H 12.32. 1H NMR: CDCl3, = 5.31-5.61 (4H, m), 3.88
SCHEME 1. Synthesis of (Z,Z)- and (E,E)-4,7-tridecadien-2-yl acetates.

SEX PHEROMONE OF C. oregonensis
2287
FIG. 1. Mass spectrum of synthetic (Z,Z)-4,7-tridecadien-2-yl acetate. Varian Saturn II
Ion Trap GC-MS.
(1H, sextet), 2.85 (2H, t), 2.28 (2H, m), 2.08 (2H, m), 1.34 (6H, m), 1.25 (3H, d),
0.92 (3H, t).
(Z,Z)-4,7-Tridecadien-2-yl Acetate (6) (Scheme 1). Overnight reaction of 5
with acetic anhydride in pyridine afforded acetate 6 (1.20 g, 5.0 mmol) in 93%
yield (20% overall yield based on 1). Anal. calcd. for C15H26O2: C 75.58; H 11.00.
1
Found: C 75.80; H 10.98. H NMR: CDCl3, = 5.28–5.54 (4H, m.), 4.92 (1H,
sextet), 2.78 (2H, t), 2.31 (2H, m), 2.04 (2H, m), 2.01 (3H, s), 1.27 (6H, m), 1.22
(3H, d), 0.88 (3H, t); mass spectrum as in Figure 1.
(E,E)-4,7-Tridecadien-2-yl Acetate (8) (Scheme 1). Overnight reaction of
3 (1.13 g, 4.00 mmol) with lithium (20-fold excess) in refluxing liq. ammonia
produced as the major product geometrically pure (E,E)-4,7-tridecadien-2-ol (7)
(60% yield). It was purified by flash chromatography (see above) and acetylated
as above to give (E,E)-4,7-tridecadien-2-yl acetate (8) (0.520 g, 2.18 mmol).
(Z,E)-4,7-Tridecadien-2-yl Acetate (13) (Scheme 2). 2-tert-Butyl-dimethyl-
silyloxy-3-pentyne (9) (5.14 g, 15.6 mmol) was coupled via cuprate with freshly
prepared (E)-2-octen-1-yl methanesulfonate (15.2 mmol) (from 2-octen-1-ol,
methanesulfonyl chloride, collidine at 0 C, 50% yield) in THF. The resulting crude
tert-butyldimethylsilyl ether of (E)-7-tridecen-4-yn-2-ol (10) was de-protected
withBu4NFinTHFaffordingalcohol11(80%pure), whichwashydrogenatedwith
Lindlar catalyst in hexanes to (Z,E)-4,7-tridecadien-2-ol (12, 1.46 g, 7.43 mmol).
Acetylation of purified 12 produced geometrically pure (Z,E)-4,7-tridecadien-2-yl
acetate (13, 1.44 g, 6.04 mmol) in 39% overall yield.

2288
GRIES ET AL.
SCHEME 2. Synthesis of (Z,E)-4,7-tridecadien-2-yl acetate.
(E,Z)-4,7-Tridecadien-2-yl Acetate (18) (Scheme 3). Compound 9 in THF
(5.70 g, 28.8 mmol) (Scheme 1) was extended by one carbon (BuLi at 70 C, then
paraformaldehyde, warm to room temperature, and stirring for 24 hr) to the mono-
protected diol 14 (95% yield). Reduction of 14 (3.1 g, 13.6 mmol) with lithium
aluminum hydride (LAH, 7 molar excess) in refluxing THF produced allylic al-
cohol 15 (1.90 g, 8.26 mmol) in 60% yield. Metylation of 15 with methanesul-
fonyl chloride in dichloromethane (0 C) in the presence of 2,4,6-collidine gave 5-
(tert-butyldimethylsilyloxy)-2-hexenyl methanesulfonate (16). Up to 50%
formation of the unwanted allylic chloride side product occurred. Reaction of
sulfonate 16 with heptynyllithium in the presence of CuI (0.1 equiv.) gave the tert-
butyldimethylsilyl ether of (E)-4-tridecen-7-yn-2-ol (17). Removal of the protec-
tive group from 17, hydrogenation, and acetylation (as described for 10 in Scheme
2) afforded after purification (E,Z)-4,7-tridecadien-2-yl acetate (18, 0.18 g, 7.56
mmol) in 9% yield based on 15.
(Z,Z)-4,8-Tridecadien-2-ol (23) (Scheme 4). Benzyl ether 1 (4.35 g,
25mmol)wascoupledat 70oCviathelithiumsaltwith1-O-tetrahydropyranyl-3-
bromopropanol (1.3 equiv.) [ 70 C to room temperature, 24 hr, THF–HMPA
(1:1)]. Subsequent removal of the THP group in methanol in the presence of triflu-
oroacetic acid afforded 7-benzyloxy-4-octyn-1-ol (19) (1.32 g, 5.71 mmol). Oxi-
dation of alcohol 19 with PCC in dichloromethane at room temperature, and imme-
diate coupling of the crude aldehyde 20 with n-pentylidene triphenylphosphorane
in THF yielded the benzyl ether of (Z)-8-tridecen-4-yn-2-ol (21). Compound 21
partially purified by flash chromatograhy was hydrogenated (4 hr) with Lindlar
catalyst in hexanes in the presence of quinoline, and the resulting diene-ether was
SCHEME 3. Synthesis of (E,Z)-4,7-tridecadien-2-yl acetate.

SEX PHEROMONE OF C. oregonensis
2289
SCHEME 4. Synthesis of (Z,Z)-4,8-tridecadien-2-yl acetate.
treated with 10 equivalents of calcium in liq. ammonia for 45 min. Usual work-up
and purification of the diunsaturated alcohol 22, followed by overnight acetylation
with acetic anhydride in pyridine, yielded (Z,Z)-4,8-tridecadien-2-yl acetate (23,
0.265 g, 1.10 mmol) in 4.5% yield based on 1.
(S)- and (R)-2-Tridecanyl Acetate (29, 34) (Scheme 5). Hydrolytic kinetic
resolution of racemic 1,2-epoxytridecane (24) (1.75 g, 8.80 mmol) with 25 mg of
(R,R)-( )-salen complex of Co(II) [Jacobsen’s catalyst; (R,R)-( )-N,N0-bis (3,5-
di-tert-butylsalicylidene)-1,2-cyclohexanediaminocobalt(II)](25)(Sigma-Aldrich,
Oakville, Ontario, Canada) and 90 l of water (Tokunaga et al., 1997; Schaus
et al., 1998; Gries et al., 2000) yielded after 3 d at room temperature (R)-1,2-
epoxytridecane (26) (0.88 g, 4.44 mmol) which was separated from (S)-1,2-
tridecanediol (27) by flash chromatography (hexanes–ether, 10:1). Ring opening
of 26 with LAH in ether afforded (S)-2-tridecanol (28) (0.84 g, 4.24 mmol). Acety-
lation of 28 gave (S)-2-tridecanyl acetate (29) (Kamezawa et al., 1994, Sharma
et al., 1996) in 47% overall yield and with an enantiomeric excess (ee) of 91.9%, as
determined by GC analysis on a Cyclodex-B column. Corresponding hydrokinetic
resolution of 24 with the (S,S)-(+)-salen complex of Co(II), and corresponding
follow-upreactions(seeabove)afforded(S)-1,2-epoxytridecane, (R)-2-tridecanol,
and finally (R)-2-tridecanyl acetate (47% overall yield, 91.3% ee).
2-Tridecenyl acetates with E and Z unsaturation at C-3 to C-12 were synthe-
sized by oxidation (PCC in dichloromethane) of the dodecenols (available in our
laboratory from previous studies) and by immediate reaction of the crude aldehydes
SCHEME 5. Synthesis of (S)-2-tridecanyl acetate.

2290
GRIES ET AL.
with methylmagnesium bromide in ether followed by acetylation (Ac2O/Py) of the
resulting 2-tridecenols.
Absolute Configuration of Pheromone. The absolute configuration of the
pheromone was determined in five steps: (1) acetyl lactate derivatization (Slessor
et al., 1985) of racemic (Z,Z)-4,7-tridecadien-2-ol (5 in Scheme 1); (2) separation
of diasteromeric derivatives by HPLC (conditions described above); (3) removal
of the acetyl lactate functionality and acetylation of the resulting enantiospecific
alcohols; (4) GC-EAD analyses (Figure 3 below) and field testing (Figure 4 below)
of (Z,Z)-4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7) and (Z,Z)-4,7-tridecadien-
(R)-2-yl acetate (2R-Z4Z7); and (5) comparison of retention times (Cyclodex-B
column) of the saturated equivalents of 2S-Z4Z7 [(S)-2-tridecanyl acetate] and
2R-Z4Z7 [(R)-2-tridecanyl acetate] with those of enantioselectively synthesized
(S)- and (R)-2-tridecanyl acetate (Scheme 5).
Field Experiments. Field experiments were conducted in Nootka, Mount
Newton, and Saanich Douglas-fir seed orchards, and the Douglas-fir seed orchard
at the Saanichton Forestry Centre, all near Saanichton, Vancouver Island, British
Columbia. Trees were 11–25 years old with spacings within and between rows
ranging from 3.8 6.1 m to 4 10 m. At 15–20-m intervals, self-made sticky
2-l milk carton traps (Gray et al., 1984) were suspended from trees 1.5 m above
ground in complete randomized blocks. Traps were baited with a gray sleeve stop-
per (The West Company, Lionville, Pennsylvania, USA) impregnated with test
chemicals in HPLC-grade hexane. All test chemicals were >93% chemically and
geometrically pure. Enantiomeric excess of 2S-Z4Z7 and 2R-Z4Z7 was 95.5%
and 97.8%, respectively, based on GC analyses on a Cyclodex-B column.
Experiment 1 tested candidate pheromone Z4Z7 at doses of 1, 10, 100, and
1,000 g. Experiments 2 and 3 tested S and R enantiomers of Z4Z7 singly and
in combination at doses of 10 and 2 g, respectively. Experiment 4 tested attrac-
tiveness of racemic Z4Z7 at 50 g versus that of caged female C. oregonensis.
Despite transformation, trap catch data were not normally distributed and
were, thus, analyzed by nonparametric analyses of variance (Friedman’s test) fol-
lowed by comparison of means by Bonferroni (Dunn) t test (Zar, 1984; SAS/STAT,
1988). In all analyses, = 0.05.
RESULTS AND DISCUSSION
Pheromone Analyses. Comparison of GC traces of abdominal tip extracts
of male and female C. oregonensis failed to disclose sex-specific volatiles.
Coupled GC-EAD analyses of female pheromone extract revealed a single EAD-
active compound (A in Figure 2) that occurred below detection threshold of the
flame ionization detector of the GC. Retention indices (RI) (Van den Dool and
Kratz, 1963) of A (DB-5: 1605; DB-210: 1872; DB-23: 1932; SP-1000: 1927;

SEX PHEROMONE OF C. oregonensis
2291
FIG. 2. Representative GC-EAD recording (8 replicates) of pheromone gland extract of
female C. oregonensis; (a) flame ionization detector (FID) chromatograph of 10 female
equivalents (10 FE) of pheromone gland extract; (b, c) corresponding recording of elec-
troantennographic detector (EAD: male C. oregonensis antenna) responses to 10 FE of
(b) untreated and (c) hydrogenated pheromone extract. Chromatography: Hewlett Packard
5890A gas chromatograph; splitless injection; injector temperature: 220 C; detector tem-
perature: 250 C; DB-23 column; temperature program: 100 C (1 min) 10 C per min to
200 C. A = (Z,Z)-4,7-tridecadien-2-yl acetate; A1 = tridecan-2-yl acetate.
Cyclodex B: 1690), and RI intercolumn differentials, suggested that it had an es-
ter (acetate) functionality. GC-EAD analysis of hydrogenated pheromone extract
revealed a new EAD-active compound (A1 in Figure 2), suggesting that hydro-
genation of A had resulted in a saturated volatile (A1) with different chromato-
graphic characteristics [RIs: 1631 (DB-5); 1890 (DB-210); 1879 (DB-23); and
1870 (SP-1000)]. Compound A1 was determined to be 2-tridecanyl acetate [an ag-
gregation pheromone component of Drosophila mulleri and D. busckii (Kamezawa
et al. 1994)], which cochromatographed with A1 on all above-referenced columns
and had comparable EAD activity as A1. Similar chromatographic characteristics
of A and A1 suggested that the former was a tridecen-2-yl acetate. However, syn-
theses and chromatography of all possible tridecen-2-yl acetates determined that
neither co-chromatographed with A, nor elicited antennal responses indicative of
a potential double bond position.
Hypothesizing then that A was a nonconjugated tridecadien-2-yl acetate,
candidate tridecadien-2-yl acetates were approximated (Gries et al., 1993) by (1)
determining retention index differentials between tridecan-2-yl acetate and each
of the tridecen-2-yl acetates (Table 1), and (2) adding these differentials to, or sub-
tracting them from, the respective tridecen-2-yl acetate. For example, the RI 1604

2292
GRIES ET AL.
TABLE 1. RETENTION INDICES AND DIFFERENTIALS (BOLD FACE TYPE)
BETWEEN SYNTHETIC TRIDECEN-2-YL ACETATES AND SYNTHETIC
TRIDECAN-2-YL ACETATE (SAT) ON GC COLUMNS COATED WITH DB-5,
DB-210, DB-23, OR SP-1000
dbpa
DB-5
1631
DB-210
1890
DB-23
1879
SP-1000
sat
1870
E 3
E 4
E 5
E 6
E 7
E 8
E 9
E 10
E 11
112
Z 3
Z 4
Z 5
Z 6
Z 7
Z 8
Z 9
Z 10
Z 11
1620: 11
1616: 15
1614: 17
1614: 17
1618: 13
1619: 12
1621: 10
1864: 26
1876: 14
1859: 31
1871: 19
1890:+11
1900: +30
1884: +14
1891: +21
1894: +24
1894: +24
1900: +30
1900: +30
1910: +40
1931: +61
1920: +50
1844: 26
1894: +24
1880: +10
1890: +20
1894: +24
1903: +33
1911: +41
1920: +50
1951: +81
1885:+6
1885:+6
1891:+12
1897: +18
1899:+20
1901:+22
1910:+31
1931:+52
1931: +52
1830: 49
1898: +19
1888: +9
1896: +17
1903: +24
1913: +34
1920: +41
1929: +50
1956: +77
1881:
9
1875: 15
1881:
1889:
9
1
1627:
1635: +4
1623:
4
1895: +5
8
1904: +14
1796: 94
1871: 19
1871: 19
1873: 17
1589: 42
1617: 14
1604: 27
1609: 22
1610: 21
1618: 13
1881:
1890:
9
0
1624:
1630:
8
1
1898: +8
1903: +13
1925: +35
1648: +17
a dbp = double bond position.
of (E,E)-4,8-tridecadien-2-yl acetate on DB-5 was predicted by subtracting the RI
differential between tridecan-2-yl acetate and (E)-8-tridecen-2-yl acetate (=12;
Table 1) from the RI of (E)-4-tridecen-2-yl acetate (1616 12 = 1604). Tak-
ing the data for all five columns into account, (Z,Z)-4,8-tridecadien-2-yl acetate
(Z4Z8) was predicted to be the most probable diene acetate to match chromato-
graphic characteristics of A. However, synthetic Z4Z8 (Scheme 4) had RIs 1-3 RI
units different from those of the target A on all five columns.
Strong EAD activity of Z4Z8 suggested that its molecular structure was sim-
ilar to that of the candidate pheromone A. An allylic double bond in C-3, instead of
C-4, would have greatly decreased the RI of the molecule (RG + GG, unpublished
data) and was discounted. Hypothesizing then that the geometry and position of the
double bond in C-8 was incorrect, (Z,Z)-4,7-tridecadien-2-yl acetate (Z4Z7) was
synthesized (Scheme 1) as an alternative structure for the candidate pheromone.
This compound, but none of its three isomers (ZE, EZ, EE) (Schemes 1–3), cochro-
matographed with A on all columns, and elicited comparable antennal responses.
In GC-EAD analyses that separated the enantiomers of Z4Z7 with base-
line resolution (Figure 3, a), antennal responses were obtained only to the early

SEX PHEROMONE OF C. oregonensis
2293
eluting enantiomer in a racemic standard (Figure 3, b) or in pheromone ex-
tract (Figure 3, c). This enantiomer was determined to have the S configura-
tion, because its hydrogenated equivalent had a retention time on Cyclodex-B
that was identical with that of enantiospecifically synthesized (S)-2-tridecanyl
acetate (29) (Scheme 5). (R)-2-Tridecanyl acetate, in contrast, eluted 0.35 min
later. Lack of response to later eluting 2R-Z4Z7 could not be attributed to a
refractory period of the antenna, because two sequential injections (at 0.5 min
intervals) of isothermally chromatographed 2S-Z4Z7 elicited two very simi-
lar antennal responses (Figure 3, d). When 2S- and 2R-Z4Z7 were analyzed
individually, only the S enantiomer was EAD-active, supporting contention
that female C. oregonensis produce 2S-Z4Z7 for pheromonal attraction of
mates.
Field Experiments. In experiment 1, a lure dose of 100 g of racemic Z4Z7
was more effective than lower or higher lure doses in attracting male C. oregonensis
(Figure 4, experiment 1), indicating that proper lure loadings are critical for optimal
attractiveness. Captures of male C. oregonensis in traps baited with 2S-Z4Z7
(Figure 4, experiments 2 and 3) were consistent with EAD-activity only of the
S enantiomer (Figure 3, b). The few males captured in traps baited with 2R-
Z4Z7 (Figure 4, experiment 2) were probably attracted to trace amounts of the S
enantiomer in the bait. The antipode 2R-Z4Z7 is not part of the C. oregonensis
pheromone because it elicited neither EAD-activity (Figure 3, b) nor behavioral
activity when tested alone at a low dose (Figure 4, experiment 3) or in combination
with pheromonal 2S-Z4Z7 (Figure 4, experiments 2 and 3). Similar numbers of
males captured in traps baited with synthetic Z4Z7 (50 g) or virgin female C.
oregonensis (Figure 4, experiment 4) indicate competitive attractiveness of natural
and synthetic pheromone.
Pheromones in Cecidomyiids and Practical Implications. Evidence for
female-produced sex pheromones has been reported for 10 cecidomyiids (reviewed
by Harris and Foster, 1999) but only four pheromones have been identified. In
pioneering studies, (E)-10-tridecen-(S)-2-yl acetate was determined to be a sex
pheromone component of the Hessian fly, Mayetiola destructor (Foster et al., 1991;
Harris and Foster, 1991; Millar et al., 1991). Attractiveness of this compound to
male M. destructor in wind-tunnel but not field experiments suggested that fe-
male M. destructor deploy additional pheromone components that have not yet
been identified (Harris and Foster, 1999). Meticulous work by Hillbur et al. (1999,
2000, 2001) deciphered the three-component pheromone of the pea midge, Con-
tarinia pisi. Synthetic (2S,11S)-diacetoxytridecane, (2S,12S)-diacetoxytridecane,
and 2-acetoxytridecane at a blend ratio as found in pheromone gland extracts
attracted male C. pisi in both wind-tunnel and field experiments. Concurrent
work by Gries et al. (2000) revealed the pheromone of the orange wheat blos-
som midge, Sitodiplosis mosellana, to be (2S,7S)-nonanediyl dibutyrate, which
attracted male S. mosellana in field experiments in Saskatchewan. While SR, RS,

2294
GRIES ET AL.
FIG. 3. Representative GC-EAD recording (5 replicates) of synthetic standards and
pheromone gland extract of female C. oregonensis; (a) flame ionization detector (FID) chro-
matogram of synthetic (Z,Z)-4,7-tridecadien-2-yl acetate (Z4Z7) with baseline resolution
of the enantiomers (Z,Z)-4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7; left) and (Z,Z)-4,7-
tridecadien-(R)-2-yl acetate (2R-Z4Z7; right); (b–d) corresponding recordings of elec-
troantennographic detector (EAD: male C. oregonensis antenna) responses to (b) synthetic
Z4Z7, (c) 10 female equivalents of pheromone gland extract and (d) two sequential injec-
tions of 2S-Z4Z7. Note: first and second injection of 2S-Z4Z7 were separated by 0.5 min.,
equivalent to the 0.5 min separation of 2S-Z4Z7 and 2R-Z4Z7 in the racemic standard (a);
lack of response to later eluting 2R-Z4Z7 (b) could not be attributed to a refractory period
of the antenna, because two sequential injections (at 0.5-min intervals) of isothermally chro-
matographed 2S-Z4Z7 elicited two very similar antennal responses (d). Chromatography:
Hewlett-Packard 5890A gas chromatograph equipped with a chiral Cyclodex-B column;
temperature program: 150 C isothermal; split injection of 25 ng per compound with 0.5 ng
reaching the EAD detector.

SEX PHEROMONE OF C. oregonensis
2295
FIG. 4. Mean (+SE) number of male C. oregonensis captured during experimental periods
in sticky Delta milk carton traps baited with various doses of racemic (Z,Z)-4,7-tridecadien-
2-yl acetate (Z4Z7) (experiment 1), (Z,Z)-4,7-tridecadien-(S)-2-yl acetate (2S-Z4Z7), and
(Z,Z)-4,7-tridecadien-(R)-2-yl acetate (2R-Z4Z7) singly and in combination (experiments
2, 3), or baited with racemic Z4Z7 or caged virgin female C. oregonensis (experiment 4).
Douglas-fir seed orchards near Saanichton, Vancouver Island, British Columbia. Bars in
each experiment with different letter superscripts are significantly different; nonparametric
analysis of variance by ranks (Friedman’s test) followed by comparison of means [Bonfer-
roni (Dunn) t test, P < 0.05] (SAS/STAT, 1988).
and RR stereoisomers of the pheromone had no adverse effects on attraction of
male S. mosellana, attraction of male C. pisi to the synthetic C. pisi pheromone
blend (see above) was strongly inhibited by the presence (20%) of the 2S,11R-
and 2R,11S-stereoisomers of diacetoxytridecane (Hillbur et al., 2001). Male C.
oregonensis, in contrast, did not perceive the antipode of pheromonal 2S-Z4Z7
(Figure 3, b) and were not inhibited by it (Figure 4, experiments 2 and 3).
The identification of the C. oregonensis pheromone should lead to improved
monitoring of C. oregonensis populations and management decisions in com-
mercial seed orchards. In both 2000 and 2001, numbers of male C. oregonensis
captured in pheromone-baited traps in seed orchards in Washington and Oregon
were correlated with numbers of both egg-infested scales in conelets and dam-
aged seed in cones (Morewood et al., 2002). Trap captures of male C. oregonensis
are, thus, predictive of seed damage at harvest, and should help orchard managers
decide whether insecticidal control of C. oregonensis populations is warranted.

2296
GRIES ET AL.
Acknowledgments—We thank Dan Rudolph, Carolyn Lohr, Cathy Cook, and Tim Crowder for
access to research sites; Dave Holden for technical assistance; Jocelyn Millar for provision of synthetic
(E)-10-tridecen-2-yl acetate; Bob Birtch for graphical art work; Harold Pierce; Jr., for reviewing the
manuscript; and two anonymous reviewers for constructive comments. The research was supported,
in part, by grants from the Natural Sciences and Engineering Research Council of Canada and the
Operational Tree Improvement Program of Forest Renewal British Columbia.
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