A male-produced aggregation pheromone blend consisting of alkanediols, terpenoids, and an aromatic alcohol from the cerambycid beetle Megacyllene caryae

Article abstract of DOI:10.1007/s10886-008-9425-3

Bioassays conducted with a Y-tube olfactometer provided evidence that both sexes of the cerambycid beetle Megacyllene caryae (Gahan) were attracted to odor produced by males. Odor collected from male M. caryae contained eight male-specific compounds: a 10:1 blend of (2S,3R)- and (2R,3S)-2,3-hexanediols (representing 3.2±1.3% of the total male-specific compounds), (S)-(-)-limonene (3.1±1.7%), 2-phenylethanol (8.0±2.4%), (-)-α-terpineol (10.0±2.8%), nerol (2.1±1.5%), neral (63.3±7.3%), and geranial (8.8±2.4%). Initial field bioassays determined that none of these compounds was attractive as a single component. Further field trials that used a subtractive bioassay strategy determined that both sexes were attracted to the complete blend of synthetic components, but the elimination of any one component resulted in a decline in trap captures. Blends that were missing (2S,3R)-2,3-hexanediol, (2R,3S)-2,3-hexanediol, or citral (a 1:1 mixture of neral and geranial) attracted no more beetles than did controls. A pheromone blend of this complexity, composed of alkanediols, terpenoids, and aromatic alcohols, is unprecedented for cerambycid species.

Full text of DOI:10.1007/s10886-008-9425-3

J Chem Ecol (2008) 34:408–417
DOI 10.1007/s10886-008-9425-3
A Male-produced Aggregation Pheromone Blend Consisting
of Alkanediols, Terpenoids, and an Aromatic Alcohol
from the Cerambycid Beetle Megacyllene caryae
Emerson S. Lacey & Jardel A. Moreira &
Jocelyn G. Millar & Lawrence M. Hanks
Received: 19 July 2007 /Revised: 20 November 2007 /Accepted: 27 December 2007 /Published online: 6 February 2008
#
Springer Science + Business Media, LLC 2008
. .
Keywords Aggregation pheromone Sex pheromone
Abstract Bioassays conducted with a Y-tube olfactometer
provided evidence that both sexes of the cerambycid beetle
Megacyllene caryae (Gahan) were attracted to odor
produced by males. Odor collected from male M. caryae
contained eight male-specific compounds: a 10:1 blend of
.
.
.
2,3-Hexanediol Longhorned beetle Wood-boring insect
.
.
.
.
.
Limonene Geranial Neral Nerol 2-Phenylethanol
α-Terpineol
(
1
2S,3R)- and (2R,3S)-2,3-hexanediols (representing 3.2±
.3% of the total male-specific compounds), (S)-(−)-
Introduction
limonene (3.1±1.7%), 2-phenylethanol (8.0±2.4%), (−)-α-
terpineol (10.0±2.8%), nerol (2.1±1.5%), neral (63.3±
Male-produced aggregation pheromones have been identi-
fied for nine species in four tribes of the cerambycid beetle
subfamily Cerambycinae (Lacey et al. 2004, 2007b; Hanks
et al. 2007). In another three species, male-produced
pheromones attract only females (reviewed by Lacey et al.
2004). The pheromones of all these species are comprised
of one to three compounds that share a similar structural
motif, consisting of molecules that are 6, 8, or 10 carbons
in length, with hydroxyl or carbonyl groups at C and C
7
.3%), and geranial (8.8±2.4%). Initial field bioassays
determined that none of these compounds was attractive as
a single component. Further field trials that used a sub-
tractive bioassay strategy determined that both sexes were
attracted to the complete blend of synthetic components,
but the elimination of any one component resulted in a
decline in trap captures. Blends that were missing (2S,3R)-
2
,3-hexanediol, (2R,3S)-2,3-hexanediol, or citral (a 1:1
2
3
mixture of neral and geranial) attracted no more beetles
than did controls. A pheromone blend of this complexity,
composed of alkanediols, terpenoids, and aromatic alco-
hols, is unprecedented for cerambycid species.
(reviewed by Lacey et al. 2004, 2007b). There are two
exceptions to this trend: the male-produced aggregation
pheromone of the cerambycine Phymatodes lecontei
Linsley, (R)-2-methylbutan-1-ol (Hanks et al. 2007), and a
component of the sex pheromone of Hylotrupes bajulus
(L.), 1-butanol (Reddy et al. 2005). Volatile pheromones of
species in the subfamily Cerambycinae apparently are
produced by glands in the prothorax and secreted through
pores lying within depressions in the cuticle (Ray et al.
:
E. S. Lacey L. M. Hanks (*)
Department of Entomology,
University of Illinois at Urbana–Champaign,
20 Morrill Hall 505 South Goodwin Ave.,
2006; Hanks et al. 2007; Lacey et al. 2007b). Pheromone
release by males of several species has been associated with
3
Urbana, IL 61801, USA
e-mail: hanks@life.uiuc.edu
a characteristic body posture, termed the “pushup stance”
(Lacey et al. 2004, 2007a, b): Males fully extend their front
E. S. Lacey
e-mail: elacey@life.uiuc.edu
legs, elevating the head and thorax, and remain motionless
for extended periods.
The cerambycine species Megacyllene caryae (Gahan) is
endemic to North America. The larvae develop in woody
tissues of stressed, moribund, and dead trees of a variety of
:
:
E. S. Lacey J. A. Moreira J. G. Millar
Department of Entomology, University of California,
Riverside, CA 92521, USA

J Chem Ecol (2008) 34:408–417
409
hardwood species (Linsley 1964). Adults are active in early
spring, and collection records from the area of our study,
east-central Illinois, range from March 29 to June 7 (Illinois
Natural History Survey, Champaign, IL). The adult beetles
reportedly feed on flowers of trees in the genus Crataegus
tions. We conducted bioassays between 11:00 and 16:00
hours for 4 d in April 2004 (skies clear, air temperatures
17–24°C). A 2-l plastic chamber containing a cylinder of
aluminum screen as a perch was attached to each arm of
the Y-tube. One chamber contained six male beetles and
the other six females. Ambient air was pulled through the
olfactometer with a vacuum cleaner connected to a variable
(
Rosaceae; Dusham 1921), but dissections of field-collected
adults of both sexes revealed that their guts contained
pollen from trees in the genus Quercus (Fagaceae;
unpublished data). In the laboratory, beetles can live for
more than 30 d if provided sugar water (Lacey, personal
observation). Adults are active from ∼11:00 to 18:00 hours.
Both sexes aggregate on larval hosts in numbers that may
exceed 40 individuals. They mate soon after emergence,
and females begin ovipositing immediately. Generation
time is usually 1 yr.
M. caryae exhibits several characteristics that have been
associated with production of volatile pheromones in other
cerambycine species. For example, adults aggregate on
larval hosts (Lacey, personal observation), males have sex-
specific gland pores on their prothoraces (Ray et al. 2006),
and males on larval hosts commonly assume the pushup
stance (Lacey, personal observation). We report here the
identification and testing of sex-specific volatile pheromone
components produced by adult male M. caryae.
−1
power supply (air speed ∼1.0 m sec ). For each trial, a
beetle was released at the base of the Y-tube and allowed
10 min to respond to an odor source by crossing a line
18 cm down one arm. Beetles that did not respond within
10 min were recorded as “no response.” Chambers were
alternated between arms of the Y-tube every three trials to
control for positional bias. Chambers and the olfactometer
were washed and rinsed with acetone each day. We
bioassayed 20 individuals of each sex and compared
2
numbers of beetles responding to treatments with a χ
goodness-of-fit test corrected for continuity (Sokal and
Rohlf 1995).
Identification of Potential Pheromone Components Volatile
compounds were collected from adult M. caryae by placing
five females and males in separate glass vacuum traps
(∼0.3 l, custom manufactured by the glass shop, School of
Chemistry, UIUC) that were lined with an aluminum screen
to provide perches. A glass tube (6 cm×4 mm inner
diameter [i.d.]) containing 100 mg of 80/100 mesh
SuperQ® (Alltech Associates, Deerfield, IL) held between
plugs of silanized glass wool was attached to one nipple of
each chamber with an 8-cm-long section of Teflon® tubing,
and charcoal-purified air was pulled through the apparatus
Methods and Materials
Source of Insects Adult M. caryae used in olfactometer
bioassays and for pheromone collections were reared from
logs of honey locust, Gleditsia triacanthos L. (Fabaceae),
that were naturally colonized by beetles from April to May
−1
with a water aspirator (0.7 l min ). Males and females
were aerated simultaneously on a laboratory windowsill
from 11:00 to 16:00 hours in April and May 2004. We
selected this period because it was the only time during the
day that males displayed the pushup stance. Collectors were
eluted with three 0.5-ml aliquots of CH Cl and the
2
003 on the campus of University of Illinois at Urbana–
Champaign (UIUC). Periodically, logs were moved to a 3×
×1-m rearing cage of window screen in a laboratory room
ambient conditions: fluorescent lighting, ∼12:12 L/D, 20°C,
0% relative humidity) during January and March 2004.
2
(
5
2
2
Most adults emerged within 14 d of moving logs into the
laboratory. Freshly emerged adults were caged individually
in 0.1-m cylindrical cages of aluminum window screen
with plastic Petri dishes at top and bottom, under ambient
laboratory conditions. They were provided 10% sucrose
solution dispensed from 8-ml vials plugged with cotton
dental rolls (Patterson Dental Supply, South Edina, MN).
Adults used in experiments appeared healthy and active.
resulting extracts analyzed at the University of California
Riverside on a Hewlett-Packard® (HP) 5973 mass selective
detector interfaced to an HP 6890 gas chromatograph, fitted
with a DB5-MS column (30 m×0.25 mm i.d., 0.25 μm film
thickness; J&W Scientific, Folsom, CA), temperature-
programmed from 40 (held for 1 min) to 250°C at
3
−1
10°C min . Injector temperature was 250°C, and injec-
tions were made in the splitless mode. Absolute configu-
ration of the insect-produced compounds was determined
by analysis of the extract on a Cyclodex-B gas chromatog-
raphy (GC) column (30 m×0.25 mm i.d., 0.25 μm film
thickness, J&W Scientific) with the GC programmed from
Testing for Attractant Pheromones Bioassays for attractants
produced by adult M. caryae were carried out with a
horizontal glass Y-tube olfactometer (6 cm diameter, main
tube 26 cm long, arm length 22 cm, 70° angle between
arms). Bioassays were conducted outdoors in partial shade
because beetles either were sedentary or appeared agitated
when placed in the olfactometer under laboratory condi-
−1
50 (held for 1 min) to 200°C at 5°C min ; injector and
detector temperatures were 100 and 200°C, respectively.
Identifications of peaks were confirmed by coinjections of
extracts with authentic standards.

4
10
J Chem Ecol (2008) 34:408–417
Synthesis of Pheromone Components The stereoisomers of
-hydroxy-3-hexanone and 2,3-hexanediol (see “Results”)
an HP 5890 GC interfaced to an HP 5970 mass selective
detector, in electron impact mode (70 eV), with helium as
the carrier gas. The GC was equipped with a DB5-MS
column (25 m×0.20 mm i.d.×0.33 μm film; J&W
Scientific). Solutions were dried over anhydrous Na SO
and concentrated under partial vacuum by rotary evapora-
tion unless otherwise stated. Crude products were purified
by flash or vacuum flash chromatography with silica gel
(230–400 mesh, EM Science, Gibbstown, NJ). Reactions
with air- or water-sensitive reagents were carried out in
oven-dried glassware under argon. GC with a chiral
stationary phase column was performed with an HP 5890
GC fitted with a Cyclodex B column (30 m×0.25 mm i.d.×
2
were synthesized as described below and shown in Fig. 1.
Racemic 2-hydroxy-3-hexanone and racemic (2R*,3S*)-
and (2R*,3R*)-2,3-hexanediols were available from previ-
ous studies (Lacey et al. 2004, 2007b; Hanks et al. 2007).
All other compounds were obtained from Sigma-Aldrich
2
4
(
St Louis, MO), including (S)-(−)-limonene (96%), 2-
phenylethanol (99%), (−)-alpha-terpineol (90%), nerol
≥90%), and citral (95%; see “Results”). Because neral
(
and geranial (see “Results”) were not readily available as
pure isomers, citral (a 1:1 mixture of neral and geranial;
Sigma-Aldrich) was used in the bioassays.
0
.25 μm, J&W Scientific), programmed from 50 (held for
−1
Tetrahydrofuran was distilled from sodium/benzophenone
1 min) to 200°C at 5°C min , with the injector at 100°C
and helium as carrier gas (20 psi).
1
13
ketyl under argon. H- and C nuclear magnetic resonance
(
(
NMR) spectra were recorded with a Varian INOVA-400
400 and 100.5 MHz, respectively) spectrometer as CDCl3
Ethyl (S)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-propanoate
[(S)-2] 3,4-Dihydro-2H-pyran (53.4 g, 635 mmol) was
added dropwise to a stirred solution of ethyl (S)-(−)-lactate
(S)-1 (50.0 g, 423 mmol; Aldrich Chem., Milwaukee, WI)
solutions. Chemical shifts were expressed in parts per
1
million relative to CDCl (7.26 and 77.23 ppm for H- and
3
1
3
C-NMR, respectively). Mass spectra were obtained with
Fig. 1 Syntheses of all four 2,3-
hexanediol stereoisomers and
the two 2-hydroxy-3-hexanone
enantiomers from ethyl
O
O
O
PTSA, DHP
5%
EtOH, LiOH.H O
2
PropylLi
O
O
OLi
9
Quantitative
-30ºC, 85%
OH
OTHP
OTHP
(S)-lactate and methyl
R)-lactate
(S)-1
(S)-2
(S)-3
(
OH
OH
O
OH
NaBH , EtOH
4
MeOH, PTSA
92%
(
2S,3R)-7
2S,3S)-7
90%
OTHP
OTHP
+
(
S)-4
(S)-6
OH
MeOH, PTSA
48%
OH
(
O
Separable by flash chromatography
OH
S)-5
(
OH
OH
+
OH
OH
O
(2R,3S)-7
(2R,3R)-7
O
OH
O
(
R)-1a
OH
R)-5
(

J Chem Ecol (2008) 34:408–417
411
1
and 100 mg p-toluenesulfonic acid in 120 ml CH Cl at
(0.1 mmHg) for 10 hr. H-NMR showed that ethanol was
still present in the salt, and so the crude lithium salt was
suspended in benzene, the benzene distilled off at atmo-
spheric pressure, and the residue again pumped under
vacuum for 10 hr, affording lithium (S)-2-[(tetrahydro-2H-
pyran-2-yl)oxy]-propanoate (S)-3 as a light yellow solid in
2
2
0
°C under argon. The mixture was warmed to room
temperature and stirred until all the starting material had
been consumed (∼5 hr). The mixture was then diluted with
diethyl ether (60 ml) and washed with saturated aqueous
NaHCO and brine, dried, and concentrated. The crude
3
1
product was Kugelrohr distilled (64–68°C, 0.25 mmHg),
yielding 81.4 g of protected alcohol (S)-2 (95%) as a
quantitative (99.7%) yield. H-NMR (major stereoisomer):
δ 1.39 (d, J=6.8 Hz, 3H), 1.42–1.92 (m, 6H), 3.40–3.55
(m, 1H), 3.82–3.92 (m, 1H), 4.18 (q, J=6.8 Hz, 1H), 4.70
1
mixture of diastereoisomers (2:1—measured by GC). H-
1
NMR (major stereoisomer): δ 1.26 (t, J=7.2 Hz, 3H), 1.44
(t, J 3.5 Hz, 1H). H NMR (minor stereoisomer): δ 1.34 (d,
(
3
d, J=7.0 Hz, 3H), 1.48–1.90 (m, 6H), 3.47–3.55 (m, 1H),
.80–3.88 (m, 1H), 4.11–4.25 (m, 3H, two quadruplets
overlapped with signals of other stereoisomer), 4.66–4.72
m, 1H, dd overlapped with signals of other stereoisomer).
J=6.8 Hz, 3H), 1.42–1.92 (m, 6H), 3.40–3.55 (m, 1H),
4.00–4.05 (m, 1H), 4.12 (q, J=7.0 Hz, 1H), 4.54 (dd, J=2.3
and 7.0 Hz, 1H). Some NMR signals from the two
diastereomers overlapped.
(
1
H-NMR (minor stereoisomer): δ 1.27 (t, J=7.2 Hz, 3H),
.38 (d, J=6.8 Hz, 3H), 1.48–1.90 (m, 6H), 3.40–3.47
m, 1H), 3.88–3.95 (m, 1H), 4.11–4.25 (m, 2H - quadruplet
overlapped with signals of other stereoisomer), 4.40 (q, J=
.0 Hz, 1H), 4.66–4.72 (m, 1H, dd overlapped with signals
1
(
Lithium (R)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-propanoate
[(R)-3] Methyl (R)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-
propanoate (R)-2a (34.20 g, 182 mmol) was converted to
lithium (R)-2-(2-tetrahydropyranyloxy)-propanoate (R)-3
7
1
of other stereoisomer); mass spectrometry (MS; m/z:
(32.70 g) as described above, in quantitative yield. H-
relative intensity): 144 (1), 130 (3), 129 (4), 101 (19), 85
NMR and mass spectra matched those of the corresponding
(
100), 73 (11), 67 (12), 57 (16), 55 (17), 45 (25), 43 (24).
(S)-enantiomer mixture.
Methyl (R)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-propanoate
(R)-2a] In the same manner as described above, methyl
R)-(+)-lactate (R)-1a (20.0 g, 192 mmol; Aldrich Chem.,
6% enantiomeric excess [ee]) was converted to a mixture
2:1—measured by GC) of methyl (R)-2-[(tetrahydro-2H-
pyran-2-yl)oxy]-propanoate diastereoisomers (R)-2a
34.24 g) in 95% yield after Kugelrohr distillation (62–
(S)-2-[(Tetrahydro-2H-pyran-2-yl)oxy]-3-hexanone [(S)-4]
Lithium wire (1% sodium content; 1.39 g, 200 mmol,
previously rinsed with hexane) was cut into small pieces
directly into a three-necked flask charged with anhydrous
diethyl ether (85 ml) under argon. A few drops of a 1-
bromopropane (12.3 g, 100 mmol) solution in 15 ml ether
were added, and the mixture was stirred at ambient
temperature until the reaction started. The mixture was
then cooled to −20°C, and the remaining solution of
bromopropane was added dropwise over 1.5 hr. When the
addition was complete, the mixture was allowed to warm to
room temperature and stirred for an additional hour before use.
[
(
9
(
(
1
6
6°C,1.3 mmHg). H-NMR (major stereoisomer): δ 1.44
(
d, J=7.0 Hz, 3H), 1.46–1.90 (m, 6H), 3.40–3.54 (m, 1H),
3
4
.72 (s, 3H), 3.76–3.93 (m, 1H), 4.41 (d, J=7.0 Hz, 1H),
.65–4.71 (m, 1H) (some signals overlapped with signals of
1
the other stereoisomer). H-NMR (minor stereoisomer): δ
1
1
1
.38 (d, J=6.8 Hz, 3H), 1.46–1.90 (m, 6H), 3.40–3.54 (m,
H), 3.72 (s, 3H), 3.76–3.93 (m, 1H), 4.19 (d, J=6.8 Hz,
H), 4.65 – 4.71 (m, 1H) (some signals overlapped with
The resulting propyllithium solution (∼1.0 M in ethyl
ether, 80 ml) was added dropwise to a suspension of lithium
(S)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-propanoate (S)-3
signals of the other stereoisomer); MS (relative intensity)
m/z: 130 (2), 129 (6), 116 (2), 101 (28), 85 (100), 73 (7), 67
(9.63 g, 53.5 mmol) in Et O (100 ml) under argon at
2
(
15), 57 (20), 55 (26), 45 (25), 43 (30).
−30°C. The reaction was allowed to warm to room tem-
perature, stirred overnight, poured into crushed ice, and
Lithium (S)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-propanoate
(S)-3] Ethyl (S)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-propa-
extracted with Et O (3×100 ml). The combined organic
2
[
layers were washed with saturated aqueous NH Cl and
4
noate (S)-2 (80.00 g, 396 mmol) was added to a stirred
brine, dried, and concentrated. The residue was purified by
vacuum flash chromatography on silica gel (hexane:
EtOAc, 9:1) affording (S)-2-[(tetrahydro-2H-pyran-2-yl)
suspension of LiOH.H O (16.60 g, 396 mmol) in ethanol
2
(
200 ml) under argon at 0°C. The reaction was stirred for
1
3
3
0 min at 0°C, warmed to room temperature, and stirred for
hr. The mixture was concentrated under vacuum, and
oxy]-3-hexanone (S)-4 (9.14 g, 85%). H-NMR (major
isomer): δ 0.90 (t, J=7.4 Hz, 3H), 1.34 (d, J=7.0 Hz, 3H),
1.45–1.95 (m, 8H), 2.40 (dt, J=7.2 and 17.3 Hz, 1H), 2.50
(dt, J=7.3 and 17.3 Hz, 1H), 3.40–3.53 (m, 1H), 2.77–3.90
(m, 1H), 4.27 (q, J=7.0 Hz, 1H), 4.54 (dd, J=3.1 and
4.5 Hz, 1H). Some NMR signals overlapped with the
hexane (50 ml) was added to the concentrate, followed by
concentration under vacuum to remove traces of ethanol
and water as azeotropes. This procedure was repeated four
times, and the residue was pumped under vacuum

4
12
J Chem Ecol (2008) 34:408–417
signals from the minor stereoisomer. MS (major stereoiso-
(S)-4 (10.00 g, 49.9 mmol) in ethanol (25 ml) was added
mer): 156 (1), 129 (3), 101 (1), 99 (1), 85 (100), 71 (9), 67
dropwise to a stirred suspension of NaBH (1.89 g, 50 mmol)
4
1
(
13), 57 (14), 55 (9), 43 (30), 41 (22). H-NMR (minor
in 25 ml ethanol at 0°C, and the mixture was stirred
overnight. The mixture was concentrated, 100 ml brine added
to the residue, and the product was extracted with ethyl
acetate (5×50 ml). The combined organic phases were dried
and concentrated. Purification by vacuum flash chromatogra-
phy (hexane/EtOAc—9:1) afforded 9.08 g (90%) of product
(S)-6. MS (m/z, rel. intensity): 158 (2), 157 (2), 129 (2), 101
(10), 85 (100), 67 (12), 57 (23), 56 (12), 55 (22), 45 (20), 43
isomer): δ 0.91 (t, J=7.4 Hz, 3H), 1.25 (d, J=6.8 Hz, 3H),
.45–1.95 (m, 8H), 2.56 (dt, J=7.2 and 17.8 Hz, 1H), 2.62
1
(
(
dt, J=7.4 and 17.9 Hz, 1H), 3.40–3.53 (m, 1H), 2.77–3.90
m, 1H), 4.07 (q, J=6.8 Hz, 1H), 4.60 (dd, J=2.8 and
5
1
4
.3 Hz, 1H). MS (minor stereoisomer): 156 (1), 129 (3),
01 (1), 99 (1), 85 (100), 71 (7), 67 (12), 57 (15), 55 (10),
3 (32), 41 (22).
(
28), 41 (29). Because there were four stereoisomers in the
1
13
(
R)-2-[(Tetrahydro-2H-pyran-2-yl)oxy]-3-hexanone
product mixture, H- and C-NMR spectra were complex
and not useful.
[
(R)-4] Lithium (R)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-
propanoate (R)-3 (10.52 g, 58.4 mmol) and n-propyllithium
0.73 M in ethyl ether, 120 ml) were reacted as described
above, giving (R)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-3-
hexanone (R)-4 (7.51g, 64%). NMR and mass spectra were
in accord with those of the (S)-2-diastereomers.
(
(R)-2-[(Tetrahydro-2H-pyran-2-yl)oxy]-3-hexanol
[(R)-6] Compound (R)-6 was obtained in similar fashion
with a 93% yield from (R)-2-[(tetrahydro-2H-pyran-2-yl)
oxy]-3-hexanone (R)-4. Mass and NMR spectra were in
accord with the (S)-6 mixture.
(
S)-2-Hydroxy-3-hexanone [(S)-5] Pyridinium-p-toluene
sulfonate (100 mg) was added to a stirred solution of (S)-
(2S,3S)-2,3-Hexanediol ((2S,3S)-7) and (2S,3R)-2,3-
hexanediol [(2S,3R)-7] A solution of (S)-2-[(tetrahydro-
2H-pyran-2-yl)oxy]-3-hexanol (S)-6 (9.05 g, 45 mmol) and
100 mg p-toluenesulfonic acid in methanol (50 ml) was
stirred overnight. After concentration, the residue was
diluted with ethyl acetate (100 ml) and washed with
2
4
-[(tetrahydro-2H-pyran-2-yl)oxy]-3-hexanone (S)-4 (9.1 g,
5.5 mmol) in methanol (100 ml) at 0°C under argon. The
reaction was allowed to warm to ambient temperature and
stirred overnight. The methanol was removed by fractional
distillation under reduced pressure. Water was added to the
residue, and the mixture was extracted with Et O (4×
saturated aqueous NaHCO and brine, dried, and concen-
2
3
3
0 ml). The organic phase was washed with saturated
trated. Purification by vacuum flash chromatography
(hexane/EtOAc—4:1) followed by Kugelrohr distillation
(90–98°C, 4.5 mmHg) gave a mixture of (2S,3S)-2,3-
hexanediol (2S,3S)-7 and (2S,3R)-2,3-hexanediol (2S,3R)-7
(48:52, respectively). The diastereoisomeric diols (6.18 g)
were separated by flash chromatography in 2-g batches
(hexane/acetone—5:1; column 32 cm long×5 cm diameter)
followed by recrystallization at 4°C (50 ml hexane/g of
diol) affording 2.55 g of pure (2S,3S)-2,3-hexanediol
(diastereomeric excess [de]>98%) and 2.89 g of (2S,3R)-
NaHCO and brine and dried, and the solvent was removed
by distillation through a Vigreux column. The residue was
purified by vacuum flash chromatography (silica gel, eluting
with pentane/Et O 9:1), removing the solvent by distillation
through a Vigreux column, followed by Kugelrohr dis-
tillation (34°C, 1.60 mmHg), giving 2.53 g (48%) of (S)-
2
primarily a result of losses during purification because of
the volatility of the compound, rather than to any problem
with the chemistry. H-NMR: δ 0.92 (t, J=7.4Hz, 3H), 1.36
3
2
-hydroxy-3-hexanone (S)-5. The moderate yield was
1
1
2,3-hexanediol (de>96%). (2S,3R)-7: H-NMR: δ 0.96 (t,
(
d, J=7.2 Hz, 3H), 1.58–1.72 (m, 2H), 2.39 (dt, J=17.0 and
J=6.8 Hz, 3H), 1.15 (d, J=6.4 Hz, 3H), 1.30–1.46 (m, 3H),
1.46–1.60 (m, 1H), 1.99 (br s, 2H), 3.62–3.67 (m, 1H),
3.76–3.84 (m, 1H). C NMR: δ 14.29, 16.84, 19.39, 34.13,
7
4
1
8
.3 Hz, 1H), 2.48 (dt, J=17.0 and 7.2 Hz, 1H), 3.57 (d, J=
1
3
13
.5 Hz, 1H), 4.22 (dq, J=4.5 and 7.2 Hz, 1H). C-NMR: δ
+
3.96, 17.29, 20.03, 39.60, 72.80, 212.80. MS: 116 (M , 1),
70.67, 74.86. MS: 103 (1), 85 (1), 75 (10), 73 (61), 72 (34),
1
7 (1), 83 (1), 74 (12), 73 (36), 72 (19), 71 (64), 55 (59), 45
57 (18), 55 (100), 45 (36), 43 (35), 41 (11). (2S,3S)-7: H
(
87), 43 (100), 41 (19).
NMR: δ 0.94 (t, J=7.2 Hz, 3H), 1.19 (d, J=6.4 Hz,
3
H),1.34–1.58 (m, 4H), 2.43 (br s, 2H), 3.31–3.37 (m, 1H),
1
3
(
R)-2-Hydroxy-3-hexanone [(R)-5] (R)-2-Hydroxy-3-
3.59 quint, J=6.2 Hz, 1H). C NMR: δ 14.26, 18.96, 19.70,
35.69, 71.13, 76.16. MS: m/z: 103 (1), 85 (1), 75 (16), 73
(59), 72 (35), 57 (21), 55 (100), 45 (38), 43 (37), 41 (12).
hexanone was obtained in 49.6% yield (2.10 g) from (R)-
-[(tetrahydro-2H-pyran-2-yl)oxy]-3-hexanone (R)-4 after
2
purification. NMR and mass spectra were identical to those
of (S)-2-hydroxy-3-hexanone.
(2R,3S)-2,3-Hexanediol ((2R,3S)-7) and (2R,3R)-2,
3hexanediol [(2R,3R)-7] In analogous fashion, a mixture
(
S)-2-[(Tetrahydro-2H-pyran-2-yl)oxy]-3-hexanol [(S)-6] A
of (2R,3S)-2,3-hexanediol (2R,3S)-7 and (2R,3R)-2,3-
hexanediol (2R,3R)-7 (51:49, respectively) was obtained
solution of (S)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-3-hexanone

J Chem Ecol (2008) 34:408–417
413
in 92% yield from (R)-2-[(tetrahydro-2H-pyran-2-yl)oxy]-
in 0.025 ml of hexanes) in uncapped 1-dram vials that were
hung in the open central slot of each trap (Lacey et al.
2007b). The study was replicated five times as described
above. Too few beetles were captured in both the 2004 and
2005 studies to warrant statistical analysis (see “Results”).
Field bioassays of blends of synthetic compounds were
conducted in an abandoned plantation of mixed tree species
∼1 km south of the campus of the UIUC in April to May
2006 (skies clear, air temperatures ∼19–28°C, wind speed
∼8–20 kph). A subtractive scheme was used to compare
activity of the complete blend with seven treatments that
each lacked a different component, as well as a solvent
control. We used the same panel traps and lures as
described above. The lures were baited with pheromone
components, each diluted in 0.025 ml hexane, in ratios that
approximated the mean relative abundances in extracts
from aerations of males (see “Results”) with two excep-
tions: (1) The relative proportions of hexanediols were
increased because other cerambycine species had responded
to relatively high quantities of similar compounds (see Lacey
et al. 2004, 2007b) and (2) the total proportion of citral was
decreased to compromise between the high proportion of
geranial and lower proportion of neral that are produced by
males (see “Results”). Lure quantities were: (2S,3R)-2,3-
hexanediol (2.5 mg), (2R,3S)-2,3-hexanediol (0.5 mg),
(S)-(−)-limonene (1.5 mg), 2-phenylethanol (5 mg), (−)-α-
terpineol (5 mg), nerol (2 mg), citral (12 mg), and the
control (0.175 ml hexane). Traps were positioned 8 m apart
in transects approximately perpendicular to the direction of
the prevailing wind. On each date, traps were baited at
10:00 hours, and beetles were collected at ∼18:00 hours.
Traps were cleaned with glass cleaner (Windex®, S. C.
Johnson & Sons, Racine, WI) and lures replaced each
day. The bioassay was replicated 14 times. Overall dif-
ferences between treatments in numbers of beetles cap-
tured were tested by one-way analysis of variance (ANOVA),
and differences between pairs of treatment means were
subsequently tested with the least significant difference
test (Analytical Software 2000). Three replicates that
captured fewer than four beetles were excluded from the
data analysis.
3
-hexanol (R)-6. After purification, 2.51 g of (2R,3S)-2,3-
hexanediol (de>97%) and 2.41 g (2R,3R)-2,3-hexanediol
de>98%) were obtained. NMR and mass spectra matched
(
those of the (2S,3R)- and (2S,3S)-enantiomers, respectively.
Field Bioassays of Synthetic Pheromone Aeration extracts
from male M. caryae contained eight male-specific com-
pounds (see “Results”). Initially, we predicted that the
hexanediols would have the greatest activity, based on
pheromones that have been identified for closely related
beetle species (see “Introduction”). Therefore, field bio-
assays of the individual components (including citral in
place of neral and geranial) were first conducted before
mixtures of compounds were tested. Bioassays were
conducted at Allerton Park (Piatt, IL), a 600-ha mixed
hardwood forest that harbors a population of M. caryae,
between 22 and 31 May 2004 (skies clear, air temperatures
∼
19–28°C, average wind speed 8–20 kph). Sticky card
traps (“mouse glue trap” cards, 12.6×22 cm, baited,
Victor® Pest Control Products, Lititz, PA) were stapled to
1
.5-m-tall wooden stakes, with the middle of the card ∼1 m
above the ground. Traps were positioned 10 m apart in a
straight line approximately perpendicular to the prevailing
wind direction. Traps were baited with a cotton dental wick
that was loaded with 5 mg of the compound in 0.1 ml of
methylene chloride and attached to the middle of the card.
There were seven treatments that were randomly assigned
to traps, as follows: racemic (2R*,3S*)-2,3-hexanediol, (S)-
(
−)-limonene, 2-phenylethanol, (−)-α-terpineol, nerol, cit-
ral, and a solvent control. Release rates of these lures were
unknown, but similar lures loaded with 2,3-hexanediols
remained attractive to N. a. acuminatus for at least 18 hr
(
Lacey et al. 2004). The study was replicated five times.
For each replication, traps were set up at 11:00 hours,
captured beetles were removed from traps the following
morning, and the numbers and sex of beetles responding to
treatments were recorded.
The 2004 bioassay was conducted late in the activity
period for M. caryae, when populations were declining, and
trap catches were low (see “Results”). Therefore, the
bioassay was repeated on 8–20 May 2005 at the same
site (skies clear, air temperatures ∼16–27°C, wind speed
8
–20 kph). By that time, we had discovered that adult
Results
beetles could extract themselves from sticky cards (Lacey
et al. 2007b) and, therefore, adopted cross-vane flight-
intercept panel traps (black, 1.2×0.30 m, Intercept™,
model PT, APTIV, Portland, OR) that had proven efficient
in capturing other species of cerambycid beetles (Lacey et
al. 2007b). Traps were positioned as described above, and
test chemicals were applied neat to cotton dental wicks
Testing for Attractant Pheromones In olfactometer bio-
assays, 100% of female and 90% of male M. caryae
responded by walking upwind. Of the 20 females that were
tested, 19 were attracted to odor emitted by males and only
one responded to odor of females. Males showed a similar
response, with 16 responding to odor of males and only two
2
(
except [2R*,3S*]-2,3-hexanediols, which were dissolved
responding to odor from females (χ 1, 18=21.8, P<0.001).

4
14
J Chem Ecol (2008) 34:408–417
Identification of Potential Pheromone Components GC-MS
analysis of volatiles produced by male M. caryae revealed
seven peaks that were absent in analogous samples of
females. The first compound to elute (Fig. 2) was ten-
tatively identified as 2,3-hexanediol, with a mass spectral
base peak at m/z 55 (100) and characteristic mass fragments
at m/z 75 (10), 73 (61), 72 (34), 45 (36), and 43 (35). The
identification was confirmed by matching the mass spec-
trum and the retention time with those of an authentic
standard of (2R*,3S*)-2,3-hexanediol; this diastereomer is
completely resolved from the (2R*,3R*)-2,3-diastereomer
on a DB5-MS column (Lacey et al. 2004). The enantio-
meric composition of the insect-produced compound was
determined to be a ∼10:1 mix of (2S,3R)- and (2R,3S)-2,3-
hexanediols by analysis on the Cyclodex-B column, with
baseline resolution of the enantiomers. These diols repre-
sented 3.2±1.3% (N=7 aeration extracts) of the total male-
specific compounds. The remaining compounds, in order of
elution (Fig. 2), were (S)-(−)-limonene (3.1±1.7%), 2-
phenylethanol (8.0±2.4%), (−)-α-terpineol (10.0±2.8%),
nerol (2.1±1.5%), neral (63.3±7.3%), and geranial (8.8±
2.4%). These compounds were tentatively identified by
mass spectral fragmentation patterns, and the identifications
were confirmed by retention time and mass spectral
matches with authentic standards. The absolute configu-
rations of those compounds that were chiral were deter-
mined by analyses and coinjections with appropriate
standards on the Cyclodex-B GC column. Relative abun-
dances of compounds varied between samples, with neral,
2-phenylethanol, (−)-α-terpineol, and geranial always being
most abundant, whereas the hexanediols, (S)-limonene, and
nerol were not detectable in some samples. We have found
that beetles of other cerambycine species also produce
pheromone sporadically under the conditions of aeration in
closed chambers (unpublished data).
Syntheses of Pheromone Components Chiral 2,3-hexanediols
were synthesized by modification and extension of the
strategy developed by Hall et al. (2006) to produce chiral
2-hydroxy-3-decanones (Fig. 1). Thus, the alcohol function
Fig. 2 Representative total ion
chromatograms of extracts of
headspace volatiles produced by
male (top) and female (bottom)
Megacyllene caryae

J Chem Ecol (2008) 34:408–417
415
of ethyl (S)-(−)-lactate (S)-1 was protected as the tetrahy-
dropyranyl (THP) ether (S)-2, followed by hydrolysis of
the ester group to give the lithium salt of the carboxylic
acid (S)-3 in quantitative yield. Alkylation of the salt
with propyllithium in ether then yielded the THP-protected
traps captured a total of five in 2005. We concluded from
this experiment that probably neither the 2,3-hexanediols
nor any of the other compounds in the volatile blend
produced by males were attractive to adult M. caryae as
single components.
2
-hydroxy-3-hexanone (S)-4. (2S,3R)- and (2S,3S)-2,3-
hexanediols were produced by reduction of THP-protected
-hydroxy-3-hexanone (S)-4 with sodium borohydride in
In bioassays that tested blends of components in 2006,
traps captured 147 adult M. caryae (76 females and 71
2
2
ethanol, giving the monoprotected diols mixture (S)-6.
Acid-catalyzed removal of the THP group gave a diaste-
reoisomeric mixture of (2S,3R)-2,3-hexanediol [(2S,3R)-7)
and (2S,3S)-2,3-hexanediol ((2S,3S)-7]. The diastereomers
were separated by flash chromatography on silica gel
followed by recrystallization from hexanes, giving the two
diols in high purity (>96% de). Analogous results were
obtained for the syntheses of (2R,3R)-2,3-hexanediol
males; sex ratio not significantly different from 1:1, χ 1, 147=
0.17, P>0.05). Treatments differed significantly in numbers
of M. caryae that were captured (Fig. 3; sexes combined,
overall ANOVA F10, 147=9.56, P<0.001). The greatest
numbers of beetles were captured in traps baited with the
complete blend of pheromone components, whereas trap
catches were reduced by ∼50% in treatments that were
missing (S)-(−)-limonene, 2-phenylethanol, (−)-α-terpineol,
or nerol and reduced to levels not significantly different from
controls in treatments missing (2S,3R)-2,3-hexanediol,
[
(2R,3R)-7) and (2R,3S)-2,3-hexanediol ((2R,3S)-7] starting
from THP-protected hydroxyketone (R)-4.
(2R,3S)-2,3-hexanediol, or citral (Fig. 3).
Alternatively, removal of the THP group from compound 4
with acid catalysis in methanol gave (R)- and (S)-2-
hydroxy-3-hexanones, (R)- and (S)-5, both of which are
known pheromone components for cerambycid beetles (see
Hanks et al. 2007). GC analysis on a Cyclodex-B column
confirmed that no isomerization or racemization (see Lacey
et al. 2007b) had occurred during the synthesis.
Discussion
Attraction of both sexes of M. caryae to odor produced by
live males in olfactometer bioassays provided the first
evidence that males produce an aggregation pheromone.
That this was an aggregation pheromone and not a sex
pheromone was confirmed during field bioassays with
synthetic pheromone, in which similar numbers of males
and females were attracted to baited traps.
Field Bioassays of Synthetic Pheromone In field bioassays
that tested the activity of individual compounds, sticky
traps captured a total of two M. caryae in 2004, and panel
Fig. 3 Relationship between
mean (±SEM) number of adult
Megacyllene caryae caught in
cross-vane panel traps (sexes
combined) and composition of
the lure. Treatments included the
complete blend of synthetic
components (All), and the
complete blend minus (2S,3R)-
2
,3-hexanediol, (2R,3S)-2,3-
hexanediol, (S)-(−)-limonene,
-phenylethanol (phoh),
2
(
(
−)-α-terpineol, nerol, and citral
a 1:1 blend of neral and
geranial), and a solvent control.
Means with different letters are
significantly different (LSD
tests; P<0.05)

4
16
J Chem Ecol (2008) 34:408–417
Although (2S,3R)- and (2R,3S)-2,3-hexanediol consti-
compound, 1-phenylethanol, is produced from metasternal
glands of a congener of M. caryae, M. robiniae, and has
been suggested to serve in defense against natural enemies
(Wheeler et al. 1988). The fact that the terpenoids of M.
caryae are produced by only one sex suggests that they are
unlikely to have a defensive function.
tuted only about 3 and 0.3%, respectively, of the total blend
of volatile compounds produced by males, absence of either
in synthetic blends resulted in a significant loss of activity.
This finding indicated that adult M. caryae are sensitive to
both components, unlike other clytine species that produce
blends of enantiomers but respond strongly to the dominant
component alone (see Hanks et al. 2007). Among the
remaining components, one or both isomers of citral (neral
or geranial) were equally as important to attraction as were
the 2,3-hexanediols. Whereas the remaining components
The preparation of (2S,3R)- and (2S,3S)-2,3-hexanediols
(and their enantiomers) in high diastereomeric and enan-
tiomeric purity took advantage of the fact that during
nonselective syntheses of mixtures of all four diol stereo-
isomers by the reduction of 2,3-hexanedione (Hanks et al.
2007), we observed that the diastereomeric 2,3-hexanediols
were readily separable in gram quantities by flash chroma-
tography. Thus, we reasoned that nonselective reduction
of the ketone function of a chiral, THP-protected 2-
hydroxy-3-hexanone precursor, in which the configuration
at the carbon bearing the protected alcohol was fixed and
known, should produce a mixture of the two diastereomers,
each in high enantiomeric purity. Reduction of THP-
protected (S)-2-hydroxy-3-hexanone (S)-4 gave an approx-
imately 1:1 ratio of the two expected alcohol products.
Removal of the THP-protecting group and separation of the
resulting diol diastereomers by flash chromatography gave
the (2S,3R)- and (2S,3S)-stereoisomers in quantities of
several grams. Analogous reaction of the THP-protected
(R)-2-hydroxy-3-hexanone (R)-4 gave the other two stereo-
isomers. Thus, this synthetic strategy provided ready access
to all four 2,3-hexanediols in high stereoisomeric purity
from readily available lactate ester synthons. In contrast,
our previous syntheses of 2,3-hexanediol stereoisomers
using the Sharpless asymmetric dihydroxylation protocols
(Kolb et al. 1994) had produced diols with only 80–90% ee
(Lacey et al. 2004). The synthetic strategy described above
also has advantages in comparison to previously published
syntheses of the 2,3-hexanediol stereoisomers, in which
each stereoisomer was synthesized individually from a
different chiral precursor (Schröder et al. 1994). It should
also be noted that exactly the same strategy can be applied
to the syntheses of the homologous 2,3-octanediols and
2,3-decanediols, compounds that also may be pheromone
components of cerambycid beetles (e.g., Hall et al. 2006).
This straightforward method of producing multigram
quantities of all four stereoisomers of 2,3-alkanediols
of any desired chain length should prove beneficial in
the unraveling of pheromone blends for other ceram-
bycid species.
(
[S]-[−]-limonene, 2-phenylethanol, [−]-α-terpineol, and
nerol) increased attraction significantly, they were not
necessary for attraction because beetles showed a partial
response to blends lacking these compounds. The increased
response of adult M. caryae to the complete blend of
synthetic compounds confirmed that all of these com-
pounds have a role in the natural pheromone blend.
The structure of the hexanediols produced by male M.
caryae is consistent with the diol/hydroxyketone structural
motif of pheromones reported for another 12 species in the
subfamily Cerambycinae (see “Introduction”). To our
knowledge, this motif is unique to cerambycine beetles.
Whereas exceptions to the motif have been identified from
two cerambycine species (see “Introduction”), the complex
blend of compounds produced by male M. caryae, in
addition to the components that are consistent with the
structural motif, is unprecedented among cerambycines that
have been studied to date.
All of the terpenoids and 2-phenylethanol produced by
male M. caryae have been identified as pheromones of
species in a broad range of insect and arachnid orders,
serving a variety of behavioral functions (see Mayer and
McLaughlin 1991). These compounds are also common
in essential oils of plants (Budavari 1996), suggesting that
beetles might acquire them from their host plants.
However, it seems more likely that beetles produce the
compounds de novo because adult male M. caryae from
which volatile compounds were collected had been reared
in the laboratory and so had not been exposed to host plants
of adults.
Previous research suggests that the compounds produced
by male M. caryae may have semiochemical functions in
other species of cerambycids. For example, in electro-
antennograph studies of potential host plant volatiles,
antennae of both sexes of Xylotrechus pyrrhoderus Bates
and Arhopalus tristis (F.) responded to citral and α-
terpineol, respectively (Iwabuchi et al. 1985; Suckling et
al. 2001). Both α-terpineol and limonene were repellent to
adult Semanotus japonicus Lacordaire in choice bioassays
Acknowledgments This project was supported by funds from the
Exotic/Invasive Pests and Diseases Research Program, University of
California, under USDA-CSREES Grant no. 2004-34439-14691, the
Alphawood Foundation, and Hatch Project no. CA-R*ENT-5181H to
JGM. This research was in partial fulfillment of a Ph.D. degree for
ESL from UIUC.
(
Yatagai et al. 2002). Male Xylotrechus quadripes Chevro-
lat produce 2-phenylethanol, although it does not appear to
be a pheromone component (Hall et al. 2006). A similar

J Chem Ecol (2008) 34:408–417
417
longhorned beetle Neoclytus mucronatus mucronatus. Entomol.
Exp. Appl. 122:171–179.
References
LINSLEY, E. G. 1964. The Cerambycidae of North America, Part V:
Taxonomy and classification of the subfamily Cerambycinae,
Tribes Callichromini through Ancylocerini. Univ. Calif. Publ.
Entomol. 22:1–197.
MAYER, M. S., and MCLAUGHLIN, J. R. (eds.). 1991. Handbook of
Insect Pheromones and Sex Attractants. CRC, Boca Raton, FL.
RAY, A. M., LACEY, E. S., and HANKS, L. M. 2006. Predicted
taxonomic patterns in pheromone production by longhorned
beetles. Naturwissenschaften 93:543–550.
REDDY, G. V. P., FETTKÖTHER, R., NOLDT, U., and DETTNER, K. 2005.
Capture of female Hylotrupes bajulus as influenced by trap type
and pheromone blend. J. Chem. Ecol. 31:2169–2177.
SCHRÖDER, F., FETTKÖTHER, R., NOLDT, U., DETTNER, K., KÖNIG, W.
A., and FRANCKE, W. 1994. Synthesis of (3R)-3-hydroxy-2-
hexanone, (2R,3R)-2,3-hexanediol and (2S,3R)-2,3-hexanediol,
the male sex pheromone of Hylotrupes bajulus and Pyrrhidium
sanguineum (Cerambycidae). Liebigs Annalen der Chemie
ANALYTICAL SOFTWARE. 2000. Statistix 7.1. Analytical Software,
Tallahassee, FL.
BUDAVARI, S. (ed.). 1996. The Merck Index. Merck, Whitehouse
Station, NJ.
DUSHAM, E. H. 1921. The painted hickory borer. Bull. Cornell Univ.
Ag. Ext. Stn. 407:170–203.
HALL, D. R., CORK, A., PHYTHIAN, S. J., CHITTAMURU, S., JAYARAMA,
B. K., VENKATESHA, M. G., SHEEDHARAN, K., VINOD KUMAR, P.
K., SEETHARAMA, H. G., and NAIDU, R. 2006. Identification of
components of male-produced pheromone of coffee white stem-
borer Xylotrechus quadripes. J. Chem. Ecol. 32:195–219.
HANKS, L. M., MILLAR, J. G., MOREIRA, J. A., BARBOUR, J. D.,
LACEY, E. S., MC ELFRESH, J. S., REUTER, F. R., and RAY, A. M.
2
007. Using generic pheromone lures to expedite identification of
aggregation pheromones for the cerambycid beetles Xylotrechus
nauticus, Phymatodes lecontii, and Neoclytus modestus modestus.
J. Chem. Ecol. 33:889–907.
1
994:1211–1218.
IWABUCHI, K., TAKAHASHI, J., NAKAGAWA, Y., and SAKAI, T. 1985.
Electroantennogram responses of grape borer Xylotrechus
pyrrhoderus Bates (Coleoptera: Cerambycidae) to its male sex
pheromone components. J. Chem. Ecol. 11:819–828.
KOLB, H. C., VANNIEUWENHZE, M. S., and SHARPLESS, K. B. 1994.
Catalytic asymmetric dihydroxylation. Chem. Rev. 94:2483–2547.
LACEY, E. S., GINZEL, M. D., MILLAR, J. G., and HANKS, L. M. 2004.
Male-produced aggregation pheromone of the cerambycid beetle
Neoclytus acuminatus acuminatus. J. Chem. Ecol. 30:1493–1507.
LACEY, E. S., RAY, A. M., and HANKS, L. M. 2007a. Calling behavior
of the cerambycid beetle Neoclytus acuminatus acuminatus. J.
Insect Behav. 20:117–128.
SOKAL, R. R., and ROHLF, F. J. 1995. Biometry, 3rd edn. Freeman,
New York, NY.
SUCKLING, D. M., GIBB, A. R., DALY, J. M., CHEN, X., and
BROCKERHOFF, E. G. 2001. Behavioral and electrophysiological
responses of Arhopalus tristis to burnt pine and other stimuli. J.
Chem. Ecol. 27:1091–1104.
WHEELER, J. W., ABRAHAM, M., HIGHET, R. J., and DUFFIELD, R. M.
1988. Metasternal gland secretion of the locust tree borer,
Megacyllene robiniae (Coleoptera: Cerambycidae). Comp. Bio-
chem. Physiol. 91B:771–775.
YATAGAI, M., MAKIHARA, H., and OBA, K. 2002. Volatile components
of Japanese cedar cultivars as repellents related to resistance to
Cryptomeria bark borer. J. Wood Sci. 48:51–55.
LACEY, E. S., MOREIRA, J. A., MILLAR, J. G., RAY, A. M., and HANKS,
L. M. 2007b. Male-produced aggregation pheromone of the