Identification, synthesis, and bioassay of a male-specific aggregation pheromone from the harlequin bug, Murgantia histrionica

J Chem Ecol (2008) 34:238–251

DOI 10.1007/s10886-007-9415-x

Identification, Synthesis, and Bioassay of a Male-Specific

Aggregation Pheromone from the Harlequin Bug,

Murgantia histrionica

Deane K. Zahn & Jardel A. Moreira & Jocelyn G. Millar

Received: 15 January 2007 /Revised: 28 October 2007 /Accepted: 4 December 2007 /Published online: 19 January 2008

#

Springer Science + Business Media, LLC 2007

Abstract Sexually mature male harlequin bugs produced

a sex-specific compound, identified as one of the

stereoisomers of the sesquiterpene epoxyalcohol 4-[3-(3,3-

dimethyloxiran-2-yl)-1-methylpropyl]-1-methylcyclohex-2-

en-1-ol (henceforth murgantiol), a compound with four

chiral centers and 16 possible stereoisomers. Production of

the compound was highest during the middle of the day.

Individual virgin male bugs in separate containers pro-

duced the compound at a higher rate than virgin males in

groups. The carbon skeleton was verified by synthesis of

several mixtures which, in total, contained all possible

isomers, one of which matched the insect-produced

compound. The relative and absolute configurations of

the insect-produced compound remain to be determined. In

laboratory bioassays, insect-produced and synthetic mur-

gantiol attracted harlequin bugs of both sexes, suggesting

that murgantiol is a male-produced aggregation phero-

mone, analogous to those found in a number of other

phytophagous bug species.

ing this and other stink bug species, such as sampling the

crop by using a sweep net or a beating tray, or by visual

inspection of damage or presence of insects, are typically

labor-intensive. Sampling at frequent intervals may be

required to detect immigration of bug populations into

crops, so that control measures can be applied before

significant damage occurs. Consequently, attractive sex or

aggregation pheromones used as baits in monitoring traps

could prove of value in integrated pest management

programs.

To date, pheromones have been identified for only a few

stink bug species. For all phytophagous pentatomids for

which sex or aggregation pheromones have been identified,

the compounds were produced by males (reviewed in

McBrien and Millar 1999; Ho and Millar 2001a, b; Millar

2005). In addition to using sex-specific pheromones,

phytophagous stink bugs use species- and sex-specific

substrate-borne vibrations for intraspecific communication

over relatively short distances (Čokl and Doberlet 2003).

The vibrational signals of M. histrionica were recently

described (Čokl et al. 2004), with males producing five

different vibrational songs and females producing only one

song. Contrary to those found for most stink bugs

investigated, the vibrational signals did not seem to play

a role in orientation toward the source of the vibration (i.e.,

mate location). Rather, the vibrational signals appeared to

function in close-range mate recognition and courtship,

suggesting that signals other than vibrational signals may

be important in bringing the two sexes together over

greater distances.

.

Keywords Aggregation pheromone Heteroptera

.

Hemiptera 4-[3-(3,3-dimethyloxiran-2-yl)-1-

.

methylpropyl]-1-methylcyclohex-2-en-1-ol Murgantiol

Introduction

The harlequin bug, Murgantia histrionica (Hahn), is an

important pest of cabbage, broccoli, and other cole crops in

the United States (McPherson 1982). Methods for monitor-

Harlequin bugs produce or sequester a variety of

chemicals. For example, the metathoracic scent gland

secretion of adult M. histrionica consists of (2E,6E)-

octadienedial and (2E,6E)-octadiene-1,8-diol diacetate

(Aldrich et al. 1996). Additionally, when adults are

:

D. K. Zahn J. A. Moreira J. G. Millar (*)

Department of Entomology, University of California,

Riverside, CA 92521, USA

e-mail: Jocelyn.millar@ucr.edu

J Chem Ecol (2008) 34:238–251

239

squeezed, a sticky froth is expelled from the margins of the

prothorax. This fluid contains aglycones of glucosinolates,

sec-butyl isothiocyanate, methylthiopropyl nitrile, and

methylthiobutyl nitrile, as well as two strong-smelling

alkylmethoxypyrazines, 2-sec-butyl-3-methoxypyrazine

and 2-isopropyl-3-methoxypyrazine, which have been

hypothesized to serve as warning odors (Aldrich et al.

1996). These compounds are likely derived from glucosi-

nolates sequestered from host plants, providing the vividly

colored aposematic bugs with a formidable chemical

defense against predation. The bugs can retain these

compounds for an extended period of time (Aliabadi et al.

2002). However, none of these compounds has been

reported to have a role as pheromones.

We describe herein the collection and analysis of volatile

compounds released by live adult M. histrionica of both

sexes. In addition to a number of compounds common to

both sexes, one major sex-specific compound was found in

the odors released by sexually mature adult males. This

compound functions as an aggregation pheromone compo-

nent for the harlequin bug.

which bugs could perch. Humidified, charcoal-filtered air

(6–14 mesh activated charcoal granules) was drawn

through the chamber by vacuum at 250 ml per min.

Volatile chemicals were collected overnight on activated

charcoal traps made from 4 mm ID glass tubes loaded with

a 0.4-cm bed of 50–200 mesh activated charcoal (Cat. #

05–690A, Fisher Scientific, Pittsburgh, PA, USA), pre-

cleaned by heating at 200°C under a flow of clean N₂

(~100 ml per minute). Aerations of groups of bugs were

conducted continuously for 2–3 weeks at ~25°C and 50%

RH with cohorts of male and female bugs of known age,

changing the food and the collectors every other day.

Aerations of individual bugs were conducted in the same

way, but using smaller, 200-ml chambers. All aeration

chambers were within the main colony rearing room.

Additional cohorts of sexed virgin bugs, of the same age

as those in the chamber, were maintained and used to

replace any bugs in the chamber that died. As a control, two

to three cauliflower florets were put in a 1-l aeration

chamber and aerated for 20 h with an air flow rate of 1 l per

minute. Trapped volatiles were stripped off the activated

charcoal by elution with methylene chloride (500 μl;

Optima grade, Cat. # D151–4, Fisher Scientific, Pittsburgh

PA, USA), and the resulting extracts stored in glass vials

with Teflon-lined screw caps at ~−20°C until needed.

Materials and Methods

Insect Colonies Adults and nymphs of M. histrionica were

collected from bladderpod, Isomeris arborea Nutt. (Caper-

aceae), from one site each in Riverside and San Diego, CA,

USA. A colony was started in the laboratory in 2003 and

augmented every year since with approximately 100 males,

females, and nymphs from each location. Voucher speci-

mens were submitted to the University of California,

Riverside Entomology Research Museum (UCRC

145863–145882). Insects were reared at 26 1°C, approx-

imately 45% RH, with a photoperiod of 16:8 h (L:D)

provided by fluorescent lights. Immature insects were

held separately from adults in cylindrical plastic contain-

ers (20×15 cm), with two 4-cm holes on opposite sides

of each container covered with brass screening. Adults

were held in 30×17×18 cm glass-topped rearing cages.

Insects were provided with Napa cabbage, cauliflower,

and broccoli three times per week. Upon eclosion, adults

were sexed and maintained for 14 d with florets of

broccoli in 175 ml transparent plastic cups, or in groups

in 3.8-l cardboard ice-cream cartons with ventilated lids,

before use.

Single-bug and group (5, 10, and 15) aerations were set

up simultaneously to determine whether the male-specific

compound was released at different rates by individuals or

groups. For this, bugs were either held individually (N=20)

or in groups (N=20 each group) for 2 weeks before use.

Traps were changed every other day and eluted with 500 μl

of CH₂Cl₂. The amounts of male-specific compound per

bug hour were analyzed by one-way ANOVA and means

were separated by Games–Howell tests.

To determine the diurnal cycle of release of the male-

specific compound, five cohorts of five virgin, sexually

mature, adult males (14–16 days old), and three florets of

cauliflower were aerated in chambers as described above.

After a cohort of bugs was confirmed to be producing the

male-specific compound, the traps were changed every 2 h

during the photophase. A single trap was used to collect

volatiles produced between 23:00 and 07:00. An external

standard of hexadecane in CH₂Cl₂(1.6 µg/ml) was used to

estimate the amount of male-specific compound present in

each extract. The total amounts of the male-specific

compound produced during each 24-h period were calcu-

lated along with the percentage of the total produced during

each 2-h interval. For the 8-h period between 23:00 and

07:00, the average amount per 2 h was calculated. The

percentage data from all cohorts were averaged, analyzed

by ANOVA, and means were separated by Student–New-

man–Keuls tests.

Collection of Insect-Produced Compounds from Live

Bugs A system for collecting volatile compounds from live

organisms was set up as previously described (Millar and

Sims 1998). Ten to fifteen virgin adult bugs and two large

cauliflower florets were put into a two-part, 400 ml

cylindrical glass aeration chamber, lined with screen upon

240

J Chem Ecol (2008) 34:238–251

General Procedure for Y-tube Bioassays Bioassays were

conducted with a vertical glass Y-tube (5 cm ID; bottom

arm=18 cm long, top arms=15 cm long, Y angle=90°)

with female ground glass joints, each fitted with male joints

terminating in a hose nipple. The Y-tube was illuminated

from above with a 1.2-m long light bank containing a

Sylvania Octron 3500K F032/735 32 W fluorescent light

and a GE F40PL/AQ Plant and Aquarium wide spectrum

fluorescent light, providing light intensities of 350 lux at

the bottom to 600 lux at the top of the Y-tube. Charcoal-

purified air was pulled through the apparatus (2.5 liter per

minute) by vacuum. Bugs were tested individually and used

only once. Each bug was gently placed inside the bottom

male joint of the Y-tube, which was then inserted into the

female joint. M. histrionica are positively phototactic and

negatively geotactic, and generally walked up toward the

light. A positive response was classified as a bug crossing a

line 12 cm up either arm from the junction. If a bug did not

move within 10 min of the start of the bioassay, it was

discarded. Each Y-tube and joint was used once, then

washed, acetone rinsed, and oven-dried at 140°C. Bio-

assays were conducted in a temperature (22–25°C) and

humidity (40–50% RH) controlled room, 4 to 8 h after the

onset of photophase. Positions of treatments were alternated

every second replicate. Choice data were analyzed with chi-

square tests with one degree of freedom and α=0.05.

diameter) mounted on wires in the top of each arm of the Y-

tube. Five bug-hours of extract contained ~30 ng of the

major male-specific component, as determined by gas

chromatography.

A pooled extract of male-produced odors was separated

into eight fractions (see below), and the fraction containing

the major male-specific compound (5 bug-hour equivalents)

was bioassayed against a CH₂Cl₂control. Equal aliquots of

the remaining seven fractions were combined and tested

against a CH₂Cl₂control. Having determined that only the

fraction containing the major male-specific compound was

attractive to females, this fraction was then tested against an

equivalent amount of crude extract.

Mixtures containing synthetic enantiomers (see below)

of the major male-specific compound were tested using 14-

day-old sexually mature adults of both sexes. The mixture

derived from (S)-citronellal was tested vs a solvent control,

and both synthetic mixtures were tested vs the crude

extract. Finally, equal doses of the two synthetic mixtures

were tested against each other. The mixtures (240 ng of

each mixture of four stereoisomers, ~60 ng/isomer), in

CH₂Cl₂, and solvent controls, were loaded onto filter paper

discs as described above.

Gas Chromatography (GC) and Mass Spectrometry (MS)

Analyses of Extracts Extracts were concentrated under a

gentle stream of nitrogen, and analyzed by splitless gas

chromatography with a Hewlett–Packard (H–P) 5890 GC

fitted with a DB-5 column (30 m×0.32 mm ID×0.25 μm

film, J&W Scientific, Folsom, CA, USA), using a temper-

ature program of 40°C for 1 min, then 15°C min⁻¹to 250°

C. The injector and detector temperatures were 250°C and

280°C, respectively, and helium was used as carrier gas.

Extracts were also analyzed by GC-MS, using an HP 5890

GC, fitted with a DB5-MS column (20 m×0.2 mm ID×

0.33 μm film), interfaced to an HP 5970B mass selective

detector (electron impact ionization, 70 eV). The same

temperature program described above was used, with

injector and transfer line temperatures of 250 and 280°C,

respectively. Injections (1 µl) were made in splitless mode.

Previously known compounds were tentatively identified

from their mass spectra, and from matches with the NBS-

NIH mass spectral database. Identifications were confirmed

by matching retention times and mass spectra with those of

authentic standards (see below).

The Y-tube olfactometer was also used to determine

which sex of adult M. histrionica produced pheromone.

The top joints of the Y-tube were connected to 200 ml glass

aeration chambers that contained five sexually mature

virgin bugs and two cauliflower florets, or two cauliflower

florets alone. Chambers were positioned above the Y-tube,

behind a cardboard screen so contents were not visible to

the bugs in the Y-tube. Ten minutes after the chambers were

set up, a test bug was introduced into the bottom of the Y-

tube and its response recorded by an observer and on video

tape (Panasonic high-density recording, time lapse video

recorder, model AG-6740p). A fresh group of five unmated,

sexually mature bugs was used as a pheromone source for

each day of bioassay. Four separate experiments were

conducted, testing the responses of females to odors from

males, females to odors from females, males to odors from

females, and males to odors from males. The responses of

40 bugs were tested in each experiment. Because females

did not attract males, but males attracted females, only

females were used as responders in the subsequent experi-

ments, except where indicated otherwise.

An aliquot of the purified male specific compound was

also analyzed by chemical ionization mass spectrometry

(NH₃reagent gas) by direct insertion on a VG-7070B

instrument.

The numbers of virgin female M. histrionica attracted to

5 bug-hour equivalents (1 bug-hour equivalent=the vola-

tiles collected from 1 bug in 1 h) of crude extract in CH₂Cl₂

vs a CH₂Cl₂blank were compared. Aliquots of extract or

solvent were loaded on half-circular filter papers (1.5 cm in

Fractionation of Extracts of Volatiles Collected from

Sexually Mature Male Bugs Thirty aeration extracts of

J Chem Ecol (2008) 34:238–251

241

sexually mature male bugs, prepared from May 2003 to

August 2005, were combined and concentrated to ~1 ml

by fractional distillation of the solvent through an 8-cm

long Vigreux column. The concentrated extract was

transferred to a vial and the solvent removed under a

gentle stream of nitrogen. The sample was reconstituted to

~250 μl with pentane, and then loaded onto a 2.8-ml solid

phase extraction (SPE) cartridge (J.T. Baker silica gel

SPE, stock #7086–01, precleaned by flushing with 3×

1 ml diethyl ether, then 3×1 ml pentane). The extract was

rinsed onto the SPE bed with two to three drops of

pentane, and eluted with 2×1 ml pentane, 2×1 ml 10%

ether in pentane, and 4×1 ml ether, collecting each elution

as a separate fraction.

NMR Analysis of the Male-Specific Compound Forty-two

extracts were combined, concentrated, and fractionated as

described above, and the fraction containing the male-

specific was compound concentrated under a gentle

stream of nitrogen, taking the solution just to dryness.

Approximately 200 µl of CD₂Cl₂were added, and the

solution was concentrated just to dryness again to remove

traces of non-deuterated solvent. The residue (estimated at

>100 µg) was then taken up in CD₂Cl₂and transferred to a

3-mm NMR tube. ¹³C, H, and H-¹H COSY spectra were

obtained with both a Varian INOVA-400 and a Brucker

600 MHz NMR spectrometer; the latter instrument was also

used to obtain NOESY, ¹³C-DEPT 135, HMBC, and HSQC

spectra.

1

Aliquots (100 µl) of crude extract were subjected to a

series of microchemical tests to ascertain characteristics of

the male-specific compound.

Chemicals Cineole, limonene, myrcene, dodecane, and

dimethyldisulfide were purchased from Aldrich Chemical

Co. (Milwaukee, WI, USA). Dimethyltrisulfide was

obtained from Alfrebro (Monroe, OH, USA). The bisabo-

lene epoxide pheromones of Acrosternum hilare were

available from previous studies (McBrien et al. 2001).

The sex-specific compound produced by male harlequin

bugs was synthesized as described below.

a) To test for the presence of a conjugated diene, an

aliquot was treated with two drops of a solution of the

dienophile, 4-methyl-1,2,4-triazolin-3,5-dione (MTAD;

Young et al. 1990) in CH₂Cl₂(2 mg/ml), producing a

pale pink solution which was analyzed immediately by

GC-MS.

b) To test for an ester function, an aliquot was concen-

trated just to dryness, and then treated with three drops

of 1 M NaOH in ethanol to water (95:5). After standing

at room temperature for 4 h, 200 µl of water were

added and the mixture extracted with 500 µl pentane.

The pentane extract was dried over Na₂SO₄, concen-

trated under a stream of nitrogen, and analyzed by GC-

MS.

c) To test for an alcohol function, an aliquot was treated

with one drop each of pyridine and acetyl chloride. The

resulting mixture was stirred for 2 h, treated with

200 µl of saturated aqueous NaHCO₃, and extracted

with 0.5 ml pentane. The pentane extract was concen-

trated and analyzed as above.

d) To test for reducible functional groups, an aliquot in

diethyl ether was treated with ~1 mg of LiAlH₄, with

stirring at room temperature for 2 h. The mixture was

quenched with 1 M HCl and extracted with 0.5 ml

pentane. The pentane extract was dried over Na₂SO₄,

concentrated, and analyzed.

For the syntheses, unless stated otherwise, all reactions

were carried out under argon. Tetrahydrofuran (THF) used

in the synthesis was distilled from sodium/benzophenone

ketyl under argon. ¹H- and ¹³C-NMR spectra were taken as

CDCl₃or CD₂Cl₂solutions, recorded on a Varian INOVA-

400 (400 and 100 MHz, respectively) spectrometer.

Chemical shifts are expressed in parts per million (ppm)

(δ-scale) relative to CDCl₃(7.26 and 77.23 ppm) and

CD₂Cl₂(5.32 and 54.00 ppm) signals for ¹H and ¹³C NMR,

respectively. Routine GC analyses were performed with an

HP 5890 series II GC, equipped with a DB-5 column

(30 m×0.25 μm ID, 0.25 μm film thickness, J&W

Scientific, programmed from 100°C for 1 min, then at 15°

C min⁻¹to 250°C) or a DB-WAX column (30 m×0.25 μm

ID, 0.25 μm film thickness, J&W Scientific), programmed

from 40°C for 1 min, then at 5°C min⁻¹to 250°C).

Preparation of (3S)-α-Methylene-3,7-dimethyl-6-octen-1-al

(2) Piperidine (5.39 g, 63.3 mmol) was added dropwise

over ~5 min to a three-necked, round-bottomed flask,

containing (S)-citronellal 1 (4.69 g, 30.4 mmol; Aldrich

Chem. Co., Cat. #37375-3, 96%), attached to a condenser.

The temperature was increased to 44°C and the mixture

became cloudy, and then cleared again. Formaldehyde

(37% aqueous solution, 7.8 ml) was added dropwise,

keeping the temperature under 40°C, followed by glacial

acetic acid (0.78 ml) dropwise. The mixture was stirred

overnight at room temperature and then heated to 80°C for

1.5 h. The mixture was cooled to room temperature, diluted

e) To test for unsaturation, an aliquot of extract and ~1 mg

of 5% Pd on carbon were placed in a 1.5-ml screwcap

vial with a Teflon-lined septum. The septum was

punctured with a syringe needle attached to a balloon

filled with hydrogen, the lid was loosened for 10 s to

flush the vial with hydrogen and resealed, and the

mixture stirred for 2 h. The reactant mixture was

filtered through a small plug of Celite and analyzed.

242

J Chem Ecol (2008) 34:238–251

with hexanes (80 ml), then washed sequentially with HCl

(2 M in water, 2×30 ml), saturated aqueous NaHCO₃

(20 ml), and brine (20 ml). The organic phase was

separated and dried with anhydrous Na₂SO₄and concen-

trated under vacuum. The crude product was Kugelrohr

distilled (64–67°C, 1.6 mm Hg), affording 4.01 g of

Preparation of 4-[(1S)-1,5-Dimethylhex-4-enyl)-1-methyl-

cyclohex-2-en-1-ol (4) A solution of cyclohexenone 3

(2.18 g, 10.6 mmol) in diethyl ether (20 ml) was cooled

to −78°C, and methyl lithium (26.4 ml of a 1.2-M solution

in hexanes, 31.7 mmol) added dropwise. The resulting

mixture was stirred 4 h, warmed to room temperature, and

stirred overnight. The reaction was quenched with dilute

aqueous NH₄Cl and extracted with ethyl acetate (4×20 ml).

The combined organic phase was washed with brine, dried

over anhydrous Na₂SO₄, and concentrated by rotary

evaporation. The residue was purified by flash chromatog-

raphy on silica gel, eluting with 5% ethyl acetate in hexane,

yielding alcohols 4 in two fractions (less polar fraction,

higher Rf, 0.681 g; more polar fraction, lower Rf, 1.272 g;

83.1% yield). Each fraction consisted of mixtures of two

diastereomers that were inseparable by flash chromatogra-

phy; each mixture eluted as a single peak on a DB-5 GC

column. Less polar diastereomers: ¹H NMR (CDCl₃): δ

0.84 (d, 1.5H, J=7.2 Hz), 0.89 (d, 1.5H, J=6.8 Hz), 1.28 (s,

3H), 1.61 (s, 3H), 1.69 (s, 3H), 1.14–1.60 (m, 7H), 1.80–

2.08 (m, 4H), 5.07–5.14 (m, 1H), 5.59–5.70 (m, 2H). MS

(m/z, rel. abundance): 222 (M⁺, 3), 207 (10), 204 (M⁺-18,

7), 189 (5), 179 (2), 161 (8), 151 (4), 148 (6), 147 (4), 138

(12), 137 (17), 133 (7), 123 (15), 121 (12), 119 (39), 109

(22), 108 (4), 107 (10), 105 (9), 95 (23), 94 (22), 93 (24),

91 (12), 82 (12), 81 (13), 79 (19), 77 (14), 69 (91), 67 (25),

55 (37), 53 (15), 43 (80), 41 (100). More polar diaster-

1

aldehyde 2 (79.3% yield). H NMR (400 MHz, CDCl₃): δ

1.07 (d, 3H, J=7.2 Hz), 1.33–1.44 (m, 1H), 1.48–1.56 (m,

1H), 1.57 (s, 3H), 1.67 (s, 3H), 1.86–2.00 (m, 2H), 2.70

(sext, 1H, J=7.0 Hz), 5.08 (br t, 1H, J=7.0 Hz), 5.99 (s,

1H), 6.23 (s, 1H), 9.53 (s, 1H). ¹³C NMR (100 MHz,

CDCl₃): δ 17.85, 19.75, 25.89, 25.96, 31.18, 35.79, 124.34,

131.88, 133.25, 155.71, 194.89. MS (m/z, rel. abundance):

166 (M⁺, 8), 151 (8), 137 (2), 135 (3), 133 (4), 124 (4), 123

(8), 121 (1), 110 (8), 109 (41), 107 (3), 106 (2), 105 (4), 96

(3), 95 (18), 93 (9), 91 (5), 84 (10), 83 (15), 81 (18), 69

(28), 67 (26), 56 (12), 55 (51), 53 (16), 43 (16), 41 (100).

Preparation of 4-[(1S)-1,5-Dimethylhex-4-enyl]cyclohex-2-

en-1-one (3) Sodium (177 mg, 7.7 mmol) was added to a

200-ml, three-necked, round-bottomed flask containing

methanol (23 ml) and attached to a condenser. After the

sodium had dissolved, a solution of methyl acetoacetate

(2.74 g, 23.6 mmol) in methanol (35 ml) was slowly added

(~20–25 min) and the mixture was stirred 30 min at room

temperature. A solution of aldehyde 2 (3.92 g, 23.6 mmol)

in methanol was added dropwise over 40 min. The resulting

mixture was stirred 2 h at room temperature and then

refluxed for 2 h. After cooling, the methanol was removed

under vacuum, the residue diluted in hexanes (100 ml),

washed with saturated aqueous NH₄Cl, water and brine,

dried over anhydrous Na₂SO₄, and concentrated under

vacuum. The crude product was purified by vacuum flash

chromatography on silica gel, eluting with hexane/ethyl

acetate (97:3). The resulting product was purified further by

Kugelrohr distillation (112–117°C, 0.35 mm Hg), affording

2.19 g of cyclohexenone 3 (45.1% yield) as a ~1:1 mixture

1

eomers: H NMR: (CDCl₃): δ 0.82 (d, 1.5H, J=6.4 Hz),

0.85 (d, 1.5H, J=7.2 Hz), 1.10–1.22 (m, 1H), 1.27 (s, 3H),

1.32–1.55 (m, 4H), 1.60 (s, 3H), 1.68 (s, 3H), 1.60–1.75

(m, 2H), 1.83–2.14 (m, 4H), 5.06–5.13 (m, 1H), 5.48–5.65

(m, 2H). MS (m/z, rel. abundance): 222 (M⁺, 2), 207 (11),

204 (M⁺-18, 10), 189 (3), 179 (2), 161 (8), 151 (4), 148 (3),

147 (3), 138 (14), 137 (20), 133 (6), 123 (20), 121 (14), 119

(38), 109 (19), 108 (4), 107 (10), 105 (9), 95 (23), 94 (11),

93 (23), 91 (12), 82 (13), 81 (13), 79 (14), 77 (14), 69 (86),

67 (24), 55 (37), 53 (15), 43 (83), 41 (100).

1

(estimated from H and ¹³C NMR spectra) of inseparable

diastereomers. ¹H NMR (CDCl₃): δ 0.90 (d, 1.5H, J=

6.8 Hz), 0.94 (d, 1.5H, J=7.2 Hz), 1.20–1.32 (m, 1H),

1.38–1.48 (m, 1H), 1.62 (s, 3H), 1.70 (s, 3H), 1.90–2.15

(m, 4H), 2.30–2.56 (m, 4H), 5.06–5.14 (m, 1), 6.02 (dt, 1H,

J=3.2 and 10.2 Hz), 6.82–6.90 (m, 1H). ¹³C NMR

(CDCl₃): d 16.23, 16.75, 17.91, 24.21, 25.92, 26.07,

34.14, 34.34, 36.29, 36.35, 37.74, 37.93, 41.30, 41.85,

124.34, 124.37, 129.91, 130.17, 132.03, 132.04, 154.54,

155.64, 200.34. Several of these peaks corresponded to

composite signals from carbons in both diastereomers. MS

(m/z, rel. abundance) 206 (M⁺, 9), 191 (4), 177 (1), 163 (5),

149 (4), 136 (6), 135 (5), 124 (5), 123 (36), 122 (30), 121

(16), 109 (13), 107 (10), 96 (18), 95 (13), 94 (16), 91 (4),

79 (16), 77 (6), 69 (61), 67 (25), 55 (31), 53 (15), 43 (10),

41 (100).

Preparation of 4-[3S-(3,3-Dimethyloxiran-2-yl)-1-methyl-

propyl]-1-methylcyclohex-2-en-1-ol ((S)-murgantiol—5) m-

Chloroperbenzoic acid (ca. 70%, 0.333 g, ~1.35 mmol) was

added in small portions over 30 min to a well-stirred

suspension of the less polar, higher Rf, fraction of alcohols

4 (0.300 g, 1.35 mmol) in an aqueous solution of NaHCO₃

(0.5 M, 10 ml) in an ice-bath (Fringuelli et al. 1992). The

mixture was stirred 3 h at 0°C, then brine (50 ml) added,

and the mixture was extracted with hexanes (4×20 ml). The

combined organic phases were dried with anhydrous

Na₂SO₄and concentrated by rotary evaporation. The

residue was purified by vacuum flash chromatography

(hexanes/ethyl acetate 5:1) to yield 0.240 g of epoxyalco-

hols 5 (74.5%) as an inseparable mixture of diastereomers.

¹H NMR (400 MHz, CD₂Cl₂): δ 0.86 (d, 1.5H, J=6.8 Hz),

J Chem Ecol (2008) 34:238–251

243

100

90

80

70

60

50

40

30

20

10

0

Treatment

Control

0.90 (d, 1.5H, J=6.0 Hz), 1.24 (S, 6H), 1.27 (S, 3H), 1.35–

1.70 (m, 9H), 1.76–1.87 (m, 1H), 1.99–2.10 (M, 1H), 2.63–

2.70 (M, 1H), 5.58–5.70 (m, 2H). The ¹³C spectrum was

complex because of the mixture of stereoisomers, but

contained peaks that matched those of insect-produced

murgantiol. MS (m/z, rel. abundance) 205 (2), 165 (9), 147

(6), 138 (10), 135 (4), 134 (13), 132 (10), 123 (10), 122 (4),

121 (12), 119 (11), 109 (15), 107 (9), 106 (6), 105 (9), 95

(15), 94 (19), 93 (17), 91 (11), 81 (15), 79 (18), 77 (11), 71

(27), 69 (12), 67 (15), 59 (20), 55 (21), 43 (100), 41 (47).

**

Whole extract

100% ether

fraction

Combined

remaining fractions

Treatment

Fig. 2 Results of vertical Y-tube bioassays testing attraction of

sexually mature, virgin female Murgantia histrionica to fractions of

an extract of odors from M. histrionica males vs a solvent control (N=

24). Each experiment was analyzed with χ²test. **P<0.001

Preparation of 4-[3R-(3,3-Dimethyloxiran-2-yl)-1-methyl-

propyl]-1-methylcyclohex-2-en-1-ol ((R)-murgantiol—5)

An analogous mixture of diastereomers containing the

other enantiomer of murgantiol was prepared in identical

fashion, and in similar yield, using (R)-citronellal (Aldrich

Chem. Co, technical grade, 90%) as the starting material.

the crude extract (N=24, χ²=0.22, P>0.05), suggesting

that the fraction containing the male-specific compound,

and possibly the compound itself, may be responsible for

all the attractiveness of the extract.

Results

Production of the Male-Specific Compound by Individual

Males or Males in Groups Males that were aerated

individually, or in groups of only five, produced more

murgantiol per bug than males aerated in groups of ten or

more (Fig. 3). Individual bugs and bugs aerated in groups

of five produced the male-specific compound at similar

rates (Games–Howell test, P=0.41; Games and Howell

1976).

Y-tube Bioassays with Odors from Live Insects, or Insect

Extracts Sexually mature females were significantly

attracted to odors from live males in Y-tube bioassays

(Fig. 1). Males were also significantly attracted to odors

from other males. In contrast, odors from live females were

not attractive to either sex (Fig. 1).

Crude extracts of male-produced odors were significant-

ly more attractive to female M. histrionica than a solvent

control (Fig. 2). Fractionation of the extract and bioassay of

the fractions showed that the fraction containing the male-

specific component (eluted from the silica gel cartridge

with the first aliquot of 100% ether) was attractive to

females (Fig. 2), whereas the recombination of the remain-

ing seven fractions appeared to be repellent (Fig. 2). In a

follow-up bioassay, the active fraction was as attractive as

Diurnal Cycle of Pheromone Production The production of

the male-specific compound followed a defined diurnal

cycle (Fig. 4), with production increasing from midmorning

and peaking in the early afternoon.

Analysis of Extracts of Odors from Adult Bugs Typical gas

chromatograms of extracts of odors from groups of 10–15

sexually mature, virgin adult bugs of both sexes are shown

in Fig. 5. Several compounds appeared in extracts from

both females and males, including dodecane, dimethyldi-

sulfide, dimethyltrisulfide, S-methylmethanethiosulfonate,

myrcene, limonene, and cineole. With the exception of S-

methylmethanethiosulfonate, for which a standard was not

available, the identifications of these compounds were

verified by comparison of their retention times and mass

spectra with those of authentic standards. Myrcene, limo-

nene, and cineole were also found in cauliflower odor

controls, whereas the sulfur-containing compounds were

only seen in the insect odor extracts.

100

Treatment

90

Control

**

80

70

60

50

40

30

20

10

0

**

ns

Stimulus:

Responders:

Male

Female

Male

Female

Male

Fig. 1 Results of vertical Y-tube bioassays that tested attraction of

sexually mature virgin Murgantia histrionica to odors from live,

sexually mature virgin conspecifics on a cauliflower floret vs odors

from a cauliflower floret control (N=24). Data were analyzed with χ²

tests. **P<0.001; ns not significant, P>0.05

Comparison of extracts collected from sexually mature

virgin males and females (>14 day old) by gas chromatog-

raphy (Fig. 5) revealed the presence of two male-specific

compounds. The first was identified by retention time and

244

J Chem Ecol (2008) 34:238–251

Fig. 3 Production of the male-

specific compound by groups of

different numbers of Murgantia

histrionica virgin males. One-

way ANOVA for group size, F=

11.48, df=3, P<0.001. Bars

surmounted by the same letter

are not significantly different

(Games–Howell test, P<0.05)

50

45

40

35

30

25

20

15

10

5

a

b

c

0

1

5

10

15

Group Size

mass spectral matches as tridecane, a common component

of stink bug odors (e.g., Ho and Millar 2001a, b). The

second compound (Kovats index, KI, 1743 on DB-5

column; Kovats 1965) was not found in aerations of

sexually immature males, nor was it a component of the

frothy defensive emissions (2-sec-butyl-3-methoxypyrazine

and 2-isopropyl-3-methoxy-pyrazine; (Aldrich et al. 1996))

or one of the two components reported from the metatho-

racic glands of male bugs ((2E,6E)-octadienedial and

(2E,6E)-octadien-1,8-diol diacetate; Aldrich et al. 1996).

The spectrum also gave no meaningful matches with any

literature spectra in the NBS-NIH mass spectral database.

Electron impact ionization GC-MS analysis (70 eV) of

the compound gave an apparent molecular ion at m/z=220,

with an ion at m/z=202 probably due to loss of water

(Fig. 6), for a possible molecular formula of C₁₅H₂₄O. The

base peak at m/z 93 suggested a methylcyclohexadiene

fragment. The spectrum bore some similarity to the mass

spectra of sesquiterpenoids, and the overall complexity of

the spectrum suggested that the compound was not

aromatic. Based upon comparison of the retention indices

of known compounds with the KI for the Murgantia

compound, it seemed unlikely that the molecular weight

was actually 220 amu. For example, the bisabolene epoxide

pheromone components (m/z=220) of Nezara viridula and

Acrosternum hilare had retention indices on a DB-5 GC

column of 1609 (trans isomer) and 1616 (cis isomer) vs the

Murgantia compound with KI 1743. Thus, we suspected that

the loss of 18 amu from m/z=220 to give an ion at m/z=202

in the 70 eV EI mass spectrum of the Murgantia compound

was not the first loss of water but the second, and that the

actual molecular ion at m/z 238, corresponding to a

molecular formula of C₁₅H₂₆O₂, was too unstable to be

seen under EI-MS conditions. This molecular formula

requires three rings or sites of unsaturation. A molecular

ion at m/z 238 also was not observed with chemical

ionization mass spectrometry (NH₃reagent gas) using a

direct insertion probe.

Microchemical tests provided further information. The

compound did not react with the dienophile 4-methyl-1,2,4-

triazoline-3,5-dione, ruling out the presence of a conjugated

diene. The compound was not changed by treatment with

NaOH in aqueous alcohol (ruling out an ester), by acetyl

chloride and pyridine in ether (ruling out primary and

Fig. 4 Diurnal rhythm of re-

lease of the male-specific com-

pound by Murgantia histrionica

males (N=5 cohorts). Two-way

ANOVA for cohort effect, F=

3.08, df=4, 39, P=.03; for time

interval effect F=107.1, df=7,

39, P<0.001. Bars surmounted

by the same letter are not sig-

nificantly different (Student–

Newman–Keuls test, P<0.05)

80

70

60

50

40

30

20

10

0

a

b

d

c

7:00 -