Electrophysiological and behavioral activity of secondary metabolites in the confused flour beetle, tribolium confusum

Article abstract of DOI:10.1007/s10886-006-9236-3

Several previous studies have addressed pheromone communication in various flour beetles (Coleoptera: Tenebrionidae), including the confused flour beetle, Tribolium confusum (du Val). Different stereoisomers of 4,8-dimethyldecanal (DMD) were reported as the only components of an aggregation pheromone, but the behavioral activity of DMD is low. In the present study, additional previously reported secondary metabolites (benzoquinones and hydrocarbons) were tested for electrophysiological activity (EAG) with both sexes of T. confusum. Two benzoquinones and three monoenic hydrocarbons elicited significant EAG activity from both male and female antennae. There was an elevated male EAG response (vs. the females) to two out of the three hydrocarbons and for both quinones. The EAG-active compounds were subsequently investigated for behavioral activity in a walking bioassay. Benzoquinones are considered toxic and have been assigned a function as alarm substances in flour beetles, but we found that methyl-1, 4-benzoquinone in intermediate concentrations was attractive to both male and female beetles and could therefore act as an aggregation pheromone component. Males were also attracted to ethyl-1,4-benzoquinone. The corresponding hydroquinones, presumed precursors of the benzoquinones, did not elicit any electrophysiological response and were not tested for behavioral activity. The unsaturated hydrocarbons (1-tetradecene, 1-pentadecene, and 1-hexadecene) elicited significant EAG responses from both male and female antennae and were also attractive in the behavioral assay. Our results show that several beetle-produced compounds, in addition to 4,8-dimethyldecanal, may be part of a complex pheromone system in flour beetles and play a role in mediating aggregation in T. confusum.

Full text of DOI:10.1007/s10886-006-9236-3

J Chem Ecol (2007) 33:525–539
DOI 10.1007/s10886-006-9236-3
Electrophysiological and Behavioral Activity
of Secondary Metabolites in the Confused
Flour Beetle, Tribolium confusum
F. Verheggen & C. Ryne & P.-O. C. Olsson & L. Arnaud &
G. Lognay & H.-E. Högberg & D. Persson &
E. Haubruge & C. Löfstedt
Received: 17 February 2006 /Revised: 31 October 2006 /Accepted: 8 November 2006 /
Published online: 31 January 2007
#
Springer Science + Business Media, LLC 2007
Abstract Several previous studies have addressed pheromone communication in various
flour beetles (Coleoptera: Tenebrionidae), including the confused flour beetle, Tribolium
confusum (du Val). Different stereoisomers of 4,8-dimethyldecanal (DMD) were reported as
the only components of an aggregation pheromone, but the behavioral activity of DMD is
low. In the present study, additional previously reported secondary metabolites (benzoqui-
nones and hydrocarbons) were tested for electrophysiological activity (EAG) with both
sexes of T. confusum. Two benzoquinones and three monoenic hydrocarbons elicited
significant EAG activity from both male and female antennae. There was an elevated male
EAG response (vs. the females) to two out of the three hydrocarbons and for both
quinones. The EAG-active compounds were subsequently investigated for behavioral
activity in a walking bioassay. Benzoquinones are considered toxic and have been
assigned a function as alarm substances in flour beetles, but we found that methyl-1, 4-
benzoquinone in intermediate concentrations was attractive to both male and female
beetles and could therefore act as an aggregation pheromone component. Males were also
attracted to ethyl-1,4-benzoquinone. The corresponding hydroquinones, presumed precursors
of the benzoquinones, did not elicit any electrophysiological response and were not tested
for behavioral activity. The unsaturated hydrocarbons (1-tetradecene, 1-pentadecene, and
:
:
:
:
F. Verheggen C. Ryne P.-O. C. Olsson L. Arnaud C. Löfstedt (*)
Department of Ecology, Lund University, Ecology building, SE-223 62 Lund, Sweden
e-mail: christer.lofstedt@ekol.lu.se
:
:
F. Verheggen L. Arnaud E. Haubruge
Department of Functional and Evolutionary Entomology, Gembloux Agricultural University,
B-5030 Gembloux, Belgium
G. Lognay
Department of General and Organic Chemistry, Gembloux Agricultural University,
B-5030 Gembloux, Belgium
:
H.-E. Högberg D. Persson
Department of Natural Science, Mid Sweden University, SE-851 70 Sundsvall, Sweden

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J Chem Ecol (2007) 33:525–539
1-hexadecene) elicited significant EAG responses from both male and female antennae
and were also attractive in the behavioral assay. Our results show that several beetle-
produced compounds, in addition to 4,8-dimethyldecanal, may be part of a complex
pheromone system in flour beetles and play a role in mediating aggregation in T.
confusum.
.
.
.
.
Keywords Tribolium confusum Tenebrionidae Electroantennography (EAG) Behavior
.
.
.
.
Chemical communication Benzoquinones Hydrocarbons Aggregation pheromone
Dimethyldecanal
Introduction
Beetles of the genus Tribolium (Coleoptera: Tenebrionidae) are cosmopolitan and common
pests in grain stores and flour mills. Numerous studies have investigated beetle-produced
volatiles and their behavioral activity in several species of this genus, including Tribolium
confusum (e.g., Ryan and O’Ceallachain, 1976), Tribolium castaneum (Herbst) (e.g.,
Loconti and Roth, 1953), and Tribolium destructor (Uyttenboogast) (e.g., Howard, 1987).
Aggregation behavior is elicited by 4,8-dimethyldecanal (DMD) in several Tribolium
species (Suzuki, 1980; Suzuki and Sugawara, 1979; Suzuki et al., 1984, 1987; Arnaud et
al., 2002). Theoretically, there are four different stereoisomers of DMD, and it has been
hypothesized that the isomeric composition may differ among species. Response rates of
both males and females of the confused flour beetle T. confusum were highest to the 4R,8R
isomer both electrophysiologically and behaviorally (Levinson and Mori, 1983), whereas
an 8:2 mixture of 4R,8R- and 4R,8S-DMD has been reported to mimic the aggregation
pheromone of T. castaneum (Suzuki et al., 1984). The behavioral response to DMD is,
however, low and seems to leave room for additional compounds to be identified as
components of an efficient pheromone communication system (Olsson et al., 2006).
Furthermore, traps baited with DMD have so far had limited success for applied purposes,
which underlines the need for development of new attractants for this important group of
insect pests.
Several previously identified beetle-produced volatiles may be pheromone component
candidates. In addition to DMD, adults of the confused flour beetle, T. confusum produce
benzoquinones (Engelhardt et al., 1965; von Endt and Wheeler, 1971; Suzuki et al., 1988)
and volatile hydrocarbons (von Endt and Wheeler, 1971; Keville and Kannowski, 1975;
Suzuki et al., 1975; Howard, 1987) (Table 1). The behavioral activity of benzoquinones has
been examined for both T. castaneum and T. confusum (Loconti and Roth, 1953; Mondal
and Port, 1984; Faustini and Burkholder, 1987). Most studies agree that these are defensive
compounds with a repellent effect on conspecific larvae and adults (Mondal, 1985). They
have also been suggested to counteract the aggregation effect of DMD (Faustini and
Burkholder, 1987). The most studied unsaturated hydrocarbon produced by Tribolium flour
beetles, 1-pentadecene, has been examined in the context of cuticular lipids and defensive
secretions of flour beetles (Keville and Kannowski, 1975; Tschinkel, 1975; Suzuki et al.,
1975, 1988; Lockey, 1978; Markarian et al., 1978). Different functions of 1-pentadecene
have been proposed, e.g., bio-solvent for benzoquinones facilitating their absorption in the
cuticle (von Endt and Wheeler, 1971; Markarian et al., 1978), repellent (Suzuki et al.,
1975), or copulation pheromone (Keville and Kannowski, 1975).
We hypothesized that the T. confusum aggregation pheromone is more complex than
what has been reported and that the previously identified quinones and unsaturated

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Table 1 Previously reported secondary metabolites produced by Tribolium confusum and relative amounts
emitted
Compound
Abbreviation
Relative Content (in %) per Class of Chemicals
Benzoquinones
Ethyl-1,4-benzoquinone
Methyl-1,4-benzoquinone
Unsaturated hydrocarbons
1-Tetradecene
EBQ
MBQ
72.0a
28.0a
1-C14:1
Tracesb
1-Pentadecene
1,6-Pentadecadiene
1-Hexadecene
1,7-Hexadecadiene
1-Heptadecene
1,8-Heptadecadiene
1,8,11-Heptadecatriene
1-C15:1
1,6Z-C15:2
1-C16:1
1,7Z-16:2
1-17:1
1,8Z-C17:2
1,8Z,11Z-C17:3
89.7b–95.2c
Tracesb-1.9c
0.4-0.5b,c
<0.01c
0.8c–9.7b
0.2b-1.3c
Tracesb-0.3c
a Pappas and Morrison (1995).
b Suzuki et al. (1975).
c Howard (1987).
hydrocarbons are potential attractive pheromone components. The activity of these
compounds to male and female beetles was examined by using electroantennography
(EAG) and an open-arena walking bioassay and compared to the activity of DMD under the
same conditions.
Methods and Materials
Insects T. confusum beetles were reared in 2.5 dm3 jars half-filled with a 4:1 (wt:wt) mix of
whole-wheat flour and Brewer’s yeast, in a climate chamber at 23°C, 60% RH, and with a
photoperiod of L17:D7. They were separated into males and females as adults (Ho, 1969) at
least 2 wk before the bioassays were performed and were between 6 mo and 1 yr old when
tested.
Chemicals Two stereoisomers of 4,8-dimethyldecanal, the (4R,8R)- and the (4R,8S)-
isomers [(4R,8R)-DMD and (4R,8S)-DMD, respectively, Fig. 1] were prepared (Suzuki,
1981; Suzuki et al., 1983, Fig. 1). Thus, (4R,8R)-DMD was obtained starting from the (R)-
enantiomer of 2-methylbutanol [(R)-1] and commercially available (S)-citronellal [(S)-2].
The alcohol (R)-1 (not commercially available) was obtained by using a lipase-catalyzed
resolution (Nordin et al., 2000) of readily available racemic 2-methylbutanol (rac-1, Fluka,
Sigma-Aldrich Sweden AB, Stockholm, Sweden). A twofold molar excess of vinyl acetate
and rac-1 (100 g) were allowed to react in pentane (1 L), catalyzed (E=2.56) by powdered
lipase PS “Amano” [10 g (Amano Enzyme Inc., Nagoya, Japan)]. The reaction was stirred
at room temperature until 92% conversion was reached. The enzyme powder was filtered
off, and the clear filtrate was poured onto a chromatography column (Silica gel 60, 230–400
mesh, Fluka, Sigma-Aldrich Sweden AB, Stockholm, Sweden). After elution with an ether/
pentane gradient (1 to >5% ether), the remaining ester reagent and the produced ester (vinyl
acetate and 2-methyl-1-butyl acetate, respectively) were collected. Gradient elution using
ether (5 to 75%) in pentane gave the remaining unreacted (R)-2-methylbutanol. Re-

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Fig. 1 Synthesis of various isomers of 4,8-dimethyldecanal. Reaction conditions: (a) Vinyl acetate,
Amano PS lipase, pentane, room temperature, 2–4 d. (b)1 (R)-1-Bromo-2-methylbutane [(R)-3], Mg,
diethylether; 2 Add (S)-2 in diethylether; 3 H3O+/H2O, 60%. (c) TsCl/pyridine. (d) LiAlH4, diethylether,
40% over two steps. (e) MCPBA, CH2Cl2, 0°C. (e) Dissolve in diethylether. Add HIO4·2H2O, in
tetrahydrofuran, 30% over two steps
chromatography of the alcohol fraction furnished (R)-1, 7 g [86% ee by GC, β−DEX™ 325
(Supelco, Sigma-Aldrich Sweden AB, Stockholm, Sweden), 30 m×0.25 mm i.d., df =
0.25 μm; carrier gas He 15 psi, split ratio 30:1, 40°C/20 min, 0.5°C/min, 50°C]. Retention
time (min): 33.5 (R)-1 and 34.9 (S)-1. The resolution experiment was repeated, but the
reaction was interrupted at 72% conversion to give a sample of (R)-1 of lower enantiomeric
purity (20 g, 60% ee). The alcohol (R)-1 was converted to the corresponding bromide (R)-3,
which was transformed into the Grignard reagent, and reacted (Suzuki, 1981; Suzuki et al.,
1983) with commercially available (S)-citronellal, [(S)-2] (Sigma-Aldrich Sweden AB,
Stockholm, Sweden), approximately 97.5% ee by GC, [β-DEX™ 225 [(Supelco, Sigma-
Aldrich Sweden AB, Stockholm, Sweden), 30 m×0.25 mm i.d., df =0.25 μm; carrier gas
He 15 psi, split ratio 30:1, 90°C/20 min, 1°C/min, 120°C. Retention time (min): 37.4 (S)-2
and 37.9 (R)-2]. This reaction sequence furnished the alcohol 4 as a mixture of
diastereomers, which was converted to the tosylate 5. This was reduced with lithium
aluminum hydride to furnish the alkene 6, which was subjected to epoxidation followed by
periodate mediated oxidative cleavage to yield (4R,8R)-DMD. Thus, (R)-1 (86% ee) gave
(4R,8R)-DMD as a stereoisomeric mixture (>99% chemical purity by GC) with a calculated
(4R,8R)/(4R,8S)/(4S,8R)/(4S,8S) ratio of 91/7/2/0 (based on the stereoisomeric purities of
the starting materials), below called 4R,8R-DMD. Similarly, (R)-1 (60% ee) furnished
(4R,8R)-DMD as a mixture with a (4R,8R)/(4R,8S)/(4S,8R)/(4S,8S) ratio of 78/20/2/0.3
(>98% chemical purity by GC), below called an 8:2 mixture of 4R,8R-DMD/4R,8S-DMD.
We prepared the other naturally occurring stereoisomer of tribolure [(4R,8S)-DMD] from
(S)-2 and (S)-2-methylbutanol [(S)-1] (both compounds from Sigma-Aldrich Sweden AB,

J Chem Ecol (2007) 33:525–539
529
Stockholm, Sweden, and both approximately 96% ee by GC), by using the same reaction
sequence, but excluding the first reaction, the resolution step (step a, Fig. 1). This resulted
in a mixture of stereoisomers (>97% chemical purity by GC) with an approximate (4R,8R)/
(4R,8S)/(4S,8R)/(4S,8S) ratio of 2/96/0/2 (based on the stereoisomeric purities of the
starting materials), below called 4R,8S-DMD. The physical properties of our samples were
identical to those described earlier (Mori et al., 1983, 1985).
Methyl-1,4-benzoquinone (MBQ) and methylhydroquinone (MHQ) were commercially
available (Sigma-Aldrich Sweden AB, Stockholm, Sweden) and were used as received.
Ethylhydroquinone, EHQ, and ethyl-1,4-benzoquinone, EBQ, were obtained (Fig. 2) from
2,5-dihydroxyacetophenone, 7 (Sigma-Aldrich Sweden, AB, Stockholm, Sweden). Thus,
when subjected to Clemmensen reduction, compound 7 gave ethylhydroquinone, EHQ.
After recrystallization from toluene, colorless crystals were obtained (approximately 99%
chemical purity by GC). M.p. 104–6°C (Alberola et al., 1980: m.p. 110°C). EHQ was
oxidized by chromium trioxide in acetic acid/water to give crude ethyl-1,4-benzoquinone,
EBQ, as a deep orange oily crystal mass. Sublimation to a cold finger in vacuo
(approximately 100 mmHg) yielded bright yellow needles (>99% purity). M.p. 34°C
(Harman and Cason, 1952: m.p. 37°C).
Four synthetic monounsaturated hydrocarbons were obtained commercially: 1-Tetrade-
cene (C14:1) (Sigma-Aldrich Germany GmbH, Germany), 1-pentadecene (C15:1) (Acros
Organics, Geel, Belgium), 1-hexadecene (C16:1) (Fluka Chemika, Sigma-Aldrich Chemie
Gmbh, Steinheim, Germany), and 1-heptadecene (C17:1) (Sigma-Aldrich Chemie Gmbh,
Steinheim, Germany) (chemical purity >97% by GC).
The DMD samples and the hydrocarbons were dissolved in redistilled n-hexane, whereas
quinones and hydroquinones were dissolved in methanol (Table 2). The purities and
concentrations of the synthetic compounds were determined by coupled gas chromatog-
raphy-mass spectrometry. The gas chromatograph, HP 5890 series II was equipped with an
HP1 nonpolar cross-linked polydimethylsiloxane column [(Hewlett Packard Sverige AB,
Kista, Sweden), 30 m×0.25 mm i.d.×0.25 μm film thickness] and interfaced with an HP
5792A mass spectrometer working in scan mode. Helium was used as carrier gas. The
initial temperature was maintained at 50°C for 2 min and then increased by 10°C/min to the
final temperature of 250°C.
Electrophysiology (EAG) The T. confusum antenna was carefully excised from the head, so
that all the segments and the basal nerve were still attached to the antenna. The antenna was
mounted between two glass capillary electrodes (1.5 mm o.d., 1.2 mm i.d.) filled with
Ringer solution, in contact with a silver wire. The base of the antenna was first inserted into
the ground glass electrode. The tip of the recording electrode was bowl-shaped, and half of
the last distal antennal segment was immersed into the saline solution. The DC potential
was recorded on a computer using a custom-built amplifier (Hanson B-102), and an IDAC
A/D converter and software (Auto Spike v. 3.0) purchased from Syntech, Hilversum, The
Fig. 2 Synthesisofethylhydroqui-
none (EHQ) and ethyl-1,4-benzo-
quinone (EBQ) . Reaction
conditions: (a)1 Zn foil HgCl2 HCl
(aq., 1 M), decant; 2 Add HCl (aq.,
5 M) and compound 7, reflux 8 hr.
(b) Cpd EHQ in HOAc, add CrO3
in HOAc/H2O (1/1), sublimation

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Table 2 Quantities of benzoqui-
nones and unsaturated hydrocar-
bons used with Tribolium
confusum
Compound
Tested Doses
EAG (ng)
Behavioral Assay (ng)
Benzoquinones
Methyl-1,4-benzoquinone
Methylhydroquinone
Ethyl-1,4-benzoquinone
Ethylhydroquinone
100–106
100–106
100–105
100–105
101–107
100–104
Not tested
10−1–104
Not tested
10−1–104
Unsaturated hydrocarbons
Netherlands. Filtered and humidified air was continuously flowing over the mounted
antenna (approximately 0.5 m/s). A Pasteur pipette, containing a 0.5-cm2 piece of filter
paper impregnated with a 10-μl solution of the test chemical in a defined amount, was used
to deliver the stimulus in a 2.5-ml puff during 0.5 s by means of a stimulus controller (CS-
02, Syntech, Hilversum, The Netherlands). The solvents used for the stimuli were tested
alone and elicited minimal EAG responses, indistinguishable from responses to air. Ten
micrograms of two DMD isomers, 4R,8R- and 4R,8S-DMD, and also an 8:2 mixture of
4R,8R-DMD to 4R,8S-DMD were tested initially. The EAG responses were recorded every
3 min after the excision to test the longevity of the antennal preparation. This experiment
was followed by an EAG study of the response to different doses of the most active isomer.
In the following experiments, an active control consisting of 10 μg of 4R,8R-DMD was
applied before each test stimulus. Five seconds separated the DMD control puff and the
tested substance puff, and 10 sec separated two tested doses. Every EAG response will,
therefore, be presented as a normalized response that corresponds to the ratio between the
EAG response induced by the tested solution of benzoquinones/hydrocarbons and the
response induced by 10 μg 4R,8R-DMD. Each experiment was conducted in five replicates
for both sexes.
Behavioral Assay The open-arena olfactometer was a modified version of a walking
bioassay previously used for investigations of beetle behavior (Lanne et al., 1987; Olsson et
al., 2006). The olfactometer was 0.8 m long and 0.2 m wide. A source of compressed air
provided a laminar airflow of 0.2 m/s over the arena and an air exhaust evacuated
contaminated air to the outside of the building. The floor of the arena was made of a glass
plate on which a sheet of paper was placed. This paper was replaced between tests of
different compounds. The test compound was released from a 1-μl glass capillary (32 mm
long, 0.1 mm i.d.) (Drummond, MICROCAPS®, Drummond Scientific Co., Broomall, PA,
USA), which was suspended over, but not touching, the paper at the upwind end of the
arena. Two capillaries were used when testing the benzoquinones. Whereas all the methanol
solvent had evaporated from the capillary within 10 min, evaporation of the n-hexane re-
quired only 5 min. Actual release rates of the test compounds are not known. The laboratory
temperature and relative humidity were controlled at 22.5 0.5°C and 60 5%, respectively.
Ten beetles of the same sex were put in a vial. Ten vials, five for each sex, were used to
test one dose of a chemical. The vial was placed in the center of the olfactometer and was
left for 30 sec for beetles to acclimate. The capillary was filled with the solution and beetles
were released and allowed to respond during 5 min. At the end of the test, beetles collected
upwind from the release point were counted as responding/attracted (beetles approaching
the capillary within the 5 min were withdrawn before they could touch the odor source and

J Chem Ecol (2007) 33:525–539
531
then counted as responding). New beetles were used in each test, so that one beetle was not
tested more than once a day. The measured response in each test was calculated as the
number of beetles attracted out of the ten tested. Solvent was used as a control (methanol
for benzoquinones, n-hexane for the other compounds). An active control consisting of
1 μg of 4R,8R-DMD was tested each day to check that the beetles were behaving similarly
each experimental day.
Statistical Analyses Statistical tests were performed with the statistical software Minitab®
14.0 for Windows. Analyses of variance (ANOVA) were carried out for both the
electrophysiological and behavioral experiments. Three factors were tested initially
(antenna, sex, and dose of tested chemical) for the electrophysiological trials, whereas
two factors (sex and dose of tested chemical) were tested in the behavioral trials. General
linear model (GLM) (Wolfinger and Chang, 1995) was used instead of ANOVA when
unbalanced datasets were analyzed. Differences between means were tested for significance
with Dunnett’s (comparison with a control) or Newman and Keuls’ (comparison by groups
of means) tests.
Results
DMD, Choice of the Active Control for Subsequent Experiments Antennal activity
decreased significantly with time after the excision of the antenna in both sexes (females,
F4,60=167.14, P<0.001; males, F4,60=208.49, P<0.001) (Fig. 3). There were differences in
both female (F2,60=2.48, P=0.022) and male (F2,60=43.65, P<0.001) EAG activities
among the three stimuli. Due to their EAG activities, the two isomers and the blend were
tested behaviorally (Fig. 4). There was no difference in walking behavior between the
Fig. 3 Effect of time on mean
EAG responses ( SE) of female
and male Tribolium confusum
antennae (N=5) to 10 μg of
4R,8R-DMD, 4R,8S-DMD, or an
80/20 blend of the two isomers
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
4R, 8R DMD
4R, 8S DMD
Mix 80:20
FEMALES
MALES
3
6
9
12
15
Time after excision of antenna (min)

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J Chem Ecol (2007) 33:525–539
Fig. 4 Mean behavioral
Females
Males
responses ( SE) of Tribolium
confusum (N=5) to 1 μg of
4R,8R-DMD, 4R,8S-DMD, or an
80/20 blend of the two isomers.
n-Hexane was used as control.
* indicates significant difference
from the control within each sex
(Dunnett’s test, P<0.001)
*
6
5
4
3
2
1
0
*
Hexane
4R,8R
4R,8S
80:20
DMD solutions
sexes, but the responses to the two isomers and the blend were different (F4,40=7.42, P<
0.001), and 4R,8R-DMD was more attractive than the n-hexane control (Dunnet’s test,
P<0.001).
EAD and Behavioral Dose–response to 4R,8R-DMD 4R,8R-DMD was the most active and
attractive isomer, so it was tested in different concentrations in both EAG and behavioral
assays. There was a positive dose–response relationship in EAG for both females (F5,80
=
105.8, P<0.001) and males (F5,80=30.7, P<0.001). The two highest doses (1 and 10 μg)
elicited higher EAG responses than the n-hexane control in both females (Dunnet’s test, P<
0.001) and males (Dunnet’s test, P<0.001) (Fig. 5). The highest dose (1 μg) induced a
higher walking response in both females and males (Dunnet’s test, P<0.001), whereas
0.1 μg induced a higher response than the control (Dunnet’s test, P<0.05) for females only
(Fig. 6). There were no differences between male and female responses.
Benzoquinones Both MBQ and EBQ elicited higher EAG responses than the control
(Fig. 7) (F6,52=20.97, P<0.001 and F5,44=13.25, P<0.001, respectively). There were also
significant differences in EAG responses between the sexes for both MBQ (F1,52=4.58, P=
0.037) and EBQ (F1,44=10.25, P=0.003); males were more sensitive. In the walking assay,
MBQ attracted both sexes (GLM, F5,68=8.42, P<0.001) compared to methanol (Fig. 8).
Fig. 5 Effect of 4R,8R-DMD
dose on the antennal responses
( SE) of female and male Tri-
bolium confusum (N=5). * indi-
cates significant difference from
the n-hexane control within each
sex (Dunnett’s test, P<0.001)
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
Females
Males
*
*
*
*
n-Hexane 0.001
0.01
0.1
1
10
4R,8R- DMD Dose (µg)

J Chem Ecol (2007) 33:525–539
533
Fig. 6 Mean behavioral responses
( SE) of Tribolium confusum
(N=5) to increasing doses of
4R,8R-DMD. n-Hexane was used
as control. * indicates significant
difference from the n-hexane con-
trol within each sex (Dunnett’s test,
P<0.05)
7
6
5
4
3
2
1
0
Females
Males
*
*
*
n-Hexane
0.001
0.01
0.1
1
Dose (µg)
There was no difference in behavior between males and females in response to MBQ
(F1,68=1.61, P=0.209), whereas there was an interaction between dose and sex with EBQ
(F6,76=4.69, P<0.001). Female and male attraction were found to be dose-dependent for
EBQ (F6,38=4.05, P=0.003 and F6,38=6.03, P<0.001, respectively). The EAG experi-
Fig. 7 Effect of doses of methyl-
1,4-benzoquinone and ethyl-1,4-
benzoquinone on antennal
Females
Males
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Methyl-1,4-benzoquinone (MBQ)
responses ( SE) of female and
male Tribolium confusum (N=5).
EAG responses to benzoquinones
have been normalized to the
10 μg 4R,8R-DMD responses.
Means with the same letter within
each sex are not significantly
different (Newman and Keuls
test; α=0.05, N=5, df=24)
a
b
α
c
c
c
β
c
β
c
β
β
β
β
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0.0
a
Ethyl-1,4-benzoquinone (EBQ)
a
a
α
β
γ
b
b
b
γ
δ
δ
0.001
0.01
0.1
1
10
100
1000
Dose (µg)

534
J Chem Ecol (2007) 33:525–539
Methyl-1,4-benzoquinone (MBQ)
Fig. 8 Mean behavioral responses
( SE) of Tribolium confusum (N=
5) to increasing doses of methyl-
1,4-benzoquinone and ethyl-1,4-
benzoquinone. Methanol (1 μl) was
used as control. * indicates means
that are significantly different from
the methanol control (Dunnett’s
test, P<0.05)
7
6
5
4
3
2
1
0
Females
Males
*
*
*
*
*
Ethyl-1,4-benzoquinone (EBQ)
7
6
5
4
3
2
1
0
*
*
*
*
0.0001 0.001
0.01
0.1
1
10
Methanol
Benzoquinone doses (μg)
ment with the benzoquinone precursors, the hydroquinones, revealed no activity for any
of the doses of MHQ or EHQ. These substances were, therefore, not tested behaviorally.
1-Tetradecene Female and male antennal responses were dependent on the dose of C14:1
(F6,24=13.99, P<0.001 and F6,24=21.93, P<0.001, respectively) (Fig. 9). The male
response was higher than the female response at the higher doses. In the behavioral assay,
the beetles displayed a positive dose–response to C14:1 (F6,76=7.84, P<0.001), and there
was no difference between the sexes (Fig. 10).
1-Pentadecene Female and male antennae responded differently, generally with higher
male responses, to different doses of C15:1 in the EAG experiment (F6,24=56.03, P<0.001
and F6,24=16.86, P<0.001, respectively) (Fig. 9). In the walking assay, both sexes
displayed a dose-dependent attraction to C15:1 (F6,76=5.02, P<0.001), but no difference
between the sexes was observed (Fig. 10).
1-Hexadecene Males and females responded similarly in the EAG experiment to C16:1
(Fig. 9). The dose had an effect on the antennal responses of both sexes (F6,52=10.93, P<
0.001). The longer homolog C17:1 elicited no significant EAG response (data not shown).
Both males and females were attracted to C16:1 in the walking bioassay (GLM, F6,76=4.29,
P<0.001) (Fig. 10).

J Chem Ecol (2007) 33:525–539
535
Fig. 9 Effect of doses of 1-
tetradecene, 1-pentadecene, or
1-hexadecene on antennal
1-tetradecene
a
1.6
1.2
0.8
0.4
0
a
Females
Males
a
responses ( SE) of female and
male Tribolium confusum (N=5).
EAG responses to hydrocarbons
have been normalized to the
10 μg 4R,8R-DMD responses.
Means with the same letter within
each sex are not significantly
different (Newman and Keuls
test; α=0.05, N=5, df=24)
b
α
α
α
α
β
β
c
c
c
β
1-pentadecene
1.6
a
1.2
0.8
0.4
a
a
α
α
α
α
b
b
b
b
β
0
β
β
1-hexadecene
1.6
1.2
0.8
0.4
0
a
α
bc
bc
b
c
α
c
c
αβ
β
β
β
β
0.01
0.1
1
10
100
1000 10.000
Hydrocarbon dose (µg)
Discussion
This study of secondary metabolites from the confused flour beetle T. confusum shows that
several semiochemicals, in addition to the previously identified aggregation pheromone
DMD, elicit antennal and behavioral responses in both sexes and may comprise a more
complex pheromone system. Both electrophysiological and behavioral responses of male
and female T. confusum were highest to the 4R,8R isomer of DMD, in agreement with the
work of Levinson and Mori (1983). However, benzoquinones, commonly referred to as
defensive secretions or inhibitors of microbial growth in the flour beetles (Engelhardt et al.,
1965; Faustini and Burkholder, 1987; Mondal, 1990), were also found to be EAG-active

536
J Chem Ecol (2007) 33:525–539
Fig. 10 Mean behavioral re-
sponses ( SE) of Tribolium con-
fusum (N=5) to increasing doses
of 1-tetradecene, 1-pentadecene,
or 1-hexadecene. n-Hexane (1 μl)
was used as a control. * indicates
means significantly different from
the n-hexane control (Dunnett’s
test, P<0.05)
1-tetradecene
Females
6
5
4
3
2
1
0
*
Males
*
*
*
1-pentadecene
6
5
4
3
2
1
0
*
*
6
5
4
3
2
1
0
1-hexadecene
*
*
*
*
0.0001 0.001
0.01
0.1
1
10
Hexane
Hydrocarbon dose (μg)
and as attractive as 4R,8R DMD, especially to males. Furthermore, two unsaturated
hydrocarbons elicited antennal responses and attraction of both female and male beetles.
A dense population of flour beetles could potentially produce substantial amounts of the
benzoquinones. Engelhardt et al. (1965) estimated that when subjected to stress, e.g.,
extreme temperature, the amounts of these compounds released by T. confusum were
around 1 μg/beetle/3 hr. Our results suggest that lower concentrations attract both males
and females, whereas higher concentrations reduce attraction. The reduced attraction to the
high concentrations is in agreement with Faustini and Burkholder (1987), who reported that
higher concentrations can cause the beetles to disperse. It seems adaptive that the beetles
have evolved different behavioral responses to different concentrations of benzoquinones,
and these compounds may serve as a spacing pheromone at higher concentrations, similar
to what has been reported for other compounds in aphid chemical ecology (e.g., Quiroz et al.,
1997; Gonzales et al., 1999).
Antennal receptors in T. confusum responded to the three unsaturated hydrocarbons
(C14:1, C15:1, C16:1). Male antennae had stronger responses to the highest doses of the

J Chem Ecol (2007) 33:525–539
537
C14:1 and C15:1 than female antennae. Both EAG and behavioral responses decreased
with increasing chain length, and the attraction to C16:1 is low in both sexes, which could
partly be explained by the corresponding decrease in volatility of the stimuli. The attraction
of males (and females) to the hydrocarbons agrees with the male response to pentadecene
and heptadecene reported by Keville and Kannowski (1975). They found that males
mounted other males and females upon stimulation with these volatiles. In contrast, they
did not find any female response to the hydrocarbons, whereas in our study, females
responded both electrophysiologically and behaviorally. Suzuki et al. (1975) reported that
the hydrocarbons in question were repulsive when 10 μg were deposited on a filter paper.
Our results do not explicitly support this claim, but the maximal dose used in our
experiments (10 μg) did, at least, not elicit any significant attraction. Comparisons between
tests using different dispensers are difficult to make as application of similar doses does not
necessarily imply the same actual emission. We conclude that, similarly to the
benzoquinones, the three hydrocarbons (C14:1, C15:1, and C16:1) were as attractive as
the previously reported aggregation pheromone DMD in the behavioral assay. Unsaturated
hydrocarbons could thus play an aggregative role when released at low doses
complementary to the benzoquinones.
Gas chromatography in combination with electroantennographic detection (GC-EAD)
should have been a suitable method to test the relative activity of precisely quantified
amounts of various pheromone candidates and also to examine insect extracts for potential
pheromone component candidates. However, we could not use GC-EAD for measuring the
activity of different compounds at different concentrations as the lifetime of antennal
preparations was short and variable. Regular use of this approach would have allowed the
introduction of a better-defined amount of the stimulus, but would at the same time have
introduced new problems when it came to the recording of the actual response. GC-EAD
analyses of natural samples were also not productive. GC-EAD recordings from T.
confusum will require major improvements of the antennal preparation.
The likely mixed function of semiochemicals produced by flour beetles is similar to
what has been reported for secretions of sternal glands in caddisflies (Trichoptera). Several
compounds produced in these glands show high fumigant activity and have been assigned a
function in chemical defense (Ansteeg and Dettner, 1991). Compounds produced in the
same glands, and in some cases, even the same compounds as those reported by Ansteeg
and Dettner (1991) have been confirmed as components of sex and aggregation
pheromones, respectively, in Rhyacophila fasciata and Hydropsyche angustipennis
(Löfstedt et al., 1994).
The chemical communication of the Tribolium flour beetles appears complex, and
several classes of chemicals are involved in their behavior. The present study shows that
DMD is not the only compound produced and attractive for T. confusum. This raises the
question of the natural composition of the aggregation pheromone of these beetles. Indeed,
T. confusum aggregation pheromone seems to be composed of a blend of chemicals, which
depending on emitted amounts, are able to attract the beetles as efficiently as DMD. Further
studies should focus on experiments involving several of the active chemicals to highlight
increased attraction or even possible synergy.
Acknowledgments The authors thank the Swedish Foundation for Strategic Environmental Research
(MISTRA), the Cerealia R&D Foundation and Erasmus for funding this project. We thank Amano Enzyme
Inc., Japan, for a generous gift of lipases. We are also grateful to Dr. Y. Brostaux (Department of Statistic and
Computer Sciences, Gembloux Agricultural University) for his advice on statistical analysis. Ludovic
Arnaud was funded by the FNRS (Fonds National de la Recherche Scientifique), OECD (Organisation for
Economic Cooperation and Development).

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J Chem Ecol (2007) 33:525–539
References
ALBEROLA, A., GONZALES NOGAL, A. M., and MARTINEZ DE ILARDUYA, J. A. 1980. Alkylaluminums.
XVII. Behaviour of p-benzoquinones with trialkylaluminium. Anales de Química, Serie C: Química
Organica Bioquímica 76:118–122.
ANSTEEG, O. and DETTNER, K. 1991. Chemistry and possible biological significance of secretions from a
gland discharging at the 5th abdominal sternite of adult caddisflies (Trichoptera). Entomol. Gen.
156:303–312.
ARNAUD, L., LOGNAY, G., VERSCHEURE, M., LEENAERS, L., GASPAR, C., and HAUBRUGE, E. 2002. Is
dimethyldecanal a common aggregation pheromone of Tribolium flour beetles? J. Chem. Ecol. 28:523–
532.
ENGELHARDT, M., RAPOPORT, H., and SOKOLOFF, A. 1965. Odorous secretion of normal and mutant
Tribolium confusum. Science 150:632–633.
FAUSTINI, D. L. and BURKHOLDER, W. E. 1987. Quinone-aggregation pheromone interaction in the red flour
beetle. Anim. Behav. 35:601–603.
GONZALES, W. L., FUENTES-CONTRERAS, E., and NIEMEYER, H. M. 1999. Semiochemicals associated to
spacing behaviour of the bird cherry-oat aphid Rhopalosiphum padi L. (Hemiptera, Aphididae) do not
affect the olfactometric behaviour of the cereal aphid parasitoid Aphidius rhopalosiphi De Stephani-
Pérez (Hymenoptera, Braconidae). J. Appl. Entomol. 123:413–415.
HARMAN, R. E. and CASON, J. 1952. The preparation of quinones from p-aminophenols obtained by
electrolytic reduction of aromatic nitro compounds. J. Org. Chem. 17:1058–1062.
HO, F. K. 1969. Identification of pupae of six species of Tribolium (Coleoptera: Tenebrionidae). Ann.
Entomol. Soc. Am. 62:1232–1237.
HOWARD, R. W. 1987. Chemosystematic studies of the Tribolinni (Coleoptera: Tenebrionidae): phylogenetic
interferences from the defensive chemicals of eight Tribolium spp., Palorus ratzeburgi (Wissmann), and
Latheticus oryzae (Waterhouse). Ann. Entomol. Soc. Am. 80:398–405.
KEVILLE, R. and KANNOWSKI, P. B. 1975. Sexual excitation by pheromones of the confused flour beetle.
J. Insect Physiol. 21:81–84.
LANNE, B. S., SCHLYTER, F., BYERS, J. A., LÖFQVIST, J., VAN DER PERS, J. N. C., UNELIUS, R., and NORIN, T.
1987. Differences in attraction to semiochemicals present in the sympatric pine shoot beetles Tomicus
minor and T. piniperda. J. Chem. Ecol. 13:1045–1067.
LEVINSON, H. Z. and MORI, K. 1983. Chirality determines pheromone activity for flour beetles.
Naturwissenschaften 70:190–192.
LOCKEY, K. H. 1978. Hydrocarbons of adult Tribolium castaneum Hbst. and Tribolium confusum Du V.
(Coleoptera: Tenebrionidae). Comp. Biochem. Physiol. B 61:401–407.
LOCONTI, J. D. and ROTH, L. M. 1953. Composition of the odorous secretion of Tribolium castaneum. Ann.
Entomol. Soc. Am. 46:281–289.
LÖFSTEDT, C., HANSSON, B. S., PETTERSSON, E., VALEUR, P., and RICHARDS, A. 1994. Pheromonal
secretions from glands on the 5th abdominal sternite of hydropsychid and rhyacophilid caddisflies
(Trichoptera). J. Chem. Ecol. 20:153–170.
MARKARIAN, H., FLORENTINE, G. J., and PRATT, J. J. 1978. Quinone production of some species of
Tribolium. J. Insect Physiol. 24:785–790.
MONDAL, K. A. M. S. H. 1985. Response of T. castaneum larvae to aggregation pheromone and quinones
produced by adult conspecifics. Int. Pest Control 27:64–66.
MONDAL, K. A. M. S. H. 1990. Effect of synthetic quinone and pheromone on Tribolium castaneum. Ann.
Entomol. 8:19–21.
MONDAL, K. A. M. S. H. and PORT, G. R. 1984. Repellent effect of synthetic methylquinone on larvae of
Tribolium castaneum Herbst. Int. Pest. Control 26:68–71.
MORI, K., KUWAHARA, S., and UEDA, H. 1983. Pheromone synthesis. 55. Synthesis of all of the four
possible stereoisomers of 4,8-dimethyldecanal, the aggregation pheromone of the flour beetles.
Tetrahedron 39:2439–2444.
MORI, K., KATO, M., and KUWAHARA, S. 1985. Pheromone syntheses, LXXIII. New synthesis of (4R,8R)-
4,8-dimethyldecanal, the aggregation pheromone of Tribolium castaneum, and its (4R,8S)-isomer.
Liebigs Ann. Chem. 1985:861–865.
NORDIN, O., NGUYEN, B. -V., VÖRDE, C., HEDENSTRÖM, E., and HÖGBERG, H. -E. 2000. Kinetic resolution of
primary 2-methylalcohols via Pseudomonas cepacia lipase catalysed enantioselective acylation. J. Chem.
Soc., Perkin Trans. 2000:367–376.
OLSSON, P. -O. C., RYNE, C., WALLÉN, R., ANDERBRANT, O., and LÖFSTEDT, C. 2006. Male-produced sex
pheromone in Tribolium confusum: behaviour and investigation of pheromone production locations.
J. Stored Prod. Res. 42:173–182.

J Chem Ecol (2007) 33:525–539
539
PAPPAS, P. W. and MORRISON, S. E. 1995. Benzoquinones of the beetles, Tribolium castaneum and Tribolium
confusum. Prep. Biochem. Biotechnol. 25:155–168.
QUIROZ, A., PETTERSSON, J., PICKETT, J. A., WADHAMS, L. J., and NIEMEYER, H. M. 1997. Semiochemicals
mediating spacing behavior of bird cherry-oat aphid. Rhopalosiphum padi feeding on cereals. J. Chem.
Ecol. 23:2599–2607.
RYAN, M. F. and O’CEALLACHAIN, D. P. 1976. Aggregation and sex pheromones in the beetle Tribolium
confusum. J. Insect Physiol. 22:1501–1503.
SUZUKI, T. 1980. 4,8-Dimethyldecanal: the aggregation pheromone of the flour beetles Tribolium castaneum
and T. confusum (Coleoptera: Tenebrionidae). Agric. Biol. Chem. 44:2519–2520.
SUZUKI, T. 1981. A facile synthesis of 4,8-dimethyldecanal, aggregation pheromone of flour beetles, and its
analogs. Agric. Biol. Chem. 45:2641–2643.
SUZUKI, T. and SUGAWARA, R. 1979. Isolation of an aggregation pheromone from the flour beetles,
Tribolium castaneum and T. confusum (Coleoptera: Tenebrionidae). J. Appl. Entomol. 14:228–230.
SUZUKI, T., SUZUKI, T., MANH HUYNH, V., and MUTO, T. 1975. Hydrocarbon repellents isolated from
Tribolium castaneum and T. confusum (Coleoptera: Tenebrionidae). Agric. Biol. Chem. 39:2207–2211.
SUZUKI, T., OZAKI, J., and SUGAWARA, R. 1983. Synthesis of optically active aggregation pheromone
analogs of the red flour beetle Tribolium castaneum. Agric. Biol. Chem. 47:869–875.
SUZUKI, T., KOZAKI, J., SUGAWARA, R., and MORI, K. 1984. Biological activities of the analogs of the
aggregation pheromone of Tribolium castaneum (Coleoptera: Tenebrionidae). Appl. Entomol. Zool.
19:15–20.
SUZUKI, T., NAKAKITA, H., and KUWAHARA, Y. 1987. Aggregation pheromone of Tribolium freemani Hinton
(Coleoptera: Tenebrionidae) I. Identification of the aggregation pheromone. Appl. Entomol. Zool.
22:340–347.
SUZUKI, T., NAKAKITA, H., and KUWAHARA, Y. 1988. Defensive secretions and hydrocarbons of two
Tribolium species and their hybrids (Coleoptera: Tenebrionidae). Appl. Entomol. Zool. 23:329–337.
TSCHINKEL, W. R. 1975. A comparative study of the chemical defensive system of tenebrionid beetles:
chemistry of the secretions. J. Insect Physiol. 21:753–783.
von ENDT, D. W. and WHEELER, J. W. 1971. Pentadecene production in Tribolium confusum. Science
172:60–61.
®
WOLFINGER, R. and CHANG, M. 1995. Comparing the SAS GLM and MIXED Procedures for Repeated
Measures, SAS Institute Inc., Cary, NC p. 11.