Pheromone Bouquet of the Dried Bean Beetle, Acanthoscelides obtectus (Col.: Chrysomelidae), Now Complete

Article abstract of DOI:10.1002/ejoc.201500196

Male-specific volatile components, released by the dried bean beetle, Acanthoscelides obtectus, were identified as methyl (E,R)-2,4,5-tetradecatrienoate, methyl (2E,4Z,7Z)-2,4,7-decatrienoate, methyl (2E,4Z)-2,4-decadienoate, octadecanal and the sesquiterpenes (3Z,6E)- and (3E,6E)-α-farnesene. In olfactometer bioassays, pure methyl (E,R)-2,4,5-tetradecatrienoate was only weakly attractive to unmated females. However, a blend of the six identified compounds released in physiologically relevant ratios and doses proved to be as active as headspace odours collected from live males.

Full text of DOI:10.1002/ejoc.201500196

FULL PAPER
DOI: 10.1002/ejoc.201500196
Pheromone Bouquet of the Dried Bean Beetle, Acanthoscelides obtectus
(Col.: Chrysomelidae), Now Complete
József Vuts,[a] Wittko Francke,[b] Kenji Mori,[c] Paulo H. G. Zarbin,[d] Antony M. Hooper,[a]
Jocelyn G. Millar,[e] John A. Pickett,[a] Miklós Tóth,[f] Keith Chamberlain,[a]
John C. Caulfield,[a] Christine M. Woodcock,[a] Armin G. Tröger,[b] Éva Bálintné Csonka,[f]
and Michael A. Birkett*[a]
Dedicated to the late Anna-Rose and Hermann Z. Levinson; their scientific achievements keep them among us
Keywords: Pheromones / Bioassay / Coleoptera / Acanthoscelides / Bruchinae
Male-specific volatile components, released by the dried
esene. In olfactometer bioassays, pure methyl (E,R)-2,4,5-
bean beetle, Acanthoscelides obtectus, were identified as tetradecatrienoate was only weakly attractive to unmated fe-
methyl (E,R)-2,4,5-tetradecatrienoate, methyl (2E,4Z,7Z)-
2,4,7-decatrienoate, methyl (2E,4Z)-2,4-decadienoate, octa-
decanal and the sesquiterpenes (3Z,6E)- and (3E,6E)-α-farn-
males. However, a blend of the six identified compounds re-
leased in physiologically relevant ratios and doses proved to
be as active as headspace odours collected from live males.
2,4,5-tetradecatrienoate by Horler,[5] and it was shown to
have the (4R)-configuration.[6] However, no successful bio-
assays using the synthetic compound have been published.
Horler[5] and Halstead[7] had noted that sections of thin
layer chromatograms containing the allenic ester did not
consistently evoke behavioural responses from females.
Horler proposed that “this could mean that the attractant
is a similar material which is usually present in these frac-
tions” and that “there are probably at least two closely re-
lated compounds”. Recently, octadecanal was identified in
solvent extracts of males and found to synergize the activity
of the ester as an attractant for females.[8] In addition, un-
specified C16 and C18 methyl and ethyl esters were re-
ported from solvent extracts of both sexes, along with a
stereochemically undefined α-farnesene.[9]
Introduction
The dried bean beetle, Acanthoscelides obtectus Say
(Coleoptera: Chrysomelidae, Bruchinae), is an economi-
cally important pest of stored dried beans, Phaseolus vul-
garis L. (Fabaceae), worldwide.[1,2] Feeding larvae are well
protected within the seeds, and so management strategies
target adult beetles.[3] Because semiochemicals constitute
valuable tools for the detection and monitoring of A. ob-
tectus populations, attempts to isolate volatile attractants
were started more than 45 years ago. Hope et al. first re-
ported a single sex-specific compound isolated from hexane
surface extracts of males and proposed it to either stimulate
the emergence of females or to be a sex attractant.[4] This
compound was later identified as the allenic methyl (2E)-
In view of these fragmentary data, we reinvestigated the
composition of volatiles released by male beetles to set the
stage for the development of pheromone-based manage-
ment strategies. Here, we report on the structure elucidation
and biological activity of volatile compounds from air en-
trainment collections of A. obtectus males, in addition to
the known allenic methyl (E,R)-2,4,5-tetradecatrienoate.
[a] Department of Biological Chemistry and Crop Protection,
Rothamsted Research,
AL5 2JQ Harpenden, United Kingdom
E-mail: mike.birkett@rothamsted.ac.uk
http://www.rothamsted.ac.uk/
[b] Institute of Organic Chemistry, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
[c] Photosensitive Materials Research Center, Toyo Gosei Co.,
Ltd.,
4-2-1 Wakahagi, Inzai City, Chiba 270-1609, Japan
[d] Department of Chemistry, Federal University of Parana,
81531-990, Curitiba-PR, Brazil
[e] Department of Entomology, University of California,
Riverside, CA 92521, United States of America
[f] Centre for Agricultural Research, Plant Protection Institute
HAS,
Results and Discussion
Because initial studies suggested dynamic headspace col-
lection to be more efficient than solvent extraction to ob-
tain sex-specific volatiles from male A. obtectus, we applied
this technique to collect samples from unmated males, using
15 Herman O. u., 1022 Budapest, Hungary
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500196.
Eur. J. Org. Chem. 2015, 4843–4846
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M. A. Birkett et al.
FULL PAPER
Porapak Q as the adsorbent. Gas chromatographic (GC)
analysis of diethyl ether extracts of the adsorbent on a non-
polar HP-1 column revealed six major components to be
consistently present in a ratio of 4.5:10:3.5:4.5:14:2, respec-
tively (see Figure 1). No peaks of beetle origin were de-
tected in headspace extracts of virgin sexually mature fe-
males. Structural assignments of the target compounds were
based on comparison of their analytical data with those of
authentic reference substances. The last eluting component
(compound 6) proved to be octadecanal,[8] whereas the
main component (compound 5) was the previously known
methyl (E,R)-2,4,5-tetradecatrienoate.[5] A reinvestigation
of the enantiomeric composition of the natural product by
enantioselective gas chromatography confirmed Pirkle’s ge-
neral assignment[6] and showed an R/S ratio of 93–94:7–6.
Minor constituents, volatiles 3 and 4, proved to be the com-
mon sesquiterpenes (3Z,6E)- and (3E,6E)-α-farnesene.[10]
The second most abundant A. obtectus volatile, component
Figure 2. 70 eV EI mass spectrum of compound 1 in Figure 1, in-
cluding a hypothetical structure for m/z 111.
2 in Figure 1, was conclusively identified as methyl (2E,4Z)- indicated by a pronounced signal at m/z 111 in the mass
2,4-decadienoate.[10] This ester had earlier been identified as spectrum (Figure 2), which was also present in those of
a component of the aggregation pheromone of the bark methyl (2E,4Z)-2,4-decadienoate (2)[10] and methyl
beetle Pityogenes chalcographus L. (Coleoptera: Scolytidae) (2E,4E,7Z)-2,4,7-decatrienoate.[14] This fragment, C6H7O2,
[11]
and several species of Euschistus stink bugs (Heterop- may represent the methoxycarbonyl group and the subse-
tera: Pentatomidae).[12]
quent four carbons of the chain as suggested in Figure 2.
GC coupled with Fourier-transform infrared spectroscopy
showed a strong absorption between 1715–1730 cm–1 as ex-
pected for an α,β-unsaturated ester. However, no absorption
in the 1930–1950 cm–1 region could be observed, which ex-
cluded the possibility of an allenic system.
As a result, the determination of the position and geome-
try of the third double bond in the target compound needed
further efforts. Preparative GC, using a DB-WAX column,
resulted in the isolation of 12 μg of the methyl decatrienoate
1
from headspace collections of males. H 1D NMR spec-
troscopy of the sample revealed the presence of a doublet
of doublets at δ = 7.66 ppm, a triplet at δ = 6.17 ppm and
a doublet at δ = 5.92 ppm, showing almost identical multi-
plicity and chemical shift to H-3, H-4 and H-2 of methyl
(2E,4Z)-2,4-decadienoate,[18] which implied the presence of
the same moiety in the unknown ester. Other salient reso-
Figure 1. Gas chromatogram of a diethyl ether extract of Pora-
pak Q trapped volatiles released by males of the dried bean beetle.
The structure of component 1 was unknown. Its 70 eV nances included a triplet at δ = 3.09 ppm that was shown
EI mass spectrum (Figure 2) showed a base peak at m/z 79 to be coupled to resonances at δ = 5.86 and 5.35 ppm by 2D
amu and a molecular ion at m/z 180 amu. Diagnostic frag- correlation spectroscopy. The chemical shift and adjacent
ments at m/z 59 (representing a methoxycarbonyl group), downfield protons identified this to be a bisallylic methyl-
m/z 121 (M+ –59, loss of a methoxycarbonyl group), and ene group at C-6, indicating a double bond at C-7. To avoid
m/z 149 (M+ –31, loss of a methoxy group) indicated the any traces of acid that might originate from chloroform and
compound to be the methyl ester of a C10-carboxylic acid could endanger the tiny amounts of the unknown natural
with three double bond equivalents.
product, we used d2-dichloromethane as the NMR solvent,
After catalytic hydrogenation[13] of a crude extract of A. which unfortunately obscured H7. However, the H-8 mul-
obtectus males, coupled GC-mass spectrometry (GC/MS) tiplet revealed a coupling constant of 10.3 Hz, suggesting
analysis showed the presence of methyl tetradecanoate the (Z)-configuration for the double bond. The alkenyl pro-
[hydrogenation product of methyl (E,R)-2,4,5-tetradecatri- ton at H-8 was coupled to a quintet at δ = 2.13 ppm, which
enoate], 2,6,10-trimethyldodecane (hydrogenation product coupled to a triplet at δ = 1.01 ppm, indicating a distal ethyl
of the farnesenes), and methyl decanoate [hydrogenation group. In sum, these data suggested the unknown com-
product of methyl (2E,4Z)-2,4-decadienoate and of the un- pound to be methyl (2E,4Z,7Z)-2,4,7-decatrienoate.
known methyl ester], revealing the unknown compound to
For reference and bioassays, methyl (2E,4Z,7Z)-2,4,7-
be the methyl ester of a straight-chain decatrienoic acid. decatrienoate was prepared according to Scheme 1, starting
The sub-structure of a conjugated methyl 2,4-dienoate was from commercially available 1-bromo-2-pentyne (7). Alkyl-
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The Complete Pheromone Bouquet of the Dried Bean Beetle
ation of the tetrahydropyranyl derivative 8 of propargyl hyde have been described as components of the scent of a
alcohol with 7 was executed as reported by Kobayashi et South American orchid.[20]
al.[15] to give 2,5-octadiyn-1-ol (9) in 90% yield after depro-
In olfactometer assays, the test compounds were applied
tection. Reduction of 9 to 10 was best carried out by onto filter paper strips in proportions and doses in such a
Brandsma’s method with Zn/Cu and ethanol yielding way that the amounts released per hour were similar to
(2Z,5Z)-2,5-octadien-1-ol (10) in 99% purity and 70% yield those emitted by one male beetle over 1 h. In test 1, methyl
after distillation.[16] The final step was the tandem oxid- (E,R)-2,4,5-tetradecatrienoate was significantly more at-
ation-olefination to convert 10 to methyl (2E,4Z,7Z)-2,4,7- tractive to virgin female A. obtectus than a hexane control
decatrienoate using Barrett’s method.[17] Treatment of 10 (Table 1). Addition of the two C10 methyl esters or octade-
with Dess–Martin periodinane and (methoxycarbonylmeth- canal to the allenic methyl ester did not enhance beetle at-
ylene)triphenyl phosphorane in the presence of benzoic acid traction. The two farnesenes, however, synergized the ac-
in dichloromethane/dimethyl sulfoxide gave crude methyl tivity of the allenic methyl ester, indicating that the farnes-
2,4,7-decatrienoate (1) in 71% yield. GC/MS analysis re- enes may play a more important role than the other three
vealed the product to contain 85% of the desired compounds. A complete blend containing all six male-spe-
(2E,4Z,7Z)-isomer, 6% of the (2Z,4Z,7Z)-isomer, and 2% cific compounds proved to be much more attractive than
of the (2E,4E,7Z)-isomer. Purification on silica gel impreg- the allenic ester alone (Table 1). Comparison of a blend
nated with silver nitrate (elution with hexane/ethyl acetate, containing the allenic methyl ester with one lacking it (test
100:1, v/v) yielded methyl (2E,4Z,7Z)-2,4,7-decatrienoate as 6, Table 1) revealed that all 6 male-specific components
a colourless oil of 99% purity. A synthetic sample co-eluted were necessary for full activity. A blend of the 6 compounds
with the natural product on nonpolar HP-1 and polar DB-
WAX GC columns and showed the same mass spectrum
and the same NMR spectroscopic data. A detailed descrip-
tion of the synthesis of methyl (2E,4Z,7Z)-2,4,7-decatrieno-
ate and a new route to methyl (E)-2,4,5-tetradecatrienoate
will be published separately.[19]
Table 1. Behavioural responses of virgin Acanthoscelides obtectus
females in 4-arm olfactometer bioassays to synthetic blends. Com-
position of the complete male blend (ng/μL in hexane) used in the
bioassays (10 μL offered on filter paper): methyl (2E,4Z,7Z)-deca-
trienoate (5), methyl (2E,4Z)-2,4-decadienoate (40), (3Z,6E)-α-far-
nesene (43), (3E,6E)-α-farnesene (50), methyl (E,R)-2,4,5-tetra-
decatrienoate (390), octadecanal (5). Results shown in test 7 were
obtained with 7.2 μg of a mixture of compounds 1–6 in the ratio
of 55:110:55:55:390:55 in hexane. Relative proportions in this mix-
ture were found to be closer to the natural composition of the bou-
quet than the “complete blend” used in tests 2–6 (see Figure 1).
Means with the same letters are not significantly different at α =
0.05 (ANOVA), followed by Fisher’s LSD-test (see also Supporting
Information).
Test Treatment
Mean time spent [min] Ϯ SE Significance
1
2
allenic ester
solvent (control)
7.25Ϯ0.58
1.44Ϯ0.33
P Ͻ 0.001
complete male blend
allenic ester
solvent (control)
6.01Ϯ1.43
2.02Ϯ0.65
2.67Ϯ0.72
b
a
a
Scheme 1. Synthesis of methyl (2E,4Z,7Z)-2,4,7-decatrieonate.
Reagents: (a) EtMgBr, CuI, THF; (b) aq. HCl, MeOH (90%, two
steps); (c) Zn, Br(CH2)2Br, CuBr, LiBr, EtOH, 100 °C (6 h), 80 °C
(16 h) [70% (99% purity)]; (d) Dess–Martin periodinane,
Ph3P=CHCO2Me, PhC2H, DMSO, CH2Cl2 (60%).
3
complete male blend
allenic ester + other
methyl esters
6.86Ϯ1.22
2.81Ϯ0.89
b
a
solvent (control)
0.98Ϯ0.25
a
The biosynthesis of (2E,4Z,7Z)-2,4,7-decatrienoic acid,
i.e. the acid part of the novel ester, can be rationalized either
via lipoxygenase-mediated cleavage of (9Z,12Z,15Z)-
9,12,15-octadecatrienoic acid (α-linolenic acid) or by a se-
quence of four β-oxidation steps and rearrangement of the
same precursor. The 12Z- and 15Z-double bonds of lino-
lenic acid would become the 4Z- and 7Z-unsaturations,
whereas the original 9Z-double bond would be transformed
to the 2E-double bond in the chain-shortened product.
Starting from (9Z,12Z)-9,12-octadecadienoic acid (linoleic
acid), a corresponding biosynthetic pathway would yield
(2E,4Z)-2,4-decadienoic acid, the precursor of methyl deca-
dienoate 2.
4
5
6
complete male blend
allenic ester + α-farnesenes
solvent (control)
5.13Ϯ0.93
3.58Ϯ1.12
0.43Ϯ0.12
b
b
a
complete male blend
allenic ester + octadecanal
solvent (control)
5.53Ϯ1.29
2.56Ϯ0.57
1.16Ϯ0.26
b
a
a
complete male blend
allenic ester
complete male blend with-
out allenic ester (control)
4.37Ϯ0.57
1.93Ϯ0.44
2.06Ϯ0.36
b
a
a
7
complete male blend
male extract (1 male equiv-
alent)
3.94Ϯ0.60
3.36Ϯ0.75
b
b
To the best of our knowledge, methyl (2E,4Z,7Z)-2,4,7-
decatrienoate is
a
new natural product. However,
solvent (control)
0.92Ϯ0.16
a
(2E,4Z,7Z)-2,4,7-decatrienol and the corresponding alde-
Eur. J. Org. Chem. 2015, 4843–4846
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M. A. Birkett et al.
FULL PAPER
the Biotechnology and Biological Sciences Research Council
(BBSRC), United Kingdom.
proved to be as attractive to virgin female A. obtectus as
the corresponding equivalent of a headspace extract ob-
tained from live males (test 7, Table 1).
Our data suggest that the newly identified compounds
are Horler’s missing pheromone components. We believe
that after 45 years, our results will finally form the basis for
development of effective semiochemical-based pest manage-
ment tools for use against A. obtectus.
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Experimental Section
General: Beetles were obtained from a laboratory population and
reared on dry Ph. vulgaris “Cannellini” beans under artificial light-
ing at 20 °C/60% RH with a 16:8 h L:D photoperiod. In order to
obtain virgin insects, seeds were kept individually in wells of a plas-
tic rack until emergence of adults, when sexes were separated imme-
diately. To obtain volatile compounds from 15 eight-day old male
A. obtectus, dynamic headspace collection (air entrainment) was
used.[21]
Methyl (E,R)-2,4,5-tetradecatrienoate and methyl (2E,4Z)-2,4-de-
cadienoate were synthesized as described by Mori.[18] The sesqui-
terpenes (3Z,6E)- and (3E,6E)-α-farnesene were prepared accord-
ing to known methods.[22,23] Octadecanal was obtained by TPAP-
mediated oxidation of octadecanol.[24]
[14] W. H. Gerwick, P. J. Proteau, D. G. Nagle, M. L. Wise, Z. D.
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[16] M. H. P. J. Aerssens, R. van der Heiden, M. Heus, L.
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[17] A. G. M. Barrett, D. Hamprecht, M. Ohkubo, J. Org. Chem.
1997, 62, 9376–9378.
To investigate the behavioural responses of virgin female A. ob-
tectus to the male-derived compounds, a Perspex four-arm olfac-
tometer was used.[25,26] Two arms were used to test synthetic com-
pounds (except in Test 1), two were used for the control, hexane
(except in Test 1 and Test 6). The time spent in each arm by a
single beetle over a 16-min period was recorded using custom-made
software (OLFA, Udine, Italy). Data were analyzed statistically by
ANOVA at α = 0.05, followed by Fisher’s LSD test (GenStat, 11th
edition, VSN International Ltd.). Ten replicates per test were done.
[18] K. Mori, Tetrahedron 2012, 68, 1936–1946.
[19] K. Mori, Tetrahedron in press.
[20] R. Kaiser, Chimia 2000, 346–363.
For detailed experimental procedures such as GC, GC/MS or GC/
FT-IR, NMR, the isolation of natural methyl (2E,4Z,7Z)-2,4,7- [21] N. Agelopoulos, J. A. Pickett, J. Chem. Ecol. 1998, 24, 1161–
1172.
decatrienoate, and its synthesis and bioassays, see Supporting infor-
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[22] P. Ramaiah, J. J. Pegram, J. G. Millar, J. Org. Chem. 1995, 60,
6211–6213.
[23] A. Svatos, A. B. Attygalle, Anal. Chem. 1997, 69, 1827–1836.
˘
[24] W. P. Griffith, S. V. Ley, G. P. Whitombe, A. D. White, J. Chem.
Soc., Chem. Commun. 1987, 1625–1627.
Acknowledgments
[25] J. Pettersson, Insect Syst. Evol. 1970, 1, 63–73.
[26] S. Y. Dewhirst, M. A. Birkett, J. D. Fitzgerald, A. Stewart-
Jones, L. J. Wadhams, C. M. Woodcock, J. Hardie, J. A. Pick-
ett, J. Chem. Ecol. 2008, 34, 1575–1583.
This work was in part supported by the Hungarian Scientific Re-
search Fund (OTKA) (grant number K81494), by the Hungarian
Research and Technology Innovation Fund (grant number OMFB-
00609/2010) and by the Royal Society, United Kingdom (grant
IE111142 to J. V.). Rothamsted receives grant-aided support from
Received: February 10, 2015
Published Online: May 27, 2015
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