The sex pheromone of the wasp spider Argiope bruennichi

Article abstract of DOI:10.1002/anie.200906311

(Figure Presented) Wasp spider looking for a mate: Female wasp spiders (see picture) use trimethyl methylcitrate as a volatile cue to attract males. The experiments were performed on a sunny meadow, showing for the first time that spider traps can be used to trap spiders in the field (photo: Helen Sandford).

Full text of DOI:10.1002/anie.200906311

Angewandte
Chemie
DOI: 10.1002/anie.200906311
Pheromones (1)
The Sex Pheromone of the Wasp Spider Argiope bruennichi**
Satya P. Chinta, Stephan Goller, Julia Lux, Sebastian Funke, Gabriele Uhl,* and Stefan Schulz*
Current biochemical research on spiders focuses on the two
characteristic traits common to spiders, namely silk and
toxins. Nevertheless, chemical communication over longer
distances is also vital: potential mates that are solitary and
predatory need to find members of the same species
wander through the meadow to find a mate, probably guided
by a female pheromone. Herein, we report on the identifica-
tion, synthesis, and activity of this pheromone in the field.
Headspace sampling of odors emitted by individual
female spiders was performed with activated charcoal traps
using glass chambers containing the spiders in three devel-
opmental and reproductive states (subadult, virgin, and
mated). The adsorbent was extracted with dichloromethane
and the extracts analyzed by GC-MS. Only the extract of
virgin adult spiders contained a compound A that was absent
from subadult or mated females (Figure 1).
(
conspecifics) over large distances during the reproductive
season. The production of a volatile signal that helps to attract
mating partners is vital, but to date only few of the respective
[1]
pheromones are known (see the following Communication).
Herein we report the identification of the first pheromone
from an orb weaver (Araneidae), namely the wasp spider,
Argiope bruennichi, and show for the first time that a spider
pheromone can be used to trap spiders in the field.
Orb weavers are one of most successful spider families
worldwide, with over 2600 known species. Early experiments
showed that Cyrtophora cicatrosa emits a volatile pheromone
that attracts males and induces courtship in higher concen-
[
2]
trations. Cages with females of Araneus trifolium and
[3]
Argiope trifasciata were able to attract conspecifics. The
latter also attracted Argiope aurantia males, suggesting the
[
4]
presence of a common sex pheromone in both species. Sex
pheromones that attract conspecific males over a distance to
the web of a partner has so far only been reported from the
desert spider Agelenopsis aperta (Agelenidae) that uses 8-
Figure 1. Gas chromatograms from headspace analysis of Argiope
bruennichi. a) Virgin female; b) adult female; c) sub-adult female.
[5]
methyl-2-nonanone.
We investigated the pheromone system of the very
characteristic wasp spider Argiope bruennichi, which lives
on meadows in the Mediterranean but occurs now also in the
temperate zone of Middle Europe. When adult, the large
females build webs in the grass, whilst the much smaller males
Extracts of webs were also analyzed, and these again
showed the presence of A in webs of virgin females only, but
additionally
a second, female specific compound (B;
Figure 2). Although A was present in all the samples of
virgin females, compound B did not occur in all samples, and
was also found in those from mated females. Therefore, we
concentrated on the biological activity of compound A in the
field experiments.
[*] S. P. Chinta, Dr. S. Goller, Prof. Dr. S. Schulz
Institut fꢁr Organische Chemie
Technische Universitꢂt Braunschweig
Hagenring 30, 38106 Braunschweig (Germany)
Fax: (+49)531-391-5272
E-mail: stefan.schulz@tu-bs.de
Homepage: http://aks7.org-chem.nat.tu-bs.de/
J. Lux, S. Funke, Prof. Dr. G. Uhl
Institut fꢁr Zoologie
Rheinische Friedrich Wilhelms Universitꢂt Bonn
Endenicher Allee 11–13, 53115 Bonn (Germany)
Prof. Dr. G. Uhl
Zoologisches Institut und Museum
Ernst Moritz Arndt Universitꢂt Greifswald
Anklamer Strasse 20, 17489 Greifswald (Germany)
Fax: (+49)3834-864-252
Figure 2. Gas chromatograms of silk extracts of Argiope bruennichi.
a) Virgin female; b) sub-adult female. ꢀ=impurity.
E-mail: gabriele.uhl@uni-greifswald.de
Homepage: http://www.mnf.uni-greifswald.de/fr-biologie/zool-
institut-museum/allgemeine-und-systematische-
zoologie.html
The mass spectrum of compound A is similar in appear-
ance to that of trimethyl citrate (Figure 3), but each major
fragment ion is 14 mass units larger, thus indicating the
[**] This work was supported by the Fonds der Chemischen Industrie.
presence of an additional CH group at an unknown position.
2
The derivatization with MSTFA (N-methyl-N-(trimethylsi-
lyl)trifluoroacetamide) provided evidence for the presence of
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200906311.
Angew. Chem. Int. Ed. 2010, 49, 2033 –2036
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
2033

Communications
contains two stereogenic centers, it was necessary to synthe-
size stereoisomers to prove our structural proposal, to clarify
the stereochemistry of the natural compound, and to provide
material for biological testing. It is well-known that stereo-
chemical aspects play an important role in pheromone
[6]
research, and that unnatural stereoisomers can suppress
[1]
reactions in bioassays (see the following Communication).
The chiral building block (S)-malic acid (1) was used as
starting material (Scheme 1). After conversion into the
[
7]
methyl ester 2, methylation using LDA as base furnished
Scheme 1. Synthesis of trimethyl (2R,3S)-methylcitrate (9). a) HCl,
MeOH, 808C; b) LDA, MeI, À788C; c) 2m KOH, THF/MeOH (1:1);
d) H SO , pivalaldehyde; e) LiHMDS, À788C, allyl bromide;
2
4
f) BF ·OEt , MeOH; g) RuCl , NaIO , CCl , MeCN, H O; h) EDC,
3
2
3
4
4
2
MeOH. EDC=N’-(3-dimethylaminopropyl)-N-ethyl carbodiimide,
LDA=lithium diisopropylamide, LiHMDS=lithium hexamethyldisila-
zanide.
Figure 3. Mass spectra of a) trimethyl methylcitrate (A); b) 3-octanoyl-
oxy-4-butanolide (B); c) trimethyl citrate.
methylated dimethyl malate 3 in a diastereomeric ratio of 6:1
[
8]
in favor of the 2S,3R isomer. Hydrolysis of 3 gave the
[
9]
one labile hydrogen atom in the molecule and indicated the
molecular ion of the parent compound A to be m/z 248.
Mixed ethyl methyl esters of citric acid have completely
different mass spectra, leaving only two structures with an
additional carbon atom, one being trimethyl methylcitrate (by
methylation of one of the methylene groups), and the other
requiring extension of the carbon chain that seems to be
biosynthetically unlikely.
respective acid 4, which was then treated with pivalaldehyde
to convert it into the thermodynamically favored cis-dioxo-
lanone 5
[10]
that was subsequently alkylated with allyl
bromide, yielding 6 according to the chiral relay method
developed by Seebach et al. Treatment with BF ·OEt in
methanol cleaved the acetal group and simultaneously
esterified the acid to give 7. Oxidative cleavage of 7 with
RuO furnished the acid 8,
[
11]
3
2
[
12,13]
[14]
which was esterified with
4
The mass spectrum of compound A is in full agreement
with the trimethyl methylcitrate structure and is closely
related to that of trimethyl citrate. The fragmentation can be
explained by a-cleavage of the methoxycarbonyl group to
form the ion m/z 189. An elimination of methanol provided
the base ion m/z 157; elimination of methyl acetate (74 amu)
or methyl propionate (88 amu) by a McLafferty rearrange-
ment and loss of the methoxycarbonyl furnished the ions m/z
methanol to yield trimethyl (2R,3S)-methylcitrate as the
major enantiomer. Because the Seebach method used here is
[11]
highly stereoselective, the minor stereoisomer present was
assigned the 2S,3S configuration. The major stereoisomer
synthesized proved to be identical in mass spectrum and gas
chromatographic retention time to the major natural product.
Chiral gas chromatography was then performed to clarify
the absolute configuration of the natural product. The
separation of the four enantiomers of trimethyl methylcitrate
was partly possible on a chiral hydrodex-6-TBDMS column
(TBDMS = tert-butyldimethylsilyl; Figure 4). The analysis of
natural extracts showed that the 2R,3S and 2S,3S enantiomers
occurred naturally in a ratio between 6:1 to 25:1. The major
isomer proved to be identical to the major synthesized
compound, trimethyl (2R,3S)-methylcitrate, whilst the minor
1
(
01 and 115 from the molecular ion (not observed) at m/z 248
Figure 3).
Compound A occurred in a diastereomeric ratio of
between 6:1 and 25:1 in all extracts of virgin females. The
major diastereomer showed a gas chromatographic retention
index (RI) of 1518 on an apolar GC phase, whereas the minor
diastereomer had a RI of 1527. As trimethyl methylcitrate
2
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Angewandte
Chemie
[
a]
Table 1: Results of bioassays performed in the field.
Amount [mg]
Trials
Successful trials (%)
Attracted males
2
1
5.00
2.50
10
10
5
10
4
10 (100)
8 (80)
3 (60)
5 (50)
4 (100)
4 (40)
1 (14)
34
19
7
6
5
2
1
0
0
0
.25
.25
.625
.30
.15
10
7
4
1
[a] Traps containing differing amounts of the 6:1 mixture of (2R,3S)- and
(2S,3S)-trimethyl methylcitrate (9) was tested against traps with
dichloromethane only (control). The number of test trials, number of
trials in which attraction occurred, and total number of males attracted
over all trials are given. Control traps never attracted spiders.
related to the female sex pheromone of the tropical wander-
[
15]
ing spider Cupiennius salei,
unsymmetrical (S)-dimethyl
citrate, that induces courtship behavior in males. Attraction of
spiders to traps in the field by use of a pheromone has been
shown herein for the first time; 8-methyl-2-nonanone, the sex
[5]
pheromone of Agenelopsis aperta, is the only other highly
volatile spider pheromone known to date. Methylcitric acid
occurs naturally in many organisms; the 2R,3S and 2S,3S
isomers are usually formed by action of a si-citrate synthase in
Figure 4. Gas chromatographic separation of trimethyl methylcitrate
on a chiral hydrodex-6-TBDMS phase. Temperature program: 508C for
À1
À1
5
min, then with 0.28Cmin to 2008Cmin . a) Natural extract;
b) synthetic 9; c) co-injection.
[16]
animals, such as pigs and humans, whereas the other two
isomers are formed by bacteria and yeasts using re-citrate
[
17]
synthase.
The use of derivatives of citric acid, a typical
isomer is the minor synthetic product, trimethyl (2S,3S)-
methylcitrate. Compound B exhibited a molecular ion at m/z
primary metabolite, as pheromones has not been reported
from other animals. Whether this close connection to primary
metabolites is typical for pheromones of spiders will need
further exploration.
In summary, we have identified and synthesized the sex
pheromone of the wasp spider Argiope bruennichi as a
mixture of the 2R,3S and 2S,3S isomers of trimethyl methyl-
citrate. This pheromone is able to attract male spiders in the
field in a concentration-dependent manner. Another female
specific spider compound, 3-octanoyloxy-g-butyrolactone,
has been identified, but its function needs to be established.
2
28. Its mass spectrum (Figure 3) was characterized by the ion
m/z 85, which is characteristic for a butyrolactone ring. Other
characteristic ions at m/z 127 (octanoyl), 144 (McLafferty
rearrangement product), and 157 (b-cleavage to carbonyl
group) and comparison with synthetic reference material
showed that this compound is 3-octanoyloxy-g-butyrolactone.
The synthesized 6:1 mixture of (2R,3S)- and (2S,3S)-
trimethyl methylcitrate was then evaluated for its activity in
the field. Tripod traps containing trimethyl methylcitrate and
control traps were placed in an open meadow field during a
dry and hot summer period. The test compound was highly
attractive to males of A. bruennichi: during the 30 minute test
period for each trial, 34 males were attracted to 10 traps
containing 25 mg per trap. These males also showed typical
courtship behavior, such as application of silk strands from
the trap to the vegetation, jerking, and abdomen vibration.
Control traps were never approached. The attractivity of the
pheromone tended to decrease with the concentration;
response to less than 1 mg was low, but still observable
Received: November 9, 2009
Published online: February 12, 2010
Keywords: araneae · chemical communication · mate attraction ·
.
natural products · pheromones
[
1] E. Jerhot, J. A. Stoltz, M. C. B. Andrade, S. Schulz, Angew.
Chem. 2010, 122, 2081 – 2084; Angew. Chem. Int. Ed. 2010, 49,
(
Table 1). Higher concentrations were more attractive than
2037 – 2040.
lower ones, as shown by competition experiments (see
Experiment 2 in the Supporting Information). We also
investigated whether the ratio of the two enantiomers present
in the natural pheromone is important for attraction. Both a
[
[
2] R. Blanke, Naturwissenschaften 1973, 60, 481.
3] C. W. Olive, J. Arachnol. 1982, 10, 241 – 245.
[4] F. Enders, Br. Arachnol. Soc. News 1975, 13, 5 – 6.
[5] M. D. Papke, S. E. Riechert, S. Schulz, Anim. Behav. 2001, 61,
877 – 886.
6
:1 and a 2:1 mixture of (2R,3S)- and (2S,3S)-trimethyl
[
[
6] K. Mori, Bioorg. Med. Chem. 2007, 15, 7505 – 7523.
7] J. L. Herrmann, R. H. Schlessinger, J. Chem. Soc. Chem.
Commun. 1973, 711 – 712.
methylcitrate were equally attractive to the spiders (see
Experiment 3 in the Supporting Information).
The results clearly demonstrate that trimethyl methylci-
trate is the sex pheromone of female Argiope bruennichi
spiders that is used to attract males. This compound is closely
[
[
8] P. Wipf, Y. Uto, S. Yoshimura, Chem. Eur. J. 2002, 8, 1670 – 1681.
9] I. T. Lim, S. O. Meroueh, M. Lee, M. J. Heeg, S. Mobashery, J.
Am. Chem. Soc. 2004, 126, 10271 – 10277.
Angew. Chem. Int. Ed. 2010, 49, 2033 –2036
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
2035

Communications
[
[
[
[
[
10] S. A. A. El Bialy, H. Braun, L. F. Tietze, Eur. J. Org. Chem. 2005,
965 – 2972.
11] D. Seebach, R. Naef, G. Calderari, Tetrahedron 1984, 40, 1313 –
324.
12] P. H. J. Carlsen, T. Katsuki, V. S. Martin, K. B. Sharpless, J. Org.
Chem. 1981, 46, 3936 – 3938.
[15] H. Tichy, E. Gingl, R. Ehn, M. Papke, S. Schulz, J. Comp.
Physiol. A 2001, 187, 75 – 78.
2
[16] F. Podebrad, M. Heil, A. Scharrer, S. Feldmer, O. Schulte-Mꢀter,
A. Mosandl, A. C. Sewell, H. J. Bꢁhles, J. High Resol. Chroma-
togr. 1999, 22, 604 – 608; S. Brandꢀnge, S. Josephson, A. Mahlen,
L. Mꢁrch, L. Sweetman, S. Vallen, Acta Chem. Scand. Ser. B
1977, 31, 628 – 630.
[17] G. Gottschalk, A. H. Barker, Biochemistry 1967, 6, 1027 – 1034;
T. Tabuchi, N. Serizawa, S. Ohmomo, Agric. Biol. Chem. 1974,
38, 2565 – 2566.
1
13] B. E. Rossiter, T. Katsuki, K. B. Sharpless, J. Am. Chem. Soc.
1
981, 103, 464 – 465.
14] J. Patel, J. C. Hoyt, R. J. Parry, Tetrahedron 1998, 54, 15927 –
5936.
1
2
036 www.angewandte.org
ꢀ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2010, 49, 2033 –2036