610 Experientia 48 (1992), Birkh/iuser Verlag, CH-4010 Basel/Switzerland
17 Girard, J. E., Madhaven, K., McMorris, T. C., De Loof, A., Chong,
J., Arunachlam, V., Schneiderman, H. A., and Meinwald, J., Insect
Biochem. 6 (1976) 347.
18 Schooley, D. A., Judy, K. J., Bergot, B. J., Hall, M. S., and Jennings,
R. C., in: The Juvenile Hormones, p. 101. Ed. L. I. Gilbert. Plenum
Press, New York 1976.
19 Baehr, J.-C., Poreheron, P., Papillon, M., and Dray, E, J. Insect Phys-
20 Deleurance, S., Baehr, J.-C., Porcheron, P., and Cassier, P., C. r.
Acad. Sci. Paris
21 Madhaven, K., Ohta, T., Bowers, W. S., and Schneiderman, H. A.,
22 Bergot, B. J., Schooley, D. A., and De Kort, C. A. D., Experientia
23 De Kort, C. A. D., Bergot, B. J., and Schooley, D. A., J. Insect Physi-
24 Baker, E C., Lanzrein, B., Miller, C. A., Tsai, L. W., Jamieson, G. C.,
and Schooley, D. A., Life Sci.
25 King, D. S., in: Endocrinology of Insects, p. 57. Eds R.G.H.
Downer and H. Laufer. Alan R. Liss Inc., New York 1983.
26 Schooley, D. A., and Baker, E C., in: Comprehensive Insect Physiol-
ogy, Biochemistry and Pharmacology, vol. 7, Endocrinology I, p. 363.
Eds G. A. Kerkut and L. I. Gilbert. Pergamon Press, Oxford 1985.
27 Dahm, K.H., Bhaskaran, G., Peter, M.G., Shirk, P. D., Seshan,
K.R., and R611er, H., in: The Juvenile Hormones, p. 19. Ed.
L. I. Gilbert. Plenum Press, New York 1976.
28 Baker, F. C., Hagedorn, H. H., Schooley, D. A., and Wheelock, G., J.
29 Koeppe, J. K., Fuchs, M., Chen, T. T., Hunt, L.-M., Kovalick, G. E.,
and Briers, T., in: Comprehensive Insect Physiology, Biochemistry
and Pharmacology, vol. 8, Endocrinology II, p. 165. Eds
G. A. Kerkut and L. I. Gilbert. Pergamon Press, Oxford 1985.
30 Chinzei, Y., Haruna, T., Miura, K., Numata, H., and Nakayama, S.,
31 Chinzei, Y., Nishi, A., Miura, K., Shinoda, T., and Numata, H., In-
0014-4754/92/060606-0551.50 + 0.20/0
9 Birkh~iuser Verlag Basel, 1992
Pheromone components of the female elephant hawk-moth, Deilephila elpenor, and the silver-striped
hawk-moth, Hippotion celerio 1
H. J. Bestmann, J. Erler, W. Garbe, F. Kern, V. Martischonok, D. Sch/ifer, O. Vostrowsky and * L. T. Wasserthal
Department of Organic Chemistry, University Erlangen-Niirnberg, Henkestr. 42, D-8520 Erlangen, and * Institute of
Zoology, University Erlangen-Niirnberg, Staudtstr. 5, D-8520 Erlangen (Germany)
Received 27 March 1991; accepted 29 November 1991
By means of gas chromatographic and mass spectroscopic methods, and combined GC-electroantennogram
and electrosensillogram techniques, (E)-I 1-hexadecenal and (10 E, 12 E)-I 0,12-hexadecadienal [(E,E)-bombykal]
were identified as components of the sex pheromone of the female sphingid moth
bykal is also the main constituent of the pheromone of the silver-striped hawk-moth
activity of the substances was demonstrated with electroantennogram and single cell recording, and the physiological
efficacy of the different hexadecadienal isomers compared.
Key words. DeiIephila elpenor ; Hippotion Celerio ;
Sphingidae; pheromone components; (E,E)-bombykal; (E)-I 1-hex-
The elephant hawk-moth (German: Mittlerer Wein-
L. (Lepidoptera, Sphingi-
dae), is one of the most common sphingid species of
Central Europe, with a distribution throughout the
palearctic region. The silver-striped hawk-moth (Ger-
man: Grosser Weinschw/irmer),
Hippotion celerio L.
(Sphingidae), is a sphingid species of the tropics and
subtropics of the old world, occasionally migrating to
Already in 1979 Starrat et al. described the identification
of (10E, 12Z)-10,12-hexadecadienal [bombykal] as an
active component of the sex attractant of the female
tobacco hornworm moth,
dae), using an electroantennogram biosasay z. Bombykal
was originally found as a sex pheromone component
in the silkworm moth
A more detailed analysis of solvent rinses of pheromone
revealed the presence of a series
of twelve saturated, mono-, di- and triunsaturated
and Cls-aldehydes4; (10E, 12Z)-10,12-hexadecadienal
[bombykal] and (10E, 12E, 14Z)-10,12,14-hexadeca-
trienal represented the active principle required to stimu-
late a 4
complete behavioral sequence . This is, to the best
of our knowledge, the only pheromone from any member
of the sphingid family of Lepidoptera of which the com-
position is known.
Materials and methods
Insect rearing. D. elpenor
L. were caught at Erlangen
(FRG), and subsequent generations reared on wil-
(Onagraceae) in the laboratory. H.
L. were collected on the Canary Island "la
Gomera" and also reared on
species in sum-
mer and on vine
(Vitaceae) in winter, un-
der a 14 : 10 h light: dark regime. Pupae were sexed and
separated a few days before hatching.
HO ~ OH
HO PPh3 1
Experientia 48 (1992), Birkh/iuser Verlag, CH-4010 Basel/Switzerland
HBr HO ~ Br PPh3
1. PhLi, LiBr; 2. O ~ 5 ;
--. -x CHzOH PCC
Synthesis of (10 E, 12 E)-10,12-hexadecadiena1 1 [E,E-bombykal].
3. PhLi, LiBr; 4. HC1, ether;
~, "~ CH=O
Isolation and identification. From newly-hatched calling
virgin females the eight and ninth abdominal segments
with the corresponding intersegmental membrane, the
pheromone gland, were excised under a stereo micro-
scope, and each preparation extracted with 15 ~tl hexane
or CS2 for GC analysis. In addition separated glands
were sealed in glass capillaries and analyzed gas-chro-
matographically using the solid sampling technique 5.
The GC analyses were performed on a HP model 5890A
gas chromatograph equipped with a splitless injector, a
flame ionization detector [FID] and a solid sampler 5.
The volatiles were chromatographed on a 25-m FSCC
SE 54 capillary column (0.25 mmID). Injection port and
detector temperatures were 200~ and 240~ respec-
tively, and the column was programmed from 80 ~ ini-
tial temperature (3 min) to 230~ final temperature at
4~ Carrier gas (N2) flow rate was 1 ml/min.
GCMS analyses were conducted with a Finnigan
MAT90 GC-mass spectrometer in electron impact [EI]
mode, 70 eV, with splitless injection, 25 m FSCC SP2340
(0.25 mmID), injector 200 ~ carrier gas He, 2 ml/min.
Electrophysiologieal bioassays. Electroantennograms
[EAG] were recorded with separated male antennae, us-
ing filter paper loaded with test chemicals as stimulus
source. For the electrosensillograms [ESG], single-cell
recording was performed according to Kaissling's
method 6 by cutting off the tip of a sensillum trichodeum
and inserting it into the "different" electrode, the "indif-
ferent" one being inserted into the base of the antenna.
GLC-coupled EAG and ESG recordings were made us-
ing a Packard United Technologies A49 model with a
1 effluent splitter which partitioned the column efflu-
ent to FID and electroantennogram detector. Injector
240 ~ FID detector 260 ~ column 25 m FSCC SP2340
(0.25 mmID), temperature program 5 min at 70 ~ 70-
195~ at 4~ carrier gas N z.
Synthesis of E, E-bombykal. ( I O E, 12E)-10,12-Hexadeca-
dienal 1 [E,E-bombykal] was synthesized according to
Schlosser's method of (E)-stereoselective carbonyl olefi-
nation 7, starting from 1,10-decandiol 2. Reaction with
HBr and subsequent with triphenyl phosphane yielded
the 10-hydroxydecyl triphenylphosphonium bromide 4,
which was converted into the corresponding ylide with
PhLi and olefinated with (E)-2-hexenal 5. Treatment of
the reaction mixture with additional PhLi and LiBr, HC1
and KO-tert-Bu gave (10E, 12E)-10,12-hexadecadien-1-
ol 6 [36%, Kp 115~ Torr (bath temp.), IR (film):
3350, 1670, 980cm-1; 1H-NMR (CDC13): 0.90-2.40
(23aliph. H), 3.15 (s, OH, H-D), 3.48 (t, J = 6Hz,
CH/O) and 4.89-6.44 (mc, 4 CH=) ppm; 13C-NMR
(CDC13): 13.95 (CH3), 22.45, 25.60, 27.54, 29.08, 29.20,
29.25, 29.31, 31.32, 32.60, 32.75 (10 CH/), 62.67 (CH20),
125.57, 128.47, 129.98 and 134.36 (4CH) ppm; MS
(70 eV): 238 (M+), 220 ( - H20), 67 (100)], from which
E,E-bombykal 1 [33 %, Kp 115~ (bath temp.); IR
(film): 1740, 1670, 980 cm-1; 1H-NMR (CDC13): 0.90-
2.40 (25 aliph. H), 4.90-6.35 (mc, 4 CH=) 9.93 (t, 2 Hz,
CHO); 13C-NMR (CDC13): 13.81 (CH3), 22.63, 23.95,
26.91, 28.80, 29.02, 29.31, 29.44, 37.82, 43.66 (all CH2),
126.03, 128.51, 129.11,132.3 (4 CH) and 204.22 (CHO)
ppm; MS (70 eV): 236 (6, M+), 192(1), 67 (100)] was
obtained by pyridinium chlorochromate [PCC] oxidation
lO,12-Hexadecadienal isomers as GC standards. (10E,
12 Z)-10,12-Hexadecadienal [bombykal] and (10 Z,
12E)-10,12-hexadecadienal [Z,E-bombykal] were avail-
able in our laboratory from previous work s. (10 Z, 12 Z)-
10,12-Hexadecadienal [Z,Z-bombykal] was obtained as a
minor isomeric reaction product only, resulting from an
attempt to synthesize it starting from commercially avail-
able (Z)-2-hexenol. The mixture, mainly comprising
(Z,E)-bombykal, still contained enough of the (Z,Z)-iso-
mer for determination of GC retention and comparison.
Results and discussion
About 8 h after onset of the scotophase female D. elpenor
exhibited calling behavior, that is, they visibly protruded
their ovipositors. From groups of two or three female
insects, the 8th and 9th abdominal segments together
with the common intersegmental membrane were dissect-
ed and extracted with solvent. In initial attempts to iden-
tify pheromonally active compounds in the gland extract,
the extract equivalent to two females was analyzed by
gas chromatography with synchronous EAG-detection
[EAD] 9, using a male antenna. These are most senSitive
towards the conspecific sex pheromone. Two physiologi-
cally active compounds (a and c in fig. 1 A) were found to
Experientia 48 (1992), Birkh~iuser Verlag, CH-4010 Basel/Switzerland
Deilephila elpenor 2 fe L0-
a I[ [ 0
of four isomeric
I , I ~ I
900 1000 -- scan no. 1, 1100
--ret. time ~ rain
Figure 1. Structure elucidation of female D. elpenor sex pheromone com-
ponents. A Gas chromatogram with electroantennogram detection
[EAD] of volatiles of two female gland equivalents [re]; B GCMS recon-
structed gas chromatogram [RGC] of three insects' glands [re]; a) (E)-I 1-
hexadecenal, b) hexadecanal, c) (lOE, 12E)-lO,12-hexadecadienal;
C RGC chromatogram of the four synthetic isomers of 10,12-hexadecadi-
enal: d) (10Z, 12E)-10,12-hexadecadienal, e) (10E, 12Z)-10,12-hex-
adecadienal, f) (10Z, 12Z)-10,12-hexadecadienal and c) (10E, 12E)-
have the retention time: of oxygenated C16-compounds.
Recording the GC mass spectra, in the corresponding
elution range the chromatogram of the gland extract of
three female insects revealed three peaks (a, b and c in
fig. 1 B). The two which eluted first were identified as
(E)-ll-hexadecenal (a) and hexadecanal (b) by compar-
ing their mass spectrum and retention time with those of
authentic compounds. The spectrum of c was almost
identical with that of bombykal, but c had a slightly
longer retention time. Because of this retention differ-
ence, substance c was thought to be an isomer of bom-
bykal. Since originally only (10E, 12Z)- [bombykal] and
(10Z, 12E)-10,12-hexadecadienal [Z,E-bombykal] were
available in our laboratory, and the retention time of the
(10Z, 12E)-isomer also differed from that of c,
(10E, 12E)-10,12-hexadecadienal 1 [E,E-bombykal] had
to be prepared according to the formula scheme.
The mixture of the four geometrical isomers of 10,12-
hexadecadienal was separated gas-chromatographically
under the same GC conditions, and the retention se-
quence determined with (10Z, 12E) (peak d in the fig-
ure), (10E, 12Z) (e), (10Z, lZZ) (f) and (10E, 12E) (c),
as shown in figure 1 C. The mass spectrum and the reten-
tion time of compound c were in full agreement with
those of 1, defining the natural hexadecadienal of D.
elpenor as (10E, 12E)-10,12-hexadecadienal 1.
DeilephilaEAG elpenor /
(n = s)
/i/ i ~ 6 
0.2- / | [EIO,ZI2"I6:AI 1 e
0.001 O.O1 0.1 1 10 100 10~
stimulus source loading
Figure 2. Normalized electroantennogram [EAG] dose response curves
of (E)-ll-hexadecenal [Ell-16:AI a ( = 1.0)], (10E, 12E)-10,12-hex-
adecadienal [El0 E12-16:A1 c] and the 10,12-hexadecadienal isomers
ZIOE12-16:AI d and EIOZ12-16:A1 e obtained from male D.
elpenor antennae (mean n of 5 recordings). Stimulus loading is given in
Both synthetic substances, (E)-ll-hexadecenal a and
(10E, 12E)-10,12-hexadecadienal c (1), were cochro-
matographed with an extract equivalent on a polar
SP 2340 column with EAD detection for final identifica-
tion. Additionally, comparative EAGs were recorded
with (E)-ll-hexadecenal a together with the hexadecadi-
enal isomers c-e. The (E,E)-bombykal c was about ten
times less active than the monounsaturated aldehyde a,
both giving typically sigmoid dose response curves
(fig. 2). The other bombykal isomers d and e revealed no
significant electrophysiological activity at all. The recep-
tor potential responses to a and c recorded by single cell
measurements (ESG) reflected similar dose response
types like the EAG experiments.
Since for the analysis of the female H. celerio pheromone
an even smaller number of moths was available, the iden-
tification had to be based on more specific electrophysio-
logical techniques. An extract of pheromone glands was
produced by similar methods to those used before, gas-
chromatographed, and monitored with a male antenna
as a species-specific detector. The chromatogram of the
extract revealed the presence of one physiologically ac-
tive compound only. Its retention time was the same as
that of (10E, 12E)-10,12-hexadecadienal c (1), as it had
already been determined for the sex pheromone compo-
nent of D. elpenor. To ascertain this identification, (E,E)-
bombykal (1, c) was monitored with a male moth anten-
na, and gave a high response signal (fig. 3 A). A sensillum
trichodeum, the pheromone-specific olfactory hair of a
male antenna, was then prepared for an electrosensillo-
gram (ESG, single cell recording) 1~ and this most
specific detector used in the GC analysis of the
pheromone extract. With this ESG detector, only one
type of spike was observed, reflecting the occurrence
of only one physiologically active component in the
Experientia 48 (1992), Birkhfiuser Verlag, CH-4010 Basel/Switzerland 613
- ~ celerio
GC coupled ESG
I~ gland solid sampling
T I r
--.ret. time .~ rain
Figure 3. Pheromone identification of female
moths. A Gas
chromatogram of (E,E)-bombykal e monitored with a male insect anten-
na; B chromatogram of the pheromone extract monitored by single cell
recording (ESG) (receptor potential) with a male olfactory hair.
(~ = 7)
Se~.e~ e e
........---O v~ LEI0,E12-16:AIJ e
I l I I I I
0.001 0.01 0.1 1 10 100 1000 /.tg
stimulus source loading
Figure 4. Normalized dose response curves of EAG tests obtained from
antenna and the bombykal isomers E 10 E 12-16
16:At (d) and El0 Z12-16:A1 (e) (mean n of 7 recordings),
stimulus source loading in gg.
pheromone extract. The spike signal could be correlated
with one significant receptor response in the extract
(fig. 3B), again at the retention time of (10E, 12E)-
10,12-hexadecadienal 1. Finally, the mass spectrum was
recorded and the exact retention time determined, and
the spectrum as well as the retention time of the physio-
logically active compound were found to be identical
with those of authentic (E,E)-bombykal 1.
In additional electrophysiological experiments, the same
spike type of response was obtained from a male H.
celerio antenna with (10E, 12E)-10,12-hexadecadienal 1
by separate ESG recordings. The spike frequency reflec-
ted a typical dose-response dependence. Comparing the
electrophysiological activities of the isomeric bombykals
using the electroantennogram test (EAG), the highest
antennal responses were found (fig. 4) for (E,E)-bom-
Acknowledgment. We want to thank Martin Grund, Claudia Obermeier
and Regina Walther for their help in rearing the caterpillars. We thank the
Deutsche Forschungsgemeinschaft and the Volkswagen-Stiftung for fi-
nancial assistance. V.M. is indebted to the Deutscher Akademischer
Austauschdienst for awarding a fellowship, D. S. for one from the Stu-
dienstiftung des Deutschen Volkes.
1 Pheromones, 79; as Pheromones, 78 is taken: Wu, Cai-Hong, and
Bestmann, H.J., Chinese Science Bulletin
Pheromones, 77: Attygalle, A. B., Steghaus-Kovac, S., Ahmed, V. U.,
Maschwitz, U., Vostrowsky, O., and Bestmann, H. J., Naturwissen-
2 Starrat, A.N., Dahm, K.H., Allen, N., Hildebrand, J. G., Payne,
T. L., and Roeller, H., Z. Naturforsch.
3 Kasang, G., Kaissling, K. E., Vostrowsky, O., and Bestmann, H. J.,
(1978) 74; int. edn, Engl.
4 Tumlinson, J. H., Brennan, M. M., Doolittle, R. E., Mitchell, E. R.,
Brabham, A., Mazomenos, B.E., Baumhover, A.H., and Jack-
son, D. M., Arch. Insect Biochem. Physiol.
5 Attygalle, A. B., Herrig, M., Vosrowsky, O., and Bestmann, H. J., J.
6 Kaissling, K. E., in: Biochemistry of Sensory Functions, p. 243. Ed.
L. Jaenicke. Springer Verlag, Berlin/Heidelberg/New York 1974.
7 Schlosser, M., Tuong, H. B., and Schaub, B., Tetrahedron Lett.
8 Bestmann, H. J., Siiss, J., and Vostrowsky, O., Liebigs Ann. Chem.
9 Struble, D., and Arn, H., in: Techniques in Pheromone Research,
p. 161. Eds H. E. Hummel and T. A. Miller. Springer Verlag, New
10 Wadhams, L. J., in: Techniques in Pheromone Research, p. 179. Eds
H. E. Hummel and T. A. Miller. Springer Verlag, New York 1984.
0014-4754/92/060610-0451.50 + 0.20/0
9 Birkh~iuser Verlag Basel, 1992