Dr Sandrine A.
This study is part of a
research program in which biological data are
incorporated into phylogenetic studies in an
attempt to formulate effect analyses for processes
Dichrorampha belongs to the lepidopteran
family of Tortricidae, subfamily of Olethreutinae,
tribe of Grapholitini. The genus
Dichrorampha consists of approximately 115
species, of which 14 occur in the Netherlands. The
distribution of the genus is Holarctic
Most of the 14
Dichrorampha species occurring in the
Netherlands are sympatric and have overlapping
flight periods. The larvae feed on a restricted
number of host plant species, all of which belong
to the family Asteraceae. For many closely related
sympatric tortricids host plant association shows
to be the key to reproductive isolation. However,
the host plant associations amongst the
Dichrorampha species are not mutually
exclusive, so that they cannot function as
isolating mechanisms (Ulenberg et al., 1993). The
daily sexual activity patterns of the different
species are also overlapping (data unpubl.). Thus,
the answer to the question how reproductive
isolation is maintained points to the pheromone
system. We, therefore, propound female sex
pheromone variation between the 14 different
species of Dichrorampha most probably is the
only isolating mechanism suggesting that female sex
pheromone variation played a major role in the
speciation events within
The aim of this study is
to characterise the female sex pheromone variation
among these 14 species of Dichrorampha and
to interpret the pheromone variation in view of the
projects in this study:
communication in the genus Dichrorampha
by Jeroen J.N. van den
Boezem1, Sandrine A. Ulenberg1 and Teris A. van
1 University of
Amsterdam, Institute for Systematics and Population
Biology, Department of Entomology, Plantage
Middenlaan 64, 1018 DH, Amsterdam.
Agricultural University, Laboratory of Organic
Chemistry, Phytochemical Section, Dreijenplein 8,
6703 HB, Wageningen.
Many moth species have
reproductively isolating host plant associations,
but the genus Dichrorampha (Olethreutinae,
Grapholitini) does not. Also flight period,
circadian rhythm, lifecycle and morphology of the
sympatric Dichrorampha species show striking
similarities and do overlap.A Therefore,
we propound sex pheromone variation is the only
isolating mechanism suggesting that sex pheromone
changes played a major role in the events from
which new species originated (speciation) within
the genus Dichrorampha.
Dichrorampha and Epiblema :
a.D. alpinana; b. D.
simpliciana ; c. D.
The aim of this project is
the determination of the female sex pheromones of
14 Dichrorampha species, occurring in the
Netherlands, and the deduction of the most probable
sequence of changes in the composition of the sex
pheromone on basis of the reconstruction of their
evolution (phylogeny). We have chosen Epiblema
foenella (Eucosmini) as outgroup
Pheromone is isolated by
solvent extraction from the pheromone gland, Solid
Phase Micro Extraction (SPME) or Thermal Desorption
(TD) from females from both the field and rearing.
Methods for optimisation SPME and TD are still
By 2-dimensional Gas
Chromatography (2d-GC) in combination with
Electro-Antennographical Detection (EAD) the
pheromone gland extracts are analysed (figure
2. The 2d-GC/EAD output of an extract of
the pheromone glands of female D.
Horz.: 1.0min/div. Channel 1: EAG
response; 0.5mV/div. (Filt. 8), Channel 2:
2d-GC output; 0.5mV/div. (Filt.
Major EAD-response is
found at retention time (37.51 min.) of
E9,11-dodecadien-1-yl acetate (E9,11-12:Ac) for the
species complex D. plumbana/D.
sedatana. Other EAD-responses correspond to
retention times of other possible components
(figure 2). Similar experiments on D.
simpliciana showed the same results.
Off-line Electro-Antennography (EAG) experiments
are conducted with reference chemicals (figure
3. Off-line EAG results on D.
flavidorsana (green), D.
simpliciana (red) and D.
Remarkable in the genus
Dichrorampha is the high and general
response to E9,11-12:Ac. E.
foenella's major sex pheromone components
and other components of many other members of the
Olethreutinae are unsaturated at positions 8 and 10
(D8D10, D8 and D10).C Other mono- (D9
and D8) and double-unsaturated (D8D10) -12:Ac's and
-12:OH's seem to play a crucial role in the sex
pheromone variation among Dichrorampha
species. These differences are subtle, but probably
the key to reproductive isolation within the genus
Dichrorampha (figure 2 and 3). In the future
sex pheromones of all 14 Dichrorampha
species will be investigated with these techniques
- Ulenberg, Minks &
van der Pers, Proc. of the Section Exp. and
Applied Entomology of the NEV, Vol. 4, p.
- Van den Boezem &
Ulenberg, Proc. of the Section Exp. and Applied
Entomology of the NEV, Vol. 8, p. 109-113,
Chemoecology, 7:13-23, 2555, 1996
orana as a model insect for Dichrorampha
Jeroen J.N. van den
Boezem, Sandrine A. Ulenberg and Teris A. van
orana (Lepidoptera, Tortricidae) has been
chosen as a model insect to optimise the analytical
techniques. Adoxophyes orana's sex
pheromone has been fully elucidated from extracts,
volatile collections and sex glands1 and
its components are commercially available.
Adoxophyes orana can be reared. These
are all characteristics Dichrorampha does
2 HP 5890 Series II GC's,
equipped with FID's and respectively a DB-1 and
DB-wax column, are connected to a Syntech
(EAD)2. The connection between the
second GC and the EAD is made through a retention
reservoir. Female sex glands of Adoxophyes
orana are thermally desorbed at 150°C
in the Cooled Injection System (CIS) of the first
electro-antennographical signals (Ch.1) of peak 1
and 2 in the chromatogram (Ch.2) we can conclude
that Z9-14:Ac and Z11-14:Ac respectively induce a
physiological response in male Adoxophyes
figure 1: DB-wax
column, 2 sex glands. Ch.1 (black); 1mV/div, Ch.2
(blue); 50mV/div, Horz.; 50s/div, filtered
A HP 5890 GC, equipped
with FID, is connected to a Gerstel Thermal
Desorption System (TDS 2). Female sex glands and
male abdomen are thermally desorbed in TDS 2 at
50-100°C and volatiles are injected into the
CIS. Experiments are also conducted with whole
insects, alive just before flash
Male abdomen are compared
to female sex glands. From these chromatograms we
can conclude that peaks 1 and 2 are the major
components of the sex pheromone of Adoxophyes
orana, respectively Z9-14:Ac and Z11-14:Ac in a
ratio of 11 to 1.
figure 2 and 3: DB-1 and
DB-wax column, male abdomen (black) and 2 sex
figure 2: TDS 2;
25ºC -> 25ºC/min, 100ºC, 5min,
splitless: CIS; -150ºC ->
350ºC, 5min, splitless: Oven; 150ºC, 4min
-> 10ºC/min, 238ºC, 10 min.
A HP 6890 Series GC is
connected to a HP 5973 Mass Selective Detector
(MSD). Female sex glands are thermally desorbed in
the liner of the GC at 30 to 150°C, while at
the beginning of the column some focusing takes
place at an oven temperature of
From the mass spectra we
can conclude that peaks 1-5 are: 1. Z9-14:OH, 2.
Z9-14:Ac, 3. E11-14:Ac, 4. Z11-14:Ac and 5.
Z11-16:Ac. These compounds are known to be among
the largest of the components of the sex pheromone
of Adoxophyes orana.
figure 4: HP-5
column, 2 sex glands. Liner; 30ºC -> ca.
25ºC /min, 150ºC, 2min, splitless: Oven;
40ºC, ca. 5min ->10ºC/min, 240ºC,
All these experiments
confirm the composition of the female sex pheromone
of Adoxophyes orana. The same
techniques will be applied for the elucidation of
the sex pheromones of Dichrorampha species. After
optimisation it should be possible to identify the
minor, as well as the major, components with
2d-GC/EAD. Surely the male antenna is sensitive
enough. GC/MSD and TDS/GC can be used for
conformation. GC/MSD can lead to identification of
both minor and major pheromone components from its
mass spectra. TDS/GC shows the components that only
the female insect produces. These are strong hints
that compounds are in fact pheromone
- P. M. Guerin, H. Arn,
H. R. Buser and P. J. Charmillot. Sex pheromone
of Adoxophyes orana: additional components and
variability in ratio of Z-9- and
Z-11-tetradecenyl acetate. Journal of Chemical
Ecology, Vol. 12, No. 3, pp. 763-772,
- 2J. J. N. van den
Boezem and S. A. Ulenberg. A methodological
approach to determination of female sex
pheromone of the genus Dichrorampha
Guenée. Proceedings of the section
experimental and applied entomology of the
Netherlands Entomological Society, Vol. 8, pp.
- 3T. A. van Beek, F. P.
Drijfhout and J. J. N. van den Boezem. On-line
collection and GC analysis of pheromones from
intact insects. Proceedings of the 2nd
International Symposium on Insect Pheromones 30
March-3 April 1998.
pheromones of Dichrorampha
Jeroen van den Boezem,
Sandrine Ulenberg and Teris van Beek
association, flight periods and circadian rhythm
overlap, sex pheromone variation is the only
plausible isolating mechanism suggesting that sex
pheromone changes played a major role in speciation
within the sympatric genus Dichrorampha
(Lepidoptera, Tortricidae, Olethreutinae,
The possible pheromone
composition of D.
are discussed here. For more information, click
The aim of this project is
the determination of the female sex pheromones of
14 Western-European Dichrorampha species and the
deduction of the most probable sequence of changes
in the composition of the sex pheromones on basis
of their phylogeny.
On-line GC/EAD experiment with extract of single
calling 2-5 day old, virgin female D..flavidorsana
on DB-wax, 60m, id 0.25mm in Varian 6000 GC
connected to Syntech EAG equipment. Ch.1 (EAD)
1mV/div, Ch.2 (FID) 2mV/div, Horz.
Off-line EAG experiments with single antennae of
2-5 day old male D. simpliciana with Syntech EAG
equipment. Experiments are conducted with 20(l of
solution on filterpaper in a Pasteur
The relative amounts of
the pheromone components in the extract of D.
flavidorsana are: E10-12:Ac 10%, Z10-12:Ac 85%
and E- or Z9,11-12:Ac 5%. The structure of the
components is verified with GC/MSD. Sensitivity is
highest for E/Z9,11-12:Ac. D. flavidorsana
does show low sensitivity to compounds mono
unsaturated at the 9th position. No distinction can
be made between the 2 isomers of ~9,11-12:Ac, since
they have not yet been separated.
antennae are mainly sensitive to E-, 26%Z, and
Z9,11-12:Ac, 40%E (white). Also to compounds mono
unsaturated at the 9th and 11th position (blue).
Typically compounds mono-unsaturated at the 7th and
8th position (yellow) do not reach their
equilibrium in this concentration range, while all
others do (black). E- and/or Z9,11-12:Ac has been
shown with GC/EAD in earlier experiments in gland
extracts of D. simpliciana (click
for more information).
The phylogeny on the
tribal level of the Olethreutinae is not very
clear. The sensitivity to E/Z9,11-12:Ac, however,
is restricted to the genus Dichrorampha and thus
can be regarded as apomorphic character that
separates Dichrorampha from other Olethreutinae.
Almost all other Olethreutinae use unsaturated
compounds at the 8th and/or 10th
The fact that the D.
flavidorsana female extract holds E- and
Z10-12:Ac as its pheromone components and D.
simpliciana does not show any significant
sensitivity to these compounds in off-line EAG
experiments should be reagarded as a plesiomorphic
character in D. flavidorsana and could
account for the separation at that specific moment
of speciation of the two species in the phylogeny
of the genus Dichrorampha.
The phylogeny of
Dichrorampha will thouroughly be
investigated. The sex pheromones of more
Dichrorampha species need to be elucidated
and tested in the field to make further comments
about speciation events within the