SEX PHEROMONES IN Dichrorampha SPECIES
Jeroen J.N. van den Boezem1, Sandrine A. Ulenberg1 and Teris A. van Beek2
1 University of Amsterdam, Institute for Systematics and Ecology,
Plantage Middenlaan 64, 1018 DH Amsterdam
2 Wageningen Agricultural University, Laboratory of Organic Chemistry,
Dreijenplein 8, 6703 HB Wageningen
INTRODUCTION
Since flight periods, circadian rhythms and host plant associations of the sympatric Dichrorampha species (Lepidoptera, Tortricidae, Olethreutinae, Grapholitini) in Western Europe overlap, sex pheromone variation is the only probable isolating mechanism suggesting that sex pheromone changes played a major role in speciation between the sympatric species of the genus Dichrorampha (Ulenberg, 1992).
The aim of this project is the determination of the female sex pheromones of 14 sympatric Dichrorampha species and interpretation in order to deduce the most probable sequence of changes in the composition of the sex pheromones. Epiblema foenella (Olethreutinae, Eucosmini) has been chosen as outgroup species.
Now the first steps towards the elucidation of the sex pheromones of some of these species have been taken. Gas Chromatography (GC), Electro-Antennographical Detection (EAD) and Mass Spectrometry (MS) play major roles in this kind of research. The elucidation of the sex pheromones of D. flavidorsana and D. simpliciana will be discussed and results will be interpreted in view of the phylogeny of the sub-family and of the genus (Figure 1&2).
Figure 1. Phylogeny of the family Olethreutinae (Horak & Brown, 1991)
Figure 2. Tentative phylogeny of the genus Dichrorampha.
MATERIAL & METHODS
Insects: Specimens from Dichrorampha species were reared from rootstocks and roots of Tanacetum vulgare and Artemisia vulgaris. These members of the family of Asteraceae are the main host plants of respectively D. flavidorsana and D. simpliciana. Every day specimens, that had emerged from the roots and rootstocks, were collected from the cages. Pheromone was isolated after 1-5 days and analysed. Males for EAD-analysis were also collected from the field. Dichrorampha species were collected on Tanacetum vulgare or Artemisia vulgaris and Lathronympha strigana on St. John's wort, Hypericum perforatum.
Isolation: The ovipositor of virgin, calling females was excised and extracted in distilled hexane. The ovipositor of tortricids is known to hold the pheromone gland.
Analysis: Antennas of 1-3 day old male insects were used for electro-antennographical analysis. For on-line EAD the Syntech equipment was connected to a Varian Vista 6000 Series gas chromatograph through the chromatograph's modified second injection port. Humidified air was led through ice and a cooler, which was cooled to 0ºC. Antennas were analysed at 13ºC. For the chromatographic separation several columns were used, e.g. DB-wax (60m id 0.25mm) and DB-23 (40m id 0.20mm), both manufactured by J&W Scientific. All reference compounds were 12 carbon straight-chained acetates (12:Ac) or alcohols (12:OH) with C-C double bonds at different positions in the chain. Off-line stimuli in GC/EAD experiments were given by Pasteur pipette with a piece of filterpaper loaded with a mixture of reference compounds, which are possible pheromone components of Dichrorampha.
ISOLATION AND ANALYSIS
Due to the availability of virgin females of these species, D. flavidorsana and D. simpliciana were investigated in most detail. A specimen of Lathronympha strigana was analysed with on-line GC/EAD with reference compounds only.
On-line GC/EAD experiments with extracts of D. flavidorsana pointed out E10-12:Ac, Z10-12:Ac and E- and/or Z9,11-12:Ac (E/Z9,11-12:Ac) as possible sex pheromone components (Figure 3).
Figure 3. On-line GC/EAD of the extract of a single, 2-5 days old female of D. flavidorsana on DB-wax. Ch.1 (EAD) 1mV/div, Ch.2 (FID) 2mV/div, Horz. 15s/div.
The relative amounts of the different pheromone components in the extract of the ovipositor of a single D. flavidorsana were: E10-12:Ac 10%, Z10-12:Ac 85% and E- or Z9,11-12:Ac 5%. The structures of these components were elucidated from the same extract with MS, spiking and retention indices. The sensitivity of the male antenna was highest to E/Z9,11-12:Ac. This species also showed sensitivity for reference compounds mono unsaturated at the 9th position. A DB-23 column should be able to separate the mixture of E- and Z9,11-12:Ac.
D. simpliciana was thoroughly investigated with off-line GC/EAD. Possible pheromone components were characterised with the help of a dose-response curve. Because many natural compounds induce a response at a high concentration, the sensitivity was investigated. At low concentrations (fg-pg /ml) the antenna shows response only to biologically active compounds like pheromone components.
D. simpliciana's antennas mainly responded to different mixtures E- and Z9,11-12:Ac and to compounds mono unsaturated at the 11th position (Figure 4). Z10- and E10-12:Ac showed the same pattern. Both did not show significant responses at lower concentrations. Compounds, mono unsaturated at the 7th and 8th position did not reach equilibrium at their maximum response in this concentration range, while other compounds did. No conclusions could be drawn in respect to the differnece in sensititvity to E- or Z9,11-12:Ac.
Figure 4. Off-line GC/EAD of antennas of 1-3 days old male D. simpliciana. The standard was 0.1% hexanol solution in hexane.
E/Z9,11-12:Ac was identified before by GC/EAD in extracts of several female abdomen of D. simpliciana (Van den Boezem et al., 1997). No other active compounds were found in that extract (Figure 5).
Figure 5. Old set-up 2d-GC/EAD (2x HP5890, Syntech EAD) on DB-1 and DB-WAX of an extract of 18 female abdomen of D. simpliciana, gathered from the field. Ch.1 (EAD) 1mV/div, Ch.2 (FID) 2mV/div, Horz. 2min/div.
A specimen of Lathronympha strigana was analysed with on-line GC/EAD. The genus Lathronympha is also a member of the Grapholitini and it is therefore interesting to compare its sex pheromone to the sex pheromones of other members of this family, like the genera Cydia, Epiblema and Dichrorampha (Peter Witzgall et al, 1996).
The response of Lathronympha strigana to a mixture of reference compounds showed high sensitivity to E8E10-, Z10-, E10- and E8-12:Ac and almost none to E/Z9,11- and E- and Z9-12:Ac (Figure 6). The amounts of these reference compounds ranged from 1 to 10ng.
Figure 6. On-line GC/EAD of Lathronympha strigana. Reference mixture of different 12:Ac's on DB-23. Ch.1 (EAD) 1mV/div, Ch.2 (FID) 2mV/div, Horz. 15s/div.
CONCLUSIONS & DISCUSSION
The phylogeny on the tribal level of the Olethreutinae is not completely clear. The sensitivity for and presence in the extract of the pheromone gland of E/Z9,11-12:Ac is, however, restricted to the genus Dichrorampha and can only be considered an apomorphic character that separates Dichrorampha from other Olethreutinae (Ulenberg et al, 1993). Almost all other Olethreutinae use compounds mono unsaturated at the 8th and 10th position as sex pheromone components, like Lathronympha strigana (Peter Witzgall et al, 1996).
D. flavidorsana's extract of the pheromone gland contains 95% of E10- and Z10-12:Ac which can be considered a plesiomorf character in D. flavidorsana. Only 5% of the extract of the pheromone gland consists of the apomorphic character E/Z9,11-12:Ac. D. simpliciana shows no sensitivity to E- and Z10-12:Ac in off-line EAD experiments. This difference in sensitivity for these components, albeit a plesiomorphy, will most probably account for the isolation of the two species. Perhaps the plesiomorphy played a role in the separation of the two species.
More experiments need to be done to establish the presence of E10-, Z10-12:Ac and E/Z9,11-12:Ac in the sex pheromone of D. flavidorsana.
Furthermore Dichrorampha's phylogeny will be thoroughly investigated. The sex pheromones of more Dichrorampha species need to be elucidated and tested in the field to be able to draw more firm conclusions about pheromone variation within the genus. Such experiments are on going.
More information about this project can be gathered from the Internet at http://ip24.eti.uva.nl/ZMA/departments/entomol/DichroinlN.html or http://www.spb.wau.nl/oc/index.html
REFERENCES
Boezem, van den, J.N., S.A. Ulenberg and T.A. van Beek,1997. A methodological approach to determination of female sex pheromones of the genus Dichrorampha Guenée. Proc. Exper. & Appl. Entomol. N.E.V. Amsterdam 8, 109-113.
Horak, M. & R.L. Brown, 1991. Taxonomy and Phylogeny. In: Tortricid Pests, Their Biology, Natural enemies and Control (L.P.S. van der Geest & H.H. Evenhuis, eds.: 23-48. Elsevier Science Publishers, Amsterdam.
Ulenberg, S.A., A.K. Minks and J.N.C. van der Pers, 1993. Sex attractants and the taxonomic status of Dichrorampha Guenée. Proc. Exper. & Appl. Entomol. N.E.V. Amsterdam 4, 211-216.
Ulenberg, S.A., 1992. The significance of host plant association in the speciation of Dichrorampha (Lepidoptera, Tortricidae). Proc. Exper. & Appl. Entomol. N.E.V. Amsterdam 3, 123-128.
Witzgall, P., J. Chambon, M. Bengtsson, C. Rikard Unelius, M. Appelgren, G. Makranczy, N. Muraleedharan, D.W. Reed, K. Hellrig, H. Buser, E. Hallberg, G. Bergström, M. Toth, C. Löfstedt and J. Löfqvist, 1996. Sex pheromones and attractants in the Eucosmini and Grapholitini (Lepidoptera, Tortricidae). Chemoecology 7:13-23.