IOBC wprs Bulletin Vol 22(9), 1999

Chemicals involved in the mating process of the almond seed wasp Eurytoma amygdali

Fragoulis D. Krokos, Maria A. Konstantopoulou and Basilis E. Mazomenos

Institute of Biology NCSR "Demokritos", Aghia Paraskevi Attikis, Greece

Abstract: Volatile chemicals collected from virgin Eurytoma amygdali females by extracting the whole or parts of their bodies in methylene chloride were attractive to males in laboratory bioassays. The same attraction was found when volatile compounds were collected by the SMPE fibre. Comparative GC-MS analysis of male and female extracts indicated differences which corresponded to a number of peaks present only in the female extract. Preparative GC fractionation of these peaks and subsequent bioassays elicited male response. These peaks correspond to mono-unsaturated and di-unsaturated long chain hydrocarbons as indicated by mass spectrometric analysis.

Key words: Almond seed wasp, Eurytoma amygdali, sex pheromone



The almond seed wasp Eurytoma amygdali Enderlein, (Hymenoptera:Eurytomidae) is considered as one of the most important pests of almonds in a number of southeastern countries, the Middle East and some of the countries of the former Soviet Union. The damage caused to the crop can reach up to 90% by mummifying the infested almond. Chemical control of this pest is usually accomplished by the application of a systemic insecticide. Since time of application is critical, monitoring of adult emergence is carried out by placing cages containing infested almonds from the previous year in the field and counting the emerging adults. Pittara and Katsoyannos have reported male attraction to virgin females in E. amygdali (Pittara and Katsoyannos 1985). Katsoyannos et al. have used traps baited with virgin females for monitoring the population of E. amygdali (Katsoyannos et al. 1992). Due to the nature of this trap set-up (living female wasps), its use is limited and can be applied only from trained personnel. Since the chemical structure and composition of the sex pheromone has not been elucidated yet, the use of synthetic sex pheromone traps which could improve the monitoring process is not yet possible. To the best of our knowledge there is no reference in the literature so far for determination of sex pheromones in Eurytomidae.

This work reports on the chemical composition of cuticular extracts from male and female E. amygdali and the potent chemicals which are involved in the mating process of this wasp.


Material and methods

The insects used in this study were obtained from infested almonds collected from the field and stored at 4 - 6 ° C in the dark. Adults were obtained from almonds that had been transferred from the cold room to the lab which was kept at 25 ± 2 ° C and a 14:10 hr light-dark regime. Adults were sex separated as soon as they emerged and kept in cages under the conditions mentioned above. Wasps were provided with a 10% sucrose solution.

Laboratory Bioassays
Bioassays were performed in a 50 x 40 x 30 cm screen cage; 10-20, 1 to 5 days old males were present in the cage during the bioassay. All bioassays were conducted 3 to 4 hr after the onset of the photoperiod. A 2 x 2 cm Whatman No 1 filter paper impregnated with the extract to be tested (2-3 female equivalent) was hung 5 cm below the top of the cage and served as the source. A fan was used to generate the air stream. Males exhibiting wing raising, lateral swing of the body, attraction and movement towards the source and source touching was the response criterion. The SPME fibre that had been used to collect analytes from E. amygdali was also evaluated for male response. The SPME holder was standing on the top of the cage in such a place that allowed the exposed fibre to penetrate ca. 5 cm inside the cage from the top part. The rest of the experimental set-up was kept the same.

Solvent Extraction
Cuticular compounds were extracted with methylene chloride for 20 min from groups of male or female individuals using the whole body or different parts of their bodies (head, thorax and abdomen).

Solid Phase Micro Extraction (SPME)
Collection of chemicals from the insects cuticle was performed by rubbing a SPME fibre (polydimethylsiloxane, SUPELCO, USA) onto the insect body. Two individuals were used for each collection. The same SPME fibre was also used to collect airborn volatiles by headspace extraction. In this case, four individuals were placed in a 4ml vial and the fibre was exposed to the volatiles for two hours.

Preparative Gas Chromatography
Preparative gas chromatography was carried out on a Varian 3400 gas chromatograph equipped with a FID detector and a 30 m x 0.53 mm x 1 µm film thickness DB-5 column (J&W Scientific). The oven temperature program was 50 °C for 2 min, then 5 °C/min to 280 °C hold for 60 min. Helium was used as the carrier gas at a flow rate of 3 ml/min. On column injections were made (1 µl) at an injector temperature of 250 °C and a detector temperature of 300 °C. The outlet of the column was split by a manually operated metering valve between the detector (10%) and the collecting capillary (90%). The fractions were collected in ice cooled fused silica uncoated capillaries and were subsequently washed with a few µls of methylene chloride.

Gas Chromatography - Mass Spectrometry
Gas chromatography-mass spectrometry analysis was carried out on a Hewlett Packard 5890 Series II gas chromatograph interfaced to a Fisons VG Trio 1000 (Manchester M23 9BE, UK) quadrupole mass spectrometer. Electron impact ionisation was used, with an electron energy of 70 eV and a trap current of 200 µA. All extracts were chromatographed on a 60 m x 0.25 mm x 0.1 µm film thickness DB-5 column (J&W Scientific). The oven temperature program was 50 °C for 2 min, then 5 °C/min to 250 °C hold for 1 min, then 2 °C/min to 280 °C and hold for 50 min. Helium was used as the carrier gas at a flow rate of 1 ml/min. Splitless injections were made (1 µl) at an injector temperature of 250 °C and a splitless period of 90 s. For the SPME analysis, the fibre was desorbed into the injection port for 5 min.

Results and discussion
Extracts of E. amygdali females from all three techniques evaluated elicited male response when tested in laboratory bioassays. On the contrary male response to male whole-body extract was not significantly different from the control. In order to identify the female body part(s) involved in the release of those chemicals responsible for the mating process of E. amygdali, male attraction to extracts of head, thorax and abdomen was evaluated in the lab. It was found that thorax extracts elicited significantly greater response than head and abdomen extracts (Fig. 1). This is in accordance with the work described recently by Leal et al. for Bephratelloides pomorum (Hymenoptera, Eurotomidae) (Leal et al. 1997).

Figure 1. E. amygdali male response (%) to extracts of different parts of female body and whole body.

Comparative GC-MS analysis of female extracts with all three techniques tested, exhibited a similar chromatographic profile with mainly quantitative rather than qualitative differences. The same applies to male extracts. The results indicate that the SPME technique compares well with the conventional solvent extraction approach and offers a fast, solventless alternative to it. Differences were observed though, between male and female extracts regardless the technique used. The vast majority of compounds identified are long chain saturated, mono-unsaturated and di-unsaturated hydrocarbons. The saturated hydrocarbon chromatographic profile from each sex was qualitatively similar with some quantitative differences. Most of the alkenes and both alkadienes identified were present only in the female extract (Fig. 2). It has been reported that for Cardiochiles nigriceps (Hymenoptera: Braconidae) alkadienes which are present only in the female extract were responsible for mediating courtship behaviour (Syvertsen et al., 1995). For this species, the alkanes fraction from each sex were qualitatively simiral and to a lesser extent the same was applied for the alkenes fraction. Further fractionation of the crude female extract on preparative GC led to the collection of three distinct fractions containing compounds that had been detected only in the female extract. Fraction 1 contained tricosene and tricosadiene, fraction 2 contained pentacosene and pentacosadiene and fraction 3 contained triacontene and a coeluting saturated hydrocarbon (Fig. 2). Bioassays performed with these fractions revealed that the highest male response was elicited by fraction 1 and the lowest by fraction 3. The sex pheromones isolated from some parasitic Hymenoptera have been determined to consist of multiple compounds (Shu and Jones 1993; Swedenborg and Jones 1992; Swedenborg et al. 1993). Our results suggest that alkadienes and to a lesser extent alkenes identified in the female extract of E. amygdali are potent chemicals responsible for the mating process of this wasp.


Figure 2. Main differences between male (A) and female (B) E. amygdali whole body extracts (in methylene chloride). The three fractions collected by preparative GC are also indicated.



The chemical composition of male and female E. amygdali cuticular extracts has been determined. The major differences between the chemical composition of the two sexes are alkenes and alkadienes which are present only in the female extract. The results presented in this work suggest that mono-unsaturated and di-unsaturated long chain hydrocarbons elicit male response. Further work will include complete elucidation of the structure of these compounds and bioassays to unambiguously determine the compound(s) which are responsible for the mating process of the almond seed wasp.



Katsoyannos, B.I., Kouloussis, N.A. & Bassiliou, A. 1992: Monitoring populations of the almond seed wasp, Eurytoma amygdali, with sex pheromone traps and other means, and optimal timing of chemical control. Entomol. Exp. Appl. 62: 9-16.

Leal, W.S., Maoura, J.I.L., Bento, J.M.S., Vilela, E.F. & Pereira, P.B. 1997: Electrophysiological and behavioral evidence for a sex pheromone in the wasp Bephratelloides pomorum. J. Chem. Ecol. 23: 1281-1289.

Pittara, I.S. & Katsoyannos, B.I. 1985: Male attraction to virgin females in the almond seed wasp Eurytoma amygdali Enderlein (Hymenoptera, Eyrotomidae). Entomol. Hellenica 3: 43-46.

Syvertsen, T.C., Jackson, L.L., Blomquist, G.L. & Vinson, S.B. 1995: Alkadienes mediating courtship in the parasitoid Cardiochiles nigriceps (Hymenoptera : Braconidae). J. Chem. Ecol. 21: 1971-1989.

Shu, S. & Jones, R.L. 1993: Evidence for a multicomponent sex pheromone in Eriborus terebraus (Gravenhorst) (Hym., Ichneumonidae), a larval parasitoid of the European corn borer. J. Chem. Ecol. 19: 2563-2576.

Swedenborg, P.D. & Jones, R.L. 1992: Multicomponent sex pheromone in Macrocentrus grandii (Goidanich) (Hymenoptera: Braconidae). J. Chem. Ecol. 18: 1901-1911.

Swedenborg, P.D., Jones, R.L., Liu, H. & Krick, T.P. 1993: (3R*, 5S*, 6R*)-3,d-Dimethyl-56-(methylethyl)-3,4,5,6-tetrahydropyran-2-one, a third sex pheromone component for Macrocentrus grandii (Goidanich) (Hymenoptera: Braconidae) and evidence for its utility at eclosion. J. Chem. Ecol. 19: 485-502.

table of contents