IOBC wprs Bulletin Vol 22(9), 1999

Identification of volatiles from Ceratitis capitata (Wiedemann) (Diptera: Tephritidae) by means of thermal desorption of solid samples coupled to GC-MS

Víctor Casaña-Giner and Jaime Primo-Millo

Abstract: Thermal desorption of biological samples has been employed for the identification of semiochemicals in Ceratitis capitata (Medfly). Anal ampoules of male medflies, whole females and eggs were introduced in a thermal desorber, coupled to a gas chromatograph-mass spectrometer. Previously described as well as new male volatiles were identified. A new compound, 2-dimethylamino ethyl acetate, was identified from female medflies. A new compound from eggs, 2-pentadecanone, was shown to have oviposition deterrent activity.

Key words: Ceratitis capitata, GC-MS, volatiles, oviposition deterring pheromone

Introduction

A considerable number of studies have been undertaken to identify the volatiles in male medflies (Jacobson et al.1973; Ohinata et al.1977; Baker et al.1985; Jang et al.1989; Cossé et al.1995), with in every case, sample collection been effected by means of extraction or aireation techniques. Up to 77 compounds have been identified in male volatiles using these techniques. Nevertheless, efforts to identify biological active female volatiles has uptill now failed, and no volatiles from medfly eggs have been described, although the existence of an oviposition deterring pheromone has been demonstrated.

In this work, we have identified male volatiles from anal ampoules by thermal desorption of the samples coupled to GC-MS, as well as a female volatile and a compound from their eggs which has oviposition deterring activity.

Material and methods

The Ceratitis capitata flies were reared in our laboratory (on a photoperiod of 16:8 (L:D) h; and at 28ºC and 50% RH). Adult flies were fed with a mixture of autolysed yeast and sucrose (1:4 wt/wt). The larvae were reared in a mixture of wheat bran: sucrose: beer yeast: nipagin: nipasol: water: hydrochloric acid (20:5:1:0.5:0.5:10:0.1).

For the desorption of biological samples, a thermal desorber was used (Dynatherm analytical Instruments Inc., mod. 890). The gas chromatographs were obtained using a VARIAN chromatograph, mod. Star 3400, and the mass spectrometer employed was a VARIAN SATURN II (with an ion-trap detector). The CG column used was an HP-5 type (25m * 0.2 mm * 0.52 µm). The GC employed column program was: init. temp.:40ºC, time: 4 min, rate: 3ºC/min and final temp.: 240ºC. Fresh samples (ca. 25 mg) were introduced in a quarz tube of 9 mm * 4 mm i.d. (Supelco) and then immediately desorbed.

Oviposition deterring activity was investigated by two-choice trials in cages with 500 gravid females. Both a control polyethylene cup covered with a cloth that let oviposition through it, and a treated cup with a cloth which was impregned, by means of a pencil, with a solution of 2-pentadecanone at 1% in acetone were used. After ten hours of exposure time, the laid eggs were counted in each cup. Three repetitions were made.

Results

The chromatograph of volatiles from thermally desorbed male C. capitata anal ampoules is shown in Fig. 1.

Figure 1. Chromatogram of thermal desorbed male anal ampoules. Compounds identified were (underlined compouds have been previously described): 3) acetic acid 5) furfuryl alcohol 7) 2,6-dimethyl-1-H-indol 8) geranyl acetate 9) (E,E)-a-farnesene 10) (E)-3-ethyl octenoate 11) propyl-4-hydroxybenzoate 12) 1-dodecene 13) ethyl dodecanoate 15) hexadecanoic acid, 1-methyletil ester 19) tetradecanoic acid 20) hexadecanoic acid.

The compounds identified were as follows (those underlined represent compounds that have been previously described): acetic acid; furfuryl alcohol; 2,6-dimethyl-1-H-indol; geranyl acetate; (E,E)-a-farnesene; (E)-3-ethyl octenoate; propyl-4-hydroxybenzoate; 1-dodecene; ethyl dodecanoate; hexadecanoic acid, 1-methylethyl ester; tetradecanoic acid and hexadecanoic acid.

The chromatograph of volatiles from thermally desorbed whole females is shown in Fig. 2. In this case we have identified 2-dimethylamino ethyl acetate, previously not described in C. capitata (at a retention time of 6 min). Several acids and methyl esters (C12, C14, C16, C18 esters; C14, C16 acids) are found also in a similar way to male volatiles.

Figure 2. Chromatogram of volatiles from thermally desorbed whole females.

Figure 3. Chromatogram of volatiles from thermally desorbed C. capitata eggs between 15 and 35 min, with particular emphasis in detection of 2-pentadecanone (up: total ion plot; middle: ion 58; down: ion 226 -molecular weight).

Fig. 3 shows the chromatogram of volatiles from thermally desorbed whole eggs. Because of the low quantities of 2-pentadecanone in C. capitata eggs, in order to obtain a good spectrum, we have found it necessary to monitor the characteristical ions of this compound. After substraction of the background, the spectrum is found to be acceptable and is shown in Fig. 4. Kovats Index of standard 2-pentadecanone matched with that of our sample. We have made behavioural assays of oviposition deterring activity, as described in the materials and methods section. In every case, the females avoid egg laying in the treated cups (0) while the mean quantity of laid eggs in the control cups was 487.6 ± 25.3.

Figure 4. Spectrum of 2-pentadecanone, identified in C. capitata eggs, in comparison with the recorded spectrum for 2-pentadecanone in the NIST library of spectra.

Discussion

Identification of known male volatiles by this technique (like (E,E)-a-farnesene and geranyl acetate) could be considered as a validation of the method. With this technique, time for the analysis is short, with no time employed in the areation and extraction. Moreover, biological samples of few mg can be used, implying that we only need a single individual for tentative identification of its pheromone. This technique could be employed in other insects or animals (for example, desorption of lepidopteran females pheromone glands).

Results concerning deterring of oviposition by 2-pentadecanone are been carried out, determining the minimal effective doses and its activity when applied in fruits. 2-pentadecanone could be one of the compounds from the C. capitata oviposition deterring pheromone, which is responsable for its biological activity, in spite of its minimal pressence in our analysis

It is worthy to note that in the three chromatograms (anal ampoules of males, females and eggs) appear some peaks of considerable area that are compounds possibly formed during thermal desorption (specially furanones and pyranones). 2-pentadecanone could be also a degradation product formed due to the high temperatures, so it would be needed to identify this compound by other means to assure its presence in the eggs, in spite of having observed its biological activity.

References

Baker, R., Herbert, R.H. & Grant, G.G. 1985: Isolation and identification of the sex pheromone of the Mediterranean fruit fly, Ceratitis capitata (Wied). J. Chem. Soc. Chem. Commun. : 824-825.

Cossé, A.A., Todd, J.L., Millar, J.G., Martinez, L.A. & Baker, T.C. 1995: Electroantennographic and coupled gas chromatographic - electroantennographic responses of the Mediterranean fruit fly, Ceratitis capitata, to male-produced volatiles and mango odor. J. Chem. Ecol. 21: 1832-1836.

Jacobson, M., Ohinata, K., Chambers, D.L., Jones, W.A. & Fujimoto, M.S. 1973: Insect sex attractants. 13. Isolation, identification and synthesis of sex pheromones of the male mediterranean fruit fly. J. Medic. Chem. 16: 248-251.

Jang, E.B., Light, D.M., Flath, R.A., Nagata, J.T. & Mon, T.R. 1989: Electroantennogram responses of the mediterranean fruit fly, Ceratitis capitata to identified volatile constituents from calling males. Entomol. Exp. Appl. 50:, 7-19.

Ohinata, B., Jacobson, M., Nakagava, S., Fujimoto, M. & Higa, H. 1977: Mediterranean fruit fly: Laboratory and field evaluations of synthetic sex pheromones. J. Environ. Sci. Health A12(3): 67-78.

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