Phyllonorycter Sylvella Moths

Solid Phase Micro Extraction Technique Used for Collecting Semiochemicals. Identification of Volatiles Released by Individual Signalling Phyllonorycter sylvella Moths Anna-Karin Borg-Karlsona and Raimondas Mozuraitisa b a The Royal Institute of Technology, Department of Chemistry, Organic Chemistry, S-100 44 Stockholm, Sweden b Institute of Ecology, Section of Chemical Ecology, Academijos 2, Vilnius, Lithuania Z. Naturforsch. 51c, 599-602 (1996); received January 26/March 7, 1996 Moth, Calling Behaviour, Z10-Tetradecenyl Acetate, Pheromone, Solid Phase Micro Extraction The SPME (solid phase micro extraction) technique was used in the collection of volatiles released by calling females of the 4 -6 mm long tentiform leafminer mothPhyllonorycter sylvella (Lepidoptera, Gracillariidae). The volatiles released by the callingP. sylvella females were identified by GC-MS as a mixture of Z10-tetradecenyl acetate (92%), £10-tetradecenyl acetate (2%) and Z8-tetradecenyl acetate 6(%). The amount of volatiles released by one calling female during three hours and collected on a polydimethylsiloxane fibre, was as large as the amount extracted from the glands of 20 females. The SPME technique gives the oppor tunity of continuously following the release of behaviour mediated signals from weak scented living organisms. Introduction 1995). A number of applications have already There has long been a need for simple and sensi been published, dealing with environmental re search, volatiles in food and beverage, fungi, and tive odour collection techniques for studying in et al., sect semiochemicals. The entrainment technique, water contaminants (Mindrup, 1995; Pelusio in which the volatiles released from the biological 1994 and references therein). objects are concentrated, via an air stream, on a The SPME technique gives us the opportunity porous polymer as Porapak Q, Tenax GC or active to collect volatiles from an individual insect, even charcoal (Borg-Karlson, 1990), has freqently been a very small one. The scent collection from small Phyllonor used also for the collection and enrichment of moths such as species from the genus ycter moth volatiles. The polymers are desorbed by heat has earlier been extremely time- and indivi dual-consuming, as the volatiles have been col or by rinsing the polymer with an organic solvent. lected by dissections of the glands of numerous The latter procedure gives a sample that can be used repeatedly both for analyses and for biologi females, followed by extraction in super-clean or cal tests. However, the need for long periods of ganic solvents (Mozuraitis, unpublished results). In this paper we present our first results using the entrainment, the possible discrimination of com SPME technique on signalling Phyllonorycter pounds of low volatility, and the large solvent peak limit the range of detectable volatiles and the pos moths. sibility to study short time changes in living organisms. The Biological Object Recently, the solid phase micro extraction tech Most of the leafminer moths of the genus Phyl nique (SPME) has been developed and made com lonorycter are monophagous or oligophagous in mercially available (Zang and Pawliszyn, 1993; sects, feeding on one or a few host-plant species Zang and Pawliszyn, 1995; Görecki and Pawliszyn, (Kuznetzov, 1975). The Phyllonorycter moths are convenient model objects for investigations of in terspecific interactions, as the form, colour, and lo Reprint requests to Dr. A.-K. Borg-Karlson. cation of a mine on a host leaf make it possible to Fax; +4687912333. identify the species before adult emergence and 0939-5075/96/0700-0599 $ 06.00 © 1996 Verlag der Zeitschrift für Naturforschung. All rights reserved. D 600 A.-K. Borg-Karlson-R. Mozuraitis • Extraction Technique for Semiochemicals collect pupae of single species for experimental for 2-3 hours at a temperature of 12-14 °C. When needs. Unlike in many other moth species, the ca the sorption from the females was finished, dode- terpillars of the phyllonoryctids feed inside the cane (100 ng) and an undecenyl acetate (5 ng) dis leaf and are not able to change the nourishment solved in hexane were added by syringe onto the place selected by the adult female. This biological inner wall of the glass tube as internal standards feature makes it possible in advance to determine and collected on the SPME fibre for six minutes. the approximate population density and the vari The volatiles from five calling females were col ous species occurring at the place where field tests lected and analysed one by one. will be performed. Little is known about the chem ical communication systems, of phyllonoryctids, Solvent extraction of abdominal glands of thus only for a few species of this genus the phero- Phyllonorycter moths mones are identified (Mozuraitis, unpublished re sults; Arn et al., 1992). Due to the size of the moth The abdominal glands of 100 calling females 10 great efforts are needed to identify pheromone were excised, cut and extracted twice in //I of components using conventional extraction pentane for 15 min at room temperature. The ex techniques. tract was concentrated and a few drops of hexane (pa Merck) were added. The solution was stored at -20 °C until GC-MS-analyses were performed. Materials and Methods Voucher specimens are kept at the Department of Phyllonorycter sylvella (Hbn.) (Lepidoptera, Chemistry, Organic Chemistry, KTH. Gracillariidae) was selected as a model species for collection of the volatiles released by the females Desorption and cleaning procedure of the during the peak of their signalling activity, using SPM E fibre the solid phase micro extraction technique. P. syl vella moths feed on different species in the genus The reference compound, 10 ng Z10-tetrade- A cer (Ivinskis et al., 1985). Leaves with P. sylvella cenyl acetate was dissolved in hexane and added mines were collected from the maple A cer plat- by syringe onto the inner wall of a glass vial of anoides L. near Vilnius (Eastern Lithuania) in Oc the same size as for the sorption of moth volatiles. tober 1994, just before the shedding of leaves. The Sorption was made during 15 min. Z10-Tetrade- leaves collected were placed in wooden boxes on cenyl acetate was totally desorbed from the fibre a 6 - 8 cm layer of moistened peat and were kept at injector temperatures of 200, 225, and 250 °C outdoors during the winter. In the spring, the pu and splitless times of 30 and 60 seconds. Before pae in the mines were placed in vials of a volume the collection periods, the purity of the SPME fi of 32 cm3 and activated in laboratory conditions at bre was checked through two subsequent injec a temperature of 14±2 °C during scotophase and tions in a split/splitless G C-injector for 30 seconds. 20±2 °C during photophase. The light:dark regime Routine conditioning of the fibre was done at was 14:10 hours. Following emergence, the adults 225 °C for 10 min in a GC injector, splittless mode were collected daily immediately after the begin with helium as the carrier gas. ning of the photophase and were then sexed and placed in individual holding vials provided with a Identification of pheromone components solution of 5% (w/v) sucrose in water. The constituents released were identified by using the Finnigan SSQ 7000 GC-MS system, in Sorption o f volatiles from Phyllonorycter moths cluding a Varian 3400 GC. One DB-5 and one DB- Pheromone components from single P. sylvella wax silica capillary column (30 m, id 0.25 mm, film females were collected as follows: The moth (4-6 thickness 0.25 ,«m) were used with a temperature mm long) was placed in a glass tube (10x80 mm). programme of 80 °C (1 min), followed by 10 °C/ The tip of the syringe with the cleaned (see below) min to 140 °C, and thereafter by 1 °C/min up to SPME fibre (100 am polydimethylsiloxane) was 200 °C. Splitless injection for 30 seconds at an in placed a few mm from the protruded abdominal jector temperature at 220 °C (helium 5 psi). After glands of the calling female. The sorption went on injection, the fibre was heated at 225 °C for 10 A.-K. Borg-K arlson-R . Mozuraitis • Extraction Techniqi for Semiochemicals 601 minutes. Peaks from non-calling and calling fe talis (Kumata) (Ando et al., 1977), P. klemanella males were compared and only those coming from (F.) (Booij and Voerman, 1984) and P. ringoniella signalling moths were measured. Mass spectral (Matsumura) (Sugie et al., 1986). With the last two data and retention times of selected peaks on both species the compound is more effective in binary columns were compared with corresponding data mixtures with either of Ell-tetradecenyl acetate for synthetic standards. Z8 -Tetradecenyl acetate, and £4, Z10-tetradecadienyl acetate. Other spe £10-tetradecenyl acetate, and Z10-tetradecenyl cies in the genus Phyllonorycter release related acetate were purchased from Flora Co., (Tartu, Es mono- and di-unsaturated acetates with twelve tonia) and the SPME-equipment from Supelco- and fourteen hydrocarbons (Mozuraitis et al. un Aldrich. published results). However, different host plants and different signalling rhythms of the moth spe cies with the same pheromone may prevent com Results and Discussions petition and confusion among species. Z8 - and Three compounds from the signalling P. sylvella £10-tetradecenyl acetate was neither found as an females were identified for the first time (Table I). attractant nor identified earlier in the genus Phyl The sorption of volatiles released by one calling lonorycter. female during three hours gave a mixture of Z8 - The SPME technique is shown to be a most tetradecenyl acetate (6 %), ElO-tetradecenyl ace valuable tool for collection of volatiles produced tate (2%), and Z10-tetradecenyl acetate (92%). In by weak scented moths. This makes it possible to all, five females were analysed.

Load more