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Journal of Chromatography A, 1180 (2008) 165–170

Semiochemicals related to the aphid Cinara pilicornis and its host,Picea abies: A method to assign nepetalactone diastereomers

Marie Pettersson a, C. Rikard Unelius b, Irena Valterova c, Anna-Karin Borg-Karlson a,∗a KTH, School of Chemical Science and Engineering, Department of Chemistry, Ecological Chemistry Group, SE-100 44 Stockholm, Sweden

b School of Pure and Applied Natural Sciences, University of Kalmar, SE-391 82 Kalmar, Swedenc Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic, Flemingovo nam. 2, 166 10 Praha 6, Czech Republic

Received 22 August 2007; received in revised form 29 November 2007; accepted 7 December 2007Available online 15 December 2007

bstract

Volatiles released by seedlings of Norway spruce infested with the aphid Cinara pilicornis were analyzed using SPME–GC–MS. Among thetress-induced compounds released by the host plant, citronellol, cis–trans-nepetalactone and cis–trans-nepetalactol was found. These compoundsriginated from the aphids and they were assumed to be pheromone components for this aphid species. To determine the relative stereochemistry

f the nepetalactone, a diagnostic method was developed. The method was based on multivariate analysis of tabulated relative intensities of massragments of the four nepetalactone diastereomers. In the practical method described, a few pairs of fragments in the mass spectra were comparednd, in combination with the Kovat’s index, were used to unambiguously identify the relative stereochemistry of the nepetalactone. 2007 Elsevier B.V. All rights reserved.

GC–M

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eywords: Aphid; Conifer; Pheromone; Nepetalactone; Diastereomer; SPME–

. Introduction

Conifer trees are long-lived organisms, which during theirife must withstand attack from a number of herbivores and fun-al pathogens. To survive, the trees have developed elaboratedhemical defences that can be divided into a constitutive andn induced part. The constitutive defences consist of chemicalompounds such as phenolics and terpenoids, which are stored inesin ducts and released upon mechanical damage of the plant1]. The induced defences are initiated during infestation andesult in an increased production of compounds and changef chemical composition in the tissues (for recent reviews see2–4]). Many insect species are known to influence the volatilemission of conifers during infestation [5–7]. Plant volatilesre important to aphids in their host location [8] and increasehe attraction of male aphids to the pheromones of conspecificemale alatae [9]. The spruce shoot aphids, Cinara pilicornis

Hartig), live on Norway spruce, Picea abies (L.) Karst., andere we characterize the volatile blend of the aphids and theirost during infestation.

∗ Corresponding author. Tel.: +46 8 790 84 49; fax: +46 8 791 23 33.E-mail address: akbk@kth.se (A.-K. Borg-Karlson).

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021-9673/$ – see front matter © 2007 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2007.12.020

S; Semiochemicals

There exist eight stereoisomers of nepetalactone, fouriastereomers and their corresponding enantiomers. With a fewxceptions, the 7S diastereomers are the ones found in natu-al sources (Fig. 1) [10]. The diastereomers of nepetalactonesive rise to very similar mass spectra (Fig. 2) and without ref-rence compounds it has hitherto not been possible to assignhe stereochemical configuration of an unknown nepetalac-one found in an insect or a plant by mass spectrometry (MS)nly.

As the commercial libraries we tested were not usable foreliable assignment of the stereochemistry of nepetalactoneiastereomers, we developed a diagnostic method to achieve thisy GC–MS. The new method was tested in the identification ofis–trans-nepetalactone emitted by the spruce shoot aphid whilstnfesting its host. The emission of cis–trans-nepetalactone from. pilicornis as far as we know has not been described before.

. Experimental

.1. Biological material

Plants of the Norway spruce (P. abies) clone 1321 originat-ng from the clone archives of Skogforsk (the Forestry Research

166 M. Pettersson et al. / J. Chromatogr. A 1180 (2008) 165–170

omers

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Fig. 1. The four 7S diastereomers of nepetalactone. In this study the diaster

nstitute of Sweden) were used in this study. Volatiles fromn uninfested plant (n = 1) were compared with volatiles fromlants (n = 2) infested by the spruce shoot aphid C. pilicornis.n addition, conventional seedlings of Norway spruce (Nassjaursery, Sweden) [11] were artificially infested. Ten aphidsere manually transferred to each plant by tweezers; they wererovoked to climb up on the tweezers and were thereafter trans-erred to the new plant. Emissions from infested plants (n = 2)nd control plant (n = 1) were sampled and analyzed 1 weekater.

.2. Chemical material

Nepetalactone and nepetalactol standards were prepared orsolated according to Liblikas et al. [10], and all four geomet-ical isomers of �-farnesene were synthesized from nerolidol

12]. Other reference compounds were obtained from com-ercial sources: citronellol (Aldrich, 95%), (E)-�-farnesene

Bedoukian, 95%) and methyl salicylate (Lancaster, 98%), allurities refer to the sum of enantiomers.

cfi82

ig. 2. Mass spectra of the four diastereomers of nepetalactone. (I) Trans–trans-nepis–cis-nepetalactone.

were not separated from their enantiomers (corresponding 7R-structures).

.3. Nepetalactone analysis

Analyses of the four isomers of nepetalactone were per-ormed using nine GC–MS instruments. Six quadrupolenstruments (Fison MD 800, HP 5972 MSD, Agilent 5973

SD, Agilent 5975B inert XL MSD and two Finnigan SSQ000), two ion traps (Finnigan GCQ and Varian Saturn 2000)nd one time-of-flight (TOF) instrument (Leco Pegasus 4D,C × GC × TOF-MS) were used, all equipped with ion sourcesorking in electron ionization mode. To find characteristic MS-

ragments and to investigate possible concentration effects, atudy of the four nepetalactone diasteromers in a mixture (pro-ortions 5:1:12:4 of trans–trans:cis–trans:trans–cis:cis–cis)as done on one of the Finnigan SSQ 7000 instruments (fourilutions of the mixture, five replicates per dilution).

For separation of the nepetalactone isomers on a DB-wax

olumn (J&W Scientific, 30 m, 0.25 mm i.d. and 0.15 �mlm thickness) the following temperature program was used:0 ◦C (1 min), 10 ◦C/min, 160 ◦C (11 min), 4 ◦C/min, 200 ◦C,0 ◦C/min, 225 ◦C (5 min). Injection temperature was 220 ◦C

etalactone, (II) cis–trans-nepetalactone, (III) trans–cis-nepetalactone and (IV)

omat

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The mixture of four nepetalactone isomers was run on dif-ferent columns together with a mixture of n-alkanes. Retentionindices were calculated and are presented in Table 1.

Table 1Kovat’s retention indices of nepetalactones on three different columns (thenepetalactones eluted within 30 min on all three columns)

Column T (◦C) Trans–trans I Cis–trans II Trans–cis III Cis–cis IV

M. Pettersson et al. / J. Chr

split/splitless 30 s), and the transfer line was held at 230 ◦C.elium was used as carrier gas (inlet pressure 0.69 bar).Retention indices were calculated in relation to the reten-

ion times of an n-alkane series (C9–C23, 50 ng/�l) on threeolumns of different polarity. (1) DB-wax column at 140 ◦C,nstrument and column described above. (2) CPBTM-1 columnt 90 ◦C, instrument and column described under Section 2.5. (3)B-5 column (J&W Scientific, 30 m, 0.25 mm i.d. and 0.25 �mlm thickness), at 100 ◦C in a HP 5890 Series II GC, injection

emperature 225 ◦C (split/splitless, splitless 0.5 min) and detec-or temperature 225 ◦C (FID). To get proper Kovat’s retentionndices, isothermal conditions were applied, although tempera-ure programs were used in volatile analysis to achieve optimaleparation of the nepetalactones as well as of other compounds.

.4. Volatile collection

Volatiles were collected using solid-phase micro-extractionSPME) with polydimethylsiloxane/divinylbenzene fibresSupelco) [11]. Plants were placed in plexiglas (seedlings) orlass vessels (clone plants) depending on size, the SPME fibreere inserted through a small drilled hole or, if the smallerlass vessels were used, plunged through the aluminium foilovering the openings, and adsorption continued during 18 h foraturally infested plants and 22 h for artificially infested plants4-h dark period). Volatiles emitted by spruce shoot aphidsithout odour background of host plant were monitored from

ix aphids (alatea) placed on a moist filter paper in a 3.5 mllass vial sealed with aluminium foil and the SPME fibres werexposed to the headspace for 1–3 h (five replicates with newphids in each replicate). As control, background volatiles wereimultaneously collected from a vial containing only a moistlter paper.

.5. Chemical analysis

Volatiles collected were separated and identified by GC–MS.Varian 3400 GC equipped with a CPBTM-1 column (Supelco,

0 m, 0.25 mm i.d. and 0.25 �m film thickness) was programmeds follows: 40 ◦C for 2 min then increased with 4 ◦C/min until80 ◦C (held 0.01 min) followed by 20 ◦C/min to 225 ◦C andept constant for 5 min (Tinj: 220 ◦C, Taux: 230 ◦C). Identifica-ions were validated on an identical instrument equipped withDB-wax column (described under Section 2.3). Helium at an

nlet pressure of 0.69 bar was the carrier gas for both instruments.The GC-instruments were connected to Finnigan 7000 MS-

nstruments with electron ionization (ion source temperature:50 ◦C, 70 eV). Identifications were accomplished by com-arison of MS-spectra and retention indices with commercialibraries (NIST and Massfinder 3) and were validated by GC–MSnalysis of reference compounds.

.6. Statistical analysis

Multivariate analyses were performed with the softwareanoco for Windows Version 4.54 (Ter Braak and Smilauer,iometris Plant Research International, The Netherlands). The

DDC

ogr. A 1180 (2008) 165–170 167

ntensities were normalized for each nepetalactone spectrumefore statistical analysis so that the base peaks intensity became00%. Principal component analysis (PCA) [13] was used tonvestigate the data from spectra obtained from the concentrationtudy. m/z-Fragments between 50 and 169 which were present inll spectra of at least one diastereomer were included in the ordi-ation. Selection of m/z-fragments for further PCA was obtainedy ocular investigation of the mass spectra. The selection wasonfirmed by a constrained linear ordination, redundancy anal-sis (RDA) [14].

. Results

.1. Infested plants

The naturally infested spruce plants emitted around 50 com-ounds in a blend that differed from the blend of compoundsmitted by uninfested plants of the same clone (Fig. 3). Thehree most characteristic substances emitted by infested plantsere (E)-�-farnesene, methyl salicylate and (E,E)-�-farnesene.he cis–trans-nepetalactone and cis–trans-nepetalactol were

wo other compounds present in the headspace of the infestedlants but were found to be emitted by the spruce shoot aphidsnd not by the spruce plants (Fig. 3).

Another trial with artificial infestation of two seedlings of. abies with C. pilicornis in the beginning of autumn resultedn a large increase of methyl salicylate emission 1 week afternfestation. The emission of farnesenes also increased but at thisime of the season (August) no nepetalactone or other compoundas emitted by the aphids.

.2. Emission of volatiles by C. pilicornis

The emission of the aphids (Fig. 3) consisted of a mixture ofis–trans-nepetalactone (88.0 ± 2.6%), cis–trans-nepetalactol6.1 ± 0.8%) and citronellol (6.0 ± 2.6%, n = 5, ±SD). Tracemounts of citronellal were also detected. We were only able toetect the nepetalactones in the emission of the infested sprucesuring the development of alate aphids in September–November005–2006, a 3-week period each year. No (E)-�-farnesene waseleased by the aphids, not even when the aphids were manipu-ated to excrete cuculiar drops.

.3. Separation of nepetalactone diastereomers

B-wax 140 1954 1972 2019 2038B-5 100 1347 1355 1382 1386PBTM-1 90 1302 1310 1334 1337

168 M. Pettersson et al. / J. Chromatogr. A 1180 (2008) 165–170

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cliitmmof m/z 111 was characteristic for cis–cis-nepetalactone. The PCAplot had the effect that the cis–cis-nepetalactone samples couldbe found further to the right than the other isomers. Axis two

ig. 3. Chromatograms of headspace samples of: (1) spruce shoot aphids (top)ithout infestation (bottom). Three peaks in the top chromatogram were emitte

Analyses of a mixture of four nepetalactone diastereomers atour different concentrations, with five repeated measurementsf each concentration were summarized in the PCA score ploteen in Fig. 4. Spectra from isomers with cis configuration at theing-junction were grouped separately from the trans-isomers.

hen only selected fragments (m/z 85, 111, 137, 138 and 151,ound by RDA and ocular investigation of mass spectra) werencluded in the ordination all four diastereomers were separatedrom each other in the biplot (Fig. 5). This indicated that theelative abundances of these fragments could be used to identify

he diastereomers of nepetalactone.

The influence of the variables on the principal componentsould be investigated through the variable vectors in Fig. 5. Axisne (principal component 1, PC1) in Fig. 5 mainly separated

ig. 4. PCA score plot of relative abundances of m/z 50–169 from spectraf four nepetalactone isomers. (♦) Trans–trans-nepetalactone, (�) cis–trans-epetalactone, (�) trans–cis-nepetalactone and (×) cis–cis-nepetalactone. Fourilutions of a nepetalactone mixture were analyzed, the bigger the symbol thetronger the concentration. Note that the concentrations of the four nepeta-actones were not similar (proportions 5:1:12:4) and the size represents theoncentration of the dilution, not the individual compounds. The largest squaresepresent spectra of cis–trans-nepetalactone of a concentration lower than theowest concentration of any of the other diastereomers.

Fdnp(f

phid infested spruce plant of clone 1321 (middle) and (3) plant of clone 1321he spruce shoot aphids, the rest belonged to the background.

is–trans- and the cis–cis-isomer from the other two nepeta-actone isomers. The m/z-fragment 85 had the highest positivempact on PC1, indicating that it had a higher relative abundancen the MS-spectra of the cis–trans- and cis–cis-nepetalactoneshan in the spectra of the trans–cis and trans–trans-isomers./z-Fragment 111 also had a high effect on PC1. From the chro-atograms, it could be concluded that a high relative abundance

ig. 5. PCA biplot of nepetalactone isomer samples based on relative abun-ances of five selected m/z-fragments (denoted by vectors in the plot). The aphidepetalactones were not included in the ordination but plotted into the PCAlot afterwards. (♦) Trans–trans-nepetalactone, (�) cis–trans-nepetalactone,�) trans–cis-nepetalactone, (×) cis–cis-nepetalactone and (+) nepetalactonerom aphids. Each nepetalactone isomer has been encircled in the plot.

M. Pettersson et al. / J. Chromat

Table 2Characteristic m/z-fragments for the nepetalactone isomers

Trans–trans I Cis–trans II Trans–cis III Cis–cis IV

Nepetalactone isomers85 < 137 85 > 137 85 < 137 85 > 137110 > 111 110 > 111 110 > 111 110 < 111

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PC2) separated cis–trans- and trans–cis-nepetalactone fromrans–trans- and cis–cis-nepetalactone. The differences betweenhe samples were mainly due to m/z 137 and 138. The two for-

er had a higher relative abundance of m/z 138 and lower of m/z37 as well as of m/z 151, the two latter showed the oppositeattern.

.4. Method to determine the relative stereochemistry of annknown nepetalactone diastereomer

This method is valid for quadropol instruments:

. Inject a standard alkane series and calculate the index values.Use Table 1 to exclude either the trans–trans and cis–trans orthe trans–cis and cis–cis isomers from the possible isomers,i.e. the isomers with the hydrogen at position 7a and 7 trans

to each other elute before the cis-isomers.

. Use Table 2 to pairwise compare m/z-fragments 85/137,137/138 and 138/151 to determine whether the unknown iso-mer is the cis or trans ring-junction isomer of the remaining

tbic

Fig. 6. Mass spectrum of nepetalacton

ogr. A 1180 (2008) 165–170 169

candidate isomers. If one fragment pair is in contradiction tothe other pairs, make the determination based on the majortrend.

. Check whether the m/z 111 fragment is bigger than them/z 110 fragment, if m/z 110 < 111 the isomer is a cis–cis-nepetalactone.

The method was tested on spectra from the six quadropolnstruments and gave the correct result for all spectra (27 spectranalyzed) but was not usable for MS instruments with otherass analyzers. The method was applied to the spectra of the

nknown nepetalactone emitted by spruce shoot aphids (Fig. 6).n total 10 spectra were tested, recorded on the Finnigan SSQ000 instruments. They all fulfilled the criteria for cis–trans-epetalactone, which was also in accordance with their retentionndices on two columns. They were plotted into the PCA plotn Fig. 5 and, as predicted, they all ended up in the cis–trans-epetalactone area of the plot.

. Discussion

During infestation the spruce shoot aphid alter the volatilerofile of the spruce seedlings. The altered emission resembledhe emissions of spruce plants infested by other organisms7,15]. Well-known stress-induced compounds like the farne-ene isomers and methyl salicylate dominate the volatile blend of

he plant 1 week after infestation. Methyl salicylate has recentlyeen shown to enhance the response to the sex pheromonen two aphid species [9]. The biosynthesis of stress-inducedompounds have been shown to be triggered by several species

e emitted by Cinara pilicornis.

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[Entomol. Exp. Appl. 36 (1984) 197.

[25] G.W. Dawson, D.C. Griffiths, L.A. Merritt, A. Mudd, J.A. Pickett, L.J.Wadhams, C.M. Woodcock, J. Chem. Ecol. 16 (1990) 3019.

[26] G.W. Dawson, J.A. Pickett, D.W.M. Smiley, Bioorg. Med. Chem. 4 (1996)351.

70 M. Pettersson et al. / J. Chro

s well as by treatment with methyl jasmonate [5–7,16,17],owever, there are indications that the volatile profiles differetween plants during attack by different species [15,18].his could be of advantage for other insects, e.g. parasitoidsearching for a host or a predator searching for a specific prey.

Some plants successfully deter aphids by releasing (E)-�-arnesene [19,20]. With this in mind, it is interesting to notehat the main compound emitted by the aphid-infested plantsas (E)-�-farnesene and that stressed or manipulated spruce

hoot aphids did not emit (E)-�-farnesene, the common aphidlarm pheromone [21,22]. No information could be found onhe behaviour of the spruce shoot aphid to (E)-�-farnesene.owever, Xiangyu et al. tested (E)-�-farnesene as an alarmheromone on 59 different aphid species whereof two belongedo the Cinara spp. Unlike 41 of the other species the twolosely related Cinara species did not react at exposure to (E)--farnesene [23]. One could speculate whether our observation

s a result of co-evolution wherein the spruce first evolved thetrategy to release the aphid alarm pheromone when attackednd that the aphids later, as a response, have evolved negligenceowards this odour cue. Some other aphid species have appar-ntly evolved an ability to use other host plant cues to distinguishE)-�-farnesene-containing emission from the host plant fromhe alarm pheromone emitted by conspecies [24].

Cis–trans-nepetalactone together with cis–trans-nepetalactolnd citronellol were detected from the spruce shoot aphid C.ilicornis. Nepetalactones forms part of the sex pheromones forany aphid species [25] and it is likely that the nepetalactone and

actol found in C. pilicornis have a similar function. The detecteditronellol may not be a semiochemical as it has been suggestedo be the precursor of nepetalactone and nepetalactol pheromoneomponents [26]. Facts that support this idea are that citronellolhowed neither electrophysiological nor behavioural activity onhe aphid species on which this has been analyzed [26].

In our investigation, spectra of all four nepetalactone diastere-mers from nine GC–MS instruments were compared. The typef MS analyzer affected the fragmentation pattern since only theix quadropol instruments could be used for an unambiguousdentification. The quadropol instruments were of five different

odels and may not be a representative sample of all the GC–MSnstruments in use over the world but the quick method presentedere seems to be a trustworthy method to identify diastereomersf nepetalactone by GC–MS. No clear differences between thepectra of the four diastereomers could be found based on theajor m/z-fragments. Instead, the diagnostic fragments were

ome of the smaller ones.In summary, the characteristic and diagnostic fragments iden-

ified in the spectra of the four nepetalactone diastereomersTable 2) can be used, together with retention indices (Table 2), todentify unknown nepetalactones found in natural sources, evenhen all four compounds are not available as references. By thisethod we successfully determined the major compound in the

mission of Cinara pilicornis to be cis–trans-nepetalactone.

r. A 1180 (2008) 165–170

cknowledgements

Bjorn Bohman (University of Kalmar, Sweden), Dr. Sachaegrand (University of Kalmar, Sweden), Dr. Ellen Santangelo

KTH, Sweden), Dr. Joseph Youssefi (Firmenich, Switzerland)nd Dr. Junwei Zhu (Iowa State University, USA) have gener-usly provided mass spectra of the nepetalactone diastereomers.oy Danielsson, Museum of Zoology, Lund University, iscknowledged for determining the aphid species to Cinara pil-cornis.

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