Journal of Chemical Ecology, Vol. 25, No. 8, 1999

LEAF VOLATILES FROM NONHOST DECIDUOUSTREES: VARIATION BY TREE SPECIES, SEASON AND

TEMPERATURE, AND ELECTROPHYSIOLOGICALACTIVITY IN Ips typographus

QING-HE ZHANG,1,* GORAN BIRGERSSON,2 JUNWEI ZHU,3,4

CHRISTER LOFSTEDT,4 JAN LOFQVIST,1 and FREDRIK SCHLYTER1

1 Chemical Ecology, Department of Plant Protection SciencesSwedish University of Agricultural Sciences

P.O. Box 44, S-230 53 Alnarp, Sweden

2Chemical Ecology, Department of BotanyGoteborg University, S-405 30 Goteborg, Sweden

4Department of Ecology, Ecology BuildingLund University, S-223 62 Lund, Sweden

(Received September 21, 1998; accepted April 12, 1999)

Abstract—The leaf volatiles emitted from four nonhost tree species ofIps typographus, i.e. Betula pendula, B. pubescens, Populus tremula, andSambucus nigra, were collected outdoors by headspace sampling in situ andanalyzed by GC-MS. Three major classes of compounds, aliphatics [mainlygreen-leaf volatiles (GLVs)], monoterpenes, and sesquiterpenes, existed in allthe deciduous tree species investigated. In June, when the bark beetles aresearching in flight for host trees, GLVs mainly consisting of (Z)-3-hexenylacetate and (Z)-3-hexen-1-ol were the dominant constituents in B. pendulaand S. nigra. In B. pubescens and P. tremula, sesquiterpenes (and theirderivatives) and monoterpenes made up the major part of whole volatileblends, respectively. Surprisingly, sesquiterpene alcohols and other oxidesreleased from B. pubescens in considerable amounts were not found in theclosely related species, B. pendula. By August, both the total volatiles andindividual compounds significantly decreased, mainly due to the maturationof leaves, since the light intensity and temperatures during sampling were thesame as in June. There were almost no volatiles detected from P. tremulaand S. nigra leaves in August. The total emissions from these deciduousspecies were significantly different among the species, with B. pubescensreleasing 5-10 times more than other species. Under the conditions of

* To whom correspondence should be addressed.3 Present address: Department of Entomology, Iowa State University, Ames, Iowa 50011.

1923

0098-0331/99/0800-1923$16.00/0 © 1999 Plenum Publishing Corporation

constant light intensity and humidity, emissions of both total volatiles andmost individual components of severed B. pendula and S. nigra branches(with fresh leaves) increased according to a saturation curve from 16°C to40°C. Ips typographus antennae responded strongly to green leaf alcohols:(Z)-3-hexen-1-ol, 1-hexanol, and (E)-2-hexen-1-ol, but not to aldehydes oracetates in GC-EAD analyses of B. pendula and B. pubescens leaf volatiles.No antennal responses to monoterpenes, sesquiterpenes, or sesquiterpeneoxides were found. These three antennally active GLVs emitted from nonhosttree leaves might be indicators of a wrong habitat in the host selection ofconifer bark beetles.

Key Words—Birch, Betula pendula (=verrucosa), B. pubescens, Betulaceae,aspen, Populus tremula, Salicaceae, elder, Sambucus nigra, Caprifoliaceae,green-leaf volatiles, monoterpene, sesquiterpene, (Z)-3-hexen-1-ol, 1-hexanol,(E)-2-hexen-1-ol, seasonal variation, temperature effect, host selection,habitat, Ips typographus, Coleoptera, Scolytidae.

INTRODUCTION

The eight-spined spruce bark beetle, Ips typographus, is one of the most destruc-tive forest insects in Europe and northern Asia (Postner, 1974). Mass attacks onstanding and healthy spruce trees are believed to be initiated by an attractivesignal, the aggregation pheromone produced by the pioneer males when bor-ing into suitable host trees (Bakke, 1970; Birgersson et al., 1984; Schlyter andLofqvist, 1986, Schlyter et al., 1987b). Either by primary attraction (Austara etal., 1986; Lindelow et al., 1992) or random landing (Schlyter and Birgersson,1999), pioneer males find suitable hosts of Norway spruce, Picea abies. How-ever, attraction to pheromone is reduced in the presence of decadent or fullycolonized hosts, due to the release of volatiles such as verbenone that act in den-sity regulation and host suitability signals (Bakke, 1981; Schlyter et al., 1987a,1989). Guerrero et al. (1997) reported that benzyl alcohol, a nonhost compound,inhibited the positive responses of Tomicus destruens (Woll.) to pine volatiles.Host selection in phytophagous insects has long been hypothesized to involveboth attraction to the host and avoidance of nonhosts (Visser, 1986; Byers, 1995).

Little is known about the mechanisms underlying rejection by bark beetlesof nonhost tree species. Rejection could be based on a lack of host volatile char-acteristics or the presence of repellent or deterrent stimuli (Poland et al., 1998).Ambrosia beetles, Trypodendron domesticum and Xyleborus (Anisandrus) dis-par, that attack broad-leaved trees respond negatively to A-pinene, one of themajor monoterpene constituents of both Scots pine, Pinus sylvestris, and Nor-way spruce, P. abies (Nijholt and Schonherr, 1976; Schroeder and Lindelow,1989). In contrast, the coniferphagous bark beetles, Dendroctonus frontalis, D.ponderosae, D. brevicomis, D. rufipennis, I. grandicollis, I. avulsus, and theambrosia beetles, T. lineatum, Gnathotrichus sulcatus, and G. retusus, avoid non-

1924 ZHANG ET AL.

host trees in part because they are repelled by green-leaf volatiles (GLVs), six-carbon primary alcohols, aldehydes, and derivative esters commonly found ingreen plants (Visser, 1986; Dickens et al., 1991, 1992 Wilson et al. 1996 Bor-den et al., 1997; Poland et al., 1998; Deglow and Borden, 1998a,b). Schlyter etal. (1995) found that I. typographus, I. duplicatus, and Tomicus pinperda wererepelled by a blend of five GLVs and the terpene alcohol, linalool. In the pres-ence of certain nonhost substances, Scolytus multistriatus rejected host-tree twigssuitable for maturation feeding (Gilbert and Norris, 1968). Attraction of both T.piniperda and Hylurgops palliatus to ethanol was reduced by odors from cut logsof nonhost birch (Betula pendula) and aspen (Populus tremula) trees (Schroeder,1992). Single cell responses to the odor from birch bark have been demonstratedin T. lineatum and I. typographus (TPmmeras, 1989, TPmmeras and Mustaparta,1989). Mountain pine beetles, D. ponderosae, were repelled in an additive andredundant manner by four antennally active volatiles from the bark of Populustremuloides: 1-hexanol, benzylalcohol, benzaldehyde, and nonanal (Borden etal., 1998). Our recent study showed that attraction of I. typographus and Pityo-genes chalcographus (L.) to their pheromone traps was significantly reduced bythe presence of fresh birch bark and leaves (Byers et al., 1998).

European birches [B. pendula Roth. (=B. verrucosa Ehrh.) and B. pubescensEhrh.], aspen (P. tremula L.) and elder (Sambucus nigra L.) are common decid-uous tree species in Scandinavia and are often found in mixed stands with Nor-way spruce. Some of the volatiles released from these nonhost trees have beenidentified (Inki and Vaisanen, 1980; Isidorov et al., 1985; Konig et al., 1995),but any effects on I. typographus have not been studied.

Our objectives were to: (1) identify and quantify the volatiles emitted fromintact leaves of the above-mentioned nonhost tree species in both June andAugust; (2) study the effects of temperature on volatile emissions from severedbranches with fresh leaves of B. pendula and S. nigra in laboratory; and (3)determine the electrophysiologically active compounds emitted from the non-host trees by using coupled gas chromatographic-electroantennographic detec-tion (GC-EAD).

METHODS AND MATERIALS

Collection of Volatiles. Volatiles released from intact leaves were collectedfrom four nonhost trees species—B. pendula, B. pubescens, P. tremula, and S.nigra—in the field in southern Sweden by headspace sampling in June andAugust 1997 (Table 1). For each species, samples were taken from two neigh-boring trees of similar size. Three branches in June and two in August (35-40cm long), at 1.5—1.7 m high were sampled separately from each experimentaltree.

LEAF VOLATILES FROM NONHOST TREES 1925

1926 ZHANG ET AL.

Intact branches with leaves were enclosed in a polyester cooking bag (MenyToppits, Terinex Ltd, England, 35 x 43 cm) with a charcoal filter tube in the inlet.Coupled gas chromatographic-mass spectrometry (GC-MS) analysis showed noobvious release of volatiles from the blank bag. Air was drawn by vacuum for 2.5hr over the branch of 150 ml/min and through a Teflon tube (3.0 mm ID x 35mm) containing 30 mg of Porapak Q, mesh 50-80 (Supelco), with polypropylenewool and nylon stopper (1.68 mm ID) at both ends and connected by plastic tub-ing (4 mm ID, 80 cm long) to a mini-pump (5 x 6.5 x 3.5 cm; GroTech, Goteborg,Sweden) driven by a rechargeable battery (12 V) (Figure 1). The total weight ofall equipment excluding the battery was only 450 g and with a sealed lead batterysufficient for several days of operation was <1.8 kg, which is significantly lighterthan the apparatus Turgeon et al. (1998) used for capturing volatiles from conifercones. Earlier versions of this setup were used effectively for capturing volatilesfrom individual insects (Birgersson and Bergstrom, 1989) and intact plants (Knud-sen et al., 1993). The Porapak Q trap was attached to the branch by cotton twineand wrapped with aluminum foil to protect it from direct sunlight. Air tempera-tures inside and outside of sampling bags were recorded during the aerations witha digital Min-Max thermometer. After the aerations, the sample branches were cut,the leaves counted, and the fresh and dry (65°C, 72 hr) weight determined. Pora-pak Q traps were sealed with aluminum foil. An ambient air control sample wasalso collected for each of the sampling trees by using the same technique (withoutenclosure of the plastic bag) (see Figure 1).

As soon as possible after the field collection, the Porapak Q traps were

FIG. 1. Aeration setup for collection of volatiles from the intact leaves.

LEAF VOLATILES FROM NONHOST TREES 1927

1928 ZHANG ET AL.

rinsed with 300 ul diethyl ether (Fluka puriss p.a.) and collected into the inserttube (cone-shaped) of Hewlett Packard 2-ml vials for the GC autoinjector.Another 300 ul diethyl ether was added to the vial, outside of the insert tube,which prevented the eluate from evaporating from the insert. The sample vialswere kept at -20°C. As an internal standard, 1 ug of octyl acetate was addedto the June samples. No octyl acetate was added to the August samples becausethe diethyl ether stabilizer was found to be a satisfactory internal standard.

Aerations of newly severed branches of S. nigra and B. pendula, with thecut ends in water, were also carried out within 30 min after cutting at fourand two temperature levels, respectively, with five branches per level (Table 2).Otherwise, procedures were similar to those used for in situ sampling.

Chemical Analysis. Chemical analyses were made by a HP 5890 series IIgas chromatograph coupled with a HP 5972 mass selective detector (GC-MSD).The GC was equipped with a 25-m x 0.25-mm fused silica column, coated withCP-wax 58 (PEG). Samples were injected by using a HP 7673 auto injector(2 ul/injection). Helium was used as the carrier gas at a constant flow of 31cm/sec at 50°C. The injector temperature was 200°C. Oven temperature for Juneand temperature effect samples was 50°C for the first 2 min, rising to 210°C at10°C/min, and then held for 5 min, and for August samples was 30°C for thefirst 3 min, rising to 200°C at 10°C/min, and held for 2 min. Volatiles wereidentified by comparison of retention times and mass spectra with those of syn-thetic compounds, with computer data in the NBS75K library, and with our ownKEM-EKOL library.

Coupled Gas Chromatographic-Electroantennographic Detection (GC-EAD) Analysis. Four microliters of leaf aeration June samples (B. pendula orB. pubescens) were injected splitless into a HP 5890 GC equipped with a HP-Innowax column (30 m x 0.25 mm x 0.25 um) and a 1:1 effuent splitter that

TABLE 2. SAMPLES FOR EXPERIMENT TESTING EFFECT OF TEMPERATURE DURING ExSITU SAMPLING IN 1997 ON VOLATILES FROM BRANCHES OF B. pendula AND S. nigra

IN LABORATORYa

Temperature (°C)

Species

B. pendula

S. nigra

Inside bag

16.7-17.332.6-32.816.4-16.824.9-25.232.8-33.239.2-39.9

Outside bag

15.7-16.431.9-32.215.9-16.324.4-24.831.9-32.039.1-39.6

Leavesper sample

(N)

100-130200-30050-7050-7050-70

105-150

Dry weight

(g)

4.16-5.719.78-19.14.50-5.523.60-5.314.50-5.406.67-18.02

Date

May 30June 10May 30May 28June 10June 11

aN = 5 branches per sample, light intensity held at 1000 lux.

allowed simultaneous flame ionization detection (FID) and EAD of the sepa-rated volatile compounds (Gries, 1995). Hydrogen was used as carrier gas, andthe injector temperature was 225°C. The column temperature was 40°C for 2 minrising to 230°C at 10°C/min. The outlet for the EAD was held in a humidifiedairstream flowing at 0.5 m/sec over an Ips typographus antennal preparation.A glass capillary indifferent electrode filled with Beadle-Ephrussi Ringer andgrounded via an Ag-AgCl wire was inserted into the severed beetle's head withthe antennae. A similar recording electrode connected to a high-impedance DCamplifier with automatic baseline drift compensation was placed in contact withthe distal end of the antennal club.

Adult I. typographus for analysis were obtained from continuous culturesmaintained on Norway spruce (Schlyter and Anderbrant, 1993). They were takenfrom generation 94 (T94) of a strain originally collected from Lardal, Norway in1983, or from a second generation (WW2) of a strain collected in Torsby in 1997.The antennal responses of both sexes (Schlyter and Cederhom, 1981) was mea-sured.

Quantification and Statistical Analysis. For GC-MS, relative amounts ofindividual compounds were calculated as percentages of whole blends. Absoluteamounts of selected compounds were obtained by comparison to the internalstandard or the stabilizer. Means were compared by either t tests (two-tailed) orone-way ANOVA of data transformed by log (X + 1), followed by the Ryan,Einot, Gabriel, Welsh (REGW) multiple Q test (SPSS 8.0; Day and Quinn, 1989).In all cases, a = 0.05.

RESULTS

Leaf Volatiles and Seasonal Variation

In no case did GC-MSD analyses of ambient air reveal any plant volatilesat detectable levels.

Betula pendula. Monoterpene hydrocarbons, aliphatics (GLVs), and ses-quiterpenes were identified from both June and August samples (Table 3). GLVscomprised ca. 45% of the June and 22% of the August samples. The maincomponents in June samples, (Z)-3-hexenyl acetate (Z3C6Ac), (Z)-3-hexen-1-ol (Z3C6OH), (E)-B-ocimene, bourbonene, methyl salicylate, A-cubebene, andcaryophellene, were also found in similar proportions in August, but at signifi-cantly lower levels. The sums of all volatiles also differed between seasons (Fig-ure 2). Some monoterpenes (B-pinene, sabinene), GLVs (hexanal, 1-hexanol),and sesquiterpenes (A-cubebene, bourbonene, and B-cubebene) were present inJune samples in relatively large amounts, but were not detected in August. Incontrast, (Z)-B-ocimene (5%), butanoic acid (1.9%) and pentanoic acid (34%)were found in August, but not in June. The emission of individual GLVs signif-icantly declined from June to August (P < 0.05; t test; Table 3).

LEAF VOLATILES FROM NONHOST TREES 1929

1934 ZHANG ET AL.

FIG. 2. Seasonal changes in total volatile emissions from in situ leaves of four deciduoustree species. Differences of mean total emission rates between June (N = 5-6) and August(N = 4) samples for each tree species compared with t-tests (two-tailed) indicated by*P < 0.05; **P < 0.01, and ***P < 0.001 levels. Bars for June samples with differentletters are significantly different, ANOVA followed by REGW Q tests, P < 0.05.

Betula pubescens. Unlike B. pendula, B. pubescens emitted considerablenumbers of sesquiterpene alcohols and other oxides at high levels in bothJune (35%) and August (18%) samples (Table 3). The most abundant com-ponents in June were (E)-B-ocimene, (Z)-3-hexenyl acetate, (Z)-3-hexen-l-ol,A-copaene, B-caryophyllene, an unidentified sesquiterpene (MW = 206),caryophyllene oxide, and two unidentified sesquiterpene oxides (m/z 91, 105,131, 159, 187, 202, 220; and m/z 69, 109, 202, 205, 220). GLVs made up onlyca. 17% and 3.6% of the blends in June and August, respectively. Both the totalvolatiles (Figure 2) and amounts of the dominant components (Table 3), includ-ing two major GLVs, (Z)-3-hexenyl acetate, and (Z)-3-hexen-1-ol, declined sig-nificantly in August. Some GLVs (hexanal and 1-hexanol), sesquiterpenes, andsesquiterpene oxides were detected in June, but not in August. However, mono-terpenes present in June as minor components (some of them in trace amounts)became the major constituents (53%) in August, with sabinene being the mostdominant component at 40.6% (125 times higher than in June).

Populus tremula. Similar to B. pendula, mainly monoterpenes, GLVs andsesquiterpenes were found from June samples of P. tremula, with monoterpenesbeing the most dominant (37%) (Table 3). GLVs including (Z)-3-hexenyl acetate,(Z)-3-hexen-l-ol, 1-hexanol, and (E)-2-hexen-1-ol (E2C6OH), made up only8.8% of the total blend in June and were not detected in August except for atrace of (E)-2-hexen-ol. The major components in summer were (E)-B-ocimene,

LEAF VOLATILES FROM NONHOST TREES 1935

3-carene, A-pinene, (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol, A-farnesene, andB-caryophyllene. The total amount of volatiles in August was significantly lowerthan in June (Figure 2). Only a few compounds were detected in August, againwith monoterpenes as the major constituents (42%). However, butanoic acid wasfound only in August as 9.6% of the blend.

Sambucus nigra. Compared to the other species, S. nigra emitted volatilesat significantly lower levels in both June and August (Figure 2). The majorcomponents were (E)-B-ocimene, (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol,l,anunidentified sesquiterpene (bp = m/z 43 > 105, 119, 161), A-farnesene, andB-caryophyllene. GLVs made up 14% of total volatiles in June. In August, mostof components found in June were absent (Table 3).

Effects of Temperature on Emission of Leaf Volatiles

The number and the total amount of volatiles released from B. pendula andS. nigra were higher at 32°C than at 16°C (Figures 3 and 4). For B. pendula, theonly compounds that were not captured at significantly different levels at the twotemperatures were hexanal, (E)-2-hexenal, A-pinene, sabinene, and B-cubebene(Figure 3). For S. nigra, the total emission of volatiles, as well as the (Z)-3-hexen-1-ol, (Z)-3-hexenyl acetate, and B-caryophyllene increased significantlyfrom 16°C to 24°C (P < 0.05, ANOVA), after which the release rate plateaued(Figure 4). The release rate of a few minor components, e.g., toluene, 3-carene,nonanal, and decanal, did not change with temperature.

GC-EAD Analysis

Three GLVs, (Z)-3-hexen-1-ol, 1-hexanol, and (E)-2-hexen-1-ol, from bothB. pendula and B. pubescens leaves consistently elicited strong responses fromfive I. typographus antennae (Figure 4). The amounts of these compounds fromB. pendula and B. pubescens samples directed toward the antenna were estimatedas: 2.33 and 28 ng, 0.43 and 2.97 ng, and 0.40 and 0.71 ng, respectively. Twoother compounds from B. pendula, linalool and geranyl acetone, were antennallyactive in only one GC-EAD run. No responses were detected to any other com-pounds. No differences in EAD responses were found between males (N = 3)and females (N - 2).

DISCUSSION

Leaf Volatiles and Seasonal Variation. The absence of plant volatiles inambient air samples indicates that volatiles found in the enclosure samples wereemitted from the plants. Although monoterpenes, aliphatics, and sesquiterpeneswere produced by all four species, there were pronounced interspecific differ-ences. In particular, B. pendula and B. pubescens are sympatric and morphologi-

1936 ZHANG ET AL.

FIG. 3. Effects of temperature on volatile emission from B. pendula leaves sampled exsitu. (A) Total volatile and GLVs emissions; (B) major monoterpenes and sesquiterpenes.Differences in mean emissions between 16°C (N = 5) and 32°C (N = 5) compared witht tests (two-tailed) indicated by *P < 0.05; **P < 0.01, and ***P < 0.001 levels.

LEAF VOLATILES FROM NONHOST TREES 1937

FIG. 4. Relationship between temperature (x) and volatile emissions (y) from S. nigraleaves sampled ex situ fitted by Verhulst nonlinear regression: y = In (emission rate + 1)= b1/[1 + b3 * exp(-b2 * X)]. (A) All volatiles: y = 6.1/[1 + 11.9 * exp(-0.21x)], r2 =0.84; (B) (Z)-3-hexen-l-ol: y = 2.3/(l + 480 * exp(-0.37 * x)], r2 = 0.52; (C) (Z)-3-hexenyl acetate: y = 3.9/(l + 13 * exp(-0.22 * x), r2 = 0.43; (D) B-caryophyllene: y =3.6/(l + 2300 * exp(-0.36 * x), r2 = 0.86.

cally and ecologically similar in Scandinavia, but the compositions of their leafvolatiles, especially the occurrence of sesquiterpene alcohols and other oxidesin the latter species, are strikingly different. It is not clear that the differenceshave a genetic basis. However, B. pendula has a diploid chromosome number(2n = 28), while B. pubescens has a polyploid chromosome number (2n = 56)(Inki and Vaisanen, 1980). In June, B. pubescens released 5, 10, and 100 timesmore volatiles than P. tremula, B. pendula, and S. nigra, respectively (Figure 2).Similar volatile profiles and emission rates were also found in June 1998 sam-ples from B. pendula, B. pubescens, and P. tremula (4 samples/species) in Asa,ca. 250 km NE of the 1997 sampling sites (Zhang et al., unpublished data).

The predominance of GLVs released from B. pendula leaves is in agreementwith the results of Isidorov et al. (1985) and Konig et al. (1995). Konig et al.(1995) found similar release rates of (Z)-S-hexenyl acetate and (Z)-3-hexen-l-olas in our study, but neither Isidorov et al. (1985) nor Konig et al. (1995) detectedthe release of any other GLVs. They did detect monoterpenes and sesquiterpenesbut with less diversity than in our samples.

Conifers exhibit a strong seasonal variation in both composition and amountof needle volatiles, mainly monoterpenes (von Rudloff, 1972; Hrutfiord et al.,1974; Schonwitz et al., 1990), depending on temperature (Tingey et al., 1980;Yukouchi and Ambe, 1984), light intensity (Yukouchi and Ambe, 1984), andphytogenic effects like foliage drop and blooming (Arey et al., 1991). Moreover,the conifer volatile spectrum may increase in richness in current-year foliageas the season progresses (Brooks et al., 1987). In contrast, all four angiospermspecies tested here had significantly lower numbers and amounts of volatilesemitted in August than in June (Figure 2, Table 3). This variation may be dueto maturation of leaves because temperature and light intensity during the aer-ations in both June and August were almost the same (Table 1). The higherconcentration of volatiles released in the early summer might be an adaptivetrait associated with deterrence of phytophagous insects.

Temperature Effects. Temperature is one of many factors believed to influ-ence the volatile emission of plants (Charron et al., 1995). Our ex situ studyexposed cut branches to temperature ranges wide enough to cover natural con-ditions in early and mid-summer (Table 2) and revealed significant increases inthe release of monoterpenes, GLVs, and sesquiterpenes from B. pendula from16°C to 32°C, the two temperatures tested (Figure 3). In S. nigra, the increasein release of all the above classes of compounds occurred between 16°C and24°C, with only a slight increase above 24°C, a trend described well by Verhulstnonlinear regression curves (Figure 4). The absence of pronounced increases inemissions at high temperature levels might be due to the defense system of theplant, including closing the leaf stomata.

It was reported that monoterpene emissions increased exponentially withtemperature from leaves of Eucalyptus globulus L. (Guenther et al., 1991),Quercus ilex L. (Staudt and Seufert, 1995), and from foliage of many coniferspecies, with little shift in the relative proportions of individual monoterpenes(Dement et al., 1975; Kamiyama et al., 1978; Tingey et al., 1980, 1991; Yu-kouchi and Ambe, 1984; Lamb et al., 1985; Juuti et al., 1990). These emis-sion rates increase more rapidly than do comparable changes in vapor pressureand may be explained by alterations of the pathway conductance as well as thechange in vapor pressure (Tingey et al., 1991). Almost no data are available onthe effects of temperature on GLV and sesquiterpene emissions (Guenther et al.,1991; Winer et al., 1992).

Electrophysiological Activity. Our results clearly showed for the first time that three GLV alcohols(Z)-3-hexen-1-ol,l, 1-hexanol, and (E)-2-hexen-1-ol,emitted from both B. pendula and B. pubescens intact leaves induced a verystrong EAD response by I. typographus (Figure 5). In one GC-EAD run, linalooland geranyl acetone from B. pendula also produced slight responses. OtherGLVs from the nonhost leaves, including the most dominant GLV, (Z)-3-hex-enyl acetate, did not elicit any EAD response. These three antennally active

1938 ZHANG ET AL.

LEAF VOLATILES FROM NONHOST TREES 1939

FIG. 5. Simultaneously recorded flame ionization detector (FID) and electroantenno-graphic detector (EAD) responses using antennae of Ips typographus in response to leafvolatiles of B. pendula (upper trace) and B. pubescens (lower trace). No difference inGC-EAD responses between males (N= 3) and females (N = 2) to the leaf volatiles.

GLVs were identified as well from P. tremula, S. nigra (Table 3), and S. race-mosa (Zhang, unpublished data). 1-Hexanol was also detected in trace amountsfrom the female flowers of Picea abies and Pinus sylvestris, but not from twigswith needles (Borg-Karson et al., 1985). Our further GC-MS analyses did notfind any of these antennally active GLVs at detectable levels from aeartion sam-ples of fresh bark and branches with needles of host tree Picea abies (Zhanget al., unpublished data). A blend of five GLVs, including the three active alco-hols above, and linalool, had pronounced inhibitory effects on the efficiency ofattractant-baited traps to I. typographus, I. duplicatus, and T. piniperda (Schlyteret al., 1995).

Tommeras (1989) and Tommeras and Mustaparta (1989) found some recep-tor neurons from I. typographus and T. lineatum antennae, that responded tounknown birch bark volatiles. In agreement with our results, Wilson et al. (1996)found that Dendroctonus ponderosae antennae responded in GC-EAD analysisto 30-ng doses of all five green leaf alcohols tested [1-hexanol, (E)-2-hexen-1-ol, (Z)-2-hexen-1-ol, (E)-3-hexen-1-ol and (Z)-3-hexen-1-ol], but not to thealdehydes, hexanal, or (E)-2-hexenal, or to alcohol or aldehyde homologs withmore or fewer than six carbon atoms.

GLVs are produced by plants as a product of oxidation of leaf surface lipids.They have been assigned several roles in insect behavior, including: (1) hostplant finding by phytophagous insects (Visser, 1986); (2) enhancement of attrac-

tion to insect pheromones (Dickens et al., 1990); (3) host finding by parasitoidsof lepidopterous larvae (Whitman and Eller, 1990); and (4) interruption of aggre-gation pheromone response in conifer bark and ambrosia beetles, as reviewedby Deglow and Borden (1998a). Hexanal and hexan-1-ol disrupted attraction ofD. frontalis Zimm, to traps baited with attractant semiochemical, and hexanalhad a similar effect on Ips grandicollis (Eichhoff) and I. avulsus (Eichhoff)(Dickens et al., 1992). A blend of four green leaf alcohols interrupted attractionby D. ponderosae Hopkins, to pheromone-baited traps, whereas a blend of twogreen leaf aldehydes was inactive (Wilson et al., 1996). Similar to D. frontalis,hexanal, (E)-2-hexenal, and 1-hexanol were disruptive in D. rufipennis and (E)-2-hexenal and two GLV alcohols, (Z)-2-hexen-1-ol and (E)-2-hexen-1-ol signifi-cantly reduced the numbers of D. brevicomis captured (Poland et al., 1998). GLValcohols have also been shown in trapping experiments to disrupt the responseto aggregation pheromones by conifer-infesting ambrosia beetles, including T.lineatum (Borden et al., 1997), G. sulcatus, and G. retusus (Deglow and Bor-den, 1998a,b). European field experiments with 1-hexanol and (Z)-3-hexen-1-oltested singly (Byers et al., 1998) and further a GC-EAD study with a nine-com-ponent synthetic GLV mixture containing two aldehydes, one acetate, and sixalcohols and a walking bioassay as well as field trapping tests that showed repel-lency only by the alcohols (Zhang et al., in preparation), support the results ofWilson et al. (1996) and corroborate our GC-EAD findings.

CONCLUSION

We have shown that angiosperm trees, commonly existing in mixed forests,emit GLVs, monoterpenes, and sesquiterpenes in early and mid-summer whenI. typographus in flight are searching for hosts. The significant EAD activity ofthe green leaf alcohols emitted from nonhost trees suggests that they may directhost-seeking I. typographus away from habitats predominating in nonhost trees,e.g., B. pendula, B. pubescens, and P. tremula. Leaf odors may also help Ipsand Tomicus spp. to detect and fly above a deciduous understory, e.g., S. nigraand S. racemosa. As postulated by several authors (Borden et al., 1998, Byers etal., 1998; Schlyter and Birgersson, 1999), bark beetles may have evolved thesebehaviors to avoid the dangers of prolonged dispersal associated with investi-gating habitats with nonhosts, or even nonhost trees themselves.

Acknowledgments—We thank J. Jonsson for maintenance and adoptions for running of highertemperature of the climate chambers and E. Marling for help with bark beetle breeding. This researchwas supported by grant 23.0521/96 from the Swedish Council for Forestry and Agricultural Research(SJFR). Drs. J. A. Byers and P. Anderson helped in review of the manuscript.

1940 ZHANG ET AL.

REFERENCES

AREY, J., WINER, A. M., ATKINSON, R., ASCHMANN, S. M, LONG, W. D., and MORRISON, C. L. 1991.The emission of (Z)-3-hexen-1-ol, (Z)-3-hexenylacetate and other oxygenated hydrocarbonsfrom agricultural plant species. Atmos. Environ. 25A: 1063-1075.

AUSTARA, P., BAKKE, A., and MIDTGARD, F. 1986. Response in Ips typographus to logging wasteodours and synthetic pheromones. J. Appl. Entomol. 101:194-198.

BAKKE, A. 1970. Evidence of a population aggregation pheromone in Ips typographus. Contrib.Boyce Thompson Inst. 24:309-310.

BAKKE, A. 1981. Inhibition of the response in Ips typographus to the aggregation pheromone: Fieldevaluation of verbenone and ipsenol. J. Appl. Entomol. 92:172-177.

BIRGERSSON, G., and BERGSTROM, G. 1989. Volatiles released from individual spruce bark bee-tle entrance holes: Quantitative variations during the first week of attack. J. Chem. Ecol.15:2465-2483.

BIRGERSSON, G., SCHLYTER, F., LOFQVIST, J., and BERGSTROM, G. 1984. Quantitative variation ofpheromone components in the spruce bark beetle Ips typographus from different attack phases.J. Chem. Ecol. 10:1029-1055.

BORDEN, J. H., CHONG, L. J., SAVOIE, A., and WILSON, I. M. 1997. Responses to green leaf volatilesin two biogeoclimatic zones by striped ambrosia beetle, Trypodendron lineatum. J. Chem. Ecol.23:2479-2491.

BORDEN, J. H., WILSON, I. M., GRIES, R., CHONG, L. J., PIERCE, H. D., and GRIES, G. 1998.Volatiles from bark of trembling aspen, Populus tremuloides Michx. (Salicaceae) disrupt sec-ondary attraction by the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera:Scolytidae). Chemoecology. 8:69-75.

BORG-KARSON, A.-K., EIDMANN, H. H., LINDSTROM, M., NORIN, T., and WIERSMA, N. 1985. Odor-iferous compounds from the flowers of the conifers Picea abies, Pinus sylvetris and Lam sibi-rica. Phytochemistry 24:455-456.

BROOKS, J. E., BORDEN, J. H., PIERCE, H. D., JR., LISTER, G. R. 1987. Seasonal variation in foliarand bud monoterpenes in Sitka spruce. Can. J. Bot. 65:1249-1252.

BYERS, J. A. 1995. Host tree chemistry affecting colonization in bark beetles, pp. 154-213, in R. T.Carde and W. J. Bell (eds.). Chemical Ecology of Insects 2. Chapman and Hall, New York.

BYERS, J. A., ZHANG, Q.-H., SCHLYTER, F., and BIRGERSSON, B. 1998. Volatiles from nonhostbirch trees inhibit pheromone response in spruce bark beetles. Naturwissenschaften. 85:557-561.

CHARRON, C. S., CANTLIFFE, D. J., and HEATH, R. R. 1995. Volatile emissions from plants. Hortic.Rev. 17:43-72.

DAY, R. W., and QUINN, G. P. 1989. Comparisons of treatments after an analysis of variance inecology. Ecol. Monogr. 59:433-463.

DEMENT, W. A., TYSON, B. J., and MOONEY, H. A. 1975. Mechanism of monoterpene volatilizationin Salvia meltifera. Phytochemistry 14:2555-2557.

DEGLOW, E. K., and BORDEN, J. H. 1998a. Green leaf volatiles disrupt and enhance response toaggregation pheromones by the ambrosia beetle, Gnathotrichus sulcatus (LeConte) (Coleoptera:Scolytidae). Can. J. For. Res. 28:1697-1705.

DEGLOW, E. K., and BORDEN, J. H. 1998b. Green leaf volatiles disrupt and enhance response bythe ambrosia beetle, Gnathotrichus retusus (LeConte) (Coleoptera: Scolytidae) to pheromone-baited traps. J. Entomol. Soc. B.C. 95:In press.

DICKENS, J. C., JANG, E. B., LIGHT, D. M., and ALFORD, A. R. 1990. Enhancement of insectpheromone responses by green leaf volatiles. Naturwissenschaften 77:29-31.

DICKENS, J. C., BILLINGS, R. F., and PAYNE, T. L. 1991. Green leaf volatiles: A ubiquitous chemical

LEAF VOLATILES FROM NONHOST TREES 1941

signal modifies insect pheromone responses, pp. 277-280, in I. Hrdy (ed.). Insect ChemicalEcology. Acadamia Praha, Prague.

DICKENS, J. C., BILLINGS, R. F., and PAYNE, T. L. 1992. Green leaf volatiles interrupt aggregationpheromone response in bark beetles infecting pines. Experientia 48:523-524.

GILBERT, B. L., and NORRIS, D. M. 1968. A chemical basis for bark beetle (Scolytus) distinctionbetween host and nonhost trees. J. Insect Physiol. 14:1063-1068.

GRIES, G. 1995. Prospects of new semiochemicals and technologies, pp. 44-47, in S. M. Salom andK. R. Hobson (eds.). Application of Semiochemicals for Management of Bark Beetle Infesta-tions. Proceedings of an Informal Conference. USDA For Serv Gen Tech Rep INT-GTR-318.

GUENTHER, A. B., MONSON, R. K., and FALL, R. 1991. Isoprene and monoterpene emission ratevariability: Observations with eucalyptus and emission rate variability: Observations with euca-lyptus and emission rate algorithm development. J. Geophys. Res. 96(D6): 10799-10808.

GUERRERO, A., FEIXAS, J., PAJARES, J., WADHAMS, L. J., PICKETT, J. A., and WOODCOCK, C. M. 1997.Semiochemically induced inhibition of behaviour of Tomicus destruens (Woll.) (Coleoptera:Scolytidae). Naturwissenschaften 84:155-157.

HRUTFIORD, B. F., HOPLEY, S. M., and GARA, R. I. 1974. Monoterpenes in Sitka spruce: Within treeand seasonal variation. Phytochemistry 13:2167-2170.

INKI, M., and VAISANEN, L. 1980. Essential oils in Betula tortuosa Ledeb. And in some other Betulaspecies and hybrids. Rep. Kevo Subarctic Res. Stat. 16:38-44.

ISIDOROV, V. A., ZENKEVICH, I. G., and IOFFE, B. V. 1985. Volatile organic compounds in the atmo-sphere of forests. Atmos. Environ. 19:1-8.

JUUTI, S., AREY, J., and ATKINSON, R. 1990. Monoterpene emission rate measurements from a Mon-terey pine. J. Geophys. Res. 95(D6):7515-7519.

KAMIYAMA, K., TAKAI, T., and YAMANAKA, Y. 1978. Correlation between volatile substances re-leased from plants and meteorological conditions, pp. 365-372, in E. T. White, P. Hethering-ton, B. R. Thiele (eds.). Proceedings from the International Clean Air Conference. Ann ArborScience Publishers, Ann Arbor, Michigan.

KNUDSON, J. T., TOLLSTEN, C., and BERGSTROM, G. 1993. Floral scents—a checklist of volatilecompounds isolated by head-space techniques. Phytochemistry 33:253-280.

KONIG, G., BRUNDA, M., PUXBAUM, H., HEWITT, C. N., DUCKHAM, S. C., and RUDOLPH, J. 1995. Rel-ative contribution of oxygenated hydrocarbons to the total biogenic VOC emissions of selectedmid-European agricultural and natural plant species. Atmos. Environ. 29:861-874.

LAMB, B., WESTBERG, H., and ALLWINE, G. 1985. Biogenic hydrocarbon emissions from deciduousand coniferous trees in the United States. J. Geophys. Res. 90 (D1):2380-2390.

LINDELOW, A., RISBERG, B., and SJODIN, K. 1992. Attraction during flight of scolytids and otherbark and wood-dwelling beetles to volatiles from fresh and stored spruce wood. Can. J. For.Res. 22:224-228.

NIJHOLT, W. W., and SCHONHERR, J. 1976. Chemical response behaviour of scolytids in West Ger-many and western Canada. Can. For. Serv. Bi-Month. Res. Notes 32:31-32.

POLAND, T. M., BORDEN, J. H., STOCK, A. J., and CHONG, L. J. 1998. Green leaf volatiles disruptresponses by the spruce beetle, Dendroctonus rufipennis, and the western pine beetle, Dendroc-tonus brevicomis (Coleoptera: Scolytidae) to attractant-baited traps. J. Entomol. Soc. B.C. 95:Inpress.

POSTNER, M. 1974. Scolytidae, Borkenkafer, pp. 334-482, in W. Schwencke (ed.). DieForstschadlinge Europas, 2. Paul Parey, Hamburg.

SCHLYTER, F., and BIRGERSSON, G. 1999. Forest beetles, pp. 113-148, In R. J. Hardie and A. Minks(eds.). Pheromones of Non-Lepidopteran Insects Associated with Agricultural Plants. CABInternational, Wallingford, UK.

SCHLYTER, F., and CEDERHOLM, I. 1981. Separation of the sexes of living spruce bark beetle, Ipstypographus (L.) (Coleoptera: Scolytidae). J. Appl. Entomol. 92:42-47.

1942 ZHANG ET AL.

SCHLYTER, F., and LOFQVIST, J. 1986. Response of walking spruce bark beetles Ips typographus topheromone produced in different attack phases. Entomol. Exp. Appl. 41:219-230.

SCHLYTER, F., BYERS, J. A., and LOFQVIST, J. 1987a. Attraction to pheromone sources of differentquantity, quality, and spacing: Density-regulation mechanisms in bark beetle Ips typographus.J. Chem. Ecol. 13:1503-1523.

SCHLYTER, F., BIRGERSSON, G., BYERS, J. A., LOFQVIST, J., and BERGSTROM, G. 1987b. Fieldresponse of spruce bark beetle, Ips typographus, to aggregation pheromone candidates. J. Chem.Ecol. 13:701-716.

SCHLYTER, F., LEUFVEN, A., and BIRGERSSON, G. 1989. Inhibition of attraction to aggregationpheromone by verbenone and ipsenol: Density regulation mechanisms in bark beetle Ipstypographus. J. Chem. Ecol. 15:2263-2277.

SCHLYTER, F., LOFVIST, J., and JAKUS, R. 1995. Green leaf volatiles and verbonone modify attractionof European Tomicus, Hylurgops, and Ips bark beetles, pp. 29—44, in F. P. Hain, S. M. Salom,W. F. Ravlin, T. L. Payne, and K. F. Raffa (eds.). Behavior, Population Dynamics, and Controlof Forest Insects, Proceedings of a Joint IUFRO Working Party Conference, February 1994,Ohio State University, OARDC.

SCHONWITZ, R., LOHWASSER, K., KLOOS, M., and ZIEGLER, H. 1990. Seasonal variation in the mono-terpenes in needles of Picea abies (L.) Karst. Trees 4:34-40.

SCHROEDER, L. M. 1992. Olfactory recognization of nonhost and birch by conifer bark beetles Tomi-cus piniperda and Hylurgops palliatus. J. Chem. Ecol. 18:1583-1593.

SCHROEDER, L. M., and LINDELOW, A. 1989. Attraction of scolytids and associated beetles by dif-ferent absolute amounts and proportions of A-pinene and ethanol. J. Chem. Ecol. 15:807-817.

STAUDT, M., and SEUFERT, G. 1995. Light-dependent emission of monoterpenes by holm oak (Quer-cus ilex L.). Naturwissenschaften 82:89-92.

TINGEY, D. T., MANNING, M., RATSCH, H. C., BURNS, W. F., GROTHAUS, L. C., and FIELD, R. W.1980. Influence of light and temperature on monoterpene emission rates from slash pine. PlantPhysiol. 65:797-801.

TINGEY, D. T., TURNER, D. P., and WEBER, J. A. 1991. Factors controlling the emissions of mono-terpenes and other volatile organic compounds, pp. 93-119, in T. D. Sharkey, E. A. Holland,and H. A. Mooney (eds.). Trace Gas Emissions by Plants. Academic Press, San Diego.

T0MMERAS, B. A. 1989. Host selection by odourous compounds from host and non-host trees inbark beetles. Fauna Norv. Ser. B 36:75-79.

T0MMERAS, B. A., and MUSTAPARTA, H. 1989. Single cell responses to pheromones, host and non-host volatiles in the ambrosia beetle Trypodendron lineatum. Entomol. Exp. Appl. 52:141-148.

TURGEON, J. J., BROCKERHOFF, E. G., LOMBARDO, D. A., MACDONALD, L., and GRANT, G. G. 1998.Differences in composition and release rate of volatiles emitted by black spruce seed conessampled in situ vs ex situ. Can. J. For. Res. 28:311-316.

VISSER, J. H. 1986. Host odor perception in phytophagous insects. Annu. Rev. Entomol. 31:121-144.VON RUDLOFF, E. 1972. Seasonal variation in the composition of the volatile oil of the leaves, buds,

and twigs of white spruce (Picea glauca). Can. J. Bot. 50:1595-1603.WHITMAN, D. W., and ELLER, F. J. 1990. Parasitic wasps orient to green leaf volatiles. Chemoecology

1:69-75.WILSON, I. M., BORDEN, J. H., GRIES, R., and GRIES, G. 1996. Green leaf volatiles as antiaggregants

for the mountain pine beetle, Dendroctonus ponderosae Hopkins (Coleoptera: Scolytidae). J.Chem. Ecol. 22:1861-1875.

WINER, A. M., AREY, J., ATKINSON, R., ASCHMANN, S. M., LONG, W. D., MORRISON, C. L., andOLSZYK, D. M. 1992. Emission rates of organics from vegetation in California Central Valley.Atmos. Environ. 26(A):2647-2659.

YUKOUCHI, Y., and AMBE, Y. 1984. Factors affecting the emission of monoterpenes from red pine(Pinus densiflora). Plant Physiol. 75:1009-1012.

LEAF VOLATILES FROM NONHOST TREES 1943