Constitutive and herbivore-induced monoterpenes emitted by Populus × euroamericana leaves are key volatiles that orient Chrysomela populi beetles


F. Loreto. Fax: +39 06 9064492; e-mail:


Chrysomela populi beetles feed on poplar leaves and extensively damage plantations. We investigated whether olfactory cues orientate landing and feeding. Young, unexpanded leaves of hybrid poplar emit constitutively a blend of monoterpenes, primarily (E)-β-ocimene and linalool. This blend attracts inexperienced adults of C. populi that were not previously fed with poplar leaves. In mature leaves constitutively emitting isoprene, insect attack induces biosynthesis and emission of the same blend of monoterpenes, but in larger amount than in young leaves. The olfactometric test indicates that inexperienced beetles are more attracted by adult than by young attacked leaves, suggesting that attraction by induced monoterpenes is dose dependent. The blend does not attract adults that previously fed on poplar leaves. Insect-induced emission of monoterpenes peaks 4 d after the attack, and is also detected in non-attacked leaves. Induced monoterpene emission is associated in mature leaves with a larger decrease of isoprene emission. The reduction of isoprene emission is faster than photosynthesis reduction in attacked leaves, and also occurs in non-attacked leaves. Insect-induced monoterpenes are quickly and completely labelled by 13C. It is speculated that photosynthetic carbon preferentially allocated to constitutive isoprene in healthy leaves is in part diverted to induced monoterpenes after the insect attack.


The interaction between plants and herbivores is often mediated by visual or olfactory cues. Among the olfactory cues, the emission of constitutive biogenic volatile organic compounds (BVOCs) can play a major direct role in attracting or deterring herbivores (Visser 1986; Dicke, Agrawal & Bruin 2003). In response to the attack by herbivores, BVOC emission can also be induced. Numerous studies demonstrated that induced BVOCs act as infochemicals, allowing plant communication with other organisms, and indirect defence against herbivores (Dicke & Sabelis 1988). Induced BVOCs are often the main components of the bouquet that recruits parasitoids or predators of the herbivores (reviewed by Baldwin et al. 2006; Gershenzon & Dudareva 2007). These BVOCs are frequently induced by chemicals released by the herbivore (e.g. Mattiacci, Dicke & Posthumus 1995; Mithöfer & Boland 2008) and the induction is often systemic (i.e. it is not limited to the attacked leaves, but is spread to the entire plant) (e.g. Röse et al. 1996). The spectrum of induced BVOCs is often different from that of constitutive BVOCs (Pare' & Tumlinson 1999) and can be also different from the BVOC induced by mechanical stresses.

Two classes of induced BVOCs have been generally separated. BVOCs emitted immediately (within few minutes) after wounding are characterized by products of membrane breakdown, under the action of lipoxygenase and hydroperoxide lyase (Hatanaka 1993). This family of C-6 compounds is often referred to as ‘green leaf volatiles’ for its typical fragrance. Green leaf volatiles are not specific to herbivore feeding because their emission is also induced by mechanical wounding (Röse et al. 1996). However, they can prime inducible defence responses and the release of other classes of volatiles (Farag et al. 2005; Ruther & Kleier 2005).

A second class of BVOCs is emitted hours to days after herbivore feeding. Phenolic compounds (Mumm & Hilker 2006), methyl salicylate, nitriles and indoles (Van Poecke, Posthumus & Dicke 2001) and methyl jasmonate (Pare' & Tumlinson 1999; Orozco-Cárdenas, Narváez-Vásquez & Ryan 2001) are often components of this blend, and may have a key role in the activation of defensive pathways. Monoterpenes and sesquiterpenes (Kessler & Baldwin 2001), and in particular acyclic isoprenoids [e.g. (E)-β-ocimene, (E)-β-farnesene, linalool] (Röse et al. 1996; De Moraes et al. 1998; Heil & Silva Bueno 2007) are also main constituents of induced BVOCs. Delay with respect to herbivore feeding reflects gene induction (Arimura et al. 2000) and terpene synthases formation (Bohlmann et al. 1998; Arimura, Huber & Bohlmann 2004a). Induced isoprenoids are perhaps the most powerful mediators of plant herbivore interactions. As recently, exhaustively reviewed (Gershenzon & Dudareva 2007), volatile isoprenoids are toxic, may deter a large number of pathogens and herbivores or may attract numerous enemies of the herbivores.

Isoprene (2-methyl-1,3-butadiene) is the simplest isoprenoid, and accounts for more than 50% of the biogenic emission of isoprenoids (Guenther et al. 1995). Poplars are large emitters of isoprene with rates often exceeding 5–10% of the carbon fixed photosynthetically. The emission of isoprene is widespread in North American and European poplar species, with no reported exceptions (Kesselmeier & Staudt 1999). The role of isoprene has been clearly related to protection against environmental stresses (Sharkey & Yeh 2001), while no clear impact on plant–herbivore interactions is known. Preliminary results show that isoprene emission may deter caterpillars from feeding on Nicotiana transgenic leaves (Laothawornkitkul et al. 2008), and may also repel parasitic wasp (Loivamäki et al. 2008). A typical feature of isoprene-emitting plants is that they do not generally emit substantial amounts of other isoprenoids, namely monoterpenes (Kesselmeier & Staudt 1999). Accordingly, no healthy leaves of poplar species have been reported to constitutively emit a substantial amount of monoterpenes; minor emissions of monoterpenes having been only measured in young aspen (Populus tremula) leaves (Hakola, Rinne & Laurila 1998). On the contrary, the induced emission of monoterpenes, sesquiterpenes and homoterpenes has been reported from leaves of the hybrid Populus trichocarpa × deltoides, following the attack by forest tent caterpillars (Arimura et al. 2004a), and from leaves of the hybrid P. tremula × tremuloides, following the attack by Phyllobius piri or Epirrita autumnata (Blande et al. 2007).

Chrysomela (syn: Melasoma) spp. are among the main herbivores of poplars worldwide. Both larvae and adult beetles attack the leaves. Typical of some Chrysomela species is the production of volatiles that deter predatory attacks (Pasteels, Duffey & Rowell-Rahier 1990). Both emission of iridoids and sequestration of the phenol glucoside salicin from plants and subsequent release of salicylaldehyde are reported (Burse et al. 2007). The high foliar damage and production losses caused by this insect have driven research on BVOC-mediated poplar–Chrysomela interactions, aiming at a biological control of the infestations. Kendrick & Raffa (2006) found that Chrysomela scripta adults are attracted by host plants, as also observed for other chrysomelid beetles (Tansey et al. 2005). Behavioural assays suggested that the attractive cue was olfactory, but did not reveal the exact nature of the attractant. The observation that Chrysomela adults preferentially land and feed on young leaves, a choice that is not associated to differences in anatomical, morphological and biochemical traits (Harrell et al. 1981; Fernandez & Hilker 2007), led us to investigate whether the pattern of isoprenoids emitted by young and adult leaves plays a role in insect attraction. We tested the hypotheses that young and adult leaves emit different isoprenoids, and that volatiles orient Chrysomela insects in their choice of young poplar leaves.


Plant material and experimental conditions

Cuttings of Populus × euroamericana Dode (Gunier) (P. deltoides Bart. ex Marsh × Populus nigra L.), clone I214, were used. The cuttings were rooted and grown in 20 L pots with commercial soil, and irrigated daily to full soil capacity, while fertilized once a week with full strength Hoagland's solution. Ten plants were placed at the beginning of summer in a 12 m2 cage covered with a finely meshed net to prevent insects from flying away (Fig. 1). The relative humidity and air temperature were not affected by the enclosure, while the light intensity was cut by about 45%. The experiment was run in summer with daily air temperatures of 30–35 °C, relative humidity of 30–50% and photosynthetic photon flux density (PPFD) of 600–900 µmol m−2 s−1. At the time of the experiment, there were 18–22 leaves per plant, on a single stem. Two leaf classes were selected. Young leaves were defined as leaves whose expansion was lower than 30% of full leaf expansion. This size was generally reached by the third leaf expanding from the vegetative apex. Mature leaves were fully expanded and generally placed in the middle of the stem (10–12 leaves from the apex).

Figure 1.

Representation of two phases of the experiment. In (a), a group of 30–40 insects were launched in the cage containing the poplar plants. The group was formed by either inexperienced adults that never fed after emerging from the pupal stage, or by adults that fed on poplar leaves before being used. In (b), the observed distribution of insect landing, 20–30 min after launching, is shown. Inexperienced adults were launched three times on new and unattacked poplar plants (total insects launched n = 110), and the same procedure was used for adults that previously fed on poplar leaves. The bar graph shows the observed percentage of insects landing on the two leaf types, young or mature, as defined in the text. Landing of the two insect types was significantly different (t-test, P < 0.01).

Gas exchange measurements

Gas exchange measurements were carried out in the laboratory, less than 100 m away from the cage. Single plants were moved to the laboratory few minutes before measurements. A single leaf (10–50 cm2, depending on the leaf class) was carefully enclosed in a 0.5 L gas exchange cuvette without detaching it from the plant. The leaf was exposed to a flux of 0.5 L min−1 of synthetic air deprived of contaminants and made by mixing N2 (80%), O2 (20%) and CO2 (370 µmol mol−1). Measurements of photosynthesis, stomatal conductance and BVOC emissions were carried out simultaneously as described in detail elsewhere (Loreto et al. 2006), maintaining the leaves at a temperature of 30 °C, an air humidity of 40% and a PPFD of 1000 µmol m−2 s−1. Photosynthesis and stomatal conductance were calculated from CO2 absorption and H2O release by the leaf, as measured by an infrared gas analyser (LI-6262; Li-Cor, Lincoln, NE, USA). BVOCs were analysed both online (every 10 s) on a part of the flux (0.15 L min−1) of the air exiting the gas exchange cuvette by proton transfer reaction–mass spectrometry (PTR–MS) (Ionicon, Innsbruck, Austria), and after concentrating 8 L of the air exiting from the cuvette, in a cartridge filled with Tenax (Alltech, Deerfield, IL, USA) and Carbograph 5 (Supelco, Bellefonte, PA, USA). The cartridges were then thermally desorbed, and the released compounds were detected by gas chromatography–mass spectrometry (GC–MS) (HP5890; Hewlett-Packard, Palo Alto, CA, USA) as shown in detail by Loreto et al. (1996). The PTR–MS was operated in a single-ion mode to detect isoprene (protonated m/z = 69), monoterpenes (protonated m/z = 137 for all) and linalool (protonated m/z = 155). The GC–MS was operated also in scan mode to separate isobaric monoterpenes and to detect other BVOCs (particularly salicylaldehyde, m/z = 122) up to m/z = 350. Calibrations using isoprene and monoterpene gaseous standards (60 nL L−1) were performed daily before measurements.

To determine whether the BVOCs were emitted by the plants or by the insects, measurements were repeated on both young and mature leaves, sampling: (1) the leaf only, after removing insects and insect faeces; (2) the leaf and adults of the insect while feeding; and (3) the adult insects only. Gas exchange measurements were made before insect feeding (day = 0) and 1, 4 and 7 d after insects started feeding. The insects were not allowed to feed on leaves after day 0 measurements. Measurements were also carried out on the leaf proximal to that on which the insects were feeding, but which was not yet attacked by insects, and on leaves of plants that were grown in the same conditions, but were not exposed to insect feeding.

All gas exchange measurements presented in the different figures were repeated at least four times on different leaves of different plants. Differences between young and mature leaves were statistically analysed using a Student's t-test (**P < 0.01). Differences between means of gas exchange parameters recorded at different days, before and after the induction, were statistically assessed with a Tukey's post hoc test.

To detect whether freshly fixed carbon was incorporated in the monoterpenes whose emission was induced by insects, 12CO2 (99% of the CO2 in nature) was replaced within 2 s by pure 13CO2 (Aldrich, Milwaukee, WI, USA) in the air entering the cuvette, maintaining the CO2 concentration at an ambient level (370 µmol mol−1), as described by Loreto et al. (1996, 2004a). The labelling experiment was carried out for 60 min. The percentage of 13C labelling was calculated from the ratio between unlabelled monoterpenes (protonated m/z of the most abundant fragment = 81) and the sum of all labelled monoterpenes (protonated m/z = 82–88), irrespective of the number of carbon atoms effectively labelled, as shown by Loreto et al. (1996). This measurement was carried out when the highest emission of induced monoterpenes was observed (day 4 after feeding). This experiment was repeated four times on different mature leaves.

Insect growth and behavioural tests

Individuals of Chrysomela populi at different stages were collected from a local poplar plantation. These insects were transferred to the laboratory where they were reared under natural light and humidity conditions at temperature ranging from 25 to 30 °C. Insects were allowed to mate, and emerging larvae were also fed with poplar leaves until pupation. After emergence from the pupal stage, two groups of 30–40 adults were launched, positioning them on the floor of the cages where plants were grown (Fig. 1). In the first group, only adults collected immediately after emerging and that were not fed with poplars (i.e. inexperienced adults) were used. In the second group, adults were fed for 3 d with poplar leaves before launching them in the cage. This experiment was repeated three times for each group of insects, each time with new sets of insects and plants, and results obtained with adults and inexperienced adults were statistically separated by t-test (P < 0.01).

For the olfactometric behavioural test, couples of mating adults were captured in the field, and were allowed to lay eggs and develop the progeny on poplar plants, grown as before under controlled conditions. Adults that freely fed on the poplar plants, or that just emerged from the pupal stage, were used in this test. Both adults and newly emerged adults were further divided into two groups, one of which was not fed. Precisely, a group of adults was starved for 3 d before the test, and a group of newly emerged adults (also called, as in the field test, inexperienced adults) was never fed before the test.

A glass Y-tube olfactometer manufactured by Marbaglass (Rome, Italy) was used. The Y-tube had a 60° angle between the 15-cm-long, 2.5-cm-wide lateral arms, and was fixed vertically on a metal pole. Both Y-tube and the metal support were placed into an open-topped cardboard box with a door-like frontal side from which the insects were introduced in the common arm of the olfactometer. The internal faces of the box were covered with green cardboard to avoid the interference of visual cues in driving insect choice.

A low flow of air (400 mL min−1, equally split in the two arms of the Y-tube) was aspired by a pump located at the end of the common arm. Air was filtered by an activated charcoal cartridge and then humidified by bubbling it into a glass humidifier. The flow rate was regulated by a rotameter.

In the air passing through one of the lateral arms, a blend of 1 µmol mol−1 (E)-β-ocimene and 1 µmol mol−1 (-)-linalool (both chemicals from Sigma-Aldrich, St Louis, MO, USA) was added to simulate the blend and the concentration of monoterpenes emitted by poplar leaves. The same procedure was replicated for isoprene. The two monoterpenes and isoprene were contained as diluted gases in certified cylinders (one for the two monoterpenes, the second for isoprene) at a concentration of 100 µmol mol−1. They were mixed and further diluted before reaching the olfactometer by finely regulating the flow with a mass flow controller (Matheson, Newark, CA, USA).

The behavioural test was carried out on a single insect at each time. The insect was placed in the common arm of the olfactometer and first exposed to a complete blank test with only filtered and humidified air flowing through the lateral arms of the Y-tube. This was done to ascertain that the insect did not walk preferentially towards one direction, in the absence of the presumed attractant. The insect was then given the choice between pure air and the blend of monoterpenes. To avoid again the possibility that the insect walked one direction independently of the attractant, the Y-tube was inverted, both for the blank and the choice test, in 50% of the tests. At the end of each test, the Y-tube was carefully washed and cleaned to remove insect frass and secretions. In two additional experiments, poplar leaves, just excised from the tree and maintained alive with petioles immersed in water, were offered to the insect at the two sides of the olfactometer. Each time the insect was allowed to choose between a young leaf emitting prevalently monoterpenes and a mature leaf emitting isoprene, or between a young and a mature leaf, both of which were attacked by adult insects and were emitting different amounts of monoterpenes.

Only those individuals which showed a preference for one of the two lateral arms within 10 min and walked up to the end of the arm were considered as successfully tested. Tests were repeated on at least 30 individuals per each of the eight groups. We observed that insects often showed a tendency to prefer one of the arms even when pure air was supplied on both arms of the olfactometer (blank test). This was also observed elsewhere in beetles (Girling, Hassall & Turner 2007), and prompted us to use a statistical test that takes care, on a case-by-case basis, of the observed insect behaviour in the absence of volatile cues. A non-parametric χ2 test, with Yates correction to account for the only degree of freedom and for the limited number of observations, was used. This test required merging data obtained for the blank test and for the test with synthetic monoterpene blend, or with different leaf types. The test did not need to be run with a paired number of replicates, but required the following conditions to be satisfied for n (the number of total observations, including both blanks and synthetic blends or leaves) and p (the faction of insects that walked towards the leaves or synthetic blend): n > 30; np > 5 and n (1 − p) > 5. Statistically significant differences were tested at P < 0.05 (*) and P < 0.01 (**).


Field behavioural test

Adults of Chrysomela were placed in a cage where whole poplar plants were grown (Fig. 1), to study whether a preferential feeding behaviour could be detected in a natural environment. Two classes of insects were launched, and launches were repeated three times on each class. Within 3 h from launching at the bottom of the cage (Fig. 1a), 78 ± 9% of the inexperienced adults landed and started feeding on young apical leaves of caged trees (Fig. 1b). The remaining inexperienced insects of this group started feeding on all mature leaves without a clear preference about leaf position in the stem. On the contrary, when adults that were previously fed with poplar leaves were launched, the insects did not show a preferential behaviour as they landed and started feeding on both young and mature leaves of the canopies (data not shown).

Volatile emissions and physiological properties of leaves

The gas exchange properties of young and mature poplar leaves were examined to understand whether BVOCs could have influenced orientation and landing of insects. Photosynthesis and stomatal conductance were similar in young and mature leaves, but the emission of BVOCs was different in the two leaf types (Fig. 2). Young leaves emitted 10 times more monoterpenes than did mature leaves, and total emission by young leaves was higher than 0.3 nmol m−2 s−1 (Fig. 2a). About 50% of the monoterpene blend emitted by young leaves was made up by (E)-β-ocimene and linalool, in a 1:1 ratio. These two monoterpenes were also the main compounds emitted in mature leaves, but relative emission of (E)-β-ocimene was about three times more than linalool emission. At least 11 other different monoterpenes were detected in both young and mature leaves. High levels of isoprene emission, typical of poplars, were measured in mature leaves, whereas in young leaves the emission rate was significantly lower (Fig. 2b).

Figure 2.

Total emission of monoterpenes and distribution between the different monoterpenes emitted in young and mature leaves of poplar (a). In (b), the rates of isoprene emission, photosynthesis and stomatal conductance (means ± SE) are reported for the two leaf types. For actual rates, differences between young and mature leaves were statistically analysed using a Student's t-test (n = 5, **P < 0.01). For percent distribution among different monoterpenes, SE was always <10% of shown mean value.

Insect attack to mature leaves induced a large emission of monoterpenes (Fig. 3a). The emission significantly increased already 1 d after the attack and peaked 4 d after the attack, reaching rates of 2.0 nmol m−2 s−1, or two orders of magnitude higher than in the same leaves before feeding. Monoterpene emission rates decreased 7 d after the attack, suggesting that the insect-induced stimulation of the emission was not permanent. (E)-β-ocimene and linalool constituted the two main compounds emitted, and (E)-β-ocimene, in particular, constituted about 80% of the total monoterpenes emitted 4–7 d after the attack (Fig. 3b). Insect feeding on young leaves also caused an increase of monoterpenes already emitted by these leaves, with the same temporal pattern as observed in mature leaves (Fig. 3a). However, monoterpene emission by young leaves was not enhanced up to the same level observed in mature leaves. Monoterpene emission was associated with a decrease of isoprene emission in mature leaves (Fig. 3c). The strongest reduction of isoprene emission occurred as soon as 1 d after the attack. At this early stage, the reduction of isoprene emission was not associated to changes in photosynthesis and stomatal conductance. However, these two physiological parameters were also negatively affected by the attack after 4–7 d. Isoprene emission started to increase again 7 d after the attack, when monoterpene emission started to decrease, and photosynthesis and stomatal conductance had not yet recovered. All gas exchange parameters were recalculated on a leaf-area basis, and their values did not depend on the area actually preserved after the insect attack.

Figure 3.

Total emission of monoterpenes in mature (bars) and young poplar leaves (filled circles) (a), and percent of the two principal monoterpenes of the emitted blend (b) in mature leaves before (time = 0) and 1, 4 and 7 d after insect attack. In (c), the rates of isoprene emission, photosynthesis and stomatal conductance are reported for the same treatment in mature leaves. In all panels, means ± SE are shown (n = 4). Differences between means recorded at different days were statistically assessed with a Tukey's post hoc test. Different letters (small = mature leaves; capital = young leaves) identify differences of isoprenoid emission statistically significant at P < 0.01. Significantly different rates of photosynthesis and stomatal conductance at days 4 and 7, with respect to prior to insect attack, are labelled by ** (P < 0.01).

Peak emission of monoterpenes, 4 d after the attack, was largely and quickly labelled by 13C, indicating biosynthesis from freshly fixed carbon. The 12C composition of monoterpenes dropped within 20 min after switching to a 100% 13CO2 source, and a similar time-course was observed for labelling washout, once the natural composition of CO2 (99% 12CO2) was restored (Fig. 4). When labelling was complete, about 80% of the monoterpenes incorporated labelled carbon, as calculated from the ratio between unlabelled carbon and the sum of all labelled carbons of the molecules (Loreto et al. 1996).

Figure 4.

Amount of unlabelled (12C) emitted monoterpenes (solid line) and percent of 13C incorporation in the same molecules (bars) after a 60 min labelling with pure 13CO2. The experiment was carried out in mature leaves emitting induced monoterpenes 4 d after the insect attack (n = 4).

Monoterpene emission was also induced in leaves that were not attacked by the insects, but were the nearest and with non-orthostichous connectivity to the attacked leaves. The emission of the first leaf above or lateral to the attacked leaf was three to four times higher than before insects started feeding (Fig. 5a). However, the emission measured 4 d after the attack was only about 0.10 nmol m−2 s−1, or about 20 times lower than the emission measured after the same time-course in leaves directly attacked. Again, (E)-β-ocimene and linalool were the major compounds emitted, representing 60–80% of the total monoterpene emission in leaves before and after insect attack (Fig. 5b). However, the ratio between these two compounds was about 2.5 compared to the ratio of 20 in directly attacked leaves, and did not change following the attack. Isoprene emission dropped in leaves that were not directly attacked by the insect (Fig. 5c), but the largest reduction occurred 1–4 d after the attack, that is, more slowly than in directly attacked leaves (compare with Fig. 3). Photosynthesis and stomatal conductance were not affected in leaves that were not directly attacked, again indicating that less carbon is invested in isoprene biosynthesis as compared to the carbon fraction invested before insect attack (Fig. 5c).

Figure 5.

Total emission of monoterpenes (a) and percent of the two principal monoterpenes of the emitted blend (b) in unattacked mature leaves of poplar nearest to those attacked by the insects. Measurements were taken before (time = 0), and 1 and 4 d after insect attack (a). In (c), the rates of isoprene emission, photosynthesis and stomatal conductance are reported for the same treatment. Differences between means of gas exchange parameters (n = 4) recorded at different days were statistically assessed with a Tukey's post hoc test. Different letters identify differences statistically significant at P < 0.01 (single letters) or 0.05 (double letters) for isoprene and monoterpene rates of emission. Rates of photosynthesis and stomatal conductance were not significantly different along the experiment.

The experiments quite clearly revealed that isoprene reduction was associated with the increase of insect-induced monoterpenes in all leaf types. Isoprene reduction was much stronger and occurred slightly earlier than monoterpene induction, but when isoprene and monoterpene emissions by all sampled leaves were plotted against each other on a log–log scale that describes better slow biological processes (Fig. 6), a clear indication was found that the emission of the two classes of volatile isoprenoids is inversely correlated after the herbivore attack.

Figure 6.

Relationship between the emission of monoterpenes and isoprene in poplar leaves. The emissions measured in leaves showed in Figs 2 (young versus mature leaves); 3 (mature leaves before herbivore attack, and 1, 4 and 7 d after the herbivore attack); and 5 (mature leaves proximal to those attacked by the herbivores, before herbivore attack and 1 and 4 d after the herbivore attack) are shown.

Laboratory behavioural tests

In a laboratory-based behavioural test, adults and newly emerged adults were introduced in a Y-tube olfactometer to repeat with excised leaves the experiment carried out with whole-caged plants, and in particular to verify whether insects were attracted by the two monoterpenes that constituted the majority of the blend emitted by young leaves, or that were induced by insect feeding in mature leaves. Adult insects that were offered a choice between clean air and a synthetic mixture of (E)-β-ocimene and (-)-linalool, in a 1:1 ratio, were not attracted by the monoterpene blend, even after experiencing a 3 d starvation (Fig. 7a). Newly emerged, inexperienced adults showed a more complex behaviour. When these inexperienced insects were not fed before the assay, they were largely attracted by the monoterpene blend, whereas this behaviour was not observed anymore after feeding the insects with poplar leaves. Addition to the synthetic blend of one or more of the minor induced monoterpenes emitted after the attack did not alter the insect behaviour (data not shown). Newly emerged insects were not attracted or repelled by isoprene, when this compound was either offered alone (Fig. 7a) or in combination with the two major monoterpenes (data not shown) in the olfactometric test.

Figure 7.

Results of the behavioural test carried out in a Y-tube olfactometer using adults or newly emerged adults. On both groups, some individuals were starved (adults) or unfed (newly emerged or inexperienced adults) before the experiment. In (a), white, black and striped bars represent, respectively, the percent of insects that walked towards pure air or a monoterpene blend made by the two main compounds emitted by young poplar leaves [(E)-β-ocimene and (-)-linalool, 1 µmol mol−1 each), or isoprene emitted by mature unattacked leaves. Data are expressed conventionally considering the monoterpene blend as flowing always from the right arm of the Y-tube, although half of the tests were carried out offering the monoterpene blend from the left arm. In (b), results of an experiment in which newly emerged adults were offered unattacked young and mature leaves (upper figure) or attacked young and mature leaves (bottom figure) at the two arms of the olfactometer are shown. As for the monoterpene blend, the two leaf types were actually offered alternatively from the two sides of the olfactometer, and results are conventionally expressed as if young and mature leaves were only offered from one arm. The percent of insects walking towards the two arms when offered poplar leaves are represented by the two tones of grey bars. The experiment was also repeated offering to insects the choice when the arms were both flushed with pure air only. This experiment (not shown) served as blank to test statistically significant effects of each trial, using a χ2 test, with Yates correction. Each trial was carried out on at least 30 individuals. The experiments on which insects were attracted by the monoterpene blend in a statistically significant way are labelled by * (P < 0.05) or ** (P < 0.01). Non-statistically significant behaviours are labelled as n.s.

We further investigated the behaviour of inexperienced adults that were never fed after emerging from the pupal stage. When given a choice between a mature leaf and a young leaf that were just excised and offered at the two extremities of the olfactometer, these inexperienced insects significantly preferred the monoterpene blend emitted by young leaves (Fig. 7b). When offered a young leaf and a mature leaf, both of which had been attacked by other adults of Chrysomela at the two extremities of the olfactometer (Fig. 7b), inexperienced insects again showed a clear preference for mature leaves that were emitting three times more monoterpenes than young leaves, as previously shown (Fig. 3a).


Young poplar leaves constitutively emit a monoterpene blend

Poplars are among the strongest isoprene-emitting species. Poplars are not reported to constitutively emit monoterpenes (Kesselmeier & Staudt 1999), but a low emission of monoterpenes was found in field measurements on young aspen leaves by Hakola et al. (1998). We found that poplars can emit detectable amounts of constitutive monoterpenes from young leaves that do not experience biotic or abiotic stresses. Interestingly, in mature leaves, the emission of monoterpenes seems to be replaced by the emission of isoprene. The carbon emitted as isoprene by mature leaves is at least five times more abundant than that emitted as monoterpenes by young leaves. This indicates that the amount of carbon invested in the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway to form volatile isoprenoids increases with leaf age. It should be studied whether the early emission of monoterpenes is a ubiquitous occurrence in isoprene-emitting plants, because this may largely change the contribution of vegetation to atmospheric processes (e.g. during spring). Monoterpenes are by far more reactive than isoprene (Atkinson 1997), and may more efficiently scavenge ozone (Fares et al. 2008) or contribute to aerosol and particulate formation (Kanakidou et al. 2005). Their strong antioxidant action in leaves (Loreto et al. 2004b) might contribute to protect young leaves from oxidative stresses.

Monoterpene emission is induced by Chrysomela attack in mature poplar leaves

The acyclic monoterpenes have been shown to be emitted in response to stressful conditions (Röse et al. 1996) and to act as defensive compounds also against biotic stresses (Pare' & Tumlinson 1999). (E)-β-ocimene may attract predators of herbivores (Dicke et al. 1990) or may signal the presence of infestation to unattacked plants that up-regulate defensive pathways (e.g. jasmonic acid and ethylene; Arimura et al. 2000). More interestingly to this study, electroantennograms showed that Chrysomelidae insects respond to (E)-β-ocimene emitted by attacked Salix viminalis leaves (Fernandez et al. 2007). Linalool may decrease oviposition by some insects (Kessler & Baldwin 2001). Consistently, with these and other studies, we have found that the emission of monoterpenes was induced by the attack of adult insects of C. populi in mature poplar leaves that were emitting trivial amounts of the same monoterpenes before being attacked. Arimura et al. (2004a) also found induction of (E)-β-ocimene, linalool and other isoprenoids (e.g. homoterpenes and sesquiterpenes) in poplar leaves attacked by forest tent caterpillars. The emission of (E)-β-ocimene and other isoprenoids is also induced in poplar leaves in response to feeding by gypsy moth larvae (Frost et al. 2007) and autumnal moth larvae (Blande et al. 2007). Our study indicates that in poplar leaves, the induction of monoterpene emission is a general response to herbivores, also including adult beetles.

The emission of isoprenoids in response to poplar caterpillars was found to be induced in a systemic way (i.e. also in non-attacked leaves; Arimura et al. 2004a). We also observed that the emission could be induced systemically, and even in leaves showing non-orthostichous connection with the attacked leaves. This is another typical feature of herbivore-induced emissions. These emissions may attract parasitoids (Turlings et al. 1995; Röse et al. 1996) or prime production of other defensive compounds (Ton et al. 2007), activating the signalling system, the signal transduction pathway and the biosynthesis of active molecules at whole-plant level, and perhaps also in neighbouring plants.

To activate the biosynthesis of monoterpenes, a certain lag-time is required, which may explain the delay with which the emission occurs with respect to the moment of the attack. Isoprenoid emission by poplar leaves was found to reach a maximum 1–3 d after the attack by caterpillars (Arimura et al. 2004a), and the peak emission of (E)-β-ocimene by Lotus japonicus leaves occurred only 1 d after the attack by spider mites (Arimura et al. 2004b). In both cases, the emission rates matched the increase of transcript levels of the terpene synthases, confirming an activation of the entire metabolic pathway. We extended the period of measurements and observed that monoterpene [particularly (E)-β-ocimene] emission induced by herbivores may remain high for at least 4 d after the attack, and may still be above constitutive levels even 7 d after the attack. This implies a sustained change of plant metabolism and a prolonged activation of the MEP pathway producing the emitted compounds.

Monoterpenes in Chrysomela-attacked leaves are formed using the carbon allocated to isoprene synthesis and emission

All previous studies reporting monoterpene induction in response to herbivores did not consider the possible relationship of these compounds with isoprene, the first volatile molecule that can be formed through the MEP pathway and the main BVOC emitted in the biosphere (Sharkey & Yeh 2001). The function of isoprene emission in response to environmental stress has been elucidated. This lipophylic compound may stabilize the chloroplast membrane when exposed to thermal (Singsaas et al. 1997) or oxidative stress (Loreto & Velikova 2001), making plants more resistant to these constraints. However, isoprene may also be opportunistically emitted when the chloroplastic biosynthesis of essential isoprenoids (e.g. carotenoids) does not require the whole carbon flux through the MEP pathway (Owen & Penuelas 2005). The trade-off observed in our study between constitutive or induced monoterpenes and isoprene emissions (cfr. Fig. 6) supports to some extent the opportunistic hypothesis, as it indicates that carbon may be readily shifted from isoprene to volatiles that are likely needed for plant defence. Consistently, it was found that the carbon fixed in herbivore-induced monoterpenes rapidly incorporates 13C labelling, as is also the case for isoprene (Delwiche & Sharkey 1993) and constitutive monoterpenes (Loreto et al. 1996). (E)-β-ocimene and linalool fast and complete labelling was reported earlier (Pare' & Tumlinson 1997). The chloroplastic MEP pathway seems to be the main source of carbon for the formation of these induced compounds. This explains why emission of monoterpenes is induced at a lower level in young leaves that do not produce and emit large amounts of isoprene (Fig. 3a). In young leaves, the MEP pathway probably does not supply enough carbon that can be shifted from isoprene production to the synthesis of the induced monoterpenes. It remains unclear why the drop of isoprene emission was much larger than the observed induced emission of monoterpenes. Monoterpenes are less volatile, and the emission may not represent as well the actual content of these compounds as it is the case for isoprene. In addition, the synthesis of other compounds that were not detected with our instrumentation might also have been induced using carbon allocated to isoprene biosynthesis in intact leaves.

Isoprene emission is predominantly, although not exclusively, dependent on photosynthesis (Sharkey & Yeh 2001). However, the reduction of isoprene emission following insect feeding precedes the drop in photosynthesis and is already very clear 1 d after feeding in mature leaves. This reduction also occurs systemically in unattacked leaves in which the photosynthetic rates remain unaffected. This is interpreted as a further indication of reallocation of carbon into the skeleton of monoterpenes. Isoprene emission was also found to decrease independently of photosynthesis in manually defoliated plants, but this was interpreted as the consequence of total carbon available for isoprene biosynthesis (Funk, Jones & Lerdau 1999). It is also unclear why photosynthesis of the leaf portions that were not eaten by the insects is inhibited as well. It has been suggested that this may also be a consequence of the cost of the biosynthesis of defensive compounds (Zangerl et al. 2002).

Constitutive monoterpenes attract inexperienced adults of Chrysomela

The observation that inexperienced adult insects predominantly landed on young leaves, as shown in this study and in Harrell et al. (1981), and the finding that young poplar leaves naturally emit higher rates of monoterpenes, led us to formulate the hypothesis that the monoterpene blend emitted by these leaves could attract the insects, or that isoprene emitted by mature leaves could repel the same insects. Because our experiments did not show any repellent effect of isoprene on insect behaviour, we speculated that insects co-evolving with plants learned to use monoterpenes, emitted for different and yet not clarified purposes by young leaves, as an olfactory cue to locate the most suitable leaves where to land and feed. The behavioural olfactometric test confirmed our hypothesis. This test showed a clear preference of inexperienced insects for young leaves when offered unattacked young and mature leaves at the two arms of the olfactometer, and for mature leaves when offered attacked leaves in which monoterpene emission was more induced than in young leaves. The test also showed that inexperienced insects were attracted by the synthetic blend reconstituting the two major compounds emitted by leaves. It should be mentioned that synthetic blends often fail to reproduce natural emissions because of the different racemic mixture as compared to natural emissions. Emission of the (-)-linalool isomer is prevalently induced by insect feeding (Miller et al. 2005), and (-)-linalool was therefore offered to insects in our experiment. We do not know whether other racemic mixtures could modify insect behaviour, although there are reports showing that antennae neurons can be also activated by a (±)-linalool mixture (Heinbockel & Kaissling 1996).

Interestingly, the olfactometric test indicated that volatiles emitted by young leaves lost the capacity to act as an olfactory cue once the insects fed on poplar leaves. In the larval stages of some Chrysomelidae, preference for young leaves relies on their high content of phenolic glucosides (Bingaman & Hart 1993), which are precursors for the production of salicylaldehyde containing defensive larval secretion (Brückmann et al. 2002). The unexpected behaviour of C. populi adults, however, was, to our knowledge, never described, and the processes underlying the changing behaviour of insects are yet undisclosed. Larvae of C. populi preferably pupate on the abaxial side of leaves of non-host plants, such as herbaceous weeds (our unpublished observations). Newly emerged adults may therefore require a suitable olfactory cue to locate the host plants or their more suitable parts. Once the target sites have been reached, the cue is not needed anymore, and adults may become habituated to the odour stimuli which lead them to a decreasing olfactory response. Habituation to olfactory cues is not such a rare process in plant–insect interactions (Bernays & Chapman 1994) and was also reported in response to synthetic attractants (Martel, Alford & Dickens 2005). Alternatively, the behaviour of adult insects might be explained if additional compounds are emitted by attacked leaves that mask the attractive monoterpene blend. Several reports have indicated that in poplar leaves also sesquiterpene emissions are induced by, and may actively influence communication with insects (Blande et al. 2007; Frost et al. 2007). No sesquiterpene emission was detectable at fluxes above 0.01 nmol m−2 s−1 in our experiment. However, sesquiterpene detection could be made difficult by the very fast reactivity of these compounds during collection and analysis. In addition, even minimal emissions of sesquiterpenes and other volatiles can sensibly affect insect behaviour.

A question that arises from our study is why the monoterpene blend induced by herbivores replicates the blend constitutively emitted by young leaves. Plants would be expected to evolve mechanisms deterring herbivores from feeding rather than attracting them. Emission of unspecified compounds was also found to drive Chrysomela scripta on cottonwood leaves (Kendrick & Raffa 2006). It may be speculated that the same monoterpene blend that attract Chrysomela insects also attracts parasitoids or predators of the herbivore, or that monoterpenes are emitted by attacked plants for protection against other pathogens or abiotic stresses, or that monoterpene stimulation in old leaves is a plant strategy to disguise young leaves, an interesting strategy to preserve plant resources, increase fitness and decrease the morbidity of the attack. For example, larvae emerging on mature leaves probably feed less than on soft, young leaves, and may be predated more easily, because the poor phenolic glucoside content of mature leaves diet may not allow sufficient biosynthesis of deterrents against predators. Further work will be needed to verify these hypotheses.


This work was supported by the European Commission Marie Curie project ‘Ecological and Physiological Functions of Biogenic Isoprenoids and Their Impact on the Environment’ (ISONET, MRTNCT–2003–504720), and by the European Science Foundation scientific programme Volatile Organic Compounds in the Biosphere – Atmosphere System (VOCBAS). The experiments comply with the current laws of the country in which they were performed.