Induction of Phenolic Glycosides by Quaking Aspen (Populus tremuloides) Leaves in Relation to Extrafloral Nectaries and Epidermal Leaf Mining

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1 J Chem Ecol (2010) 36: DOI /s Induction of Phenolic Glycosides by Quaking Aspen (Populus tremuloides) Leaves in Relation to Extrafloral Nectaries and Epidermal Leaf Mining Brian Young & Diane Wagner & Patricia Doak & Thomas Clausen Received: 11 October 2009 / Revised: 24 January 2010 / Accepted: 10 February 2010 / Published online: 31 March 2010 # Springer Science+Business Media, LLC 2010 Abstract We studied the effect of epidermal leaf mining on the leaf chemistry of quaking aspen, Populus tremuloides, during an outbreak of the aspen leaf miner, Phyllocnistis populiella, in the boreal forest of interior Alaska. Phyllocnistis populiella feeds on the epidermal cells of P. tremuloides leaves. Eleven days after the onset of leaf mining, concentrations of the phenolic glycosides tremulacin and salicortin were significantly higher in aspen leaves that had received natural levels of leaf mining than in leaves sprayed with insecticide to reduce mining damage. In a second experiment, we examined the time course of induction in more detail. The levels of foliar phenolic glycosides in naturally mined ramets increased relative to the levels in insecticide-treated ramets on the ninth day following the onset of leaf mining. Induction occurred while some leaf miner larvae were still feeding and when leaves had sustained mining over 5% of the leaf surface. Leaves with extrafloral nectaries (EFNs) had significantly higher constitutive and induced levels of phenolic glycosides than leaves lacking EFNs, but there was no difference in the B. Young : D. Wagner : P. Doak Institute of Arctic Biology and Department of Biology & Wildlife, University of Alaska Fairbanks, Fairbanks, AK , USA T. Clausen Department of Chemistry and Biochemistry, University of Alaska Fairbanks, Fairbanks, AK , USA Present Address: B. Young (*) Department of Forest Sciences, University of Alaska Fairbanks, Fairbanks, AK , USA bdyoung@alaska.edu ability of leaves with and without EFNs to induce phenolic glycosides in response to mining. Previous work showed that the extent of leaf mining damage was negatively related to the total foliar phenolic glycoside concentration, suggesting that phenolic glycosides deter or reduce mining damage. The results presented here demonstrate that induction of phenolic glycosides can be triggered by relatively small amounts of mining damage confined to the epidermal tissue, and that these changes in leaf chemistry occur while a subset of leaf miners are still feeding within the leaf. Key Words Populus tremuloides. Phyllocnistis populiella. Epidermal leaf mining. Induced defense. Phenolic glycosides. Extrafloral nectaries. Alaska Introduction Some plants respond to herbivore damage by increasing production of chemical, physical, or biotic defenses, responses that can help protect the remaining tissue against further damage (Karban and Baldwin 1997). Induction of resistance traits may have evolved in response to the costs of resistance. Such costs may include tradeoffs in resource allocation between growth and resistance and enhanced apparency to specialist herbivores that use resistance traits to locate hosts (Rhoades 1979; Strauss et al. 2002). For trees such as immature Populus spp., which must grow quickly to avoid being shaded by competitors or consumed by browsers (Bokalo et al. 2007), cost savings may allow a greater proportion of resources to be invested in early growth and survival. Quaking aspen (Populus tremuloides Michx.) is a fastgrowing forest tree subject to attack by a variety of vertebrate and invertebrate herbivores, including several

2 370 J Chem Ecol (2010) 36: insect species subject to large-scale population outbreaks (Mattson et al. 2001). Since the late 1990s and continuing through 2009, aspen in Alaska and western Canada have experienced a severe outbreak of the aspen leaf miner (Phyllocnistis populiella Chambers, Lepidoptera: Gracillariidae) (Fig. 1a) (U.S. Forest Service 2006, 2007, 2008). Unlike most leaf miner taxa, which feed on the photosynthetic tissue of the mesophyll, P. populiella feeds only on the cells of the leaf epidermis, removing very little leaf biomass (Condrashoff 1964) (Fig.1b). Although feeding damage is restricted to the epidermis, at high density P. populiella can reduce photosynthesis and slow the growth of aspen ramets (Wagner et al. 2008). The leaves of aspen trees produce phenolic glycosides, which can provide resistance against some herbivores (Philippe and Bohlmann 2007). Four types of phenolic glycosides appear in aspen leaves, salicin, salicortin, tremuloidin, and tremulacin, with salicortin and tremulacin having greater biological activity than the others (Lindroth et al. 1987). Phenolic glycosides can reduce the growth rate and survivorship of several of aspen s most devastating pests, including the large aspen tortrix (Choristoneura conflicta), the forest tent caterpillar (Malacosoma disstria), and the gypsy moth (Lymantria dispar) (Bryant et al. 1987; Lindroth and Hemming 1990; Lindroth and Hwang 1996a, b; Osier et al. 2000; Osier and Lindroth 2001; Donaldson and Lindroth 2007). Average foliar phenolic glycoside concentrations vary widely among aspen genotypes (Hwang and Lindroth 1997; Osier and Lindroth 2006; Donaldson and Lindroth 2007), and expression also can vary within genets in response to leaf age (Lindroth et al. 1987; Bingaman and Hart 1993; Kleiner et al. 2003), resource availability (Hemming and Lindroth 1999; Donaldson and Lindroth 2007), and herbivory (Clausen et al. 1989a; Lindroth and Kinney 1998). In addition to phenolic glycosides, aspen also produce extrafloral nectaries (EFNs). Studies of other plant species indicate that EFNs can function as an indirect defense by attracting arthropod predators and parasitoids of herbivores to the plant (Bentley 1977; Koptur 1992; Röse et al. 2006). In some populations, EFNs increase plant fitness by reducing tissue loss to herbivores (Heil et al. 2004; Rudgers et al. 2004). EFN expression in aspen is extremely variable. EFNs occur on only a subset of aspen leaves, and the frequency of EFN expression varies among shoots, ramets, and genets (Doak et al. 2007). At the level of the leaf, the expression of EFNs and phenolic glycoside concentrations co-vary (Young et al. 2010). After controlling for mean differences among sites, ramets, and shoots, concentrations of foliar phenolic glycosides are about 30% higher in leaves with EFNs than in leaves lacking them (Young et al. 2010). On short aspen ramets, leaves bearing EFNs sustain lower levels of epidermal leaf mining than leaves lacking EFNs (Doak et al. 2007), but this difference may be caused by the higher phenolic glycoside content of EFN-bearing leaves, rather than by the indirect defense of EFN-bearing leaves by the leaf miner s natural enemies (Mortensen 2009; Young et al. 2010). In this study, we tested whether leaf mining damage restricted to the epidermis induces the production of phenolic glycosides salicortin and tremulacin in leaves with and without EFNs. By comparing the phenolic glycoside concentrations of leaves collected serially from ramets with natural and reduced levels of leaf mining, we tracked the time course of phenolic glycoside expression in young aspen leaves and investigated whether induction has the potential to impact the performance of P. populiella. Methods and Materials Fig. 1 a Aspen leaf miner moth and eggs on a leaf with two EFNs at the petiole. b Epidermal leaf mining by the aspen leaf miner on a leaf without EFNs Natural History Quaking aspen is widely distributed in North America (Mitton and Grant 1996) and reproduces both sexually and asexually, resulting in the formation of clonal stands. In the interior of Alaska, it tends to grow on south facing hillsides and along ridgelines.

3 J Chem Ecol (2010) 36: The EFNs of P. tremuloides are located at the junction of the petiole and the leaf (Trelease 1881). The number of EFNs on a leaf can vary from 0 to 6, with most leaves possessing 0 or 2 (Doak et al. 2007). Among the preformed leaves, the first 5 to 8 leaves to emerge in spring (Critchfield 1960), EFNs are consistently expressed on the most proximal leaf, with frequency of expression decreasing distally along the shoot (Doak et al. 2007). In contrast, the neoformed leaves, which emerge later and extend the growing shoot, typically all possess EFNs (Doak et al. 2007).Wefocusedonthepreformedleavesin this study. The aspen leaf miner P. populiella overwinters as an adult and emerges in early to mid May, prior to bud break. Oviposition begins as soon as the protective bud scales are shed, exposing the furled aspen leaves, and continues for about 10 d. Eggs are laid singly on both the upper and lower surfaces of young leaves. The eggs sink into the leaf tissue, and about a week after oviposition the larvae hatch directly into the epidermal tissue. The developing larvae feed on the cells of the leaf epidermis, leaving obvious tracks or mines. Larvae remain on the leaf surface on which they hatched and feed for 9 15 d (Condrashoff 1964). In the final instar, which lasts 2 d, larvae cannot feed and form leaf folds in which to pupate (Condrashoff 1964). During the years of this study (2006 and 2007), the median preformed leaf at the study site hosted a single leaf fold (Doak and Wagner, unpublished survey data). Field Method To investigate the effects of epidermal leaf mining on aspen phenolic glycoside concentrations, we first (2006) experimentally reduced leaf miner densities on a set of aspen ramets by using an insecticide, and we contrasted the mean foliar phenolic glycoside concentration of insecticide-sprayed and naturally-mined ramets at a single point in time. The following year (2007), we measured the concentration of phenolic glycosides on insecticide-sprayed and naturally-mined ramets at intervals during the period of active feeding by the leaf miner. In early June of 2006, we randomly chose a set of 12 small ramets ( m in height) near the summit of Ester Dome ( N, W, elevation 720 m) near Fairbanks, Alaska. Ramets were located within a m area and exhibited similar physical characteristics; however, we cannot be certain that they all belonged to a single clone. Of the 12 ramets, half were assigned at random to receive an insecticide treatment. Just as the P. populiella eggs began to hatch, the treatment ramets were sprayed with the insecticide spinosad (Conserve; Dow AgroSciences, Indianapolis, IN.USA; concentration 1.56 ml l 1 ; applied with a hand-powered pump sprayer untilrunoff).theinsecticidewasreapplied6dlater.when the treatment ramets were sprayed with the insecticide, the remaining ramets were sprayed with an equal quantity of water. This experiment relied on the assumption that the application of insecticide does not prevent aspen from producing an induced response to damage. To test this assumption, we took advantage of the ability of aspen leaves to mount a rapid, localized induction of phenolic glycosides in response to mechanical leaf damage (Clausen et al. 1989a). One day before we planned to harvest the insecticide-sprayed and naturally-mined leaves, we damaged 6 randomly-chosen leaves, from leaf positions 1 5 from two separate shoots, on each of the ramets that had been sprayed with insecticide by cutting them in half diagonally across the mid vein with scissors. We harvested all leaves (naturally-mined, sprayed, and sprayed and mechanically damaged) 11 d following the approximate onset of leaf mining. At this point, 80 90% of the leaves on the naturally-mined ramets possessed one or more pupal folds, indicating that most of the aspen leaf miner larvae had ceased to feed (Condrashoff 1964). We sampled preformed leaves from the 5 most proximal leaf positions from 3 randomly-chosen shoots per ramet. Leaves were snipped at the petiole and immediately immersed in 10 ml of 50% aqueous MeOH. Cold 50% aqueous MeOH effectively extracts phenolic glycosides from aspen leaves (Bryant et al. 1987; Lindroth and Pajutee 1987). We maintained the leaf samples in solution at 2 C±1 C for 24 h. The leaves were then removed from the extract solution and pressed in a plant press for subsequent analysis of herbivore damage. The extract was stored at 40 C until analysis was conducted. For each leaf, we counted the number of EFNs and visually estimated leaf mining on the top and bottom surfaces of the leaf and the percent of leaf tissue missing and/or damaged by other herbivores to the nearest 5.0 %. Our visual estimates of mining correlated strongly with measurements made using image analysis software (R 2 > 0.9, Doak et al. 2007). For simplicity, we report % total leaf surface mined, calculated as the average of % mining on the top and bottom surfaces. Leaves were scanned using a desktop scanner and leaf area was measured using the image analysis program Scion Image (Fredrick, MD, USA). Leaves were dried at 60 C for 1 wk and weighed to the nearest 0.1 mg. To determine how foliar phenolic glycoside concentrations changed on a finer time scale during the period of feeding by P. populiella, we conducted a second experiment at the same study area in May We chose 42 ramets ranging in size from 0.5 to 1.6 m in height; all located within a m area adjacent to the 2006 site. Again, the genetic identity of the plants was not known. We assigned half of the ramets at random to receive an insecticide treatment. On the day that it appeared that the majority of

4 372 J Chem Ecol (2010) 36: leaf miner eggs had hatched, we applied insecticide to treatment ramets and water to the others. The method of insecticide application was identical to the previous year. To accommodate repeated sampling while avoiding the removal of excessive numbers of leaves from individual ramets, we used a larger number of ramets than in 2006 and sampled each ramet on only two occasions. Leaf samples were collected on days 1, 2, 4, 5, 6, 9, and 12 after the onset of leaf mining. On each sampling day, we sampled preformed leaves from leaf positions 2 and 3 on a single shoot from 6 naturally-mined and 6 insecticide-sprayed ramets, chosen at random from the set of ramets that had not yet been sampled. Leaves were again sampled by cleanly snipping leaves at the petioles. Mattson and Palmer (1988) did not find a chemical response of the ramet when the leaf was sampled in this manner. When each ramet had been sampled once, we resampled each ramet a second time, using a different shoot. All leaf samples were handled and processed as in the previous year. Chemical Analysis We prepared the aspen leaf extracts for analysis by filtering a 1.0 ml aliquot of extract through a 0.45 μm pore poyproplyene membrane syrine filter (Acrodisc, Thermo Fisher Scientific, Waltham, MA, USA). Samples were injected onto a mm XDB-C8 column (Agilent) attached to a High Performance Liquid Chromatography (HPLC) (Agilent 1100) equipped with a UV/VIS diode array detector (Agilent) and analyzed at 230 nm. The phenolic glycosides were separated using a mobile phase gradient of acetonitrile (CH 3 CN) and H 2 0 with a constant flow rate of 1.0 ml/min. The gradient elution was: 1% CH 3 CN (0 4 min), 1 60% CH 3 CN (4 10 min), 60 80% CH 3 CN (10 15 min), 100% CH 3 CN (15 22 min), and 100 1% CH 3 CN (22 27 min). This was followed by a 10 min period at 1% CH 3 CN prior to the injection of next sample. Salicortin and tremulacin were quantified using purified reference standards (Clausen et al. 1989b). Statistical Analyses For both 2006 and 2007 data sets, we compared the % leaf mining damage, and the % leaf damage caused by other herbivores, on insecticide-sprayed vs. naturally-mined ramets using Wilcoxon tests. Preliminary data analysis showed that concentrations of salicortin and tremulacin were highly correlated within leaves (R=0.8, df=166, P<0.01), responded similarly to treatment, and changed in similar ways over time. For simplicity, we therefore report total phenolic glycoside concentrations, calculated as the sum of salicortin and tremulacin. To test whether insecticide treatment prevented induction of phenolic glycosides in response to damage, we compared the phenolic glycoside concentration of experimentally cut and uncut leaves (2006 data) using a mixed model analysis of covariance (ANCOVA) with mechanical damage as a fixed effect, leaf position as a covariate, and ramet and shoot as random effects. To test the effect of leaf mining on foliar phenolic glycoside concentration (2006 data, leaves mined for 11 d) we again used ANCOVA, with insecticide treatment, EFNs (dichotomous: absence vs. presence), and their interaction as fixed effects, leaf position as a covariate, and shoot and ramet as random effects. We tested when induction occurred (2007 data) by comparing the phenolic glycoside concentrations of leaves from naturally-mined and insecticide-sprayed ramets within each sampling date. We first ran a mixed model ANCOVA with insecticide treatment, day sampled, and their interaction as fixed effects, leaf position as a covariate, and shoot and ramet as random effects. We then conducted planned contrasts of the mean foliar phenolic glycoside concentration of naturally-mined and insecticide-sprayed ramets within each sampling day. Phenolic glycoside concentrations were log transformed to meet parametric assumptions. Mixed model analyses applied the restricted maximum likelihood method and were conducted using JMP IN version using the Kenward and Roger (1997) method to calculate denominator degrees of freedom (SAS Institute, Cary, NC, USA). Results The insecticide treatment prevented all detectable leaf mining damage by P. populiella on the treated ramets during both 2006 and 2007 (Table 1). Across all experimental trees, leaf mining represented by far the greatest source of leaf damage, although a small amount of chewing, skeletonizing, and galling damage were also observed (Table 1). Leaf damage due to herbivores other than P. populiella was not reduced significantly by the insecticide treatment (Table 1). Foliar phenolic glycosides were induced in insecticidesprayed leaves in response to mechanical leaf damage. One day after leaves on sprayed ramets were cut with scissors, the cut leaves had higher concentrations of phenolic glycosides than un-cut leaves (F 1, 113 =6.45, P=0.012). The average phenolic glycoside concentration of the cut leaves was 37% higher then the undamaged leaves (back-transformed least square means; damaged leaves=27.3 mg/g, undamaged leaves=19.9 mg/g). Leaf mining by P. populiella induced foliar phenolic glycosides. Eleven days after the approximate onset of leaf mining in 2006, the foliar phenolic glycoside concentration was, on average, 125% higher for ramets that had sustained natural levels of leaf mining damage than for ramets

5 J Chem Ecol (2010) 36: Table 1 Percent leaf damage (mean±se) by Phyllocnistis populiella and other herbivore taxa after experimental reduction in leaf miner abundance 11 days post treatment. Other damage is the sum of chewing, skeletonizing, and galling Naturally-mined Insecticide-sprayed Naturally-mined Insecticide-sprayed Top mining 60.7± ±0.0 *** 18.1± ±0.0 *** Bottom mining 43.9± ±0.0 *** 17.3± ±0.0 *** Other damage 0.2± ±0.1 n.s. 0.2± ±0.7 n.s. Mean values for each ramet were compared with Wilcoxon signed rank tests n.s. P>0.05, *** P<0.001 sprayed with insecticide (Fig. 2; F 1, 10 =7.58, P<0.001). Across all naturally-mined and insecticide-sprayed ramets, leaves with EFNs contained on average 26% higher concentrations of phenolic glycosides than leaves lacking EFNs (Fig. 2; F 1, 148 =5.40; P=0.021). EFN presence and treatment did not interact (F 1, 145 =0.08; P=0.77), indicating that leaves with and without EFNs were similar in their ability to induce phenolic glycosides in response to mining. Foliar phenolic glycoside concentrations increased along the shoot, with the most distal leaves having the highest concentrations (Fig. 3; F 1,144 =6.14;P<0.01). By monitoring the time course of induction during the next growing season (2007), we found that induction occurred approximately 6 9 days after the onset of mining. Because on each collection day we harvested many fewer leaves (N=12) than in the 2006 experiment (N=180), we had less statistical power to detect differences between treatments. In the overall ANCOVA model, the mean phenolic glycoside content of leaves varied over time (F 6, 50=2.85, P=0.01), but there was no statistically significant effect of insecticide treatment (F 1, 37 =2.30, P=0.14) or interaction between treatment and time (F 6, 50 =1.12, P= 0.36). However, the results of planned contrasts within days provided information about the timing of induction. During the first six days, average phenolic glycoside concentrations in both naturally-mined and insecticide-sprayed leaves rose, peaking at day 4, and then declined (Fig. 4a). Contrasts within sampling day revealed no significant differences in phenolic glycoside concentration between naturally-mined and insecticide-sprayed leaves up to and including day 6 (P>0.05). However, after day 6 the two groups diverged, and the average phenolic glycoside concentration was higher in naturally-mined leaves than in insecticide-treated leaves on day 9 (Fig. 4a, F 1, 67 =4.42, P=0.04). The average phenolic glycoside concentration of both insecticide-treated and naturally mined ramets increased somewhat between days 9 and 12. While average phenolic glycoside concentration of the mined trees still exceeded that of insecticide-treated trees on day 12, the difference was no longer statistically significant (Fig. 4a, F 1, 69 =3.32, P=0.07). Leaf mining damage extended over approximately 5% of the leaf surface at the time induction was noted (Fig. 4b). Fig. 2 Concentrations of phenolic glycosides from naturally-mined and insecticide-sprayed leaves with and without EFNs during the summer of 2006 eleven days after the approximate onset of leaf mining. Values are back transformed least squared means±se. There was a significant effect of insecticide treatment (P<0.001) and EFN presence (P<0.05), and no interaction between insecticide treatment and EFNs (P>0.05) Fig. 3 Concentrations of foliar phenolic glycosides from leaf positions 1 through 5 during the summer of Values are back transformed least squared means±se

6 374 J Chem Ecol (2010) 36: Although some leaf miner larvae had ceased to feed by the time induction was detected, a subset of larvae apparently ingested leaves containing induced levels of phenolic glycosides. About 25% of sampled leaves possessed at least one pupal leaf fold (indicating that feeding had ceased) on day 6, and this percentage rose to 42% by day 9 (Fig. 4c), the day we observed induced levels of phenolic glycosides (Fig. 4a). On day 12, 92% of leaves had at least one pupal fold, indicating that approximately half of the leaves still contained feeding larvae on day 9 (Fig. 4c). Moreover, a substantial amount of feeding damage occurred post-induction (Fig. 4b), indicating that some larvae continued to feed following the damageinduced increase in phenolic glycoside concentration between days 9 and 12. Phenolic glycosides (mg/g) Leaf mining damage (%) Fraction of leaves with > one pupal fold a b c control sprayed Day sampled Fig. 4 Variation over time in a total foliar phenolic glycoside concentration (back transformed least squared means±se) of naturally-mined (filled symbols) and insecticide-sprayed (open symbols) ramets; b mining damage (means±se) on naturallymined ramets and (c). the fraction of leaves sampled from naturally-mined ramets on which at least one leaf miner had ceased to feed and formed a pupal fold. Data are from summer The asterisk indicates that, on this sampling date, the contrast between average phenolic glycosides in naturally-mined and insecticide-sprayed leaves was statistically significant at P< * 10 1 Discussion Damage to the epidermis caused by the mining activity of P. populiella led to the induction of phenolic glycosides in aspen leaf tissue. Epidermal leaf mining, while geographically widespread and ecologically important, is taxonomically restricted to a small subset of leaf miner taxa (Hering 1951). Consequently, few studies have investigated the physiological consequences of this form of herbivory for plants. In contrast, the vast majority of leaf mining species feed primarily on the photosynthetic cells of leaf mesophyll (Hering 1951). Studies investigating the effect of mesophyll mining on the chemistry of a variety of plant species report that mining can cause induced responses (Stout et al. 1994; Karban and Adler 1996; Inbar et al. 1999), although investigations of the induction of phenolic compounds in particular generally have reported negative results (Fisher et al. 2000; Ramiroetal.2006). Our results suggest that phenolic glycosides were induced while many leaf miner larvae were still feeding and growing and before the majority of the mining damage was inflicted on leaves (Fig 4). Phenolic glycosides are expressed in the epidermal tissue of aspen (Kao et al. 2002), thus it appears that P. populiella larvae ingest phenolic glycosides as they feed. Phenolic glycosides have negative effects on growth and survivorship of several generalist lepidopteran herbivores of aspen (Bryant et al. 1987; Hemming and Lindroth 1995; Hwang and Lindroth 1997; Osier and Lindroth 2001), but specialist herbivores, such as P. populiella, often are resistant to the effects of host plant secondary chemistry. However, natural patterns of leaf mining on leaves varying in phenolic glycoside concentration support the hypothesis that phenolic glycosides reduce P. populiella feeding damage (Young et al. 2010). Treatment with insecticide did not prevent aspen leaves from inducing phenolic glycosides in response to mechanical damage. One day after mechanical damage was imposed, insecticide-sprayed leaves that were cut had 37% higher phenolic glycoside concentrations than sprayed leaves that were not cut. Clausen et al. (1989a) found that mechanically damaged aspen leaves contained 15% more phenolic glycosides (salicortin and tremulacin combined) than undamaged leaves one day post-damage. The data suggest that the difference in foliar phenolic glycoside concentration between aspen ramets sprayed with insecticide and ramets naturally damaged by leaf miners reflects induction caused by leaf mining, rather than an artifact of the insecticide treatment. The concentration of phenolic glycosides was 26% higher in leaves with EFNs than in leaves lacking EFNs, in both the damaged and undamaged states. There was no

7 J Chem Ecol (2010) 36: evidence that leaves with EFNs induced phenolic glycosides more strongly than leaves without EFNs; rather, leaves with and without EFNs both increased in phenolic glycoside concentration by approximately 125% in response to leaf mining (Fig. 2). High levels of phenolic glycosides in EFNbearing leaves may help to explain the previously reported pattern of lower mining on these leaves (Doak et al. 2007). The association between EFNs and phenolic glycosides is addressed in greater detail elsewhere (Young et al. 2010). In this study, approximately 9 days of mining damage accumulated before phenolic glycoside induction was observed. The newly-unfurled leaves may have been too young to mount an induced response. Alternatively, a threshold amount of damage may be required before aspen leaves respond by up-regulating phenolic glycoside expression (Underwood 2000). At the onset of induction in our study, approximately 5% of the total leaf epidermis was mined. While low, this level of damage falls within the range of damage levels documented to trigger induced resistance among species (Karban and Baldwin 1997). Aside from the induction response, we observed considerable ontogenetic variation in the phenolic glycoside content of leaves. This also has been observed in previous studies (Lindroth et al. 1987; Osier et al. 2000). Foliar concentrations of phenolic glycosides varied more than 2-fold during the first six days of sampling and prior to the herbivore-induced response (Fig 4a). It is possible that the generally high concentrations of phenolic glycosides observed during the first four sampling days were induced by damage caused by eggs or newly hatched leaf miner larvae prior to insecticide application. However, we find this unlikely because the phenolic glycoside concentration subsequently declined between days 4 and 6 on both insecticide-sprayed and naturally-mined trees. More likely, the increase in concentration between days 1 and 4 reflected up-regulation of phenolic compounds during early leaf development, and the subsequent decrease in the concentration of phenolic glycosides after sampling day 4 reflected dilution of phenolic glycosides as the leaf gained mass (Jones and Hartley 1999). Regardless of the mechanism, high constitutive concentrations of phenolic glycosides in these newly-developing leaves, albeit short-lived, could present a challenge to some early spring herbivores. Induced defenses can be costly to deploy (Agrawal et al. 1999). For aspen, detection of a cost of allelochemical expression depends upon the environmental conditions (Stevens et al. 2007). However, tradeoffs between aspen allelochemical expression and growth have been reported by several studies (Hwang and Lindroth 1997; Osier and Lindroth 2006; Stevens et al. 2007) suggesting that the production of phenolic glycosides may be costly. Aspen ramets naturally mined by P. populiella grew more slowly than those treated with insecticide to reduce mining damage (Wagner et al. 2008). In addition to the direct costs of herbivory that stem from decreased photosynthesis and early leaf abscission in mined leaves (Wagner et al. 2008), the cost of induction of phenolic glycosides in response to P. populiella mining damage may have contributed to the slow growth of naturally-mined ramets. In summary, our results demonstrate that P. populiella mining damage to a small fraction (<10%) of the epidermis of aspen leaves induced the production of phenolic glycosides. The induction response did not occur immediately upon initiation of mining, suggesting that a threshold amount of damage was necessary to trigger the induction response. The onset of induction occurred while many of the leaf miners were still feeding; hence, induction of phenolic glycosides may increase aspen resistance to P. populiella herbivory. Higher constitutive and induced concentrations of phenolic glycosides on leaves with EFNs, relative to those without EFNs, may contribute to previouslyreported lower mining damage on leaves bearing EFNs (Doak et al. 2007). Acknowledgment We thank Shandra Miller and Sara Young for assistance with data collection. Colin McGill provided technical assistance and advice with the HPLC. We also thank Diana Wolf and Julie McIntyre for valuable insight and input. This research was funded by NSF DEB to DW and PD. B. Young was partially supported by TASK (Teaching Alaskans, Sharing Knowledge), an NSF supported GK-12 Program. References AGRAWAL, A. A., STRAUSS, S. Y., and STOUT, M. J Costs of induced responses and tolerance to herbivory in male and female fitness components of wild radish. Evolution 53: BENTLEY, B. L Extrafloral nectaries and protection by pugnacious bodyguards. Annu. Rev. Ecol. Systemat. 8: BINGAMAN, B. R., and HART, E. 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