Exposure of Solidago altissima plants to volatile emissions of an insect antagonist (Eurosta solidaginis) deters subsequent herbivory

Abstract

Recent work indicates that plants respond to environmental odors. For example, some parasitic plants grow toward volatile cues from their host plants, and other plants have been shown to exhibit enhanced defense capability after exposure to volatile emissions from herbivore-damaged neighbors. Despite such intriguing discoveries, we currently know relatively little about the occurrence and significance of plant responses to olfactory cues in natural systems. Here we explore the possibility that some plants may respond to the odors of insect antagonists. We report that tall goldenrod (Solidago altissima) plants exposed to the putative sex attractant of a closely associated herbivore, the gall-inducing fly Eurosta solidaginis, exhibit enhanced defense responses and reduced susceptibility to insect feeding damage. In a field study, egg-laying E. solidaginis females discriminated against plants previously exposed to the sex-specific volatile emissions of males; furthermore, overall rates of herbivory were reduced on exposed plants. Consistent with these findings, laboratory assays documented reduced performance of the specialist herbivore Trirhabda virgata on plants exposed to male fly emissions (or crude extracts), as well as enhanced induction of the key defense hormone jasmonic acid in exposed plants after herbivory. These unexpected findings from a classic ecological study system provide evidence for a previously unexplored class of plant–insect interactions involving plant responses to insect-derived olfactory cues.

Olfactory cues and signals play important roles in a diverse array of ecological interactions among plants and insects. The best documented of these interactions include pheromonal signaling between conspecific insects and the use of plant-derived odors as foraging cues by insect pollinators, herbivores, and predators (1–7). Recent findings demonstrate that plants themselves can also perceive and respond to environmental odors; for example, parasitic plants in the genus Cuscuta use host-derived volatiles to direct their growth toward preferred host plants (8)—apparently using host plant odors as foraging cues in much the same way that insect herbivores do (5, 6). In other systems, plants appear to perceive the characteristic odors emitted from herbivore-damaged plant tissues as warning cues indicating the presence of potential attackers (9–11). Thus, the perception of volatile chemical cues appears to play important roles in plant ecology, although our understanding of the prevalence and significance of plant olfaction in natural systems remains quite limited.

Most previous work on plant responses to odor cues has addressed “priming” of induced defense responses. Plants frequently employ defenses that are induced by environmental stimuli, rather than being expressed constitutively, in environments where the occurrence of particular antagonists (e.g., herbivores, pathogens) is not entirely predictable—presumably to conserve resources and maintain the flexibility to precisely target defense responses against specific attackers (12). Still further economy may be achieved through priming responses, in which induced defenses are made ready for deployment in response to cues reliably associated with impending attack (10, 11, 13, 14). Priming of plant defenses by olfactory cues has been documented after exposure to herbivore-induced volatiles emitted either by neighboring plants (9, 10) or by other parts of the same plant (11, 14). The latter finding has given rise to speculation that such mechanisms might have initially evolved to overcome constraints on the within-plant transmission of wound signals imposed by the discontinuous architecture of plant vascular systems, with eavesdropping by neighboring plants arising secondarily (11).

Defense priming also has been reported in response to (nonolfactory) cues directly associated with the presence of herbivores, including insect footsteps on leaves and broken trichomes (15, 16). However, direct plant perception of insect-derived olfactory cues has not been reported previously, despite the many herbivores emitting volatile chemicals that function in intraspecific communication [e.g., sex, aggregation, alarm pheromones (1–3)] or defense [e.g., predator repellents (17)]. Furthermore, these compounds are frequently released in substantial quantities and in proximity to plants on which feeding will subsequently occur (18, 19); thus, they would appear to provide a class of potentially reliable olfactory cues that plants might profitably use for defense priming or induction.

In light of these observations, in the present study we explored whether and how the antiherbivore defenses of tall goldenrod, Solidago altissima L., are influenced by exposure to chemical emissions of its specialist herbivore Eurosta solidaginis (Fitch), a tephritid fruit fly (Fig. 1A) whose larvae induce ball-shaped galls in the stems of this plant species (Fig. 1B). The interactions of these two species have been studied for decades and suggest a tightly coevolved relationship (20, 21). Moreover, gall induction and feeding by E. solidaginis greatly reduce S. altissima growth and fitness (21), implying that individual plants may benefit from efficient deployment of effective defenses.

Fig. 1.

(A) Male E. solidaginis fly perching on its host plant, S. altissima, and releasing a volatile blend attractive to potential mates. (B) Developing E. solidaginis gall on stem of S. altissima. (Photo credits: Ian Grettenberger.)

We specifically explored whether the chemical emissions of male E. solidaginis might induce or prime antiherbivore defenses in S. altissima, as the ecology of this system suggests that these emissions may provide a salient cue reliably associated with impending attack. Male E. solidaginis begin to emerge before females (during mid-May in the northeastern US), and after emergence typically perch on the upper leaves of S. altissima ramets, often for hours at a time (20, 21), and emit copious amounts (mean ± SD, ∼70 ± 20 μg over 24 h) of a volatile blend that we hypothesize functions as a sex attractant. Through GC-MS analysis, the blend was found to be dominated by spiroacetals, whose biological significance as insect-derived volatiles remains largely undocumented but with known functions as pheromones, kairomones, or allomones in some systems (22). Females begin searching for oviposition sites immediately after mating and have been observed to oviposit into stems of the same goldenrod genet on which mating occurs, frequently within 30 min (20). Eggs typically hatch 5–8 d later (20, 21), and galls become visually apparent within 3 wk; thus, it seems plausible that exposure to the volatile emissions of male flies might reliably predict subsequent oviposition and larval herbivory. Consequently, we undertook field and laboratory experiments designed to explore the ecological role of the E. solidaginis emission, in particular its potential influence on the defense responses of S. altissima.

Results

Volatile Emissions from Male E. solidaginis Flies Are Attractive to Female Flies.

In initial olfactometry assays testing the attractiveness of male and female flies to the odors of the opposite sex, male flies (which, as described above, more or less continuously release copious amounts of a volatile blend dominated by spiroacetals) were significantly attractive to females; in contrast, female flies were not significantly more attractive to males than clean air (Table 1). Although these findings are not sufficient to formally characterize the male emission as a sex pheromone, they are consistent with our hypothesis that the emission plays a role in mate attraction.

Table 1.

Olfactometry assays testing the response of E. solidaginis flies to the volatile emissions of members of the opposite sex

	Choice				Statistics
Respondents	Females	Males	Blank	No decision	χ2	P
Females	*	29	10	11	9.26	0.0023
Males	24	*	18	13	0.86	0.35

*Not tested.

Plant Exposure to Volatile-Emitting E. solidaginis Males Reduces Subsequent Rates of Ovipuncture by E. solidaginis Females and Overall Levels of Herbivory in the Field.

To test the hypothesis that exposure to the male E. solidaginis emission enhances S. altissima defense responses, we conducted a large-scale field study comparing the oviposition preferences of E. solidaginis females for plants with previous exposure to male E. solidaginis or various controls. We also assessed overall rates of herbivory on these plants. Randomly selected S. altissima plants on which male E. solidaginis were caged for 3 d subsequently demonstrated significantly reduced rates of ovipuncture by E. solidaginis females [i.e., insertion of the ovipositor into plant tissues as assessed by characteristic patterns of tissue damage (20, 21, 23, 24)] compared with control plants similarly exposed to female E. solidaginis, common houseflies, or empty cages (Fig. 2A; χ² test of independence, χ²₃ = 12.5, P = 0.006). The female E. solidaginis and housefly controls were designed to account for the effects of fly-associated cues other than the male volatile emission. The proportion of ovipunctured plants observed in the control treatments was 3.7–4.8 times higher than that of plants exposed to E. solidaginis males, whereas no significant differences were observed among the three control treatments [post hoc relative risk analysis assessing the risk of each control group receiving an ovipuncture relative to the male E. solidaginis treatment, with associated 95% confidence intervals: male vs. female: 3.80 (1.35–10.65); male vs. housefly: 4.75 (1.73–13.03); male vs. empty net: 3.68 (1.31–10.39)]. Thus, egg-laying E. solidaginis females appear to actively discriminate against plants previously exposed to male flies. Female E. solidaginis are known to assess host plant quality before oviposition by tasting bud tissue with chemoreceptors located on their feet and mouthparts, and often reject more resistant plant genotypes (21, 24, 25). Because E. solidaginis females so rarely oviposited on exposed plants in our field study, our data do not allow us to assess the effects of exposure on gall development after oviposition. However, levels of foliar damage by chewing and leaf-mining insects were dramatically reduced on plants previously exposed to E. solidaginis males compared with each of the control groups (Fig. 2B; negative binomial regression: male vs. female: t_1,109 = 3.794, P = 0.0002; male vs. housefly: t_1,106 = 2.837, P = 0.005; male vs. empty cages: t_1,107 = 3.236, P = 0.002). These findings suggest that the diminished preference of E. solidaginis females for exposed plants may reflect changes in plant quality that deter other insects as well.

Fig. 2.

(A) Rates of ovipuncture by E. solidaginis females on S. altissima after exposure to the male emission compared with the three control groups. (B) Rates of herbivory (number of damaged leaves) on plants exposed to the male E. solidaginis emission compared with the three control groups (2 wk after exposure). Boxes represent the 25th–75th percentiles, the line within each box denotes the median, the error bars indicate the 10th and 90th percentiles, and the points represent outlying values.

S.altissima Plants Exposed to Male E. solidaginis Volatile Emissions Are Less Palatable to Specialist Herbivores.

We further explored the effects of E. solidaginis emissions on S. altissima defenses through controlled insect feeding assays conducted in the laboratory. Because the galling habit of E. solidaginis larvae limits their usefulness in such assays, we assessed the effects of volatile exposure on the performance of adults and larvae of another S. altissima specialist, the goldenrod leaf beetle Trirhabda virgata, a member of the herbivore community present at our field sites (Fig. 3A). Previous studies have documented a negative association between E. solidaginis and T. virgata in the field (26) and have shown that T. virgata feeding deters subsequent oviposition by E. solidaginis (27), suggesting potential overlap in the defenses deployed against these two herbivores.

Fig. 3.

Feeding by T. virgata on S. altissima. (A) T. virgata adult feeding on S. altissima foliage (Photo credit: Ian Grettenberger). (B) Amount of feeding by T. virgata adults on S. altissima plants exposed or not exposed to the emission released by adult male E. solidaginis. (C) Amount of feeding by T. virgata adults on S. altissima plants exposed to crude extracts of male E. solidaginis emission or solvent controls. (D) T. virgata larva feeding on S. altissima leaves. (E) Amount of feeding by T. virgata larvae on S. altissima plants exposed to solvent controls, adult male E. solidaginis, or commercially acquired western bean cutworm pheromone (ANOVA, F_2,23 = 7.8, P = 0.003). Data are shown untransformed, but statistical analyses were performed on log-transformed data.

Consistent with our observation of reduced herbivory on emission-exposed plants in the field, adult T. virgata consumed significantly less leaf tissue during 24 h of feeding on plants exposed to live male flies (over the preceding 24 h) compared with control plants (Fig. 3B; ANOVA, F_1,11 = 9.32, P = 0.012), indicating reduced palatability after exposure. To confirm that the reduced herbivory observed in our emission treatment was not explained by some other cue associated with presence of the flies, we repeated this experiment using biologically realistic doses of crude extracts of the E. solidaginis volatile emission. This design yielded results similar to those of the previous experiment; beetles feeding on emission-exposed plants ate significantly less plant tissue than those feeding on control plants (Fig. 3C; ANOVA, F_1,9 = 5.60, P = 0.046), confirming that prior exposure of plants to the volatile emission itself, in the absence of any other fly-derived cues, deterred beetle feeding. Consequently, we used live flies in subsequent experiments to mimic as closely as possible the exposure occurring in nature.

We next conducted a similar performance assay with T. virgata larvae (Fig. 3D) rather than adults. The larvae are active and feeding during the period when E. solidaginis females mate and oviposit, and both insect species appear to prefer the most vigorously growing S. altissima ramets (24). Like adult beetles, larvae consumed far less leaf tissue on plants with previous exposure to fly volatiles than on unexposed controls (Fig. 3E).

S. altissima Plants Exposed to Male E. solidaginis Volatile Emissions Exhibit More Vigorous Defense Responses.

To determine whether the lower levels of herbivory observed on volatile-exposed plants were mediated by enhanced plant defense responses, we assayed levels of the key defense-related phytohormone jasmonic acid (JA) in exposed and unexposed plants before and after (similar amounts of) feeding by adult T. virgata. JA is a key defense phytohormone that regulates the expression of genes involved in defenses against herbivores, and up-regulation of JA is frequently assayed as an indicator of defense induction (10, 28). Before herbivory, volatile-exposed and control plants had similar JA levels, but at 6 h after the initiation of feeding, exposed plants exhibited significantly higher concentrations of JA than controls (Fig. 4) (repeated-measures ANOVA: time factor, F_1,23 = 80.0, P < 0.00001; treatment × time interaction, F_1,23 = 7.4, P = 0.021), indicating that exposure to the male E. solidaginis emission enhanced JA-mediated defense responses to subsequent herbivory.

Fig. 4.

Levels of JA in S. altissima leaves after exposure to the emission of male E. solidaginis and herbivory by adult T. virgata beetles. After 6 h of damage, JA levels were significantly higher when beetles fed on plants previously exposed to the emission.

Volatile Emissions from Noncoevolved Insect Species Do Not Similarly Influence Plant Defenses.

As a further test of the hypothesis that the enhanced defense responses of S. altissima represent a specific reaction to the emission of its coevolved herbivore, we assessed (as a separate treatment in the larval feeding experiment) the effect of exposure to the sex pheromone of an unassociated herbivore, the western bean cutworm Striacosta albicosta (Smith), an agricultural pest of maize (Zea mays L.) and beans (Phaseolus vulgaris L.) (29, 30). We found no differences in the damage inflicted by T. virgata larvae on plants exposed to western bean cutworm pheromone compared with unexposed control plants (Fig. 3E). Moreover, higher damage levels were observed both in plants treated with the western bean cutworm pheromone and in control plants compared with plants exposed to E. solidaginis emission. This result suggests that the response of S. altissima to insect-derived volatiles is not broadly tuned, but more likely represents a specific response to a coevolved antagonist.

Finally, to account for the possibility that our results might be explained by nonadaptive “by-product” effects of plant exposure to the E. solidaginis emission, we conducted a parallel set of experiments in a different plant species, maize (Z. mays L.), that does not have an association with this fly. We found that the generalist caterpillar Heliothis virescens fed similarly on unexposed (control) plants and plants exposed to volatile emissions from either E. solidaginis or western bean cutworm (unexposed: 165.01 ± 37.9 cm² leaf tissue removed; E. solidaginis emission exposed: 195 ± 59 cm²; cutworm pheromone exposed: 170 ± 27 cm²; ANOVA, F_2,23 = 0.14, P = 0.87). These results indicate that volatile cues from these insect species did not induce a defensive response effective against this generalist herbivore species.

Discussion

Taken together, our results demonstrate that exposure to the volatile emissions of male E. solidaginis flies enhances the defense responses of S. altissima plants to subsequent herbivory and reduces their attractiveness to ovipositing female E. solidaginis (Figs. 2–4). Our field study revealed a 73–79% reduction in the frequency of ovipuncture by E. solidaginis females (Fig. 2A), indicating a reduced risk of feeding damage by E. solidaginis larvae, which can greatly reduce plant fitness (21). After oviposition, E. solidaginis eggs typically hatch within 5–8 d (20, 21), and previous work has documented significant mortality of early-stage E. solidaginis larvae, owing at least in part to plant defenses (24). We also observed a significant reduction in overall rates of foliar herbivory on emission-exposed plants in the field (Fig. 2B). Consistent with these results, herbivory by T. virgata adults and larvae was reduced by 41–62% relative to unexposed controls in laboratory assays (Fig. 3), a difference that might be expected to influence the significant ecological impacts of Trirhabda herbivory on Solidago, which include reductions in both aboveground and belowground biomass (31). This diminished herbivory may be explained by the priming of induced defense responses, as indicated by our finding of stronger JA induction in exposed plants (Fig. 4). Finally, the observation of no similar effects in plants exposed to the pheromones of unassociated herbivores suggests that the patterns reported here reflect adaptive responses of S. altissima to the volatile emissions of its specialist herbivore E. solidaginis.

These findings thus provide evidence for a unique class of plant-insect interactions mediated by plant perception of insect-derived olfactory cues. As noted above, other recent work has clearly demonstrated that plants can respond to environmental odors (8–11), for example, by priming induced defenses after exposure to the herbivore-induced volatile emissions of their neighbors (9–11). Nevertheless, circumspection is clearly warranted in drawing broad conclusions from the current results. Potential alternative interpretations of our findings include (i) the possibility that the fly emission might effect a biochemical manipulation of the host plant by the fly and (ii) the possibility of insect–insect interactions mediated by retention of some components of the E. solidaginis volatile blend on plant tissues. The former hypothesis is undercut by the observation that ovipositing E. solidaginis females discriminated against emission-exposed plants in the field, strongly suggesting that the quality of these plants as hosts for the fly was compromised rather than enhanced. The latter hypothesis is contradicted by our observation of strongly enhanced JA responses to herbivory in emission-exposed plants (Fig. 4). Furthermore, we observed no effect on feeding by a generalist insect herbivore (H. virescens) on maize plants exposed to the E. solidaginis emission—suggesting that the emission itself does not have general deterrence effects—but did see effects for multiple herbivores on emission-exposed S. altissima plants, including not only female E. solidaginis and T. virgata adults and larvae, but also the other herbivore species responsible for the bulk of foliar herbivory observed in our field studies (largely lepidopteran larvae and leaf miners). Moreover, there is no obvious adaptive rationale for these diverse insects to respond to a fly-associated cue in the absence of volatile-mediated changes in host plant quality. Competing herbivores might exhibit evolved strategies for avoiding competition with E. solidaginis, but this is difficult to reconcile with the observation that oviposition by the fly itself is reduced on plants exposed to the fly emission. Thus, we believe that the enhancement of plant defense responses after exposure to the emission of E. solidaginis males is by far the most parsimonious and compelling explanation for our findings.

Moreover, there are reasons to suspect that plant detection of herbivore-derived volatile emissions may occur in other systems as well. As discussed above, previous studies have described plant responses to other classes of herbivore-associated cues encountered before the initiation of feeding (15, 16, 32). Furthermore, other work has demonstrated that plants can detect volatile cues from nearby damaged or undamaged plant tissues (9–11, 33). Likewise, the volatile emissions of insect herbivores might be expected to provide reliable information about impending herbivory, particularly if such emissions are released in proximity to potential host plants. In addition, we might expect adaptive plant responses to insect-derived olfactory cues to emerge most readily in the context of tightly coevolved relationships, such as that between S. altissima and E. solidaginis, or in other systems where a particular herbivore accounts for a large portion of the feeding damage inflicted on a given plant species—as occurs, for example, with some bark beetles and aphids (34, 35). The sex attractants of specialist herbivores would seem to be particularly likely candidates to serve as cues for the priming or induction of plant defense responses, given that courtship and mating frequently occur on or near prospective host plants (18, 36), as would the aggregation pheromones that some specialist herbivores use to recruit conspecifics (37). Although plants also might prime or induce defenses in response to volatile emissions from commonly encountered generalist herbivores, we hypothesize that such responses evolve less frequently, because generalist insects tend to be more variable in host plant selection for oviposition and feeding (18, 36). In the present study, we found no evidence for priming of maize defenses in response to the pheromone of the generalist caterpillar S. albicosta.

In conclusion, our findings reveal an unexpected and apparently adaptive feature of the tightly coevolved relationship between S. altissima and E. solidaginis that likely influences the outcome of interactions between them, as well as broader community dynamics. If plant response to insect-derived olfactory cues is shown to be a more general phenomenon, it may have widespread implications for ecology, including not only plant defense strategies, but also the ecology and evolution of insect signaling systems in environments where plants potentially act as illegitimate receivers. Finally, we speculate that this class of volatile-mediated plant–insect interactions also might have applied relevance for the management of agricultural and forest ecosystems, perhaps via general or targeted priming of plant defenses against herbivorous insect pests.

Experimental Procedures

Plant and Insect Material.

Goldenrod (S. altissima) plants of the same genetic background (clone 110) were grown from rhizomes in insect-free, climate-controlled growth chambers (16-h light/8-h dark, 22 °C/21 °C, 65% relative humidity). Rhizomes were collected from an old field near State College, PA and stored at 4 °C until use. S. altissima ramets used in the experiments were ∼35 cm tall (8 wk old). Corn (Z. mays cv. Delprim) was grown from seed in insect-free, climate-controlled growth chambers (16-h light/8-h dark, 25 °C/25 °C, 65% relative humidity) until plants were in the three-leaf stage. Male E. solidaginis flies were reared from overwintering galls collected from S. altissima in the vicinity of State College, PA during the winter of 2010–2011. T. virgata larvae were also collected from S. altissima near State College in early May 2011 and reared at room temperature on growth chamber-grown goldenrod until their use in feeding trials. Adult female T. virgata were collected from the same field site between July and September 2011 and were also kept at room temperature and fed growth chamber-grown goldenrod. Tobacco budworm (H. virescens) larvae were reared from eggs in an incubator (16-h light/8-h dark, 22 °C/20 °C, 65% relative humidity) on an artificial casein-based diet.

Olfactometry Assays.

Attractiveness of the odor of male and female flies was assessed using a Y-tube olfactometer with a 1.5-cm interior diameter, 18-cm-long base tube, and 16-cm-long arms. The olfactometer was a closed system, and airflow meters regulated the movement of purified air, which was pushed through Teflon tubing past twin humidifiers, then through the two glass sample chambers containing a single male or female fly, and down to the arms of the Y-tube and simultaneously pulled through the base tube. Airflow through the apparatus was 0.6 L·min⁻¹. Individual flies were introduced into the base of the Y-tube and responded to odors by walking upwind into one of the arms. A fly recorded a response when it walked 6 cm up an arm, crossed a “decision line,” and remained beyond that line for at least 20 s Flies not reaching a decision line within 5 min were recorded as “no response.” Every five trials, the male or female fly was changed, the tube was rinsed with acetone and hexane, and treatments were switched between the arms of the Y-tube.

Field Ovipuncture and Herbivore Damage Assessment.

The effects of plant exposure to male E. solidaginis emissions on the subsequent oviposition preferences of E. solidaginis females (and overall rates of herbivory) were explored through a field study conducted in a naturally occurring S. altissima population near State College, PA. In early May 2012, undamaged plants of approximately equal height (mean, 31.7 ± 5.8 cm) and spaced ∼4-m apart were selected. Each plant was randomly assigned to one of four groups and then, based on its group membership, was subjected either to an emission exposure treatment or to one of three controls. The upper portion of each plant was contained inside a mesh net. For treatment plants, a single, newly emerged live male E. solidaginis fly was placed within this net and left for 72 h. For plants in the first control group, one newly emerged live female E. solidaginis fly was similarly confined. For plants in the second control group, one common housefly (Musca domestica) was similarly confined. For the third control group, the mesh nets were left empty.

After the 72-h exposure period, the nets were removed from all plants. Plants were then inspected weekly for 4 wk for herbivore feeding damage, as well as ovipuncture scars created by (naturally occurring) E. solidaginis females from insertion of the ovipositor into the terminal bud, which leaves characteristic and readily observable wounds (20, 21, 23, 24, 27). Herbivore leaf damage was recorded as the number of damaged leaves per plant and included both chewing damage and leaf mines. Data presented are from the survey conducted 2 wk after the exposure treatment and represent the total herbivore leaf damage accumulated during this time. These data were analyzed by fitting negative binomial regression models to compare emission-exposed plants with each of the control groups (38). Negative binomial regression models were used in place of poisson regression models because the data did not meet the assumption of equidispersion (38). The models were fit by regressing the number of damaged leaves per plant on treatment group. The first ovipuncture scars on S. altissima were recorded on May 17, 2012, and new scars continued to appear until May 31, 2012. The ovipuncture data were analyzed using a χ² test of independence and comparing the relative risks (38).

Emission Collection.

The volatile emission emitted by male E. solidaginis is not characterized in prior literature. The amount of volatile compounds released by males that we report (mean, ∼70 ± 20 μg) is based on 24-h headspace aerations of eight males. The male E. solidaginis volatile blend is dominated by three spiroacetals: (5S,7S)-7-methyl-1,6-dioxasopiro[4.5]decane, (E)-2-methyl-1,6-dioxaspiro[4.5]decane, and (Z)-2-methyl-1,6-dioxaspiro[4.5]decane. These compounds account for ∼95% of the total emission. Identification of these compounds in the emission was achieved via coupled GC-MS in collaboration with Hans Alborn (US Department of Agriculture, Gainesville, FL) and Wittko Francke (University of Hamburg, Hamburg, Germany). More work is needed to fully describe, characterize, and verify the role of these compounds for E. solidaginis and its interaction with S. altissima.

To collect emission from male E. solidaginis flies, two male flies were aerated for 24 h inside a small ground-glass sealed chamber. Filtered air was pushed through Teflon tubing into the chamber and over the emission-producing flies at 0.6 L⋅min⁻¹. Air was then pulled out of the chamber with a vacuum at 0.5 L⋅min⁻¹ and over a filter containing 45 mg of SuperQ (Alltech Associates). The filters were eluted using 150 μL of dichloromethane, and individual samples were pooled to ensure a uniform concentration of emission. The concentration of the emission extract was quantified using a gas chromatograph with a flame ionization detector.

Laboratory Emission Exposure Treatments.

Plants were exposed to the E. solidaginis emission through exposure to either live male flies or crude extract of the emission on rubber septa. Plants exposed to live flies were enclosed inside individual glass chambers together with two recently emerged male flies for 24 h; control plants were enclosed in individual glass chambers without flies. Although the time budgets of females have been well characterized (39), male flies have not received as much attention, perhaps because their emission has not been reported previously. Female flies spend ∼65% of their time resting and walking on host plants (23). It seems likely that male flies spend even more of their time perching on plants while waiting for females (20, 23); thus, our exposure levels likely are not excessively high compared with what may occur in natural settings, particularly considering that E. solidaginis can be very abundant in some fields. Each 9-L glass chamber rested on a two-piece aluminum base, which was supported by the rim of the plant’s pot. The aluminum base had a hole in the center to allow the plant stem to pass through, and each stem was wrapped in cotton where it passed through the hole to plug the gap between stem and base. To avoid the development of condensation and an unrealistic concentration of emission within the chambers, air was pulled through the chambers at 0.25 L⋅min⁻¹ with a vacuum attached to a manifold that split airflow equally among the chambers.

Plants exposed to crude emission extract were placed inside individual 9-L glass chambers, and two rubber septa, each containing a 12-h male equivalent of E. solidaginis emission (37.5 μL of crude extract) were added to the chamber. Two more rubber septa, each containing a 12-h male equivalent of emission, were added to the chamber at 12 h after the first dose. The doses were split in this way to better approximate the emission exposure experienced by plants exposed to live flies and to avoid having an initial strong concentration exposure be followed by a period of weak exposure. Control plants were enclosed in individual glass chambers containing either no flies or rubber septa dosed with a dichloromethane solvent control.

Plants exposed to western bean cutworm (S. albicosta) pheromone were placed in glass chambers each containing a synthetic pheromone lure (Suterra) comprising one rubber septum containing sufficient pheromone for 6 wk of field use (2 mg of a three-component pheromone). Given this large amount of western bean cutworm raw material, our treatment likely “overexposed” plants to this cue, likely increasing the probability that S. altissima would have responded to the pheromone had they the capacity to do so.

Feeding Assays.

For feeding assays with adult beetles, female T. virgata were starved at room temperature for 24 h. After the 24-h emission exposure period, three beetles were placed into each S. altissima-containing chamber and allowed to feed on the plant for 24 h. After this 24-h period, the beetles were removed, the plants were harvested, and the insect damage on each plant was quantified. To quantify damage, leaves were scanned, and the resulting images were imported into SigmaScan (Systat Software) to calculate the areas of leaf tissue eaten and leaf tissue remaining. For the T. virgata larval feeding assays, five larvae (each <1 cm long) were added to each S. altissima plant and allowed to feed for 10 h. After 10 h, the larvae were removed, and the feeding damage was quantified as described above. For the feeding assays with Z. mays, third-instar H. virescens were starved for 24 h at room temperature. Two larvae were placed into each Z. mays-containing chamber and allowed to feed for 24 h. After 24 h, the larvae were removed, and insect feeding damage was quantified using the previously described methods.

Quantification of JA.

Individual S. altissima leaves selected for JA analyses were of similar size and exhibited similar levels of feeding damage. A previously described protocol was used to extract and detect JA in S. altissima plants (40, 41). In brief, carboxylic acids were derivatized to methyl esters, which were isolated using vapor-phase extraction and analyzed by coupled GC-MS with isobutane chemical ionization using selected-ion monitoring. Amounts of JA were quantified using 100 ng of the internal standard dihydro-JA, which was derived from methyl dihydrojamonate (Bedoukian Research) via alkaline hydrolysis. To confirm the identity of methyl jasmonate recovered from the samples, extracts were analyzed by GC-MS with electron ionization, with retention times and spectra compared with those of the pure compound. Further samples were also processed in the absence of the derivatizing agent to confirm that endogenous methyl jasmonate was minimal.