Insects Betray Themselves in Nature to Predators by Rapid Isomerization of Green Leaf Volatiles

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Science  27 Aug 2010:
Vol. 329, Issue 5995, pp. 1075-1078
DOI: 10.1126/science.1191634


Plants emit green leaf volatiles (GLVs) in response to herbivore damage, thereby attracting predators of the herbivores as part of an indirect defense. The GLV component of this indirect defense was thought to be a general wound signal lacking herbivore-specific information. We found that Manduca sexta–infested Nicotiana attenuata attract the generalist hemipteran predator Geocoris spp. as the result of an herbivore-induced decrease in the (Z)/(E) ratio of released GLVs, and that these changes in the volatile bouquet triple the foraging efficiency of predators in nature. These (E)-isomers are produced from plant-derived (Z)-isomers but are converted by a heat-labile constituent of herbivore oral secretions. Hence, attacking herbivores initiate the release of an indirect defense a full day before the attacked plants manufacture their own defensive compounds.

Plants defend themselves against herbivore attack by producing chemical and physical defenses that decrease herbivore performance, but they also release distinct volatile bouquets when attacked. These herbivore-induced plant volatiles (HIPVs) can function as indirect defenses by attracting carnivores. Although the attraction of natural enemies to HIPVs has frequently been observed in laboratory studies (1), few studies have demonstrated that HIPVs attract carnivores in nature (13).

Elicitors in herbivore oral secretions introduced into plant wounds during feeding (46), as well as the rhythm of caterpillar feeding (7), provide the plant with the information required to activate herbivore-specific defenses, including the release of specific volatiles. The composition of the HIPV blend can differ among plant and herbivore species, abiotic conditions, and over time (2, 810). HIPVs have several different metabolic origins, of which the isoprene-derived terpenoids and fatty acid–derived green leaf volatiles (GLVs) are the best-studied classes. Terpenoids are released with a delay from the whole plant, not just attacked leaves, after a few hours or with the plant’s next photosynthetic phase (i.e., often a day after the start of herbivore attack) (1113). Because of their delayed, systemic release, the terpenoids likely function in the long-distance attraction of carnivores. GLVs—which consist of six-carbon aldehydes, alcohols, and their esters—are, unlike terpenoids, immediately and likely passively released from wounded leaves (11, 14). Consequently, GLVs likely provide rapid, but nonspecific information about the exact location of a feeding herbivore. GLVs were shown to play a role in host-location of predators and parasitic wasps (1517). Although changes in constitutive GLV ratios can alter the ability of herbivores to locate their host (18), the degree to which natural enemies use induced GLVs to find plants with prey remains unclear (19, 20).

The GLV blend of mechanically wounded Nicotiana attenuata plants contains large amounts of (Z)-GLVs and proportionally smaller amounts of (E)-GLVs (Fig. 1). These different isomers result from rearrangements of the double bonds. However, plants that had been attacked for 24 hours by 1, 5, or 10 Manduca sexta neonates, a specialist herbivore of this native tobacco, released (Z)- and (E)-isomers in nearly equal amounts (Fig. 1A) (21). We compared changes in the (Z)/(E) ratio of GLVs released from mechanically wounded leaves of which the wounds were treated with water (w + w) or M. sexta oral secretions (OS) (w + OS; fig. S1) (22). We found that w + w–treated plants emitted high levels of (Z)-GLVs and low levels of (E)-GLVs, whereas treating wounds with M. sexta OS decreased emissions of (Z)-GLVs and increased those of (E)-GLVs, resulting in a distinct change in the (Z)/(E) ratio (Fig. 1, B and C, and table S1). To determine whether the OS-elicited (Z)/(E) shift is a transient response, we monitored the GLV bouquet for several hours after a single elicitation. Although the GLV burst occurred immediately after a single wounding and vanished after a few hours (fig. S2A), the OS-elicited changes in the (Z)/(E) ratio persisted for the duration of the GLV burst (fig. S2B).

Fig. 1

Herbivory and the application of Manduca sexta’s oral secretions to the wounds of wild-type Nicotiana attenuata plants lead to a marked change in GLV emissions. (A) Mean (Z)/(E) ratios, with 95% confidence limits, of N. attenuata plants attacked by 1, 5, or 10 M. sexta neonates (22); CP, caterpillar. (B) Wounded plants release GLVs with a high (Z)/(E) ratio, whereas OS-elicited plants emit a GLV bouquet with a much lower ratio (22). Bars represent the average ratio (n = 5) and their 95% confidence limits. (C) Mean (+ SE) release of GLVs in w + w–treated and w + OS–treated plants in the first 20 min after elicitation (n = 5). Values are indicated in nanograms per gram of fresh mass per 20-min period. LOX2, lipoxygenase 2; HPL, hydroperoxide lyase; ADH, alcohol dehydrogenase; AAT, alcohol acetyltransferase; 13-HPs, 13-hydroperoxides.

Saliva or OS from herbivores can elicit specific responses in a plant, and two main classes of endogenously produced elicitors have been characterized in lepidopteran larvae: fatty acid–amino acid conjugates (FACs) and enzymes such as β-glucosidase and glucose oxidase (6). In addition, OS contains elicitors of plant origin, such as inceptins, which are processed in the oral cavity and reintroduced into the plant during feeding (6). FACs are central elicitors in M. sexta’s OS. In N. attenuata they amplify the wound-induced bursts of the phytohormones, jasmonic acid (JA), and ethylene, as well as the release of the sesquiterpene (E)-α-bergamotene (5, 6). We trapped volatiles from plants that had been wounded and treated with the two most abundant FACs in M. sexta’s OS: N-linolenoyl-glutamate (18:3-Glu) or N-linolenoyl-glutamine (18:3-Gln) (5). Neither of the two elicited the OS-induced change in the (Z)/(E) ratio (fig. S3D). Because the alkaline pH of M. sexta OS (pH = 9) elicits the release of methanol during herbivory by activating pectin methyl esterases in leaves (23), we tested the influence of pH on the GLV emissions. In theory, alkaline conditions could convert the relatively unstable (Z)-hex-3-enal into its more stable (E)-isomer, (E)-hex-2-enal. However, an alkaline buffer (0.1 M Tris, pH 9, in 0.02% Tween-20) equal to the pH of M. sexta OS sufficient to elicit the methanol release did not change the (Z)/(E) ratio relative to w + w–treated plants when applied to puncture wounds (fig. S3, A and B). FACs are relatively heat-stable compounds, and heating OS to 90°C for 10 min did not reduce the concentration of FACs in OS (fig. S3, E and F). However, heated OS no longer elicited the shift in the (Z)/(E) ratio (fig. S3C). Therefore, we conclude that FACs are not involved, but that a heat-labile elicitor of M. sexta’s OS directly converts (Z)- into (E)-GLVs or indirectly activates an isomerase in the plant. Additional tests revealed that the M. sexta OS-mediated (Z)/(E) shift is independent of the plant’s JA, salicylic acid, and ethylene-dependent defense signaling pathways (fig. S4 and tables S2 and S3) (22).

To determine whether the OS elicitor responsible for the (Z)/(E) shift functions directly as an isomerase, we added (Z)-hex-3-enal to M. sexta’s OS in an in vitro system and quantified the conversion to (E)-hex-2-enal. More than 50% of the added (Z)-hex-3-enal was converted, which did not occur when water or heated OS were used as the converting solution and only slightly with an alkaline buffer (Fig. 2). Additionally, we tested two OS-derived enzymes, glucose oxidase and β-glucosidase, both of which elicit specific responses in plants (6). However, these enzymes did not increase the release of (E)-hex-2-enal. Bovine serum albumin (BSA) has lipophilic properties and increased the isomerization of cis- to trans-JA when added to plant cell cultures (24). However, BSA had no effect on the (Z) to (E) conversion of leaf aldehydes (fig. S5).

Fig. 2

Percentage conversion to (E)-hex-2-enal in vitro (n = 6; gray bars) and in vivo (n = 6; black bars) (Fig. 1) (22). Asterisks indicate significant differences from the control treatment (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001); univariate analysis of variance, F6,37 = 99.957, P ≤ 0.001, followed by a Tamhane post hoc test; oral secretions (OS), P ≤ 0.001; OS from artificial diet–fed caterpillars (OSAD), P ≤ 0.001; OS heated to 90°C for 10 min (OSboiled), P = 0.051; 1× buffer (pH 9), P ≤ 0.05; leaf juice (LJ), P = 1.000; LJ + OS, P ≤ 0.01; the control was 0.02% Tween-20 (22).

An (3Z):(2E)-enal isomerase that converts the (Z)-aldehyde to its (E) isomer has been proposed for several plant species; (3Z):(2E)-enal isomerase activity has been found in crude extracts of alfalfa and soybean (25, 26). However, we showed that a plant-derived isomerase was not responsible for the (Z) to (E) conversion of leaf aldehydes, but an unknown OS-derived, heat-unstable compound was necessary and sufficient for the conversion. The addition of crude leaf extract did not increase the conversion rate, and OS from caterpillars never fed on leaf material, but only on an artificial diet (22), converted (Z)-aldehydes to (E)-aldehydes as efficiently as OS from plant-fed caterpillars (Fig. 2). The observed (Z) to (E) conversion also appears to be species-specific, as the OS from two other generalist lepidopteran species (Spodoptera exigua and S. littoralis) that can feed on N. attenuata were not nearly as active as the OS from M. sexta larvae (fig. S6).

Whether this herbivory-specific change is relevant for tritrophic interactions hinges on whether carnivores can use these isomeric changes to augment their prey-hunting abilities. Insects are known to distinguish different isomeric forms of volatiles (27, 28), and two structural isomers elicit responses in distinct antennal olfactory receptor neurons (29). Geocoris spp. are generalist predators feeding on eggs and early larval instars of M. sexta. They use individual HIPVs, including terpenoids and GLVs, to locate their prey on herbivore-attacked plants (3, 16). We tested whether Geocoris could distinguish between (Z)- and (E)-GLVs, and used the changes in the (Z)/(E) ratio to discriminate between an herbivore-attacked and a mechanically wounded N. attenuata plant in nature.

We created two mixtures (A and B) in lanolin containing either (Z)- or (E)-GLVs (30). We also created GLV mixtures to mimic the GLV emissions of w + w–treated or w + OS–treated plants. To do so, we combined both isomeric alcohols and hexenyl esters (table S5, C and D) or all eight GLVs used for mix A and B (table S5, E and F) and added different amounts of each isomer to mimic the 1:1 (Z)/(E) ratio released by OS-elicited plants or the 9:1 ratio to mimic mechanically wounded plants.

We tested the attractiveness of these mixtures to Geocoris spp. in a native N. attenuata population in the Great Basin desert of southwest Utah by gluing three M. sexta eggs per plant to the underside of a lower stem leaf of 21 pairs of plants, as described (3). Plants were of the same size and developmental stage. Developmental stage did not influence the OS-induced (Z)/(E) shift (fig. S7) (22). On each day, two different mixes were tested by dipping a cotton swab into lanolin paste containing different GLV mixes and placing them immediately adjacent to the leaf with the M. sexta eggs.

Predated eggs were counted after 12 and 24 hours. We started with mixes that contained either (Z)- or (E)-GLVs (table S5, mixes A and B), and Geocoris spp. showed a clear preference for those scented with the (E)-GLVs. Whereas 8% of the (Z)-baited eggs were predated, 24% of the (E)-baited eggs were predated (fig. S9). These results demonstrated that Geocoris may distinguish between (Z)- and (E)-GLVs. We tested GLV mixes that consisted of both isomers (table S5C versus S5D; table S5E versus S5F) in different ratios to determine whether Geocoris detected changes in the (Z)/(E) ratio similar to those of OS-elicited plants. In both experiments, predation rates were higher on plants scented with equal amounts of (Z)- and (E)-GLVs [(Z)/(E) = 1:1] relative to those scented with a 9:1 ratio of (Z)/(E)-GLVs (Fig. 3) (22).

Fig. 3

Predation by Geocoris spp. in the field. (A) Geocoris spp. are generalist predators feeding on eggs and early-instar larvae of Manduca sexta. [Photo: M. Stitz] (B) Predation assays were performed in a native Nicotiana attenuata population in the Great Basin desert of southwest Utah. [Photo: D. Kessler] (C) Average egg predation per plant and day (±SE). Numbers in the plot denote total number of eggs predated per treatment. Treatment pairs with no predated egg were excluded before statistical analysis. Asterisks indicate significant differences between treatments (paired-sample t test, mix E versus F, t17 = 4.600, ***P ≤ 0.001; mix C versus D, t12 = 1.594, P = 0.137). The composition of the different mixes tested (mixes C, D, E, and F) are explained in table S5. n.s., not significant.

These results show that attack by the specialist herbivore M. sexta and the addition of their oral secretions to mechanical wounds elicits a rapid (Z)/(E) isomeric change in the GLV release of N. attenuata plants. This change, which lowers the (Z)/(E) ratio of the GLV blend, increases the predation rate of the generalist predator Geocoris spp., likely by betraying the location of the feeding caterpillar in a rapid, herbivore-specific, and spatially explicit manner. Why Manduca larvae would produce such an apparently maladaptive elicitor in their OS remains to be determined, but the larvae may benefit from the enhanced antimicrobial properties of a GLV blend enhanced in (E)-hex-2-enal (31).

Supporting Online Material

Materials and Methods

SOM Text

Figs. S1 to S13

Tables S1 to S6


References and Notes

  1. GLV esters were near or below the detection limit; thus, only the ratios for the aldehyde and the alcohol are shown.
  2. See supporting material on Science Online.
  3. We used the four most abundant GLVs of both isomers in N. attenuata: mix A, (Z)-hex-3-enal, (Z)-hex-3-en-1-ol, (Z)-hex-3-enyl acetate, and (Z)-hex-3-enyl butyrate; mix B, (E)-hex-2-enal, (E)-hex-2-en-1-ol, (E)-hex-2-enyl acetate, and (E)-hex-2-enyl butyrate (tables S5 and S6).
  4. Supported by the Max Planck Society. We thank M. Kant, R. Schuurink, E. Gaquerel, G. Bonaventure, and three anonymous reviewers for comments on the manuscript; S. Meldau for providing ir-sipk × ir-wipk seeds; D. Kessler and C. Diezel for help with the field experiments and enzyme assays; M. Kallenbach and A. Weinhold for analytical help; and Brigham Young University for the use of their Lytle Ranch Preserve field station.
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