Trichome-derived O-acyl sugars are a first meal for caterpillars that tags them for predation Alexander Weinhold and Ian Thomas Baldwin1 Max-Planck-Institute for Chemical Ecology, 07745 Jena, Germany Edited by Jerrold Meinwald, Cornell University, Ithaca, NY, and approved March 31, 2011 (received for review January 27, 2011) Plant glandular trichomes exude secondary metabolites with defen- survived and grew as well as larvae fed on previously washed and sive functions, but these epidermal protuberances are surprisingly AS-free wild-type (WT) sepals as well as sepals from plants the first meal of Lepidopteran herbivores on Nicotiana attenuata. transformed to silence nicotine production (Fig. S2 C and D). O-acyl sugars, the most abundant metabolite of glandular tri- Observations of neonate M. quinquemaculata and S. littoralis chomes, impart a distinct volatile profile to the body and frass of feeding behavior on native N. attenuata growing in natural habitats larvae that feed on them. The headspace composition of Manduca in Utah confirmed these laboratory observations and led us to sexta larvae is dominated by the branched chain aliphatic acids hy- conclude that trichomes and their exudates, rather than being drolyzed from ingested O-acyl sugars, which waxes and wanes rap- directly defensive, provide a sugary first meal for these Lepidop- idly with trichome ingestion. In native habitats a ground-hunting teran herbivores. Interestingly, trichome feeding has been repor- predator, the omnivorous ant Pogonomyrmex rugosus, but not ted for other Lepidopteran species and may be common (4, 17). the big-eyed bug Geocoris spp., use these volatile aliphatic acids to locate their prey. Ingestion of O-Acyl Sugars Dramatically Alters M. sexta Body Headspace. Although the ingestion of trichomes and their AS indirect defenses | dangerous body odors contents may not be directly deleterious for larvae, it dramatically alters larval body and frass volatile profiles. Trapping and GC-MS richomes, or leaf hairs, have long been considered a plant’s analysis of the headspace surrounding larvae that had fed on N. Tfirst line of defense. These epidermal protuberances are attenuata leaves and trichomes revealed that four branched chain known to function as physical and chemical barriers against aliphatic acids (BCAA), namely 2-methyl butanoic acid (I), 3- attacking herbivores and pathogens (1, 2), as well as protecting methyl butanoic acid (II), 3-methyl pentanoic acid (III), and plants against UV-B radiation, water loss, and heat stress (3). 4-methyl pentanoic acid (IV) were the most abundant con- Glandular trichomes can be found on ≈30% of all vascular plants. stituents (Fig. 1 B and D). These 4 BCAAs found in larval body These secretory hairs contribute to a plant’s resistance in nu- headspace are exactly those found from an alkaline hydrolysis of merous ways: as obstacles that thwart movement across plant N. attenuata AS (Fig. S3), and we hypothesized that BCAAs in the larval headspace originate from AS hydrolysis in the alkaline surfaces (4), as entrapment devices (5), and sources of volatile ECOLOGY and nonvolatile secondary metabolites (6, 7) or proteins (8) that midgets (18) of these larvae. AS are readily removed by washing poison or repel herbivores. leaves with water and do not occur in a species of Nicotiana that The native tobacco, Nicotiana attenuata, as it is common lacks trichomes, N. glauca (Fig. S4A). When fed on water-washed among Solanaceous plants, produces glandular trichomes on both N. attenuata or N. glauca leaves, larvae lose their characteristic adaxial and abaxial leaf surfaces (Fig. 1A and Fig. S1 A and B). body or frass volatile profile. Similarly, when fed on an artificial These trichomes contain as a minor constituent, nicotine (Fig. diet amended with AS from N. attenuata leaves, larvae produce S1C), a secondary metabolite known to inhibit growth even of a body headspace similar to that of larvae feeding on WT plants adapted larvae (6, 9). O-acyl sugars (AS), viscous liquids that (Fig. 2 A and B). The effect of AS ingestion on larval body consist of aliphatic acids of different chain lengths esterified to headspace rapidly waxes and wanes with diet; when switched sucrose, are the most abundant secondary metabolites in the from the AS-free N. glauca to N. attenuata diets, larvae rapidly glandular trichomes of Solanaceous plants (10, 11). They domi- emit the characteristic BCAAs, and these volatile acids disappear nate aqueous extracts of N. attenuata leaf surfaces and can be again when larvae are returned to AS-free N. glauca leaves (Fig. readily detected after Rhodamine B staining (Fig. 1 A and B) and 2C). We conclude that consuming AS from trichomes has a sim- by HPLC-ToFMS (Fig. S1). AS, particularly those esterified to ilar effect for larvae as the consumption of asparagus has for aliphatic acids with alkyl chains longer than eight carbons, are humans: It conveys a strongly dynamic and distinct olfactory effective defenses against psyllids (12), aphids (13), white flies, signature to bodies and excretions. Although only a potential and spider mites (14), likely by speeding the desiccation of social annoyance for humans, these emissions, which approach entrapped or anointed herbivores (15). AS from Datura wrightii, 5 μg of single BCAAs from larval bodies and 30 μg from frass although differing in their sugar moiety and aliphatic acid resi- over 2 h (Fig. 2), may have more serious consequences for dues, decrease growth of Manduca sexta larvae when applied to a caterpillar. artificial diets (16). The natural enemies of caterpillars are known to use fecal odors to locate their prey. For example, parasitic wasps can learn Results and Discussion to associate frass volatiles with prey location under laboratory Neonate Lepidopteran Larvae Consume Trichomes as a First Meal. Given the established defensive value of trichomes and their chemistry, we were surprised to observe that these trichomes and Author contributions: A.W. and I.T.B. designed research, performed research, analyzed their exudates were the first feeding choices of both generalist data, and wrote the paper. (Spodoptera littoralis and S. exigua) and specialist (M. sexta) Lep- The authors declare no conflict of interest. idopteran herbivores on N. attenuata (Fig. 1C, Fig. S2A, and Movie This article is a PNAS Direct Submission. S1). More than 70% of all larvae chose to feed on trichomes for Freely available online through the PNAS open access option. their first meal after hatching. Moreover, larvae did not suffer 1To whom correspondence should be addressed. E-mail: [email protected]. from this choice. When placed on N. attenuata sepals, which are This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. entirely covered in glandular trichomes, larvae feeding on sepals 1073/pnas.1101306108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1101306108 PNAS | May 10, 2011 | vol. 108 | no. 19 | 7855–7859 Downloaded by guest on September 28, 2021 N. attenuata leaf surface chemicals Acyl sugar and aliphatic acid N. attenuata artificial diet A B residue structures A Acyl sugars washed 0.6 CTRL CTRL R = I 6.0 +AS 2-methyl butanoic acid 0.4 R = II g/2h] 3.0 g/2h] AS-2 [ [ 3-methyl butanoic acid 0.2 Intensity Body headspace Body headspace R = III n.d. 3-methyl pentanoic acid n.d. n.d. n.d. Nicotine 0 0 I II III IV I II III IV R =IV AS-3 98765432 10 N. attenuata Time [min] 4-methyl pentanoic acid B washed N. glauca CTRL C Herbivore`s feeding choice D M. sexta body volatile profile N. attenuata Trichome III 30.0 30.0 Lamina Super Q - trap No Choice g/2h] g/2h] [ ** m/z 60 [ 80 air ** ** m/z 74 Frass headspace 10.0 Frass headspace 10.0 60 Charcoal trap IV caterpillar headspace n.d. n.d. n.d. 40 I n.d. n.d. n.d. 0 n.d. II 0 I II III IV I II III IV % feeding choice % feeding 20 BCAA BCAA 0 M. sexta S. littoralis S. exigua 25 27.5 30 32.5 35 Time [min] C 6.0 8000 fi Fig. 1. Trichomes and their exudates are commonly the rst meal of neo- g nate larvae of the three main Lepidopteran species that feed on N. 6000 3.0 attenuata.(A) HPLC-ToFMS analysis of an N. attenuata leaf surface wash. 4000 Inset shows a single trichome with the O-acyl sugars stained by Rhodamine B BCAA III 0 6030 120 180 240 300 360 dye. (B) Structures of the two classes of O-acyl sugars (ASII, III; ref. 10) found 2000 [min] in N. attenuata trichomes and the four most abundant BCAA substituents. 100 (C) Feeding choices of neonate larvae of the three most abundant Lepi- n.d. dopteran herbivores of N. attenuata in nature: M. sexta, S. littoralis, and 0 24h 24h 24h S. exigua. Choice is expressed as the mean (±SE) percent of feeding choices N. glauca N. attenuata N. glauca of larvae in four (M. sexta, n = 55), three (S. littoralis, n = 43), and one (S. exigua, n = 20) experiments. Asterisks indicate significant differences Fig. 2. Ingestion of O-acyl sugars determines the body and frass headspace 2 by χ analysis: *P < 0.05, **P < 0.01, ***P < 0.001. (D) Headspace of N. of M. sexta larvae. (A) Mean ± SE of four BCAAs trapped from M. sexta body attenuata-fed M. sexta larvae trapped on Super-Q traps (Inset) is dominated headspace from caterpillars feeding on plants with AS (CTRL) or without AS by the four BCAAs esterified to the sucrose moiety within AS-2 and AS-3. (washed) as well as artificial diets supplemented with N. attenuata AS (+AS) Dashed line represents ion-trace m/z 74 and the solid line m/z 60 (Fig.