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Home , Alucitoidea, Cecidosidae, Cimeliidae, Locastra, Locastra muscosalis, Pryeria sinica

J Chem Ecol (2010) 36:319–325 DOI 10.1007/s10886-010-9764-8

Fatty Acid-amino Acid Conjugates Diversification in Lepidopteran Caterpillars

Naoko Yoshinaga & Hans T. Alborn & Tomoaki Nakanishi & David M. Suckling & Ritsuo Nishida & James H. Tumlinson & Naoki Mori

Received: 30 September 2009 /Revised: 29 January 2010 /Accepted: 11 February 2010 /Published online: 27 February 2010 # Springer Science+Business Media, LLC 2010

Abstract Fatty acid amino acid conjugates (FACs) have the presence of FACs in lepidopteran outside these been found in noctuid as well as sphingid caterpillar oral families of agricultural interest is not well known. We con- secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)- ducted FAC screening of 29 lepidopteran species, and found L-glutamine] and its biochemical precursor, N-linolenoyl-L- them in 19 of these species. Thus, FACs are commonly glutamine, are known elicitors of induced volatile emissions synthesized through a broad range of lepidopteran cater- in corn plants. These induced volatiles, in turn, attract natural pillars. Since all FAC-containing species had N-linolenoyl-L- enemies of the caterpillars. In a previous study, we showed glutamine and/or N-linoleoyl-L-glutamine in common, and that N-linolenoyl-L-glutamine in larval Spodoptera litura the evolutionarily earliest species among them had only plays an important role in nitrogen assimilation which might these two FACs, these glutamine conjugates might be the be an explanation for caterpillars synthesizing FACs despite evolutionarily older FACs. Furthermore, some species had an increased risk of attracting natural enemies. However, glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylog- eny indicates that glutamic acid conjugates can be synthe- N. Yoshinaga : J. H. Tumlinson Center for Chemical Ecology, Department of Entomology, sized by relatively primitive species, while hydroxylation of The Pennsylvania State University, fatty acids is limited mostly to larger and more developed University Park, PA 16802, USA macrolepidopteran species.

H. T. Alborn . Center of Medical, Agricultural, and Veterinary Entomology, Keywords -produced elicitors Agricultural Research Service, Chemistry Unit, Lepidopteran phylogeny. Volicitin . Insect-plant interactions U. S. Department of Agriculture, 1600 Southwest 23rd Drive, Abbreviations Gainesville, FL 32611-0620, USA FACs fatty acid amino acid conjugates T. Nakanishi VOC volatile organic compounds Forestry and Fisheries Technology Center, ESI electrospray ionization Fruit Tree Research Institute, Tokushima Prefectural Agriculture, Katsuura-cho, Katsuura, Tokushima 773-4301, Japan

D. M. Suckling Introduction The New Zealand Institute for Plant and Food Research Limited, PB 4704, Christchurch, New Zealand It is well documented that constituents of insect oral : : N. Yoshinaga R. Nishida N. Mori (*) secretions can trigger plant responses, such as elicitation Division of Applied Life Sciences, of induced de novo synthesis and release of volatile organic Graduate School of Agriculture, Kyoto University, compounds (VOCs) (Turlings et al. 1990; Paré et al. 1998; Sakyo, Kyoto 606-8502, Japan Kessler and Baldwin 2001). The best known and most e-mail: [email protected] studied of insect-produced elicitors are the fatty acid amino 320 J Chem Ecol (2010) 36:319–325 acid conjugates (FACs), first identified from beet army- lepidopteran larvae utilize FACs. Furthermore, differences worm Spodoptera exigua larvae (Alborn et al. 1997). In a in physiology or metabolism associated with glutamine- recent study of a range of plant species, FACs and espe- FACs vs. glutamic acid-FACs are not understood. These cially volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] questions are not easy to answer, since only a limited showed the broadest effects on plant hormone levels number of insect species (mainly of agricultural interest) as well as induction of plant volatiles compared with have been investigated for FAC content so far. Consequent- caeliferin A16:0 and inceptin, two recently identified new ly, we decided to collect and analyze gut contents from an classes of insect-produced elicitors of inducible plant environmentally, taxonomically, and physiologically di- defenses (Schmelz et al. 2006, 2009; Alborn et al. 2007). verse group of lepidopteran larvae consisting of 29 species It also has been shown that application of volicitin to a from16 families. mechanically wounded site selectively and transcriptionally activated genes for indole-3-glycerol phosphate lyase (Igl) and specific sesquiterpene cyclase (stc1), and that this Materials and Methods activation also occurred systemically in undamaged leaves (Frey et al. 2000; Shen et al. 2000). However, previous Caterpillar Source Colonies of Spodoptera litura, Mythimna studies (Truitt and Paré 2004; Truitt et al. 2004) showed separate, and Hyphantria cunea were maintained succes- that volicitin did not on its own serve as a mobile sively in the laboratory, and Helicoverpa armigera was messenger for systemic VOCs emissions, but rather that a supplied by Dr. Kenji Fujisaki, Anadevidia peponis by volicitin binding protein-ligand interaction may initiate Dr. Tetsu Ando, Samia cynthia ricini by Dr. Masatoshi plant defenses in response to herbivory. Ichida, and Agrius convolvuli by Dr. Masami Shimoda. Since glutamine-based FAC components initially were Commercially available Bombyx mori were purchased from identified in oral secretions from S. exigua larvae, several Mukin Yosan System Institute (Kyoto, Japan), Agrotis other noctuid caterpillars have been reported to have the ipsilon eggs were purchased from Benzon Research Inc. same glutamine-based FACs (Pohnert et al. 1999; Mori et (PA, USA), and Manduca sexta from North Carolina al. 2001, 2003; De Moraes and Mescher 2004). In addition, University, NC, USA. Another 18 wild species were oral secretions from some noctuid larvae such as S. collected in Kyoto, Osaka, Mie, and Tokushima prefectures littoralis also contain traces of glutamic acid-based FACs. in Japan. Malacosoma americanum was caught in Pennsyl- Glutamine conjugates also are the major FACs in species of vania, USA, all identified by their morphological traits and Geometridae, and one sphingid species (Pohnert et al. 1999; food habitats. Epiphyas postvittana from New Zealand were Mori et al. 2003). In contrast, glutamic acid conjugates are from a recently-established colony fed on apple. Hy. cunea the dominant FACs in the sphingid tobacco hornworm, and An. peponis were fed on artificial diet (Insecta-LFS, Manduca sexta, (Alborn et al. 2003) and tomato hornworm, Nihon Nosan Kogyo Ltd., Yokohama, Japan), while other M. quinquemaculata (Halitschke et al. 2001). Paré et al. species, including laboratory-reared species, were fed on (1998) showed that the fatty acid moiety of the FAC their host plants. Last-instars of each species were frozen molecule originates from the diet of the caterpillar. at −4°C to extract gut contents. Consequently, the fatty acid composition of the FACs roughly reflects the fatty acid composition in the host plant, Gut Content Extraction and Sample Preparation At least although there seems to be a preference for linolenic and three were used from one species (one insect per linoleic acid in the FAC synthesis (Aboshi et al. 2007). sample). The frozen gut contents were dissected out as Since the isolation and identification of FACs as elicitors earlier described (Mori et al. 2003), placed in plastic tubes, of defensive reactions in plants, one intriguing question still and immediately boiled for 20 min to avoid enzymatic remains to be answered: How do the insects benefit from decomposition of FACs (Mori et al. 2001). To each sample producing FACs? Recently, we discovered that, at least for was added an equal volume of 50% water/acetonitrile S. litura, glutamine-containing FACs play an active role in solution (v/v) (an addition of 10–300 µl, dependent on the nitrogen assimilation by regulating the glutamine supply in amount of gut content). The samples were then roughly the larval midgut (Yoshinaga et al. 2008). Enriching homogenized with a plastic homogenizer and centrifuged at artificial diet with linolenic acid not only resulted in an 14,000g for 5 min. Ten-fold dilutions of the supernatants increased FAC synthesis, but also promoted a 50% with 50% acetonitrile solution were analyzed by LCMS. increased glutamic acid to glutamine conversion, ultimately resulting in a significantly increased amount of glutamine LC/MS Analyses Mass spectral measurements were carried in the whole body. Thus, the ability to utilize FACs in the out with an LCMS-2010A instrument (Shimadzu, Kyoto, digestive system might be one way that lepidopteran larvae Japan) combined with an HPLC system (LC-10ADvp pump, optimize their growth rate. However, it is not known if all CTO-10ACvp column oven, and SCL-10AVvp system J Chem Ecol (2010) 36:319–325 321 controller, Shimadzu). Three µl of sample solution were For some species, a few FACs were detected only in injected into a reversed-phase column (Cosmosil 5C18-AR-II, trace amounts. In such cases, we increased sample numbers, 50×2.0 mm I.D., Nacalai tesque, Kyoto, Japan) eluted for and determined an FAC was present only when it was 15 min at (0.2 ml/min) with a solvent gradient of 40–95% detected in more than three replicates.

CH3CN containing 0.08% acetic acid, in water containing 0.05% acetic acid. The column temperature was maintained at 40°C (CTO-10Avp column oven, Shimadzu), and the column eluant was monitored by continuous MS total ion Results current trace. The CDL temperature was 250°C, the voltage was 1.5 kV, the nebulizer gas flow was 1.5 l/min, and the Linolenic and linoleic acid were the dominant fatty acids in analytical mode was ESI negative scan from m/z 150–500. FACs. Although we also detected N-oleoyl-L-glutamine, and The negative ionization mass spectra gave characteristic other less abundant fatty acids conjugated with glutamine (M-1)− ions for volicitin at m/z 421, N-hydroxylinolenoyl-L- reflecting the proportion of dietary fatty acids (Aboshi et al. glutamic acid m/z 422, N-hydroxylinoleoyl-L-glutamine 2007), we focused our analysis on conjugates based on these m/z 423, N-hydroxylinoleoyl-L-glutamic acid m/z 424, acids. N-linolenoyl-L-glutamine m/z 405, N-linolenoyl-L-glutamic Figure 1 shows the reconstructed total ion chromatograms acid m/z 406, N-linoleoyl-L-glutamine m/z 407, and of gut contents of (a) muscosalis (), (b) N-linoleoyl-L-glutamic acid m/z 408. The position of Phalera flavescens (), (c) Xanthodes transversa the hydroxyl group in the hydroxylated FACs was not (), (d) Limantria dispar (Lymantriidae), (e) Agrotis determined. ipsilon (Noctuidae), and (f) Acherontia styx (),

A B 1 Linolenic acid 1 a 1’ Linoleic acid

O NH 2 NH HOOC 2 b O 1 2’ 1’ 2 1 1’ 3 c 3’ O NH OH HOOC O 3 3 d 3’ 1 1’ OH

1 O NH NH e HOOC 2 1’ O 3 3’ 2 2’ 4 4 2 f OH O NH OH 3 4’ 1 2’ HOOC O 5 10 15 20 min

Fig. 1 (A) Typical extracted ion chromatograms of gut contents from (a) 1, N-linolenoyl-L-glutamine (m/z 405); 1′, N-linoleoyl-L-glutamine (m/z ,(b)Phalera flavescens,(c)Xanthodes transversa, 407); 2, N-linolenoyl-L-glutamic acid (m/z 406); 2′, N-linoleoyl-L- (d) Lymantria dispar,(e)Agrotis ipsilon,and(f)Acherontia styx,and(B) glutamic acid (m/z 408); 3, N-hydroxylinolenoyl-L-glutamine (m/z 421); chemical structures of compounds 1-4. A reversed-phase column was 3′, N-hydroxylinoleoyl-L-glutamine (m/z 423); 4, N-hydroxylinolenoyl- eluted for 15 min with a 40–95% solvent gradient of CH3CN in water L-glutamic acid (m/z 422); 4′, N-hydroxylinoleoyl-L-glutamic acid containing 0.05% acetic acid. ESI-negative MS scan at m/z 150–500. (m/z 424) 322 J Chem Ecol (2010) 36:319–325 and chemical structures of the major FACs. Noticeable lepidopteran group but synthesized by widely different differences in FAC composition among the species are first families. As expected, closely related species in the same that L. dispar larvae (Fig. 1Ad) have more of the hydrox- family tended to have the same or very similar FAC ylated compounds (3, 3′) than glutamine conjugates (1, 1′), patterns. Interestingly, glutamine conjugates (1, 1′) were while for X. transversa (c) these ratios are reversed. found commonly in all these 19 species. Based on this, all Second, P. flavescens (b), Ag. ipsilon (e) and Ac. styx (f) FAC patterns were classified into 4 types: ① glutamine synthesize glutamic acid conjugates (2, 2′) as well as only (Fig. 1Aa), ② addition of glutamic acid (b), ③ glutamine conjugates (1, 1′), but glutamine conjugates are addition of hydroxylation (c, d), ④ addition of glutamic the major compounds in Ag. ipsilon, while glutamic acid acid and hydroxylation (e, f) (Table 1). conjugates are dominant in P. flavescens and Ac. styx. Third, as seen in Fig. 1A, the linolenic/linoleic acid ratios in FACs are parallel to those corresponding free fatty acids as Discussion expected if the ratio simply depended on dietary fatty acid compositions rather than on an enzymatic substrate prefer- In this investigation, we were interested mainly in each ence (Aboshi et al. 2007). species’ ability to synthesize various FAC compounds Table 1 shows the FAC components found in 29 lepi- regardless of diet. Considering the fact that linolenic acid- dopteran species, including 10 species in which no FACs FACs are more active as plant volatile elicitors than linoleic were found. Clearly, FACs are not specific to a certain acid-FACs, this ratio may have ecologically important

Table 1 Fatty Amino Acid Conjugate (FAC) components FAC components FAC found in lepidopteran species 1, 1′ 2, 2′ 3, 3′ 4, 4′ Type

Papilionidae Atrophaneura alcinous − Notodontidae Phalera flavescens ++ ② Lymantriidae Lymantria dispar japonica ++③ Arctiidae Hyphantria cunea − Noctuidae Arcte coerulea − Anadevidia peponis ++③ Xanthodes transversa ++③ Helicoverpa armigera ++③ Mythimna separata ++③ Spodoptera litura ++③ Agrotis ipsilon ++++④ Samia cynthia pryeri ++③ Samia cynthia ricini ++③ Sphingidae Clanis bilineata tsingtauica ++ ② Agrius convolvuli ++③ Acherontia styx ++++④ Manduca sexta ++++④ Cephonodes hylas ++③ Theretra oldenlandiae − Bombyx mori − Malacosoma americanum − Pyralidae Locastra muscosalis + ① Notarcha derogata − Pyrausta panopealis − Parasa lepida lepida − sinica − Epiphyas postvittana ++ ② Cossus insularis ++ ② Brachmia triannulella + ① J Chem Ecol (2010) 36:319–325 323 meaning for each species (De Moraes and Mescher 2004). of group ①: L. muscosalis and B. triannulella. The fact that However, we found no indication that the ratio of linolenic/ B. triannulella is evolutionarily the most ancestral species linoleic acid in insect FACs differed from the ratio of the among these FAC-containing species supports our hypoth- free fatty acids in the gut contents. This result suggests that esis that glutamine conjugates are the evolutionarily older there is no appreciable bias for fatty acid selection during FACs, although more data for ancestral species are essential FAC synthesis. Consequently, we focused this investigation to corroborate this conclusion. Equally surprising, group on the difference between glutamine and glutamic acid and the ②, characterized by glutamic acid conjugates, can be seen in hydroxylation status, rather than the fatty acids. No species relatively primitive lepidopteran families such as Cossidae have glutamic acid-FACs (2, 2′) without glutamine-FACs, nor and Tortricidae. In contrast, group ③ and ④, which include hydroxylated FACs (3, 3′) without glutamine-FACs. This hydroxylated FACs, are limited to , which is suggests that glutamine-FACs are the evolutionarily older considered to be the most advanced lepidopteran group. These FACs, and that the presence of glutamic acid conjugates, as species typically are characterized by their relatively large well as hydroxylated acyl conjugates, are two independent body size. However, since there are some large-sized species indicators of FAC diversification. For the hydroxylation, we that do not synthesize FACs at all, these compounds can not found clear differences between species, but it is worth noting be correlated simply with size and weight. Alternatively, that the ratio of hydroxylation can be variable even within a species in group ③ and ④ may be associated with a broad species. Previously, we reported hydroxylated FACs (3, 3′) range of host plants. Li et al. (2003) suggested that host plant as the major FACs in S. litura (Mori et al. 2003), but recently diversity can be related directly to P450 activity and inverse- we found that S. litura larval gut contents have more ly related to substrate specificity. It is, therefore, likely that a N-linolenoyl-L-glutamine (1) than volicitin (3) (Yoshinaga cytochrome P450 enzyme is responsible for the FAC hydrox- et al. 2005). Even within an individual larva feeding on the ylation as represented by volicitin (Ishikawa et al. 2009). same diet we have seen variation depending on the time Possibly, there is an evolutionary interplay between a course after the meal (data not shown). We also have noticed plant’s ability to detect and respond to different FACs and the that a high abundance of dietary lipid increased the relative insect’s dependence on these same compounds for maximized abundance of hydroxylated compounds in M. sexta (data not nitrogen assimilation. We have shown that glutamine-FACs shown). Thus, the hydroxylation/non-hydroxylation ratio are involved in this process, but the function of, and even the might be, at least partially, diet related. Since glutamine biosynthesis of, glutamic acid conjugates still remain un- conjugates (1, 1′) are precursors of hydroxylated FACs (3, 3′) known. In this study, glutamic acid conjugates were not (Yoshinaga et al. 2005), the composition also will be in- limited to M. sexta or M. quinquemaculata, but were found fluenced by the kinetics of 3 enzymes: conjugase, hydrolase, also in relatively primitive lepidopteran families. Further- and hydroxylase. The glutamine/glutamic acid ratio appears more, we have already reported FACs in the gut content of to be more stable. In this investigation, P. flavescens and crickets and larval fruit flies in a pattern similar to that of Ac. styx, had the same FAC proportions as M. sexta, with M. sexta or Ar. styx (Yoshinaga et al. 2007), in which glu- substantial amounts of glutamic acid-FACs, and only minor tamic acid conjugates are dominant, with only trace amounts glutamine-FACs peaks. At least for M. sexta, an insect with of glutamine conjugates. It appears that glutamine, as well which we have worked for several years, this ratio has been as glutamic acid FACs, play important roles in insects, but consistent and independent of the diet. We have no idea of we do not know if the role is the same in all these different the decisive factor for this, but it indicates a critical regulation insect species. of the proportions of these two types of FAC compounds. Despite the apparent physiological benefit of FACs, we The data we present here provide a broad base of also found 10 lepidopteran species where the gut content did information about FAC diversification in lepidopteran not contain detectable amounts of any FACs. If the main caterpillars. Figure 2 shows the distribution on the function for FACs is to maximize nitrogen uptake and lepidopteran family tree (based on the Tree of Life web subsequently maximize growth rate, then we would expect project database: http://tolweb.org/tree/) of the four FAC that these FAC-free insects all should be characterized by long types from the 29 lepidopteran species described in this developmental times, but this is not the case. Bombyx mori paper and nine other species studied earlier: three Geo- and Ar. coerulea grow fast and achieve large body size, while metridae species (Pohnert et al. 1999); S. frugiperda (③), Cossidae species (which make glutamic acid conjugates) S. littoralis (④) (Pohnert et al. 1999); Heliothis virescens grow slowly, boring into the trunk of salixes or apples, and (③), Helicoverpa zea (③) (Mori et al. 2001); Helio. require years before pupation. Curiously, there is a tendency subflexa (③) (De Moraes and Mescher 2004) in Noctuidae; for FAC-free caterpillars to have specific defensive strategies: M. quinquemaculata (④) in Sphingidae (Halitschke et al. Hyphantria cunera and Malacosoma americanum are 2001). All FAC-containing species had glutamine-based gregarious on a host tree and make nests. Two Crambidae FACs in common but, surprisingly, only two species were species also make nests by using leaves to wrap themselves. 324 J Chem Ecol (2010) 36:319–325

Papilionidae Hesperiidae Hedylidae Sematuridae Geometridae Doidae Notodontidae Macrolepidoptera Pantheidae Quadrifid Noctuoidea Lymantriidae Arctiidae Noctuidae Saturniidae Sphingidae Lemoniidae Lasiocampoidea Bombycidae Copromorphoidea Crambidae Pyralidae Zygaenidae Whallevana Tortricidae Tischeriidae Himantopteridae Anomoeotidae Aididae Megalopygidae Schreckensteinia Somabrachydae Crinopterygidae Limacodidae Neolepidoptera Cyclotornidae Phaudidae Lophocoronidae Sesiodea Dudgeoneidae Mnesarchaeidae Ericraniidae Cossidae Heterobathmiidae Palaeosetidae Agathiphagidae Neotheoridae Glyphidoceridae Anomosetidae Xylorictidae Prototheridae Amphisbatidae Peleopodidae Schistonoeidae Deoclonidae Gelechiidae Chimabachidae

Fig. 2 FAC-pattern classification and the phylogenetic relationship of Tree of Life Web Project, http://tolweb.org/.] ① gutamine conjugates . [The phylogenetic tree is based on the Tree of Life Web only; ② glutamine and glutamic acid-type FACs; ③ hydroxylated Project. 2003. Lepidoptera. and . Version 01 January glutamine-type FACs; ④ hydroxylated glutamine and glutamic acid 2003 (temporary). http://tolweb.org/Lepidoptera/8231/2003.01.01 in The type FACs; O no FACs detected

Atrophaneura alcinous, Parasa lepida lepida,andPryeria ously shaking its upper body and spitting slimy gut contents. sinica are all notoriously toxic. Pryeria sinica also has an Only domesticated silkworms have no such means of escape strategy that involves releasing thread to hang down protection, but they no longer live in nature. In contrast, most from a tree branch. Theretra oldenlandiae has a threatening of the FAC-containing species show no direct defensive bull’s-eye pattern that mimics snakes. Arcte coerulea, which reactions, with a few exceptions as shown in the case of was the only FAC-free species found in the Noctuidae so far, E. postvittana, which also uses thread to escape from para- is famous for its characteristic threatening behavior, vigor- sitoids (Suckling et al. 2001). Noctuidae and Sphingidae J Chem Ecol (2010) 36:319–325 325 species seem to especially focus on feeding and sleeping to HALITSCHKE,R.,SCHITTKO, U., POHNERT, G., BOLAND, W., and grow faster, which characterizes them as serious pests (Fig. 2). BALDWIN, I. T. 2001. Molecular interactions between the specialist herbivore Manduca sexta (Lepidoptera, Sphingidae) and its natural In summary: in this investigation we found that volicitin- host Nicotiana attenuata. III. Fatty acid-amino acid conjugates in related compounds are more commonly synthesized in herbivore oral secretions are necessary and sufficient for herbivore- lepidopteran larvae than was previously known but also that specific plant responses. Plant Physiol.125:711–717. not all Lepidoptera utilize FACs. We were able to classify the ISHIKAWA, C., YOSHINAGA,N.,ABOSHI,T.,NISHIDA,R.,andMORI,N. 2009. Efficient incorporation of free oxygen into volicitin in FAC pattern into 4 groups: 1, glutamine conjugates only; 2, Spodoptera litura common cutworm larvae. Biosci. Biotech. glutamic acid variation; 3, hydroxylated glutamine-type Biochem.73:1883–1885. FACs; and 4, hydroxylated glutamine and glutamic acid KESSLER, A., and BALDWIN, I. T. 2001. Defensive function of herbivore-induced plant volatile emissions in nature. Science type FACs. All FAC-containing species had N-linolenoyl-L- 291:2141–2144. glutamine and/or N-linoleoyl-L-glutamine, which might be LI, W., SCHULER,M.A.,andBERENBAUM, M. R. 2003. Diversification evolutionarily older FACs. Although the data provided here of furanocoumarin-metabolizing cytochrome P450 monooxyge- are biased toward noctuid and sphingid species, a compar- nases in two papilionids: Specificity and substrate encounter rate. Proc. Natl. Acad. Sci. USA 100:14593–14598. ison of the diversity of FACs with lepidopteran phylogeny MORI,N.,ALBORN,H.T.,TEAL,P.E.A.,andTUMLINSON,J.H.2001. indicates that glutamic acid conjugates can be synthesized by Enzymatic decomposition of elicitors of plant volatiles in Heliothis relatively primitive species, while hydroxylation of fatty virescens and Helicoverpa zea. J. Insect Physiol.47:749–757. acids is mostly limited to larger and more developed macro- MORI,N.,YOSHINAGA, N., SAWADA, Y., FUKUI,M.,SHIMODA,M., FUJISAKI,K.,NISHIDA,R.,andKUWAHARA,Y.2003.Identification lepidopteran species. of volicitin-related compounds from the regurgitant of lepidopteran This paper has highlighted the need to intensify our caterpillars. Biosci. Biotech. Biochem.67:1168–1171. studies of FACs in Lepidoptera as well as other insects in PARÉ, P. W., ALBORN, H. T., and TUMLINSON, J. H. 1998. Concerted order to obtain a new perspective and understanding of the biosynthesis of an insect elicitor of plant volatiles. Proc. Natl. Acad. Sci. USA 95:13971–13975. multifaceted functions of FACs in the insect world. POHNERT,G.,JUNG,V.,HAUKIOJA,E.,LEMPA,K.,andBOLNAD,W. 1999. New fatty acid amides from regurgitant of lepidopteran Acknowledgement We thank Drs. Fujisaki, K., Ando, T., Kunimi, Y., (Noctuidae, Geometridae) caterpillars. Tetrahedron 55:11275–11280. Ichida, M., and Shimoda, M. for providing insect materials. This study SCHMELZ,E.A.,CARROLL,M.J.,LECLERE, S., PHIPPS, S.M., was supported partly by Grants-in-aid for Scientific Research (no. MEREDITH,J.,CHOUREY,P.S.,ALBORN,H.T.,andTEAL,P.E.A. 15580090, 18580053, and 19580122) and by the 21st century COE 2006. Fragments of ATP synthase mediate plant perception of insect program for Innovative Food and Environmental Studies Pioneered by attack. Proc. Natl. Acad. Sci. USA 103: 8894–8899. Entomomimetic Sciences from the Ministry of Education, Culture, SCHMELZ, E. A., ENGELBERTH, J., ALBORN, H. T., TUMLINSON, J. H., Sports, Science and Technology of Japan. 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