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 species 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 Insect-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 insects 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