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 been found in noctuid as well as sphingid caterpillar oral secretions; in particular, volicitin [N-(17-hydroxylinolenoyl)L-glutamine] and its biochemical precursor, N-linolenoyl-Lglutamine, are known elicitors of induced volatile emissions in corn plants. These induced volatiles, in turn, attract natural enemies of the caterpillars. In a previous study, we showed that N-linolenoyl-L-glutamine in larval Spodoptera litura plays an important role in nitrogen assimilation which might be an explanation for caterpillars synthesizing FACs despite an increased risk of attracting natural enemies. However, N. Yoshinaga : J. H. Tumlinson Center for Chemical Ecology, Department of Entomology, The Pennsylvania State University, University Park, PA 16802, USA H. T. Alborn Center of Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, Chemistry Unit, U. S. Department of Agriculture, 1600 Southwest 23rd Drive, Gainesville, FL 32611-0620, USA T. Nakanishi Forestry and Fisheries Technology Center, Fruit Tree Research Institute, Tokushima Prefectural Agriculture, Katsuura-cho, Katsuura, Tokushima 773-4301, Japan D. M. Suckling The New Zealand Institute for Plant and Food Research Limited, PB 4704, Christchurch, New Zealand N. Yoshinaga : R. Nishida : N. Mori (*) Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo, Kyoto 606-8502, Japan e-mail:
[email protected]
the presence of FACs in lepidopteran species outside these families of agricultural interest is not well known. We conducted FAC screening of 29 lepidopteran species, and found them in 19 of these species. Thus, FACs are commonly synthesized through a broad range of lepidopteran caterpillars. Since all FAC-containing species had N-linolenoyl-Lglutamine and/or N-linoleoyl-L-glutamine in common, and the evolutionarily earliest species among them had only these two FACs, these glutamine conjugates might be the evolutionarily older FACs. Furthermore, some species had glutamic acid conjugates, and some had hydroxylated FACs. Comparing the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is limited mostly to larger and more developed macrolepidopteran species. Keywords Insect-produced elicitors . Lepidopteran phylogeny . Volicitin . Insect-plant interactions Abbreviations FACs fatty acid amino acid conjugates VOC volatile organic compounds ESI electrospray ionization
Introduction It is well documented that constituents of insect oral secretions can trigger plant responses, such as elicitation of induced de novo synthesis and release of volatile organic compounds (VOCs) (Turlings et al. 1990; Paré et al. 1998; Kessler and Baldwin 2001). The best known and most studied of insect-produced elicitors are the fatty acid amino
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acid conjugates (FACs), first identified from beet armyworm Spodoptera exigua larvae (Alborn et al. 1997). In a recent study of a range of plant species, FACs and especially volicitin [N-(17-hydroxylinolenoyl)-L-glutamine] showed the broadest effects on plant hormone levels as well as induction of plant volatiles compared with caeliferin A16:0 and inceptin, two recently identified new classes of insect-produced elicitors of inducible plant defenses (Schmelz et al. 2006, 2009; Alborn et al. 2007). It also has been shown that application of volicitin to a mechanically wounded site selectively and transcriptionally activated genes for indole-3-glycerol phosphate lyase (Igl) and specific sesquiterpene cyclase (stc1), and that this activation also occurred systemically in undamaged leaves (Frey et al. 2000; Shen et al. 2000). However, previous studies (Truitt and Paré 2004; Truitt et al. 2004) showed that volicitin did not on its own serve as a mobile messenger for systemic VOCs emissions, but rather that a volicitin binding protein-ligand interaction may initiate plant defenses in response to herbivory. Since glutamine-based FAC components initially were identified in oral secretions from S. exigua larvae, several other noctuid caterpillars have been reported to have the same glutamine-based FACs (Pohnert et al. 1999; Mori et al. 2001, 2003; De Moraes and Mescher 2004). In addition, oral secretions from some noctuid larvae such as S. littoralis also contain traces of glutamic acid-based FACs. Glutamine conjugates also are the major FACs in species of Geometridae, and one sphingid species (Pohnert et al. 1999; Mori et al. 2003). In contrast, glutamic acid conjugates are the dominant FACs in the sphingid tobacco hornworm, Manduca sexta, (Alborn et al. 2003) and tomato hornworm, M. quinquemaculata (Halitschke et al. 2001). Paré et al. (1998) showed that the fatty acid moiety of the FAC molecule originates from the diet of the caterpillar. Consequently, the fatty acid composition of the FACs roughly reflects the fatty acid composition in the host plant, although there seems to be a preference for linolenic and linoleic acid in the FAC synthesis (Aboshi et al. 2007). Since the isolation and identification of FACs as elicitors of defensive reactions in plants, one intriguing question still remains to be answered: How do the insects benefit from producing FACs? Recently, we discovered that, at least for S. litura, glutamine-containing FACs play an active role in nitrogen assimilation by regulating the glutamine supply in the larval midgut (Yoshinaga et al. 2008). Enriching artificial diet with linolenic acid not only resulted in an increased FAC synthesis, but also promoted a 50% increased glutamic acid to glutamine conversion, ultimately resulting in a significantly increased amount of glutamine in the whole body. Thus, the ability to utilize FACs in the digestive system might be one way that lepidopteran larvae optimize their growth rate. However, it is not known if all
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lepidopteran larvae utilize FACs. Furthermore, differences in physiology or metabolism associated with glutamineFACs vs. glutamic acid-FACs are not understood. These questions are not easy to answer, since only a limited number of insect species (mainly of agricultural interest) have been investigated for FAC content so far. Consequently, we decided to collect and analyze gut contents from an environmentally, taxonomically, and physiologically diverse group of lepidopteran larvae consisting of 29 species from16 families.
Materials and Methods Caterpillar Source Colonies of Spodoptera litura, Mythimna separate, and Hyphantria cunea were maintained successively in the laboratory, and Helicoverpa armigera was supplied by Dr. Kenji Fujisaki, Anadevidia peponis by Dr. Tetsu Ando, Samia cynthia ricini by Dr. Masatoshi Ichida, and Agrius convolvuli by Dr. Masami Shimoda. Commercially available Bombyx mori were purchased from Mukin Yosan System Institute (Kyoto, Japan), Agrotis ipsilon eggs were purchased from Benzon Research Inc. (PA, USA), and Manduca sexta from North Carolina University, NC, USA. Another 18 wild species were collected in Kyoto, Osaka, Mie, and Tokushima prefectures in Japan. Malacosoma americanum was caught in Pennsylvania, USA, all identified by their morphological traits and food habitats. Epiphyas postvittana from New Zealand were from a recently-established colony fed on apple. Hy. cunea and An. peponis were fed on artificial diet (Insecta-LFS, Nihon Nosan Kogyo Ltd., Yokohama, Japan), while other species, including laboratory-reared species, were fed on their host plants. Last-instars of each species were frozen at −4°C to extract gut contents. Gut Content Extraction and Sample Preparation At least three insects were used from one species (one insect per sample). The frozen gut contents were dissected out as earlier described (Mori et al. 2003), placed in plastic tubes, and immediately boiled for 20 min to avoid enzymatic decomposition of FACs (Mori et al. 2001). To each sample was added an equal volume of 50% water/acetonitrile solution (v/v) (an addition of 10–300 µl, dependent on the amount of gut content). The samples were then roughly homogenized with a plastic homogenizer and centrifuged at 14,000g for 5 min. Ten-fold dilutions of the supernatants with 50% acetonitrile solution were analyzed by LCMS. LC/MS Analyses Mass spectral measurements were carried out with an LCMS-2010A instrument (Shimadzu, Kyoto, Japan) combined with an HPLC system (LC-10ADvp pump, CTO-10ACvp column oven, and SCL-10AVvp system
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controller, Shimadzu). Three µl of sample solution were injected into a reversed-phase column (Cosmosil 5C18-AR-II, 50×2.0 mm I.D., Nacalai tesque, Kyoto, Japan) eluted for 15 min at (0.2 ml/min) with a solvent gradient of 40–95% 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 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 analytical mode was ESI negative scan from m/z 150–500. The negative ionization mass spectra gave characteristic (M-1)− ions for volicitin at m/z 421, N-hydroxylinolenoyl-Lglutamic acid m/z 422, N-hydroxylinoleoyl-L-glutamine m/z 423, N-hydroxylinoleoyl-L-glutamic acid m/z 424, N-linolenoyl-L-glutamine m/z 405, N-linolenoyl-L-glutamic acid m/z 406, N-linoleoyl-L-glutamine m/z 407, and N-linoleoyl-L-glutamic acid m/z 408. The position of the hydroxyl group in the hydroxylated FACs was not determined.
A
1
For some species, a few FACs were detected only in trace amounts. In such cases, we increased sample numbers, and determined an FAC was present only when it was detected in more than three replicates.
Results Linolenic and linoleic acid were the dominant fatty acids in FACs. Although we also detected N-oleoyl-L-glutamine, and other less abundant fatty acids conjugated with glutamine reflecting the proportion of dietary fatty acids (Aboshi et al. 2007), we focused our analysis on conjugates based on these acids. Figure 1 shows the reconstructed total ion chromatograms of gut contents of (a) Locastra muscosalis (Pyralidae), (b) Phalera flavescens (Notodontidae), (c) Xanthodes transversa (Noctuidae), (d) Limantria dispar (Lymantriidae), (e) Agrotis ipsilon (Noctuidae), and (f) Acherontia styx (Sphingidae),
B
Linolenic acid
1 a
Linoleic acid
1’
O
2
b
1
1’
1 3
c
NH NH2
HOOC O
2’
2
1’
3’
O
NH OH
HOOC O
3
3
d
3’
1
1’
OH O
1
e
NH NH2
HOOC
1’
3 3’
2
O
2’
4
2
4
OH
f
O
3
4’
1
2’
NH OH
HOOC O
5
10
15
20
Fig. 1 (A) Typical extracted ion chromatograms of gut contents from (a) Locastra muscosalis, (b) Phalera flavescens, (c) Xanthodes transversa, (d) Lymantria dispar, (e) Agrotis ipsilon, and (f) Acherontia styx, and (B) chemical structures of compounds 1-4. A reversed-phase column was eluted for 15 min with a 40–95% solvent gradient of CH3CN in water containing 0.05% acetic acid. ESI-negative MS scan at m/z 150–500.
min
1, N-linolenoyl-L-glutamine (m/z 405); 1′, N-linoleoyl-L-glutamine (m/z 407); 2, N-linolenoyl-L-glutamic acid (m/z 406); 2′, N-linoleoyl-Lglutamic acid (m/z 408); 3, N-hydroxylinolenoyl-L-glutamine (m/z 421); 3′, N-hydroxylinoleoyl-L-glutamine (m/z 423); 4, N-hydroxylinolenoylL-glutamic acid (m/z 422); 4′, N-hydroxylinoleoyl-L-glutamic acid (m/z 424)
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and chemical structures of the major FACs. Noticeable differences in FAC composition among the species are first that L. dispar larvae (Fig. 1Ad) have more of the hydroxylated compounds (3, 3′) than glutamine conjugates (1, 1′), while for X. transversa (c) these ratios are reversed. Second, P. flavescens (b), Ag. ipsilon (e) and Ac. styx (f) synthesize glutamic acid conjugates (2, 2′) as well as glutamine conjugates (1, 1′), but glutamine conjugates are the major compounds in Ag. ipsilon, while glutamic acid 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 expected if the ratio simply depended on dietary fatty acid compositions rather than on an enzymatic substrate preference (Aboshi et al. 2007). Table 1 shows the FAC components found in 29 lepidopteran species, including 10 species in which no FACs were found. Clearly, FACs are not specific to a certain
lepidopteran group but synthesized by widely different families. As expected, closely related species in the same family tended to have the same or very similar FAC patterns. Interestingly, glutamine conjugates (1, 1′) were found commonly in all these 19 species. Based on this, all FAC patterns were classified into 4 types: ① glutamine only (Fig. 1Aa), ② addition of glutamic acid (b), ③ addition of hydroxylation (c, d), ④ addition of glutamic acid and hydroxylation (e, f) (Table 1).
Discussion In this investigation, we were interested mainly in each species’ ability to synthesize various FAC compounds regardless of diet. Considering the fact that linolenic acidFACs are more active as plant volatile elicitors than linoleic acid-FACs, this ratio may have ecologically important
Table 1 Fatty Amino Acid Conjugate (FAC) components found in lepidopteran species
FAC components
Papilionidae Notodontidae Lymantriidae Arctiidae Noctuidae
Saturniidae Sphingidae
Bombycidae Lasiocampoidea Pyralidae Crambidae Limacodidae Zygaenidae Tortricidae Cossidae Gelechiidae
Atrophaneura alcinous Phalera flavescens Lymantria dispar japonica Hyphantria cunea Arcte coerulea Anadevidia peponis Xanthodes transversa Helicoverpa armigera Mythimna separata Spodoptera litura Agrotis ipsilon Samia cynthia pryeri Samia cynthia ricini Clanis bilineata tsingtauica Agrius convolvuli Acherontia styx Manduca sexta Cephonodes hylas Theretra oldenlandiae Bombyx mori Malacosoma americanum Locastra muscosalis Notarcha derogata Pyrausta panopealis Parasa lepida lepida Pryeria sinica Epiphyas postvittana Cossus insularis Brachmia triannulella
1, 1′
2, 2′
+ +
+
+ + + + + + + + + + + + +
+
3, 3′
4, 4′
+
+
+ + + + + + + +
+
+ + +
+
+ +
FAC
+ +
+ + + +
+ +
Type − ② ③ − − ③ ③ ③ ③ ③ ④ ③ ③ ② ③ ④ ④ ③ − − − ① − − − − ② ② ①
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meaning for each species (De Moraes and Mescher 2004). However, we found no indication that the ratio of linolenic/ linoleic acid in insect FACs differed from the ratio of the free fatty acids in the gut contents. This result suggests that there is no appreciable bias for fatty acid selection during FAC synthesis. Consequently, we focused this investigation on the difference between glutamine and glutamic acid and the hydroxylation status, rather than the fatty acids. No species have glutamic acid-FACs (2, 2′) without glutamine-FACs, nor hydroxylated FACs (3, 3′) without glutamine-FACs. This suggests that glutamine-FACs are the evolutionarily older FACs, and that the presence of glutamic acid conjugates, as well as hydroxylated acyl conjugates, are two independent indicators of FAC diversification. For the hydroxylation, we found clear differences between species, but it is worth noting that the ratio of hydroxylation can be variable even within a species. Previously, we reported hydroxylated FACs (3, 3′) as the major FACs in S. litura (Mori et al. 2003), but recently we found that S. litura larval gut contents have more N-linolenoyl-L-glutamine (1) than volicitin (3) (Yoshinaga et al. 2005). Even within an individual larva feeding on the same diet we have seen variation depending on the time course after the meal (data not shown). We also have noticed that a high abundance of dietary lipid increased the relative abundance of hydroxylated compounds in M. sexta (data not shown). Thus, the hydroxylation/non-hydroxylation ratio might be, at least partially, diet related. Since glutamine conjugates (1, 1′) are precursors of hydroxylated FACs (3, 3′) (Yoshinaga et al. 2005), the composition also will be influenced by the kinetics of 3 enzymes: conjugase, hydrolase, and hydroxylase. The glutamine/glutamic acid ratio appears to be more stable. In this investigation, P. flavescens and Ac. styx, had the same FAC proportions as M. sexta, with substantial amounts of glutamic acid-FACs, and only minor glutamine-FACs peaks. At least for M. sexta, an insect with which we have worked for several years, this ratio has been consistent and independent of the diet. We have no idea of the decisive factor for this, but it indicates a critical regulation of the proportions of these two types of FAC compounds. The data we present here provide a broad base of information about FAC diversification in lepidopteran caterpillars. Figure 2 shows the distribution on the lepidopteran family tree (based on the Tree of Life web project database: http://tolweb.org/tree/) of the four FAC types from the 29 lepidopteran species described in this paper and nine other species studied earlier: three Geometridae species (Pohnert et al. 1999); S. frugiperda (③), S. littoralis (④) (Pohnert et al. 1999); Heliothis virescens (③), Helicoverpa zea (③) (Mori et al. 2001); Helio. subflexa (③) (De Moraes and Mescher 2004) in Noctuidae; M. quinquemaculata (④) in Sphingidae (Halitschke et al. 2001). All FAC-containing species had glutamine-based FACs in common but, surprisingly, only two species were
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of group ①: L. muscosalis and B. triannulella. The fact that B. triannulella is evolutionarily the most ancestral species among these FAC-containing species supports our hypothesis that glutamine conjugates are the evolutionarily older FACs, although more data for ancestral species are essential to corroborate this conclusion. Equally surprising, group ②, characterized by glutamic acid conjugates, can be seen in relatively primitive lepidopteran families such as Cossidae and Tortricidae. In contrast, group ③ and ④, which include hydroxylated FACs, are limited to Macrolepidoptera, which is considered to be the most advanced lepidopteran group. These species typically are characterized by their relatively large body size. However, since there are some large-sized species that do not synthesize FACs at all, these compounds can not be correlated simply with size and weight. Alternatively, species in group ③ and ④ may be associated with a broad range of host plants. Li et al. (2003) suggested that host plant diversity can be related directly to P450 activity and inversely related to substrate specificity. It is, therefore, likely that a cytochrome P450 enzyme is responsible for the FAC hydroxylation as represented by volicitin (Ishikawa et al. 2009). Possibly, there is an evolutionary interplay between a plant’s ability to detect and respond to different FACs and the insect’s dependence on these same compounds for maximized nitrogen assimilation. We have shown that glutamine-FACs are involved in this process, but the function of, and even the biosynthesis of, glutamic acid conjugates still remain unknown. In this study, glutamic acid conjugates were not limited to M. sexta or M. quinquemaculata, but were found also in relatively primitive lepidopteran families. Furthermore, we have already reported FACs in the gut content of crickets and larval fruit flies in a pattern similar to that of M. sexta or Ar. styx (Yoshinaga et al. 2007), in which glutamic acid conjugates are dominant, with only trace amounts of glutamine conjugates. It appears that glutamine, as well as glutamic acid FACs, play important roles in insects, but we do not know if the role is the same in all these different insect species. Despite the apparent physiological benefit of FACs, we also found 10 lepidopteran species where the gut content did not contain detectable amounts of any FACs. If the main function for FACs is to maximize nitrogen uptake and subsequently maximize growth rate, then we would expect that these FAC-free insects all should be characterized by long developmental times, but this is not the case. Bombyx mori and Ar. coerulea grow fast and achieve large body size, while Cossidae species (which make glutamic acid conjugates) grow slowly, boring into the trunk of salixes or apples, and require years before pupation. Curiously, there is a tendency for FAC-free caterpillars to have specific defensive strategies: Hyphantria cunera and Malacosoma americanum are gregarious on a host tree and make nests. Two Crambidae species also make nests by using leaves to wrap themselves.
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Papilionoidea Hesperiidae Hedylidae
Geometroidea Drepanoidea Cimeliidae Callidulidae Macrolepidoptera
Noctuoidea
Quadrifid Noctuoidea
Bombycoidea Lasiocampoidea Mimallonidae Thyrididae Hyblaeidae Copromorphoidea Pyraloidea
Ditrysia Tischeriidae Palaephatidae
Neolepidoptera Neopseustidae Lophocoronidae Ericraniidae Acanthopteroctetidae Heterobathmiidae Agathiphagidae Micropterigidae
Prodoxidae Cecidosidae Incurvariidae Crinopterygidae Adelidae Heliozelidae Nepticulidae Opostegidae Mnesarchaeidae Hepialidae Palaeosetidae Neotheoridae Anomosetidae Prototheridae
Immidae Whallevana Tortricidae Zygaenoidea Pterophoridae Alucitoidea Epermeniidae Schreckensteinia Choreutidae Urodidae
Sesiodea Cossoidea
Gelechioidea Yponomeutoidea Gracillarioidea Tineoidea
Papilionidae Pieridae Riodinidae Lycaenidae Nymphalidae Sematuridae Uraniidae Geometridae Oenosandridae Doidae Notodontidae Pantheidae Nolidae Lymantriidae Arctiidae Noctuidae Saturniidae Sphingidae Lemoniidae Brahmaeidae Bombycidae Eupterotidae
Crambidae Pyralidae Zygaenidae Dalceridae Lacturidae Himantopteridae Anomoeotidae Aididae Megalopygidae Somabrachydae Limacodidae Epipyropidae Cyclotornidae Phaudidae Dudgeoneidae Cossidae Oecophoridae Batrachedridae Glyphidoceridae Xylorictidae Elachistidae Amphisbatidae Peleopodidae Schistonoeidae Deoclonidae Cosmopterigidae Gelechiidae Autostichidae Chimabachidae Lecithoceridae
Fig. 2 FAC-pattern classification and the phylogenetic relationship of Lepidoptera. [The phylogenetic tree is based on the Tree of Life Web Project. 2003. Lepidoptera. Moths and Butterflies. Version 01 January 2003 (temporary). http://tolweb.org/Lepidoptera/8231/2003.01.01 in The
Tree of Life Web Project, http://tolweb.org/.] ① gutamine conjugates only; ② glutamine and glutamic acid-type FACs; ③ hydroxylated glutamine-type FACs; ④ hydroxylated glutamine and glutamic acid type FACs; O no FACs detected
Atrophaneura alcinous, Parasa lepida lepida, and Pryeria sinica are all notoriously toxic. Pryeria sinica also has an escape strategy that involves releasing thread to hang down from a tree branch. Theretra oldenlandiae has a threatening bull’s-eye pattern that mimics snakes. Arcte coerulea, which was the only FAC-free species found in the Noctuidae so far, is famous for its characteristic threatening behavior, vigor-
ously shaking its upper body and spitting slimy gut contents. Only domesticated silkworms have no such means of protection, but they no longer live in nature. In contrast, most of the FAC-containing species show no direct defensive reactions, with a few exceptions as shown in the case of E. postvittana, which also uses thread to escape from parasitoids (Suckling et al. 2001). Noctuidae and Sphingidae
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species seem to especially focus on feeding and sleeping to grow faster, which characterizes them as serious pests (Fig. 2). In summary: in this investigation we found that volicitinrelated compounds are more commonly synthesized in lepidopteran larvae than was previously known but also that not all Lepidoptera utilize FACs. We were able to classify the FAC pattern into 4 groups: 1, glutamine conjugates only; 2, glutamic acid variation; 3, hydroxylated glutamine-type FACs; and 4, hydroxylated glutamine and glutamic acid type FACs. All FAC-containing species had N-linolenoyl-Lglutamine and/or N-linoleoyl-L-glutamine, which might be evolutionarily older FACs. Although the data provided here are biased toward noctuid and sphingid species, a comparison of the diversity of FACs with lepidopteran phylogeny indicates that glutamic acid conjugates can be synthesized by relatively primitive species, while hydroxylation of fatty acids is mostly limited to larger and more developed macrolepidopteran species. This paper has highlighted the need to intensify our studies of FACs in Lepidoptera as well as other insects in order to obtain a new perspective and understanding of the multifaceted functions of FACs in the insect world. Acknowledgement We thank Drs. Fujisaki, K., Ando, T., Kunimi, Y., Ichida, M., and Shimoda, M. for providing insect materials. This study was supported partly by Grants-in-aid for Scientific Research (no. 15580090, 18580053, and 19580122) and by the 21st century COE program for Innovative Food and Environmental Studies Pioneered by Entomomimetic Sciences from the Ministry of Education, Culture, Sports, Science and Technology of Japan. N.Y. was supported by a Postdoctoral Fellowship for Research Abroad from the Japan Society for the Promotion of Science.
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