J Chem Ecol (2013) 39:350–357 DOI 10.1007/s10886-013-0253-8

Identification of the Sex Pheromone Secreted by a Nettle Moth, Monema flavescens, Using Gas Chromatography/Fourier Transform Infrared Spectroscopy Hiroshi Shibasaki & Masanobu Yamamoto & Qi Yan & Hideshi Naka & Toshiro Suzuki & Tetsu Ando

Received: 5 October 2012 / Revised: 28 November 2012 / Accepted: 29 January 2013 / Published online: 12 February 2013 # Springer Science+Business Media New York 2013

Abstract The nettle moth Monema flavescens (Limacodidae) is a defoliator of fruit trees, such as Chinese plum and persimmon. The larvae of this species have spines containing a poison that causes serious irritation and inflammation in humans. Coupled gas chromatography-electroantennogram detection and gas chromatography/mass spectrometry analyses of a crude pheromone extract, combined with derivatization, indicated that female moths produced 8-decen-1-ol and 7,9-decadien-1-ol at a ratio of approximately 9:1. The E configuration of the double bonds was assigned for both components from infrared spectra, recorded on a gas chromatograph/Fourier transform-infrared spectrophotometer equipped with a zinc selenide disk cooled to −30 °C. The monoenyl and dienyl alcohols had absorptions characteristic of E geometry at 966 and 951 cm−1, respectively. A band chromatogram at 951 cm−1 was useful for distinguishing geometric isomers, because terminal conjugated diene are difficult to resolve, even on high polarity columns. Furthermore, we identified the Z configuration of the same 7,9-dienyl alcohol secreted by another nettle moth, Parasa lepida lepida, through the absence of this absorption. In field trials, lures baited with a 9:1 mixture of (E)-8-decen-1-ol and H. Shibasaki : M. Yamamoto : Q. Yan : T. Ando (*) Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan e-mail: [email protected] H. Naka Laboratory of Applied Entomology, Faculty of Agriculture, Tottori University, 4-101 Koyama-minami, Tottori 680-8553, Japan T. Suzuki Department of Environment, Gifu Prefectural Agricultural Management Division, Gifu, Gifu 501-1152, Japan

(E)-7,9-decadien-1-ol attracted M. flavescens males. Furthermore, the field trials indicated that contamination with the (Z)-diene reduced catches to the pheromone mixture more than did contamination with the (Z)-monoene. Keywords Female sex pheromone . Lepidoptera . Limacodidae . Oriental moth . Infrared spectrometory . GC/FT-IR . (E)-7,9-decadien-1-ol . (E)-8-decen-1-ol . Male attraction . Insect pest

Introduction Lepidopteran sex pheromones have been identified from more than 630 species (Ando, 2012; El-Sayed, 2012). To determine chemical structures of pheromone components, gas chromatography (GC) with an electroantennographic (EAG) detector (GC-EAD) and GC combined with mass spectrometry (GC/MS) are usually used (Ando et al., 2004). Many lepidopteran sex pheromones are straight-chain compounds; their structures are determined by clarification of the functional group, carbon chain length, degree of unsaturation, and position and geometry of the double bond(s). Although an infrared (IR) spectrum gives information about functional group and double-bond configuration of a compound, IR analysis rarely has been utilized for pheromone identifications, primarily because pheromones are usually composed of multiple components and are produced in low amounts by female moths. Combined use of GC and IR of vaporized compounds eluting from a GC column has been used for identification of natural products (Attygalle, 1994; Attygalle et al., 1994; Leal et al., 2001; Visser, 2002). Recently, a new type of GC/Fourier transform infrared spectrophotometer (FT-IR), equipped with a zinc selenide disk cooled to −30 to −40 °C, has been developed. The disk is turned slowly, and

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compounds eluting from a capillary column are fixed on the disk in discrete positions. The resulting FT-IR spectra, which are similar to IR spectra recorded by the usual KBr or liquidfilm methods, are measured continuously. Furthermore, the IR absorptions of a target compound can be accumulated through repeated irradiation by the IR beam after all the components of a GC run have eluted (and been scanned). This increased analytical sensitivity should facilitate application of GC/FTIR to pheromone studies of lepidopteran species, particularly for determination of double-bond configurations. Since geometric isomers of many pheromone components have similar mass spectra (Ando and Yamakawa, 2011), and GC separation of some isomers (especially terminal conjugated dienes) can be difficult (Islam et al., 2009), GC/FT-IR analysis could be an important tool for obtaining structural information unable to be defined by GC/MS. Nettle caterpillars have spines containing toxic compounds. Touching these spines can cause serious irritation and inflammation of the skin in humans. Esters with a conjugated dienyl structure at the terminal position of an acid moiety have been identified as female sex pheromones of some Indonesian and Malaysian species (Sasaerila et al., 2000a; Siderhurst et al., 2007). In addition to these novel esters, alcohol and acetate derivatives with a terminal conjugated dienyl structure are known to be pheromone components of other nettle moth species (Ando, 2012; El-Sayed, 2012), suggesting a common motif in pheromones of Limacodidae species. While more than 35 Limacodidae species inhabit Japan, a sex pheromone has been identified from only one of these species, Parasa lepida lepida Cramer (Wakamura et al., 2007; Islam et al., 2009). This species is distributed throughout Honshu, the main island of Japan, although the species is regarded as exotic. The Oriental moth, Monema flavescens Walker, is a Japanese native nettle moth that is well known for its cocoon, which looks like a small bird egg with a hard shell. This univoltine defoliator of many kinds of trees, such as Chinese plum and persimmon, is also found in China and Korea. Currently, populations of M. flavescens appear to be decreasing in Japan, and it is not easy to find wintering cocoons in orchards and parks around the Tokyo area. However, the insect still is considered a pest by fruit growers. Monitoring male moths with a synthetic pheromone should help identify infested orchards. Therefore, we determined the pheromone components of this species by various methods, including GC/FT-IR with a zinc selenide disk. This project also allowed us to explore the advantages and limitations of this instrument.

Methods and Materials Insects and Pheromone Extraction About 200 cocoons of M. flavescens were collected in orchards of Chinese plum, peach, and persimmon in Gifu Prefecture and Tottori

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Prefecture, in the autumns of 2008–2012. The cocoons were maintained outdoors over winter on the TUAT campus. Pupae were taken out of cocoons in spring, sexed, and kept in 16L: 8D at 25 °C. After eclosion, the pheromone glands of 2- or 3-d-old virgin females, which had shown calling behavior 2 h after the beginning of scotophase, were excised and immersed in hexane (50 μl/gland) for 15 min. The crude extract was used for structural analysis of pheromone components after filtration. Instruments for Analysis GC-EAD was used to identify physiologically active compounds in female gland extracts. An HP6890 Series II GC was equipped with a DB-23 column (0.25 mm ID, 30 m length, 0.25 μm film thickness, J & W Scientific, Folsom, CA, USA). The effluent from the column was split into two lines, which led to a flame ionization detector (FID) and an EAD, at a ratio of 1:1 (Inomata et al., 2005). The temperature of the FID was 220 °C. GC/MS analyses were conducted using electron impact (EI) ionization (70 eV) on an Agilent Technologies 6890/5975 instrument. The GC was equipped with the same DB-23 column as the GC-EAD. IR spectra were recorded on an FT-IR (DiscoverIR; Spectra Analytics, Marlborough, MA, USA) instrument coupled to an Agilent Technologies 7980C GC, equipped with an HP-5 capillary column (0.25 mm ID, 30 m length, 0.25 μm film thickness, J & W Scientific). A liquid nitrogen-cooled photoconductive mercury-cadmium-telluride detector was used with a FT-IR resolution of 8 cm−1. Compounds eluting from the capillary column were solidified on a zinc selenide disc at −30 °C and rotated at 3 mm/min. The distance between the column end and disc was 5 mm. The oven temperature for all GC analyses was set initially at 50 °C for 2 min. and then programmed at 10 °C.min−1 to 160 °C and 4 °C.min−1 to 220 °C. The flow rate of the carrier gas (He) was 1.0 ml.min−1, and the GC inlet temperature was 220 °C. 1H- and 13C-NMR spectra were recorded on a Jeol Delta 2 FT spectrometer (JEOL Ltd., Tokyo, Japan) at 399.8 and 100.5 MHz, respectively, as CDCl3 solutions containing TMS as an internal standard. Derivatization of Pheromone Components To reveal the double-bond position of a monoenyl component, crude pheromone extract was treated with dimethyl disulfide (DMDS) (Buser et al., 1983; Inomata et al., 2000). Briefly, the hexane was evaporated from the extract (3 female equivalents, FE), and the residue dissolved in DMDS (50 μl) including a diethyl ether solution of iodine (65 mg.ml−1, 20 μl). The mixture was reacted at 40 °C overnight. After addition of a 5 % sodium thiosulfate solution (0.5 ml), the DMDS adduct was extracted with hexane and analyzed by GC/MS. Chemicals C10 monoenyl alcohols were synthesized from 1,7-heptanediol. After one hydroxyl group of the diol was

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protected with 3,4-dihydro-2H-pyran, another hydroxyl group was iodinated, by treatment with a mixture of I2, triphenylphosphine, and imidazole, to yield a THP ether of 7-iodoheptan-1-ol. The iodide was treated with a lithium acetylide-ethylenediamine complex in DMSO to yield a THP ether of 8-nonyn-1-ol. A coupling reaction of the alkyne and iodomethane with butyllithium, in a mixed solvent of THF and HMPA, produced a THP ether of 8-decyn1-ol. (E)-8-Decen-1-ol (E8-10:OH) was prepared from the acetylene compound by Birch reduction with Li in ethylamine and deprotection. 1H-NMR: δ ~1.3 (8H, m), 1.56 (2H, tt, CH2CH2OH, J=6.5, 6.5 Hz), 1.64 (3H, dd, CH3CH=CH, J=3.3, 1.3 Hz), 1.97 (2H, m, CH2CH=CH), 3.64 (2H, t, CH2OH, J=6.5 Hz), 5.41 (2H, m, CH=CH). 13C-NMR: δ 17.9, 25.7, 29.1, 29.3, 29.5, 32.6, 32.8, 63.1, 124.6, 131.6. (Z)-8-Decen-1-ol (Z8-10:OH) was prepared from the same acetylene compound by partial hydrogenation over a Pd-BaSO4 catalyst poisoned with quinoline. 1H-NMR: δ ~1.3 (8H, m), 1.57 (2H, tt, CH2CH2OH, J=6.5, 6.5 Hz), 1.60 (3H, d, CH3CH=CH, J=6.0 Hz), 2.03 (2H, m, CH2CH= CH), 3.64 (2H, t, CH2OH, J=6.5 Hz), 5.41 (2H, m, CH=CH). 13 C-NMR: δ 12.8, 25.7, 26.8, 29.2, 29.3, 29.5, 32.8, 63.1, 123.7, 130.8. C10 dienyl alcohols, (E)-7,9-decadien-1-ol (E7,9-10:OH) and (Z)-7,9-decadien-1-ol (Z7,9-10:OH), had been synthesized in a previous study of the P. l. lepida pheromone (Islam et al., 2009), and were used in this study. Kováts retention indices of synthetic compounds: DB-23 column; E810:OH 1756, Z8-10:OH 1786, E7,9-10:OH 1891, Z7,9-10: OH 1895, HP-5 column; E8-10:OH 1277, Z8-10:OH 1288, E7,9-10:OH 1299, Z7,9-10:OH 1301. Field Tests For dispensers, the synthetic compounds (> 99 % purity) were dissolved in hexane and applied to rubber septa (white rubber, 8 mm OD; Sigma-Aldrich). Each lure was placed at the center of a sticky board trap (SE-trap®, 30× 27 cm bottom plate with a roof; Sankei Chemical Co., Tokyo, Japan), and the traps hung at a height of 1.5 m, and in intervals of 10 m. The field trial was conducted at four places in 2012: a blueberry orchard in Kanagawa Prefecture (Tsukui-gun, 35.45°N, 139.64°E), a persimmon orchard in Gifu Prefecture (Gifu-shi, 35.26°N, 136.42°E), and mixed fruit gardens, including Chinese plum, peach, and persimmon trees, in Tottori Prefecture (Tottori-shi, 35.50°N, 134.24°E; Tohaku-gun, 35.48°N, 133.77°E). The numbers of captured males were counted each week. Replicated data in each test were log-transformed before analysis by the Tukey-Kramer HSD test. The level of significance in all tests was set at 5 %.

Results GC-EAD and GC-MS Analyses of M. flavescens Pheromone GC-EAD analysis of a crude pheromone extract

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(1 FE) of M. flavescens females indicated two EAG-active components, Comp. I (RT 13.5 min) and Comp. II (RT 14.8 min), as shown in Fig. 1a. While Comp. II eluted with a similar RT to the conjugated dienyl pheromone component (Z7,9-10:OH) of P. l. lepida females, Comp. I eluted much faster. Next, the extract (5 FE) was analyzed by GC/MS. Figure 2a–c shows the total ion chromatogram (TIC) and mass spectra of Comps. I and II. Since the characteristic m/z 61 of acetates was not detected in either compound, the largest fragment, at m/z 138, of Comp. I (RT 12.05 min) was assigned as [M-18]+ of a C10 monoenyl alcohol, and the m/z 154 and 136 of Comp. II were assigned as M+ and [M-18]+, respectively, of a C10 dienyl alcohol. The mass spectrum of Comp. II (RT 13.21 min) included the diagnostic ions m/z 67 (100 %) and 54 (58 %) of terminal conjugated dienes, and was similar to that of synthetic Z7,9-10:OH (RT 13.27 min). A small difference in RTs between the natural and synthetic material indicated that Comp. II was E7,9-10:OH. The double-bond position of Comp. I was determined by DMDS derivatization. The DMDS adduct of Comp. I showed a mass spectrum with

a

I

II

EAD

0.1 mV

FID 10.0

20.0

15.0

b

RT (min)

c

EAD

0.1 mV

10.0

15.0

10.0

15.0 RT (min)

Fig. 1 Coupled gas chromatography-electroantennogram detection (GC-EAD), using flame ionization detection (FID), of: (a) sex pheromone extract of Monema flavescens females (1 female equivalent); (b) synthetic (E)-8-decen-1-ol (10 ng); and (c) synthetic (E)-7,9-decadien-1-ol (2 ng). The antenna of a M. flavescens male responded reproducibly to two components (I and II) in the extract but not to hydrocarbons that eluted after 20 min

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M+ at m/z 250 (42 %) and fragments at m/z 175 (100 %) and 75 (35 %), indicating the double bond of the original compound was at the 8-position (Fig. 2d). RTs of synthetic E8-10: OH (12.05 min) and Z8-10:OH (12.29 min), and GC/FT-IR analysis, described below, confirmed that Comp. I was E8-10: OH. The TIC in Fig. 2a indicated that the pheromone gland content is a 9:1 mixture of E8-10:OH (20 ng/female) and E7,9-10:OH. Male antennae exhibited EAG responses to the two synthetic alcohols (Fig. 1bc).

(E)-isomers of 1,2-disubstituted alkenes. By contrast, synthetic Z8-10:OH (RT 11.01 min) showed an absorption at 3012 cm−1, characteristic of C-H stretching, and a weak absorption at 973 cm−1 and no absorption at 966 cm−1 (Fig. 3b). Figure 3c shows the IR spectrum of Comp. II (RT 11.20 min). The absorption at 951 cm−1 reveals the E configuration of the double bond at the 7-position. In addition to the absorption of the 1,2-disubstitution, other absorptions were detected at 1003 and 903 cm−1, caused by C-H bending of geminal hydrogen atoms at the terminal double bond. Synthetic E7,9-10:OH had a similar GC/FT-IR spectrum. By contrast, synthetic Z7,9-10:OH (RT 11.17 min) showed characteristic absorptions at 3010 and 907 cm−1 and had a weak absorption at 969 cm−1 and no absorption at 951 cm−1 (Fig. 3d). We also focused on several characteristic IR absorptions in the crude pheromone extract (Fig. 4a). The band chromatogram at 3236 cm −1 indicated the two alcohol

GC/FT-IR Analysis of M. flavescens Pheromone Gland extract (20 FE) was analyzed by GC/FT-IR, and the doublebond configuration of the two natural pheromone components determined. Figure 3a shows the IR spectrum of Comp. I (RT 10.85 min); this spectrum is similar to that of synthetic E8-10:OH. The absorption at 966 cm−1 reveals the E configuration of the double bond; absorption at 970– 960 cm−1, caused by C-H bending, is characteristic of all

I

a

II TIC

Intensity (%)

b 100

55

20.0 RT (min)

15.0

10.0

68 81

41

95

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109

138

0 50

c Intensity (%)

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150

m/z

67

100

79 41

54 93

50

107

121

154 (M+)

136

0 50

100

150

75

d

175

100 Intensity (%)

Fig. 2 Gas chromatography/ mass spectrometry analyses of crude pheromone extract of Monema flavescens females (1 female equivalent) and extract (3 female equivalents) treated with dimethyl disulfide (DMDS): (a) total ion chromatogram (TIC), mass spectra of (b) Comp. I [(E)-8decen-1-ol], (c) Comp. II [(E)7,9-decadien-1-ol], and (d) the DMDS adduct of Comp. I

50

0 50

H3CS

75

m/z

175 SCH3

OH 250 (M+)

100

150

200

250

m/z

354

E8-10:OH 3000

b

966

OH

Absorbance

a

Absorbance

Fig. 3 Infrared spectra, obtained by gas chromatography/Fourier transform infrared spectroscopy of pheromone components of Monema flavescens females and synthetic geometric isomers: (a) Comp. I [(E)-8decen-1-ol; E8-10:OH], (b) synthetic (Z)-8-decen-1-ol (Z810:OH), (c) Comp. II [(E)-7,9decadien-1-ol; E7,9-10:OH], and (d) synthetic (Z)-7,9decadien-1-ol (Z7,9-10:OH)

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1000 (cm-1)

2000

OH

3012

973 Z8-10:OH

3000

1000

2000

(cm-1)

c

Absorbance

1003

Absorbance

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1004 969

OH

3010

907

Z7,9-10:OH

components in the extract, with the band chromatograms at 966 and 951 cm−1confirming the E configuration of Comps. I and II, respectively. Peaks also were recorded for these

a

1000

2000

3000

Fig. 4 Infrared band chromatograms at 3236, 3010, 966, and 951 cm−1 from gas chromatography/Fourier Transform infrared spectroscopy analyses of pheromone extracts of two Limacodidae spaces: (a) Monema flavescens (20 female equivalents); and (b) Parasa lepida (1 female equivalent)

951

E7,9-10:OH 3000

d

903

OH

1000 (cm-1)

2000

two components in the band chromatogram at 3010 cm−1, indicating that this absorption (C-H stretching) was not appropriate for specific detection of Z7,9-10:OH.

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E8-10:OH

Z7,9-10:OH

E7,9-10:OH

0 8.0

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3236 cm-1

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GC/FT-IR Analysis of the P. l. lepida Pheromone Pheromone extract (1 FE) of P. l. lepida was analyzed by GC/FT-IR to determine the usefulness of this technique. The IR spectrum, which was almost identical to that of synthetic Z7,9-10:OH (Fig. 3d), was recorded at the expected RT (11.17 min). Absorption at 3010 and 907 cm−1, and no absorption at 951 cm−1, is characteristic of the terminal conjugated dienyl structure with Z configuration. Figure 4b shows the band chromatograms of the GC/FT-IR analysis. The chromatogram at 3236 cm−1 indicated only one alcohol component. A small peak was apparent at 966 cm−1, but there was no peak at 951 cm−1 for the alcohol component, clearly demonstrating the Z configuration. The chromatogram at 3010 cm−1 was not useful for determining the configuration of the terminal conjugated dienes. Field Attraction of M. flavescens Males by Synthetic Lures Lures baited with E8-10:OH and E7,9-10:OH in different ratios were evaluated (Table 1). Both alcohol components, in a ratio of 9:1 (monoene diene), were necessary to attract males. While (Z)-isomers of these compounds are not produced by females, we tested the effects of these compounds added to respective (E)-isomers in order to understand tolerance for the impurities in the synthetic lure. When the ratio of Z8-10:OH was increased, the number of males caught decreased in the test in Tottori. However, no differences in catch with addition of Z8-10:OH to the pheromone blend were observed in the test in Gifu, even though half of the amount of the monoenyl component was replaced by Z8-10:OH (Table 2). Adding Z7,9-10:OH to the pheromone blend resulted in a clear reduction in trap catch (Table 3).

Table 1 Trap catches of Monema flavescens males to lures containing mixtures of (E)-8-decen-1-ol (E8-10:OH) and (E)-7,9-decadien-1-ol (E7,9-10:OH) Lure component (mg/rubber septum)

Captured males/trap a (Total)

E8-10:OH

Kanagawa

1.0 0.9 0.5 0.1 0 0

E7,9-10:OH 0 0.1 0.5 0.9 1.0 0

b

0 2.8±0.6 a (11) 0.8±0.6 b (3) 0 0 0

Tottori

c

0 12.5±2.8 a (50) 0.3±0.2 b (1) 0 0 0

a

Mean±SE. Values within each column followed by a different letter are different at P<0.05 by Tukey-Kramer Test

Table 2 Trap catches of Monema flavescens males to lures containing mixtures of (E)-8-decen-1-ol (E8-10:OH), the (Z)-isomer (Z8-10:OH), and (E)-7,9-decadien-1-ol (E7,9-10:OH) Lure component (mg/rubber septum)

Captured males/trap a (Total)

E8-10: OH

Gifu

0.90 0.81 0.63 0.45 0

E7,9-10: OH

0 0.09 0.27 0.45 0

0.10 0.10 0.10 0.10 0

b

8.7±3.0 a 9.0±1.3 a 8.7±3.3 a 8.3±2.0 a 0

Tottori

(26) (27) (26) (25)

c

15.3±8.5 a (46) 6.3±2.7 ab (19) 7.0±3.3 ab (21) 3.7±1.9 b (11) 0

a

Mean±SE. Values within each column followed by a different letter are different at P<0.05 by Tukey-Kramer Test

b Tested with three traps of each lure in Gifu prefecture (Gifu-shi) from July 13 to 26, 2012 c

Tested with three traps of each lure in Tottori prefecture (Tottori-shi and Tohaku-gun) from July 12 to 29, 2012

Discussion GC-EAD and GC/MS analyses of pheromone gland extract of M. flavescens revealed that females produced two EAGactive C10 alcohols, I and II, in a ratio of 9:1. DMDS derivatization of the monoenyl component I showed a double bond at the 8-position, and diagnostic fragment ions in the mass spectrum of the dienyl component II indicated a conjugated structure unsaturated at the 7- and 9-positions. The E configuration of both components was demonstrated by GC/FT-IR analysis; hence, we assigned E8-10:OH for I

Table 3 Trap catches of Monema flavescens males to lures containing mixtures of (E)-8-decen-1-ol (E8-10:OH), (E)-7,9-decadien-1-ol (E7,9-10:OH), and the (Z)-isomer (Z7,9-10:OH) Lure component (mg/rubber septum)

Captured males/trapa

E8-10:OH

/trapb

E7,9-10:OH

Z7,9-10:OH

Total

0.90

0.10

0

8.0±1.9 a

24

0.90 0.90 0.90 0.90 0.90 0.90 0

0.09 0.07 0.05 0.03 0.01 0 0

0.01 0.03 0.05 0.07 0.09 0.10 0

5.0±1.3 4.0±1.3 1.3±0.3 2.0±0.8 1.0±0.0 0 0

15 12 4 6 3 0 0

b Tested with four traps of each lure in Kanagawa Prefecture (Tsukuigun) from June 29 to July 16, 2012

a

c

b

Tested with four traps of each lure in Tottori prefecture (Tottori-shi and Tohaku-gun) from June 26 to July 12, 2012

Z8-10: OH

ab bc d cd d

Tested with three traps of each lure in Kanagawa Prefecture (Tsukuigun) from July 16 to August 6, 2012 Mean±SE. Values followed by a different letter are different at P< 0.05 by Tukey-Kramer Test

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and E7,9-10:OH for II. These structures as pheromone components were confirmed by field evaluation of synthetic compounds; lures baited with a 9:1 mixture of E8-10:OH and E7,9-10:OH attracted M. flavescens males. Both compounds are new pheromone components identified from insects. The minor component, which is the geometric isomer of the pheromone component (Z7,9-10:OH) of P. l. lepida (Wakamura et al., 2007; Islam et al., 2009), has the terminal conjugated dienyl structure commonly found in Limacodidae pheromones. It has been reported that Darna spp. in Limacodidae produce esters of (E)-7,9-decadienoic acid (Sasaerila et al., 2000a; Siderhurst et al., 2007). The present study shows that alcohol derivatives also are utilized as sex pheromones by nettle moths. C10 monoenyl alcohols and their acetate derivatives, unsaturated at the 3-, 4-, 5-, or 7-position, have been identified from species in the Gelechiidae, Cossidae, and Noctuidae families, while a C12 chain, unsaturated at the 8-position, is a key pheromone structure of many Olethreutinae species (Ando, 2012; ElSayed, 2012); a C10 chain compound, unsaturated at the 8position, has not yet been found in Lepidoptera. With reference to known lepidopteran sex pheromones composed of monoenyl and conjugated dienyl compounds, the combinations fall into two groups. The first group includes a monoene with the double bond located at the same position as the diene. For example, females of Setothosea asigna and Setora nitens in the Limacodidae secrete a mixture of 9dodecenal and 9,11-dodecadienal (Sasaerila et al., 1997; 2000b). If the two components are biosynthesized by a common pathway, the terminal double bond of the diene might be introduced after desaturation at the ω3-position, suggesting biosynthesis by two different desaturation steps. The second group includes a monoene with unsaturation in the middle of the two double bonds in the diene. For example, females of Deilephila elpenor and Theretra oldenlandiae in the Sphingidae secrete a mixture of 11-hexadecenal and 10,12hexadecadienal (Uehara et al., 2012). The same 10,12-dienyl structure of bombykol is biosynthesized by a single bifunctional enzyme acting as a Δ11- and a Δ10,12-desaturase (Moto et al., 2004). The combination of E8-10:OH and E7,9-10:OH produced by M. flavescens females belongs to this second group, suggesting the possible construction of the terminal conjugated dienyl structure via an intermediate unsaturated at the ω2-position. In the Limacodidae, dienyl components do not seem to be produced by a common desaturase. Elucidation of the biosynthetic pathway is an interesting subject for future research. Attraction of males in four different fields demonstrates the usefulness of a synthetic lure for monitoring adults. The field tests also demonstrated that contamination with Z7,910:OH more strongly impaired the attraction to the synthetic lure than did contamination with Z8-10:OH. A small amount of contamination by either (Z)-isomer, however,

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did not affect catches of males. Therefore, high purities of the respective (E)-isomers might not be necessary for a useful lure. In the case of P. l. lepida, 10 % contamination with the opposite geometric isomer of the pheromone component caused a 50 % decrease in male catch (Islam et al., 2009). For these studies, we used a mixture of 1.0 mg of pheromone on a rubber septum as a lure. Since the volatility of C10 alcohols is much higher than that of the typically longer-chain pheromone components of other Lepidoptera, further work is needed to clarify the best dispenser type and dosage for monitoring. Recently, solid-phase GC/FT-IR has been utilized for structure determination of male-produced sex pheromones of Heteroptera, including for methyl 4,8,12-trimethylpentadecanoate in Edessa meditabunda (Zarbin et al., 2012) and 5,9,17-trimethylhenicosane in Phthia picta (Soldi et al., 2012). The IR spectra of the former stink bug and the latter true bug confirmed the presence of an ester group and the absence of any functional groups, respectively. To our knowledge, our study is the first use of solid-phase GC/FT-IR for the identification of lepidopteran pheromones. IR spectra allowed the E configuration of the monoenyl and dienyl components to be assigned, based on the characteristic absorptions at 966 and 951 cm−1, respectively. In the GC/FT-IR analysis of the natural pheromone, the band chromatogram at 951 cm−1 was useful, because the chromatographic separation of the two geometric isomers of a terminal conjugated diene is difficult. We used the absence of absorption in same band chromatogram to confirm the Z configuration of the same 7,9-dienyl alcohol secreted by P. l. lepida females. While an extract of twenty females was required for a useful IR spectrum of the minor dienyl component in M. flavescens, we obtained a practical GC/FT-IR analysis for the P. l. lepida pheromone component from an extract of only one female. This sensitivity is probably sufficient for application of GC/FT-IR to studies of many insect pheromones, particularly for the determination of double-bond configuration. The wavenumber range of the instrument is 4000–700 cm−1, precluding it for analysis of disubstituted (Z)-monoenes, which show a broad absorption band of C-H bending at 730–675 cm−1. The (Z)-monoenes showed a band of C-H stretching at 3012 cm−1, while the configuration of the double bond can be indirectly indicated by the absence of absorption at 966 cm−1, as in the case of the (Z)-diene of P. l. lepida. We are now analyzing synthetic pheromones of many lepidopteran species by GC/FT-IR to obtain a database to aid in the identification of natural sex pheromone compounds. Acknowledgements We thank Prof. June Shimada of the Faculty of Agriculture at Tokyo University of Agriculture and Technology and Mr. Shigeru Miyagi for help with field experiments in Kanagawa Prefecture, and Messrs. Soki Kubota, Tomohiro Takasaka, Ms. Kanako Ohbayashi, Asami Oda, Mai Ohmachi and students of the Laboratory

J Chem Ecol (2013) 39:350–357 of Applied Entomology in Faculty of Agriculture at Tottori University for help with collecting cocoons.

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