Appl. Entomol. Zool. 42 (3): 375–382 (2007) http://odokon.org/

Sex pheromone of the oriental tussock Artaxa subflava (Bremer) (: Lymantriidae): Identification and field attraction

Sadao WAKAMURA,1,* Tetsuya YASUDA,1,† Yoshio HIRAI,1,‡ Hiroshi TANAKA,2 Teruo DOKI,2 Yoshitsugu NASU,2 Manabu SHIBAO,2 Akira YUNOTANI3 and Kanae KADONO3 1 Laboratory of Behavior, National Institute of Agro-Biological Sciences (NIAS); Tsukuba 305–8634, Japan 2 Agricultural, Food and Environmental Sciences Research Center of Osaka Prefecture; Habikino, Osaka 583–0862, Japan 3 Osaka Prefectural Flower Garden; Kawachinagano, Osaka 586–0036, Japan (Received 5 December 2006; Accepted 5 February 2007)

Abstract Two compounds were isolated and identified from abdominal tips of the female oriental tussock moth, Artaxa (Eu- proctis) subflava (Bremer). The major EAG-active components were identified as 10,14-dimethylpentadecyl isobu- tyrate (M10M14-15:iBu) and 14-methylpentadecyl isobutyrate (M14-15:iBu). Comparative GC-EAD analysis sug- gested that the absolute configuration of C-10 position in M10M14-15:iBu is R. Amounts of these compounds in the extract were ca. 10 and 3 ng/female, respectively. These compounds were the same compounds as those of the sex pheromone of the tea tussock moth, Arna () pseudoconspersa (Strand). In field tests, (R)-10,14-di- methylpentadecyl isobutyrate [(R)-M10M14-15:iBu] showed significant attractiveness to Art. subflava males while M14-15:iBu did not. These results confirmed that (R)-M10M14-15:iBu is a component of sex pheromone of Art. sub- flava.

Key words: Oriental tussock moth; Artaxa subflava; sex pheromone; (R)-10,14-dimethylpentadecyl isobutyrate; 14-methylpentadecyl isobutyrate

domen and anal tufts. Females are often attracted INTRODUCTION by light in the evening and enter human living The oriental tussock moth, Artaxa subflava (Bre- rooms. The fly around the illumination and mer), which had been described in Euproctis scatter the poisonous scales into the air. This also before the taxonomic revision by Holloway (1999), may be a hazard to humans. is distributed throughout Japan, Korea, China and This insect has occasionally had local outbreaks southeastern Siberia (Inoue, 1982). It feeds on a in Japan (Hattori, 1955, 1997; Hirano, 1955; variety of plant leaves (Hattori, 1955). The larvae Asahina and Ogata, 1956; Miyashita, 1961; Ito and have venomous spicules on the dorsal thorax and Takahashi, 1997). The outbreaks are sporadic and abdomen and sting anything that accidentally difficult to predict. Control of the moth has gener- brushes the plants on which they perch. Contact ally relied on chemical pesticides against the larvae can cause “spots” and/or allergy on human skin but public opinion appears directs managers to (Kawamoto et al., 1978; Su, 1981; Kubo et al., minimize chemical spray and develop alternatives 1990a, b). Furthermore, the larvae spin into co- to insecticides. coons and rubs the inside walls with the venomous The NIAS authors’ group has succeeded in scales on the back. When adults, particularly fe- chemical identification of two compounds as can- males, come out of the cocoons, they rotate their didates of sex pheromone components of Art. sub- bodies to acquire the venomous scales on their ab- flava in the mid 90’s. The compounds identified

*To whom correspondence should be addressed at: E-mail: [email protected] † Present address: National Agricultural Research Center (NARC), Kannondai 3–1–1, Tsukuba, Ibaraki 305–8666, Japan ‡ Present address: Sena 7–12–11, Aoi, Shizuoka 420–0911, Japan DOI: 10.1303/aez.2007.375

375 376 S. WAKAMURA et al. were the same compounds as those of the tea tus- liminary observation, illumination was gradually sock moth, Arna pseudoconspersa (Strand) (Waka- increased after the dark period (0.1 lx). Light in- mura et al., 1994, 1996a; Ichikawa et al., 1995; tensity was kept at 1.2 lx for 30 min and then at Zhao et al., 1996, 1998), which had been also Eu- 10.7 lx for 30 min and increased to 430 lx (light pe- proctis before Holloway (1999). We hesitated to riod) (see Fig. 1). present these facts because we failed to catch any Extraction. Solvent extracts of sex pheromone male moths with the synthetic compounds in the glands were obtained from 17 virgin females 3 d Tsukuba area, probably due to low local insect den- after emergence near the beginning of the light pe- sity. In 2005, we started a project to evaluate the riod when calling behavior was most frequently ob- synthetic sex pheromone as a monitoring tool for served (Fig. 1). The tip containing the pheromone Arna pseudoconspersa in various areas in Osaka gland was excised from the female abdomen with and Wakayama Prefectures. The Osaka group fine tweezers and soaked in hexane (5–10 tips/ found that Art. subflava males were also captured 0.5 ml) for ca. 15 min. The tips were filtered off in the traps together with Arna pseudoconspersa through a small piece of cotton to obtain an extract. males. These circumstances allowed us to conduct The tips were rinsed twice with the same volume field evaluations at several sites in Osaka and of hexane and the rinses were added to the extract. Wakayama in 2006. Extracts were concentrated to ca. 0.1 ml and This paper deals with identification and field poured onto 1 g of the Florisil (100–200 mesh, evaluation of sex pheromone components of Art. Floridin Co., USA) column and 20 ml each of subflava. Synthetic sex pheromone can provide a hexane, 5% and 15% ether in hexane was succes- useful tool for forecasting sudden outbreaks of this sively used as the eluting solvent. hazardous insect pest. Gas chromatography (GC) and electroan- tennographic detection (GC-EAD). GC analyses were conducted on a Hewlett-Packard (HP) 5890 MATERIALS AND METHODS series II gas chromatograph equipped with a flame Insect. Art. subflava larvae were collected from ionization detector (FID). A DB-1 or DB-WAX leaves of various trees in May and June of 1994 fused silica column (30 m0.25 mm I.D.0.25 mm and 1995 at Tsukuba, Ibaraki and Ôtawara and film thickness, J & W Scientific, Folsom, CA, Nishinasuno, Tochigi, Japan. The plants from USA) was used at a column head pressure of which we collected the larvae were Prunus mume 110 kPa. Injection was made directly into the capil- Sieb. & Zucc., Pourthiaea villosa var. laevis lary column through an on-column injector held at (Rosaceae), and Quercus serrata Murray, Q. 53°C and the temperature was controlled at oven acutissima Carruthers (Fagaceae). Larvae were temperature plus 3°C after injection. The tempera- reared on an artificial diet (Insecta LF, Nihon ture program (TP) for the column oven was 50°C Nosan Kogyo, Co. Ltd., Kanagawa, Japan) in the for 1 min, 50 to 150°C at 20°C/min, 150 to 240°C laboratory at 25°C and L16:D8 until emergence. (or 270°C) at 5°C/min and held at the final temper- Pupae were taken from cocoons by dissolving the ature for 5 min [abbreviation for this TP: 50(1)-20- latter with 1% sodium hypochlorite solution, and 150(0)-5-240(5) or -270(5)]. the urticating hairs were washed away with tap An electroantennographic (EAG) response from water. Pupae were sexed and maintained in sepa- the male antenna was obtained simultaneously with rate containers until emergence. the FID recording. The electroantennographic de- Observation of calling behavior. One- and 3-d- tector was set up according to Struble and Arn old females were individually placed in glass cylin- (1984). ders (9 cm diam.6 cm ht.) with a piece of mois- Coupled gas chromatography–mass spec- tened cotton. Female behavior was observed at 1-h trometry (GC-MS). GC-MS analyses were con- intervals from the beginning of the dark period, and ducted with an HP-5890II gas chromatograph con- at 15-min intervals for 2 h after the end of the dark nected to a JEOL JMS SX-102A mass spectrome- period. A dim red light from a torch was used for ter (EI mode, 70 eV). For injection to the GC, an observation during the dark period. Since calling on-column injector was used. GC was equipped by females began just after “light on” in the pre- with a DB-WAX column (30m0.25 mm I.D. Tussock Moth Pheromone 377

0.25 mm film thickness). The carrier gas was he- Chemical Co. Ltd. for the previous study on Arna lium and the column head pressure was 35 kPa. pseudoconspersa pheromone (Wakamura et al., The TP for the column oven was 50(1)-10-230(10). 1994). In certain analyses, n-alkanes with an even number Trap and field tests. Synthetic chemicals, (R)- of carbon atoms (between 20 and 26) were added M10M14-15:iBu and M14-15:iBu, were blended at as internal standards for the calculation of Kovát’s various ratios between 100 : 0 and 0 : 100 (total retention index (KI; Kovát, 1965). amount: 5 mg) in 300 ml of hexane. The solutions Derivatization to alkyl chloride. Naturally oc- were applied into the depressions of rubber septa curring esters from Art. subflava (ca. 230 ng15 (9 mm O.D., 19 mm ht., gray, West Co., Singapore). female equivalents) were hydrolyzed in 200 ml of The septa were placed in a draft chamber overnight 1% KOH in ethanol in a small sealed glass tube at to evaporate the solvent completely, and stored at 50°C for 20 h. Ethanol was evaporated under re- below 20°C until use. duced pressure after 20 ml of decane was added. A tent-shaped sticky trap (29 cm32 cm8cm The residue was then extracted with hexane and ht., SE trap (white type), Sankei Chem. Co., poured on to a silica gel column chromatograph Kagoshima, Japan), with a sticky plate (24 cm (500 mg, Wakogel® C-200, Wako Pure Chem. Ind., 30 cm), was used for field tests. Each rubber sep- Osaka). The compounds were successively eluted tum was placed on the center of a sticky plate. with 5 ml each of hexane, 5% and 20% ether in Traps were placed at about 1.2–1.8 m above the hexane and ether. Alcohols were eluted with 20% ground at 5- to 10-m intervals. Trap catches were ether in hexane and this fraction was concentrated counted at 1-week intervals from late June to early to several microliters. The chloride derivatives September 2006. Field tests were conducted from were prepared with the alcohol solution and 25 ml late June to early September at five sites in four each of methanesulfonyl chloride and dimethyl- different areas in Osaka and Wakayama Prefectures formamide in a small sealed glass tube at 65°C in 2006: (1) Shakudo, Habikino, Osaka (two sites), for 20 h (Evans et al., 1968). The reaction mixture (2) Takou, Kawachinagano, Osaka, (3) Koda, was then placed directly on a silica gel column Tondabayashi, Osaka, and (4) Nakado, Hashimoto, (500 mg) and the chloride derivatives eluted with Wakayama. 5 ml of hexane. Statistical analyses. Data were transformed to Chemicals. Authentic chemicals used for chem- (X0.5)1/2 and submitted to 2-way-layout ANOVA ical identification were the lots that had been where zero data (mean0) were omitted. The synthesized for comparative analyses of sex means were ranked by Tukey’s method when pheromone components: 14-methylpentadecyl iso- ANOVA was significant at the 5% level (Figs. 5 butyrate (M14-15:iBu) (Wakamura et al., 1994), and 6). Means accompanied by the same letter are and (R)- and (S)-10,14-dimethylpentadecyl isobu- not significantly different, and the same letter was tyrates [(R)-M10M14-15:iBu and (S)-M10M14- given to the zero data when the confidential range 15:iBu, respectively] (Ichikawa et al., 1995). The of the mean contained zero [(00.5)1/2]. When enantiometric purities of the latter enantiomers ANOVA was not significant, standard error and were 98% e.e. (Ichikawa, personal communication) confidential range of mean were calculated and the while the gas chromatographic purities of all these same letter was given to the zero data in the same compounds were more than 99%. For the field ex- manner as above. periments, (R)-M10M14-iBu was prepared accord- ing to Ichikawa et al. (1995). M14-15:iBu was syn- RESULTS thesized from 6-methylhept-1-yne and 8-bromo- octyl pyranyl ether via acetylene coupling reaction Calling time and by subsequent hydrogenation and esterifica- Calling behavior was observed just after the time tion. These also had the same purities as above. when light intensity increased to 1.2 lx (Fig. 1). 10,12-Dimethylpentadecyl isobutyrate, 10,13-di- Calling frequency increased to almost 100% when methylpentadecyl isobutyrate and 10-methylhexa- light intensity increased to 10.7 lx. This high rate decyl isobutyrate were of laboratory stock synthe- continued for about 1 h and then decreased within sized and provided by Dr. Fukumoto of Shin-Etsu 5h after the start of the light period. 378 S. WAKAMURA et al.

Fig. 1. Calling frequency of 1- and 3-d-old females of Ar- taxa subflava in the laboratory (25°C, L15:D9). Light intensity Fig. 2. GC-EAD chromatograms of (1) crude extract and was 0.1 lx during the dark period, increased to 1.2 lx at h 8, (2) 5%-ether-in-hexane fraction of Artaxa subflava virgin fe- 10.7 lx at h 8.5, and 430 lx (light period) at h 9. males. EAG for response B was ca. 0.25 mV for crude and 5% fraction, and peak sizes for compound B corresponded to that of ca. 1.5 ng and ca. 0.4 ng of authentic M10M14-15:iBu, re- Extraction and structure elucidation of EAG- spectively. GC column: DB-1, 30 m, TP: (1): 50(1)-20-150(0)- factive compounds 5-280(5), (2): 50(1)-20-150(0)-5-240(5). On-column injection. When 1 female equivalent (FE) of 3-d-old virgin female extract (17 FE) was injected into GC-EAD, two distinct EAG responses were observed at tR 17.65 min (compound A: KI 2091) and tR 18.38 min (compound B: KI 2133) on apolar DB-1 column (Fig. 2). These two EAG-active com- pounds were eluted from the Florisil column with 5% ether in hexane (5% fraction). The amounts of the compounds A and B were estimated to be ca. 10 ng/FE and ca. 3 ng/FE, respectively. In the GC- MS analyses of this fraction, mass spectra of com- pounds A and B were measured (Fig. 3). Com- pound A showed a molecular ion at m/z 312 (rela- tive intensity: 4.7%) and fragment ions at m/z 224 (M88: 18%) and m/z 89 (base). Compound B showed a molecular ion at m/z 326 (21%), diagnos- tic fragment ions at m/z 238 (M88, 5.6%) and m/z 89 (base). These spectra and the KI values were quite similar to those of the sex pheromone component of the tea tussock moth Arna pseudo- Fig. 3. Mass spectra of EAG-active components in virgin conspersa (Wakamura et al., 1994), which sug- female extract of Artaxa subflava. Tussock Moth Pheromone 379 gested that these are M14-15:iBu and M10M14- butyrate [(R)-M10M14-15:iBu], although the pos- 15:iBu. sibility of (S)-enantiomer or a mixture of the enan- In order to examine the assumptions above, alkyl tiomers cannot be completely excluded. chlorides derived from the esters (ca. 15 FE) in the 5% fraction by hydrolysis and subsequent chlorina- Field attraction tion were analyzed with GC-MS equipped with DB- When blends of M14-15:iBu and (R)-M10M14- WAX. Two alkyl chlorides were detected at a ratio 15:iBu were baited in the traps, Art. subflava males of ca. 1 : 3 and their retention times and mass spec- were captured with 25 : 75 to 0 : 100 blends signifi- tra were identical to those of authentic 1-chloro-14- cantly more than the other blends (Fig. 5), while no methylpentadecane (tR 13.71 min) and 1-chloro- individuals were recognized in the larval survey in 10,14-dimethylpentadecane (tR 14.71 min), respec- the preceding season. At Hashimoto, capture of tively [see Wakamura et al. (1994) for the mass Art. subflava males was observed with blends con- spectral data and interpretations]. These results sup- ported the identification that the original com- pounds have alcohol moieties of 14-methylpen- tadecan-1-ol and 10,14-dimethylpentadecan-1-ol. As for compound B, further comparative GC analyses were conducted on the DB-WAX column. The retention value of compound B was tR 23.37 min, identical to that for M10M14- 15:iBu. The other positional isomers showed dif- ferent retention values: tR 23.06 min and 23.25 min for the two diastereomers of 10,12-di- methylpentadecyl isobutyrate, and tR 23.62 min for 10,13-dimethylpentadecyl isobutyrate and tR 24.29 min for 10-methylhexadecyl isobutyrate. Compounds A and B were thus confirmed to be M14-15:iBu and M10M14-15:iBu, respectively. Male antennae showed stronger EAG reponse to the (R)-enantiomer than to the (S)-enantiomer (Fig. 4). This suggested that compound B has (R)-con- formation, i.e. (R)-10,14-dimethylpentadecyl iso-

Fig. 5. Capture of Artaxa subflava males with blends of 14-methylpentadecyl isobutyrate (M14) and (R)-10,14-di- methylpentadecyl isobutyrate (M10M14) (dose: 5 mg/septum). Values indicated are the means for one trap for 3–8 weeks; Hashimoto: 8 weeks (24 June–8 July and 22 July–26 August Fig. 4. Relative EAG of (R)- and (S)-10,14-dimethylpen- 2006), Kawachinagano: 3 weeks (26 June–3 July and 1 Au- tadecyl isobutyrate enantiomers. EAG values were normalized gust–14 August 2006), and Tondabayasi: 3 weeks (26 June–16 for 1.0 at 5 ng of the racemic mixture. Indicated values are the July 2006). Values presented are means and SE (bar) back- means and SE (bar) of the normalized values for four replica- transformed from (X0.5)1/2 transformation and two-way lay- tions. Means accompanied with the same letter are not signifi- out ANOVA. Means accompanied with the same letter are not cantly different by t-test (p0.01). Doses indicated are the net significantly different at p0.05 level. No males were cap- amount to be applied toward the antenna. tured at Habikino. n.t.: no tests. 380 S. WAKAMURA et al.

taining (R)-M10M14-15:iBu, and probably de- pended on the contents of this compound. In Kawachinagano, Art. subflava males were captured only with 25 : 75, 10 : 90 and 0 : 100 blends. At Tondabayashi, a similar tendency was observed, al- though the data were limited due to the trap site size. No males were captured with sole use of M14-15:iBu. At both Habikino sites, no males were captured. Arna pseudoconpersa males were also captured with the same lures (Fig. 6). Profiles of total trap catches of Art. subflava showed broad peaks at Fig. 6. Capture of Arna pseudoconspersa males with Hashimoto and Kawachinagano and a single peak blends of 14-methylpentadecyl isobutyrate (M14) and (R)- at Tondabayashi, while those of Arna pseudo- 10,14-dimethylpentadecyl isobutyrate (M10M14) (dose: cospersa had single peaks from early to mid-July 5 mg/septum). Values indicated are the means for weekly catches of three traps for 2–3 weeks at Hashimoto (2 weeks (Fig. 7). from 08 July to 22 July 2006), Habikino (3 weeks from 27 June to 18 July) and Kawachi-Nagano (2 weeks from 02 July to 18 July). Values presented are backtransformed means and DISCUSSION 1/2 SE (bar) from (X0.5) transformation and two-way layout Two EAG-active compounds were found in the ANOVA, and means accompanied with the same letter are not extract of virgin Art. subflava females and are iden- significantly different at p0.05 level.

Fig. 7. Capture of males of Artaxa subflava (solid circle) and Arna pseudoconspersa (open circle) in Osaka 2006. Values indi- cated are total number for all the traps for one week. The totals were converted to weekly values if the period was longer or shorter than 7 days. Tussock Moth Pheromone 381 tified as M14-15:iBu and M10M14-15:iBu. Com- may be limited to vicinities around camellia and parative GC-EAD analysis suggested the latter tea plants in the flight season of Arna pseudocon- compounds have (R)-chirality (Fig. 4). (R)- spersa. In general, insect habitat is limited by food M10M14-15:iBu showed attractiveness in the field availability. Food plants of Arna pseudoconspersa tests, but blending with M14-15:iBu did not en- larvae are Camellia spp.: C. japonica L. (camellia), hance this (Fig. 5). These facts lead us to consider C. sasanqua Thunb. (sasanqua camellia) and C. that (R)-M10M14-15:iBu functions as a compo- sinensis L. (tea) (Inoue, 1982) while those of Art. nent of sex pheromone of Art. subflava. However, subflava are of a wide variety of plants (Hattori, our conclusion does not completely exclude a pos- 1955). Camellia, sasanqua camellia and tea are sibility that the (S)-enantiomer might also be a planted mainly in public parks and private gardens pheromone component of this insect. Currently it is in domestic areas, while tea plant is cultivated for not possible to examine since enantiomers of tea production in rural areas in Japan. In contrast, M10M14-15:iBu are impossible to separate with host plants of Art. subflava occur in much wider chiral GC columns (Ichikawa et al., 1995; Zhao et rural and urban areas. al., 1998). Cross attraction may cause another problem on With the same compound or blends, Arna species identification when using a pheromone trap pseudoconspersa males were also captured (Fig. for population monitoring. Male Arna pseudocon- 6). The sex pheromone of Arna pseudoconspersa is spersa show color polymorphism of the wings also (R)-M10M14-15:iBu (Wakamura et al., 1994, from light yellow to dark yellow or black: most are 1996a; Ichikawa et al., 1995; Zhao et al., 1996, black or dark yellow but small numbers of light 1998). Although the enantiomer problem (Waka- yellow individuals also appear in the first flight mura et al., 1996a; Zhao et al., 1998) still remains (overwintered population) in July, while most are to be solved before further detailed discussion, light yellow in the second flight in September and cross attraction between these two species is of in- October (Inoue, 1982). Coloration of Art. subflava terest. males is similar to that of the light yellow type of Arna pseudoconspersa is bivoltine and has a Arna pseudoconspersa males. This requires the ob- short flight period between early and mid-July (Fig. server to pay special attention to identification of 7; Inoue, 1982) and the second flight period in captured moths. early and mid-October in temperate Japan (Inoue, Despite some limitations, synthetic (R)- 1982). Art. subflava is univoltine and has a longer M10M14-15:iBu would be useful for work with flight season from late June to early August (Fig. 7; occurrence of Art. subflava males. Hattori, 1955; Inoue, 1982) than the period for the ACKNOWLEDGEMENTS first flight of Arna pseudoconspersa. Consequently the flight of Art. subflava may partially overlap We thank Hiroe Yasui and Toshiharu Akino of NIAS for with the first flight period of Arna pseudocon- critical reading of the manuscript, Kiyo Hasuo of the Osaka Prefectural Flower Garden for encouraging support, and Serge spersa in July (Fig. 7, Hashimoto and Kawachi- Glushkoff for editing the manuscript. Thanks are also due to nagano). 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