Journal of Asia-Pacific Entomology 15 (2012) 413–418

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Journal of Asia-Pacific Entomology

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Sex pheromone composition of selenaria (: Geometridae) and its regional variation in Korea

Kyung San Choi a, Young Mi Park b, Kyung Hee Choi c, Dong Hwan Kim a, Dong Soon Kim d,⁎ a Agricultural Research Center for Climate Change, National Institute of Horticultural and Herbal Science, RDA, Jeju 690-150, Republic of Korea b Jungbu Regional Office, National Plant Quarantine Service, 433-1, Republic of Korea c Apple Research Station, National Institute of Horticultural and Herbal Science, RDA, Gunwi 716-812, Republic of Korea d Majors in Plant Resource Sciences & Environment, Jeju National University, Jeju 690-756, Republic of Korea article info abstract

Article history: This study was conducted to investigate sex pheromone composition of (Lepidoptera: Received 1 September 2011 Geometridae) in Korea. Two sex pheromone compounds such as (Z,Z)-6,9-cis-3,4-epoxynonadecadiene Revised 11 February 2012 (6Z,9Z-cis-3,4-epoxy-19:H) and (Z,Z,Z)-3,6,9-nonadecatriene (3Z,6Z,9Z-19:H) were identified in the glands Accepted 25 February 2012 of A. selenaria females by gas chromatography–mass spectrometry analysis. However, the component 3Z,6Z,9Z-19:H neither elicited an electroantennogram response nor increased the attractiveness for A. selenaria Keywords: males in the field. The role of 3Z,6Z,9Z-19:H seems to be as an antagonistic signal for mating behavior of A. selenaria Ascotis selenaria Sex pheromone males. (Z,Z)-6,9-cis-3,4-epoxynonadecadiene The blend ratios of two 6Z,9Z-cis-3,4-epoxy-19:H isomers such as, 6Z,9Z-cis-3R,4S-epoxy-19:H and 6Z,9Z-cis- (Z,Z,Z)-3,6,9-nonadecatriene 3S,4R-epoxy-19:H, were critical to attract A. selenaria males. The blend ratios of the two isomers showing peak EAG catch of A. selenaria males had large variations among the locations investigated. A. selenaria populations in Gunwi showed peak activity at ratios of 0.9:0.1 and 0.8:0.2, whereas the populations in Goheung, Yeongam, and Jeju (Aewol and Harye) showed peak activity at a 0.5:0.5 ratio. In Changnyeong, the peak activity occurred in a bimodal form at ratios of 0.7:0.3 and 0.4:0.6. Such variation was partially explained by geographical isolation due to mountain ranges. Consequently, the results of our study should be useful for designing a region-specific pheromone lure for successful A. selenaria monitoring. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2012. Published by Elsevier B.V. All rights reserved.

Introduction 3,4-epoxy-19:H) and (Z,Z,Z)-3,6,9-nonadecatriene (3Z,6Z,9Z-19:H) have been identified in the sex pheromone glands of A. selenaria females Ascotis selenaria is an important pest of avocado in Israel, coffee in in Japan (Ando et al., 1997) and Israel (Becker et al., 1990; Cossé et al., Kenya, and tea in Georgia, USSR and also causes severe damage to 1992). However, 3Z,6Z,9Z-19:H did not evoke any behavioral response orange, peanut, alfalfa, apple, lemon, and pecan (Wysoki, 1982). It in A. selenaria in a wind tunnel (Cossé et al., 1992) and had no attractive has been reported as a major pest in citrus orchards in Korea, causing activity in field tests (Becker et al., 1990; Ando et al., 1997). In contrast, direct damage to fruit such as gnawed and deep scars or wide holes as the two 6Z,9Z-cis-3,4-epoxy-19:H isomers such as 6Z,9Z-cis-3S,4R- well as feeding on citrus leaves (Kim et al., 2000; Choi et al., 2011b). epoxy-19:H (SR) and 6Z,9Z-cis-3R,4S-epoxy-19:H (RS), attracted A. This pest occurs in apple, bean, and carrot fields in Korea (Kim and selenaria males in the field. Japanese populations are frequently attracted Beljaev, 2001). Thus, an effective population monitoring tool is required to RS, whereas Israel populations prefer SR. It seems that the optimum for successful management of A. selenaria. pheromone blend of A. selenaria populations varies by geographical A. selenaria sex pheromones have been classified in the type-2 region. The sex pheromone compositions of several agricultural pheromone group based on hydrocarbons with a polyene and/or pests in Korea differ from those reported in the neighboring countries epoxide functional group that are typically found in four large of Japan and China (Boo, 1998; Boo and Park, 2005). Therefore, it is families: Geometridae, Noctuidae, Arctiidae, and Lymantriidae (Millar, necessary to thoroughly examine the sex pheromone composition of 2000; Byer, 2006). (Z,Z)-6,9-cis-3,4-epoxynonadecadiene (6Z,9Z-cis- native A. selenaria populations to apply field monitoring in Korea. The objective of this study was to elucidate the sex pheromone com- position of A. selenaria in Korea in terms of the blend ratios and regional ⁎ Corresponding author at: Majors in Plant Resource Sciences & Environment, Col- variations. A series of studies was carried out, including mating behavior, lege of Applied Life Sciences, Jeju National University, Jeju 690-756, Republic of Korea. Tel.: +82 64 754 3312; fax: +82 64 725 2351. sex pheromone biosynthesis, electroantennogram (EAG) responses of E-mail address: [email protected] (D.S. Kim). male antennae, and field tests of pheromone blends.

1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2012. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2012.02.006 414 K.S. Choi et al. / Journal of Asia-Pacific Entomology 15 (2012) 413–418

Materials and methods Pheromone quantification

Insects and chemicals To quantify the amount of sex pheromone produced in the female gland during scotophase, the abdominal tips of eight female adults Ascotis selenaria larvae, collected from a Jeju citrus orchard in (1-d-old) were excised and soaked individually for 15 min in 20 μl 2007, were reared on a semi-artificial diet (Choi et al., 2011a) in the n-hexane containing 50 ng/μl(Z,Z,Z)-3,6,9-octadecatrienyl acetate laboratory at 25±1°C under 16:8 h (L:D) conditions. Pupae were (3Z,6Z,9Z-18:Ac) as an internal standard at every hour after lights-off. sexed, and emerged adults were provided with a 10% sucrose solution In addition, gland extracts were prepared from 10 females (each of 0, as food. All synthetic sex pheromones (purity>95%), were purchased 1, 2, 3, and 4-d-old) at 5 h after lights-off using the same method from Chemtech B. V. (Amsterdam, the Netherlands). described above for the sex pheromone analysis study. Each 1 μlof extract was injected into the GC–MS, and pheromone quantification Emergence and mating rhythm was conducted under the same GC–MS conditions described above.

The emergence rhythms of male and female adult were EAG response examined individually (♀: 86, ♂: 114). Pupae were placed in a plastic cage (40×30×40 cm), and the number of moths that emerged was Five A. selenaria males (3-d-old) were used in the EAG tests. The checked at 1 h intervals for 24 h. antenn preparation method was the same as for GC–EAD. A charcoal- Groups of 10 females with the same age (0, 1, 2, and 3-d-old) were filtered air stream was blown continuously over the antennae via a placed in each cage (25×25×25 cm) and calling behavior was observed stainless steel pipe during the EAG test. All compounds used in the at 1 h intervals after lights-off. To examine mating rhythm, a single EAG test were dissolved in n-hexane and applied to a filter paper strip female at different ages (0, 1, 2, and 3-d-old) was placed individually (2×80 mm, Whatman No.1), which was finally inserted into a Pasteur in a cage (15×15×15 cm) with two males (1–3-d-old), and mated pipette (15 cm long). The pipette was connected to the stimulus air pairs were counted at 30 min intervals after lights-off. This experiment was replicated nine times.

Pheromone analysis

Seventy-three females (1–2-d-old) were anesthetized with CO2 and their abdominal tips were excised at 5 h after lights-off. The sex pheromone components were extracted by soaking the abdominal tips in 100 μlofn-hexane (HPLC grade) for 15 min. The extract was analyzed by gas chromatography–mass spectrometry (GC–MS) (Shimadzu QP-2010) using a capillary column (30 m×0.32 mm I.D., 0.25 um thick; Rtx5MS). Injector temperature was set to 250 °C. Oven temperature was set at 80 °C for 1 min, raised 10 °C/min to 200 °C, and maintained at 200 °C for 13 min. One microliter of the extract was injected in the splitless mode. Helium was used as the carrier gas and flowed at 2 ml/min into the column. Effluents were ionized by electron impact mass spectrometry while the temperatures of the ion source and interface were maintained at 200 and 250 °C respectively. The peaks were then analyzed by comparing the retention time and ionization patterns with those of authentic compounds. To measure the antennal responses to the sex pheromone compo- nents in the extract, GC–electroantennographic (EAD) tests were conducted with an HP 7890 GC coupled with Syntech EAD system, using a capillary column (30 m×0.32 mm I.D., 0.25 μm thick; HP-5). The end of the column was split into two paths at a 1:1 ratio using a Y-splitter (Agilent Technologies, Santa Clara, CA, USA). One of the paths was connected to a flame ionization detector, whereas the other path was connected to an EAD system through heated transfer. The tem- perature of the injector, detector, and interface was set to 250 °C. Nitro- gen was used as the carrier gas. One microliter of extract was injected in splitless mode. Oven temperature was set to 80 °C for 1 min, increased to 170 °C at 15 °C/min, raised to 200 °C at 10 °C/min, and then held at the final temperature for 15 min. To measure EAG responses, antenna of unmated male adults (1–4-d-old) with both ends cut out, was mounted between the reference and recording electrode of an EAG probe (PRG-2) using an electroconductive gel (Parker Laboratories Inc., Fairfield, NJ, USA). The mounted antenna was then placed close to the outlet of a glass tube (length, 120 mm; I.D., 8 mm). Charcoal-filtered and moisturized air was continuously blown (about 600 ml/min) through the glass tube during GC–EAD. EAG signals were recorded using the Syntech EAG 2000 program (Hilversum, The Netherlands) on a personal computer that included a probe/micromanipulator (MP- Fig. 1. Emergence, calling, and mating rhythms in Ascotis selenaria.(■: scotophase, □: 15), a data acquisition interface box (serial IDAC-232), and a stimulus photophase) (A) Emergence rhythms (♀:86,♂: 114). (B) Calling frequency of 10 females air controller (CS-55). in different age groups. (C) Mating rhythms of nine females in different age groups. K.S. Choi et al. / Journal of Asia-Pacific Entomology 15 (2012) 413–418 415

A Comp. Comp. (tR=12.415) (tR=15.465)

(x10,000,000) 1.50 TIC 1.25 B 1.00 Comp. 0.75 Comp. (tR=16.75)

0.50 (tR=13.87)

0.25

10.0 11.0 12.0 13.0 14.0 15.0 16.0 17.0 18.0 (x10,000) 1.0 79 C Comp. 41 67 0.5 55 108 93 121 262 135 149 206 0.0 163 233 243 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 (x10,000) 1.0 80 Comp.

0.5 41 67 93 55 107 278 121 135 206 0.0 152 161177 192 220 231 249 260 50.0 75.0 100.0 125.0 150.0 175.0 200.0 225.0 250.0 275.0

Fig. 2. Gas chromatography–mass spectrometry (GC–MS) and GC–electroantennographic (EAD) traces of the female Ascotis selenaria gland extracts. (A) GC–EAD trace of male an- tenna in response to the extract. (B) GC–MS total ion chromatogram of the gland extract (n=73). (C) Mass spectra of the sex pheromone components: Compound I is 3Z,6Z,9Z-19:H and Compound II is 6Z,9Z-cis-3,4-epoxy-19:H.

controller (model CS-05) (Synthech) and was used for stimulation. The To evaluate the effect 3Z,6Z,9Z-19:H and 3Z,6Z,9Z-18:H, each signals were amplified with an amplifier (UN-06) (Synthech) and dis- component (0.01, 0.05, 0.1, 0.2 mg) was added onto the lure containing played on a computer with an interface. total 1 mg of the two sex pheromone components, RS and SR, at a To compare the EAG responses with the sex pheromone components 0.5:0.5 ratio. Field attraction tests were conducted using the same and related chemicals, 200 ng of each compound was loaded on the method described above for the citrus orchard in Aewol, Jeju in 2009. filter paper strip. To measure the dose-responses, two synthetic sex pheromone compounds, RS and SR, were tested at serial dilutions from 1 to 10,000 ng on a filter paper strip.

6Z,9Z-3S,4R-epoxy-19:H a Field attraction test 6Z,9Z-3R,4S-epoxy-19:H a 6Z,9Z-19:H b Field trappings were carried out in an apple orchard in Gunwi, Gyeonbuk province and a citrus orchard in Aewol and Harye, Jeju in 3Z,6Z,9Z-19:H b 2009. A mixture of RS and SR ranging from 1.0:0 to 0:1.0 (total 3Z,6Z,9Z-18:H b 1 mg) was loaded into a rubber septum (11 mm O.D., Wheaton). 3Z,6Z,9Z-18:Ac b Wing traps (Green Agro Tech Co., Ltd. Korea) were hung on a branch Hexane b of the outer canopy of trees at 1.2–1.5 m height with 5 m intervals. Air This experiment was replicated three times. b In 2010, field trappings were conducted with the same sex phero- 0 4 8 121620 mone treatment mentioned above in the same apple orchard in EAG response (mV) Gunwi, Gyeongbuk province, two persimmon orchards in Changnyeong, Gyeongnam and in Yeongam, Jeonnam respectively, and at a citron Fig. 3. Electroantennogram (EAG) responses of male Ascotis selenaria to the synthetic pheromone and related compounds (200 ng each) (N=5, R=5). Bars with the same orchard in Goheung, Jeonnam province. Lures were prepared the same letters are not significantly different as determined by Duncan's multiple range test as in 2009 but the total amount used was 0.5 mg. (α=0.05). 416 K.S. Choi et al. / Journal of Asia-Pacific Entomology 15 (2012) 413–418

30 a 25 a 20 15 b bc 10 d cd 5 d d d d d d EAG response (mV) 0 1 1 10 10 Air 100 100 1000 1000 10000 10000 Hexane 6Z,9Z-3R4S-epoxy-19:H (ng) 6Z,9Z-3S4R-epoxy-19:H (ng)

Fig. 4. Electroantennogram (EAG) dose-responses of male Ascotis selenaria to different amounts (ng) of the two sex pheromone components (N=5, R=5). Bars with the same let- ters are not significantly different as determined by Duncan's multiple range test (α=0.05).

Data analysis Time- and age-dependent amount of pheromone

All data were analyzed with the SAS program (SAS Institute Inc., The pheromone component 6Z,9Z-cis-3,4-epoxy-19:H in the glands of 1999). Means in each experiment were compared by the Duncan's 1-d-old female started to be produced after lights-off and the amount in- multiple range test (P=0.05). creased continuously during scotophase (F=5.95; df=6, 49; Pb0.0001) (Fig. 5A). However, the component 3Z,6Z,9Z-19:H was detected at 13.2± 0.58 ng in the gland at 0 h, and the concentration was not significantly Results different throughout scotophase (F=2.13; df=6, 49; Pb0.066) (Fig. 5A). Female age affected pheromone production (Fig. 5B). Newly Emergence and mating rhythms emerged females (0-d-old) produced 113.0±14.99 ng 6Z,9Z-cis-3,4- epoxy-19:H, but it decreased rapidly to 15.8±5.47 ng in 4-d-old females Most A. selenaria adults emerged between 3 h before lights-off and (F=5.39; df=4, 45; P=0.0012), when examined at 5 h after lights-off. 3 h after lights-off. Peak times of emergence occurred at 0 h after and The concentration of 3Z,6Z,9Z-19:H in the gland of 0-d-old females was 1 h before lights-off in females and males, respectively (Fig. 1A). 9.0±1.10 ng, and it increased significantly to 25.2±8.13 ng in 3-d-old Adult females exposed their sex pheromone glands on the terminal females ( F=2.62; df=4, 45; P=0.0471). Finally, the amount of the abdominal segment to call males by arching their abdomen dorsally. two sex pheromone components was at a 1:1 ratio in 4-d-old females. Calling behavior usually started from 3 h after lights-off, peaked at 6 h after lights-off, and extended for 2 h after the scotophase (Fig. 1B). The A. selenaria mating pairs were frequently observed between 2 h and 9 h after lights-off with a peak time between 5 h and 7 h A 150 6Z,9Z-cis-3,4-epoxy-19:H a after lights-off (Fig. 1C). Two of 34 mated females mated twice with 120 3Z,6Z,9Z-19:H different males. The duration of copulation was 191.5±12.41 min ab ab ab (n=34). 90 bc 60

Analysis of pheromone components (ng/gland) ab` a` 30 bc` abc` abc` abc` Distinct EAG activity was observed from male antennae 15.465 min amounts Pheromone c c` c (component II) after the gland extract was injected into the GC 0 (Fig. 2A). Two components, known as the A. selenaria sex pheromone 0245678 components, were detected (Fig. 2B). Mass spectra of component II Hours after lights-off

(M=278) showed characteristic ions at m/z M-71 (M−[CH3CH2CH-O- + − − + B 150 CH] ), at m/z 206 and M-198 ([ CH2CH=CHCH2CH=CH ] )andat a m/z 80, which was identical to those of synthetic RS and SR, respectively ab 120 (Fig. 2C). Although clear EAD-activity was not observed in the male an- tennae at 12.415 min (Fig. 2A), mass spectra of component I (M=262) 90 + showed the characteristic ions at M-56 (M−[CH3CH2CH=CH] )at + bc m/z 206, M-154 ([−CH2CH=CHCH2CH=CH−] )atm/z 108, and 60 bc M-183 ([−CH CH=CHCH CH=CH−]+)atm/z 79 and was identified (ng/gland) a` 2 2 c as 3Z,6Z,9Z-19:H (Fig. 2C). 30 ab` ab` Pheromone amounts Pheromone b` b` 0 01234 EAG response to sex pheromones and related analogue compounds Days after emergence

Two isomers of 6Z,9Z-cis-3,4-epoxy-19:H, RS and SR, elicited signif- Fig. 5. Changes in the amounts of 6Z,9Z-cis-3,4-epoxy-19:H and 3Z,6Z,9Z-19:H pro- icantly stronger responses than those of other compounds (F=181.61; duced in the sex pheromone glands of Ascotis selenaria. (A) Temporal profile of the pheromone quantity in 1-d-old females during scotophase (n=8). (B) Effect of female df=5,24; Pb0.0001) (Fig. 3). Male antennae showed dose-dependent age on biosynthesis of the sex pheromone components in the glands extracted at 5 h fi EAG-activity to both isomers. The EAG responses increased signi cantly after lights-off (n=10). Bars with the same letters are not significantly different as de- from 200 ng (F=29.0; df=11, 48; Pb0.0001) (Fig. 4). termined by Duncan's multiple range test (α=0.05). K.S. Choi et al. / Journal of Asia-Pacific Entomology 15 (2012) 413–418 417

Table 1 Mean numbers of Ascotis selenaria males caught in traps baited with various ratios of 6Z,9Z-cis-3R,4S-epoxy-19:H (RS) and 6Z,9Z-cis-3S,4R-epoxy-19:H (SR); 1 mg per lure in 2009 and 0.5 mg per lure in 2010.

Lure composition Trap catches

Gunwi Chanynyeong Goheung Yeongam Aewol Harye

RS SR Aug. 8–Sep. 7, 2009 Jun. 10–Jul. 22, 2010 Jun. 10–Jul. 22, 2010 Jun. 9–Jul. 21, 2010 Jun. 9–Jul. 21, 2010 Jul. 21–Aug. 19, 2009 Jul. 21–Sep. 8, 2009

1.0 0.0 1.3 abc 2.7 ab 3.0 bcde 0.0 b 0.3 ef 0.0d 0.0 c 0.9 0.1 2.7 a 2.3 abc 5.0 abcd 1.7 b 0.7 def 0.3d 0.3 c 0.8 0.2 1.7 ab 3.7 a 6.0 abc 3.0 b 2.7 bcdef 0.3d 0.3 c 0.7 0.3 0.0 c 2.0 abcd 8.3 a 3.3 b 3.7 bcd 3.3 abc 1.7 c 0.6 0.4 0.0 c 2.3 abc 6.0 abc 7.7 a 4.7 abc 3.0 bc 5.3 a 0.5 0.5 0.0 c 2.0 abcd 6.7 abc 8.0 a 5.7 ab 5.3 a 4.7 ab 0.4 0.6 0.3 c 0.7 bc 8.0 ab 8.7 a 3.3 bcde 4.0 abc 2.0 bc 0.3 0.7 0.0 c 0.7 bcd 6.7 abc 2.3 b 6.7 a 4.3 ab 2.3 bc 0.2 0.8 0.0 c 1.0 bcd 4.7 abcde 1.0 b 1.7 cdef 2.0cd 1.0 c 0.1 0.9 0.0 c 0.3cd 2.0 cde 0.3 b 3.0 bcdef 0.3d 1.0 c 0.0 1.0 0.0 c 0.3cd 0.3 de 0.0 b 0.7 def 0.0d 0.0 c 0.0 0.0 0.0 c 0.0d 0.0 e 0.0 b 0.0 f 0.0d 0.0 c

Means followed by the same letters in a column are not significantly different as determined by Duncan's multiple range test (α=0.05).

Field experiments study, as reported in Japanese and Israel populations (Cossé et al., 1992; Ando et al., 1997). The trap catches of A. selenaria males differed significantly between The roles of 3Z,6Z,9Z-19:H in the pheromone communication system different blend ratios based on the binary combinations of RS and SR of A. selenaria are controversial. Witjaksono et al. (1999) reported a (Table 1). Additionally, the optimal blend for attracting males was differ- synergistic effect of 3Z,6Z,9Z-19:Hwhenitwasmixedwithepoxydiene ent among locations. The peak catches in Gunwi were observed at a ratio in a very low ratio (0.01 mg in total 1.0 mg). However, many researchers of 0.9:0.1 in 2009 and a ratio of 0.8:0.2 in 2010 (2009: F=3.45; df=11, have reported an antagonistic effect or no activity of 3Z,6Z,9Z-19:H in 24; P=0.0054, 2010: F=3.04;df=11,24;P=0.0011). In Changnyeong, the A. selenaria pheromone communication system. The addition of a wide range of pheromone compositions between 0.9:0.1 and 0.2:0.8 3Z,6Z,9Z-19:H decreases male responses in a wind tunnel and eliminates was attractive to males with bimodal peaks at 0.7:0.3 and 0.4:0.6 ratios attraction in the field (Cossé et al., 1992), and also moderately inhibits (F=3.51; df=11, 24; P=0.0049), respectively. In Goheung, Yeongam, male orientation and the mating of tethered females (Ohtani et al., Aewol, and Harye, peak catches were generally observed around a 2001). Hadar and Wysoki (1989) reported that delayed mating 2 d 0.5:0.5 ratio (Goheung: F=6.85; df=11, 24; Pb0.0001, Yeongam: after A. selenaria female emergence results in low egg hatchability, and F=5.27; df=11, 24; P=0.0003, Aewol: F=8.39; df=11, 24; no hatching when mating was delayed 3 d or more. Additionally, in Pb0.0001, Harye: F=3.76; df=11,24; P=0.0032). field trapping with virgin females, males were less attracted from the The addition of 3Z,6Z,9Z-19:H to the 0.5:0.5 (total 1 mg) ratio of 3rd day after female emergence and exhibited almost no attraction the binary blend did not affect male catches (F=0.94; df=4, 10; thereafter. In our study, the relative proportion of 3Z,6Z,9Z-19:H in the P=0.4798) (Fig. 6). sex pheromone gland of A. selenaria sharply increased in 3-d-old females. Watanabe et al. (2007) reported that the pheromone binding Discussion proteins in the male antennae of A. selenaria do not show a significant difference in affinity from the synthetic sex pheromone and related Two sex pheromone components of 6Z,9Z-cis-3,4-epoxy-19:H and compounds. Accordingly, we consider that the relative increase in 3Z,6Z,9Z-19:H were identified in the sex pheromone glands of A. selenaria 3Z,6Z,9Z-19:H acts as an antagonistic signal or a competent with 6Z,9Z- females by GC–MS analysis. In Israel and Japan, the same sex pheromone cis-3,4-epoxy-19:H in male antennae so that the mating behavior of A. components were found in A. selenaria females (Becker et al., 1990; Cossé selenaria males decreased, although further studies are required to et al., 1992; Ando et al., 1997). However, the component 3Z,6Z,9Z-19:H verify this hypothesis. neither elicited an EAG-response on the male antenna in GC–EAD studies The EAG experiment results in Figs. 3 and 4 are contradictory nor increased the attractiveness for A. selenaria males in the present because SR and RS (200 ng each) were significantly different from the other compounds and hexane in Fig. 3, but the 100 ng level of these two compounds was not significant compared to hexane or air. In the 10 experiment depicted in Fig. 4, serial dilutions of pheromone compo- a nents ranging from 1 to 10,000 ng were sequentially applied to the A. 8 selenaria antennae. Thus, the antennae may have acclimated to the a stimuli resulting in less of a response. In contrast, a sudden application 6 a of higher doses of pheromone components may have invoked strong a 4 responses in the experiment shown in Fig. 3. a Field trapping studies showed that the blend ratios of the two 2 isomers, 6Z,9Z-cis-3R,4S-epoxy-19:H and 6Z,9Z-cis-3S,4R-epoxy-19: H, were critical for the attractiveness of A. selenaria males. Millar Males capture per trap 0 0 0.01 0.05 0.1 0.2 (2000) reviewed the synergism between enantiomers in lepidopteran Amounts of 3Z,6Z,9Z-19:H (mg) pheromone blends of Semiothisa signaria, Epelis truncataria,andBleptina caradrinalis. Ando et al. (1997) first reported the natural blend of the two Fig. 6. Mean numbers of Ascotis selenaria males captured in the traps baited with isomers, RS and SR, as 47:53 in the A. selenaria female sex pheromone 3Z,6Z,9Z-19:H added onto the lure containing a 0.5:0.5 ratio of 6Z,9Z-cis-3R,4S- gland. In contrast to the composition of isomers in the sex pheromone epoxy-19:H and 6Z,9Z-cis-3S,4R-epoxy-19:H at a citrus orchard in Aewol, Jeju during September 9–29, 2009. Bars with the same letters are not significantly different as de- gland, A. selenaria males were highly attracted to lures containing a termined by Duncan's multiple range test (α=0.05). high content of the RS isomer: a ratio of 10:0 or 9:1 of RS and SR in the 418 K.S. Choi et al. / Journal of Asia-Pacific Entomology 15 (2012) 413–418

1.0 Gunwi Changnyeong 0.8 Goheung Yeongam Jeju (Aewol, Harye) 0.6

0.4

0.2

Relative proportion in attractiveness 0.0 0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 Relative rate of 6Z, 9Z-cis-3S, 4R-epoxy-19:H compared to Major mountain range 6Z, 9Z-cis-3R, 4S-epoxy-19:H Minor mountain range

Fig. 7. Geographic optimum variation blend ratio of 6Z,9Z-cis-3R,4S-epoxy-19:H and 6Z,9Z-cis-3S,4R-epoxy-19:H for Ascotis selenaria in Korea. The original trap catch data (see Table 1) were smoothed using fast Fourier transform filtering between 5 and 12% to make the location of peaks in the actual data more distinct (Jandel Scientific, 1996). The smoothed data were scaled as ratios against the peak catches to compare among localities. The surveyed localities were noted in relation to the major and minor mountain ranges.

field in Japan (Ando et al., 1997). Israeli populations show a stronger References response to the SR isomer than that to RS (Becker et al., 1990; Cossé et al., 1992), which is much different from that of Japanese populations. Ando, T., Ohtani, K., Yamamoto, M., Miyamoto, T., Qin, X.-R., Witjaksono, 1997. Sex pheromone of Japanese giant looper, Ascotis selenaria cretacea: identification and In this study, optimal blend ratios for male attraction showed some field tests. J. Chem. Ecol. 23, 2413–2423. variations among different locations in Korea (Table 1 and Fig. 7). Becker, D., Cyjon, R., Cossé, A., Moore, I., Kimmel, T., Wysoki, M., 1990. Identification The Gunwi area is located in the southeast part of the Korean and enantioselective synthesis of (Z, Z)-6,9-cis-3S,4R-epoxynonadecadiene, the major sex pheromone component of Boarmia selenaria. Tetrahedron Lett. 31, peninsula and is surrounded by a major mountain range. A. selenaria 4923–4926. populations in Gunwi were attracted to the blend with a high RS content, Boo, K.S., 1998. Variation in sex pheromone composition of a few selected lepidopteran which is similar to that of Japan. However, populations in Goheung, species. J. Asia Pac. Entomol. 1, 17–23. Boo, K.S., Park, K.C., 2005. Insect semiochemical research in Korea: overview and prospects. Yeongam, and Jeju (Aewol and Harye), located in the southwest Korean Appl. Entomol. Zool. 40, 13–29. peninsula, were attracted to 0.5:0.5 blend ratio of the two isomers, Byer, J.A., 2006. Pheromone component patterns of moth evolution revealed by computer which is different from the optimal blend of the A. selenaria population analysis of the pherolist. J. Anim. Ecol. 75, 399–407. Cossé, A.A., Cyjon, R., Moore, I., Wysoki, M., Becker, D., 1992. Sex pheromone components of in Japan and Israel as well as that of Gunwi. The Changnyeong area is the giant looper, Boarmia selenaria, Schiff. (Lepidoptera: Geometridae): identification, located between Gunwi and Goheung (also Yeongam), and is isolated synthesis, electrophysiological evaluation, and behavioral activity. J. Chem. Ecol. 18, largelyfromtworegionsbymajorand minor mountain ranges. Thus, 165–181. we speculate that there are two A. selenaria populations in terms of sex Choi, K.S., Park, Y.M., Kim, D.S., 2011a. Rearing method for Ascotis selenaria (Lepidoptera: Geometridae) using an artificial diet. Kor. J. Appl. Entomol. 50, 55–63. pheromone blend ratios, one is in the southeast part (Gunwi area) of Choi, K.S., Park, Y.M., Kim, D.S., Kim, D.S., 2011b. Seasonal occurrence and damage Korea and the other is in the southwest part (Yeonam, Goheung, Jeju of Geometrid moths with particular emphasis on Ascotis selenaria (Geo- area). Changnyeong populations of A. selenaria may consist of various metridae: Lepidoptera) in Citrus orchards in Jeju, Korea. Kor. J. Appl. Entomol. 50, 203–208. sex pheromone blend ratio populations due to geographical characteris- Hadar, D., Wysoki, M., 1989. Studies on the biology and reproduction of the giant looper tics. In many insect species, inter-population and geographic differences Boarmia (Ascotis) selenaria Schiff. Lepidoptera: Schiff. (Lepidoptera: Geometridae). in sex pheromone composition either between Korea and other Alon Hanotea 44, 195–206. Jandel Scientific, 1996. TableCurve 2D. Automated Curve Fitting and Equation Discovery: countries or within Korea are known (Boo and Park, 2005). We report Version 4.0. Jandel Scientific, San Rafael, CA, USA. the inter-population pheromone polymorphism of A. selenaria for the Kim,D.-H.,Kwon,H.-M.,Kim,K.-S.,2000.Currentstatusoftheoccurrenceofthe first time in Korea. insectpestsinthecitrusorchardinChejuIsland.Kor.J.Appl.Entomol.39, 267–274. A. selenaria is an important pest on avocado, coffee, tea, orange, Kim, S.S., Beljaev, E.A., 2001. Family Geometridae. Economic of Korea 8, In: the peanut, alfalfa, apple, lemon, and pecan. This pest is a direct fruit feed- editorial committee of INSECTA KOREANA (Eds), Koreana Suppl. 15. National Institute er on citrus fruits at Jeju (Choi et al., 2011b). Accordingly, population of Agricultural Science and Technology, Suwon, Korea. pp. 106–107. Millar, J.G., 2000. Polyene hydrocarbons and epoxides: a second major class of lepidopteran monitoring using sex pheromones will be useful to successfully sex attractant pheromones. Annu. Rev. Entomol. 45, 575–604. manage A. selenaria in the field. Furthermore, the results of our studies Ohtani, K., Witjaksono, Fukumoto, T., Mochizuki, F., Yamamoto, M., Ando, T., 2001. Mating will be useful for designing a region-specific pheromone lure to success- disruption of the Japanese giant looper in tea gardens permeated with synthetic – fully monitor A. selenaria. pheromone and related compounds. Entomol. Exp. Appl. 100, 203 209. SAS Institute Inc., 1999. SAS OnlineDoc, Version 8.02. SAS Institute, Cary, NC. Watanabe, H., Tabunoki, H., Miura, N., Sato, R., Ando, T., 2007. Analysis of odorant- binding proteins in antennae of a geometrid species, Ascotis selenaria cretacea, Acknowledgments which produces lepidopteran type II sex pheromone components. Invert. Neurosci. 7, 109–118. Witjaksono, Ohtani, K., Yamamoto, M., Miyamoto, T., Ando, T., 1999. Responses of Japanese This study was conducted with the support of the Cooperative giant looper male moth to synthetic sex pheromone and related compounds. J. Chem. Research Program (Project Title: Studies on the development of tech- Ecol. 25, 1633–1642. Wysoki, M., 1982. A bibliography of the giant lopper, Boarmia (Ascotis) selenaria nique to control and predict occurrence of Geometridae moths in citrus Schiffermüller, 1775 (Lepidoptera: Geometridae), for the years 1913–1981. Phyto- orchards; Project Number: PJ006504201007), RDA, Korea. parasitica 10, 65–70.