Appl. Entomol. Zool. 43 (2): 265–269 (2008) http://odokon.org/

Electroantennographic responses and field attraction to peach fruit odors in the fruit-piercing moth, Oraesia excavata (Butler) (Lepidoptera: Noctuidae)

Ruilin TIAN,1 Yohei IZUMI,1 Shoji SONODA,1 Hideya YOSHIDA,1 Takuma TAKANASHI,2 Kiyoshi NAKAMUTA2 and Hisaaki TSUMUKI1,* 1 Research Institute for Bioresources, Okayama University; Kurashiki 710–0046, Japan 2 Department of Forest Entomology, Forestry and Forest Products Research Institute; Tsukuba, Ibaraki 305–8687, Japan (Received 23 August 2007; Accepted 25 December 2007)

Abstract The olfactory responses of adult males and females of the fruit-piercing moth, Oraesia excavata, to lactones as spe- cific components among ripe peach fruit odors were recorded by electroantennogram (EAG) techniques and trap cap- tures in the field. Six lactones (g-hexalactone, g-octalactone, d-octalactone, g-decalactone, d-decalactone and g- dodecalactone) and a green leaf volatile compound (cis-3-hexen-1-ol) as the reference compound for normalization were used to measure EAG responses. The EAG response to g-hexalactone, shown to be the highest among the six lactones tested, did not reach as high as that to a mixture of five lactones when 10% concentrations (v/v) of the lac- tones were used. There was no significant difference between males and females in EAG responses to those com- pounds. In the field experiment, the number of moths captured by traps baited with a mixture of the five lactones (g- hexalactone, g-octalactone, g-decalactone, d-decalactone and g-dodecalactone142 : 7 : 145 : 70 : 28, v/v) was about half that captured with ripe peach fruit; however, the moths were not captured by traps with individual lactones. These results show that O. excavata is attracted by a mixture of lactones, but not by individual lactones, although individual lactones are recognized by antennal receptors.

Key words: Attractant; electroantennogram; lactone; Oraesia excavata; peach fruit odor

fruits, the moths are strongly attracted to peach INTRODUCTION fruit (Uchida et al., 1978) and are mostly captured Fruit-piercing moths are distributed worldwide in peach orchards (Park et al., 1988). In a previous (Muniappan et al., 1995). In Japan, Oraesia exca- experiment, fruit-piercing moths were captured by vata (Butler), O. emarginata (Fabricius) and Adris a trap with ripe peach fruit, showing that olfactory tyrannus amurensis (Staudinger) are the main stimuli in ripe peach fruits may play an important species that attack ripe fruits, including peach, or- role in the attraction of the moths (Tian et al., ange, grape, and pear, among others (Nomura, 2007); however, no moths were captured by a trap 1962; Omori and Mori, 1962; Uchida et al., 1978). baited with an unripe peach or were observed to These moths live in the hills around orchards in the suck unripe peach fruits in an orchard (Tian et al., daytime and fly into orchards to suck ripe fruit unpublished observation). juice for egg maturation at night (Nakajima and Several lactones, such as g-hexalactone, g-octa- Shimizu, 1956; Ohmasa et al., 1991), suggesting lactone, g-decalactone, d-decalactone and g-dode- that the moths utilize the volatile compounds emit- calactone, constituting the fruity aromas increase ted from ripe fruits to search for food. It is well rapidly in peach fruits with ripening (Horvat et al., known that fruit-piercing moths are attracted to 1990; Zhang and Jia, 2005); however, it is still various fruit odors (Kohno, 1962; Saito and Mu- unclear how the fruit-piercing moth reacts to these nakata, 1970; Miyazaki et al., 1972; Uchida et al., fruit odors. In the present study, therefore, we 1978); in particular, among the various kinds of examined electroantennogram (EAG) responses of

*To whom correspondence should be addressed at: E-mail: [email protected] DOI: 10.1303/aez.2008.265

265 266 R. TIAN et al. adult O. excavata moths to lactones contained in pounds were normalized from the decline curves of ripe peach fruit, and compared the number of the responses to the reference at the beginning and moths captured by traps using lactones and ripe end of each series. For all experimental procedures, peach fruit in the field. Because we detected d- an interstimulus interval of 60–120 seconds was octalactone in ripe and overripe peach fruit in our maintained between successive stimulations. Each preliminary experiment, we used the compound as antenna was only used once for each compound. well as five individual lactones to elicit the EAG Green leaf volatile compounds were prepared by response. dilution with paraffin oil at 1% concentration (v/v) for normalization. Lactones were dissolved in paraffin oil at concentrations of 10% and 1% (v/v). MATERIALS AND METHODS A mixture of 10% lactones was prepared by adding Insects. O. excavata larvae were reared on an one portion of the five individual lactones to five artificial diet composed mainly of dried leaf pow- portions of paraffin oil. Furthermore, the resultant der of the host plant (Cocculus trilobus DC.), bean mixture was diluted with paraffin oil for 1% con- powder, wheat germ and dried brewer’s yeast at centration. One microliter of the solution was 25°C under a photoperiod 16L : 8D (Ohmasa et al., applied to a piece of filter paper (0.6 cm1.2 cm) 1991). Furthermore, the larvae were also reared on (Toyo Roshi Kaisha, Ltd., Japan) inserted into a the leaves of the host plant under the same condi- glass Pasteur pipette. The same amount of paraffin tions. Pupae were sexed and placed in screen cages oil was used as a blank control. The tip of the (303030 cm) until adult emergence. Adult pipette was inserted into the side hole of the mix- moths were kept in the same screen cages and pro- ing tube where a continuous charcoal-filtered air vided with a whole apple fruit as food until use. flow (1.2 l/min) was blown through onto the an- The antennae of adult females and males 2 to 4 tenna. Using a stimulus controller (Type CS-55, days after eclosion were used for EAG experi- Syntech®), a 0.5-second puff of air flow (0.6 l/min) ments. was injected through the pipette to simulate the an- Chemicals. A green leaf volatile compound and tenna positioned 20 mm from the outlet of the tube. six lactones were purchased from Tokyo Chemical Field trap test. The field trap test was con- Industry Co., Ltd. (Japan). The chemical purities ducted in the surroundings of peach orchards on a were as follows: cis-3-hexen-1-ol, 98%; trans- hill located in Tamashima in Kurashiki city, Japan 2-hexenal, 98%; g-hexalactone, 98%; g-octalac- (34.3°N; 133.4°E). The plastic funnel trap (Uni- tone, 96%; d-octalactone, 97%; g-decalactone, trap, Sankei Chemical Co. Ltd., Japan) consisted of 96%; d-decalactone, 97% and g-dodecalactone, a bucket (semiclear, 16 cm dia.12.5 cm ht.) at- 95%. Paraffin oil was obtained from Nacalai tached to a funnel (yellow, top; 12 cm and bottom; Tesque, Inc. (Japan). 16 cm dia.7 cm ht.) with a round lid (green, EAG recordings. An antenna was excised at its 16 cm dia.). The eleven traps, baited with ripe base and mounted between two metal electrodes peach fruit (ripeness was determined from sugar using electrically conductive gel (Spectra® 360, content, although this quantitative standard Parker Lab., Inc., Fairfield, New Jersey 07004, changed in different years; ripe peach fruits har- USA). EAG signals through a preamplifier were vested in 2006 contained more than 13% sugars) fed into a 16-bit analog to digital converter (IDAC- and individual lactones (0.5 ml) or a mixture of five 2, Syntech®, Germany) and transferred to a PC lactones (0.5 ml) with or without the two green leaf (IBM AptivaE 2255) for further amplification and volatile compounds contained in glass vials analysis with EAG software version 2.7 (Syn- (0.8 mm dia.40 mm ht.), were hung on branches tech®). A green leaf volatile compound, cis-3- 1–1.5 m above the ground and at a distance of hexen-1-ol (1% v/v) was used as the reference about 15 m (Tian et al., 2007). Two sets of these compound for normalization of all responses rela- funnel traps were prepared in this experiment. The tive to the responses to the reference compound glass vial was placed in the bottom of the funnel (100%). Stimulation with the reference compound trap. The relative proportions of the five lactones was given at the beginning and end of each individ- (g-hexalactone : g-octalactone : g-decalactone : d- ual series of odors. Responses to individual com- decalactone : g-dodecalactone142 : 7 : 145 : 70 : Responses and Attraction to Peach Fruit Odors in O. excavata 267

28, v/v) and two green leaf volatile compounds RESULTS (cis-3-hexen-1-ol : trans-2-hexenal14 : 29, v/v) were prepared according to the report by Jia et al. EAG responses (1999). The ripe peach fruit was renewed at inter- Electroantennogram responses elicited by paraf- vals of two or three days and all chemical com- fin oil were 0.880.07 mV in females and 0.71 pounds were changed weekly. The hanging loca- 0.06 mV in males (n10). EAG amplitudes in re- tions of the traps were randomly changed at inter- sponse to (the standard stimulus) 1 ml of 1% cis-3- vals of 6 days. Traps were checked every two or hexen-1-ol were 2.960.21 mV (meanSE, n10) three days. After the number of captured moths and 2.700.15 mV (meanSE, n10) on female was recorded, the moths were removed from the and male antennae, respectively. Both sexes showed traps. The field test was conducted from 1 to 22 dose-dependent EAG responses to cis-3-hexen-1-ol August 2005 and 5 August to 5 September 2006. and trans-2-hexenal, and the response profiles were Data analysis. Normalized EAG responses (%) similar for alcohol and aldehyde (data not shown). were analyzed using ANOVA after square root The mixture of five lactones elicited stronger transformation of the data. The transformed values response than all individual lactones tested (Fig. 1). were compared by Tukey’s methods when ANOVA EAG responses to individual lactones decreased was significant at the 5% level. The numbers of with increasing carbon chain length among the six moths captured by the traps on each collecting day lactones. No significant differences in EAG re- were analyzed using ANOVA with factors of a day sponses were observed between lactone concentra- (22 times/trap) and test samples (peach fruit, a tions of 1% and 10%, except g-octalactone, in both mixture of five lactones, six individual lactones, sexes (Tukey test, p0.05). Furthermore, no signif- two individual green leaf volatiles and paraffin oil icant differences in EAG responses were observed as a control) followed by the Bonferroni test. between g-octalactone and d-octalactone, and be- tween g-decalactone and d-decalactone.

Field trap test In the experiment conducted from 1 to 22 Au-

Fig. 1. EAG responses (meanSD) on O. excavata (n10) to 1 ml of lactones at concentrations of 10% and 1% (v/v). Mix- ture, mixture of five lactones (see Materials and Methods); g-Hex., g-hexalactone; g-Oct., g-octalactone; d-Oct., d-octalactone; g- Dec., g-decalactone; d-Dec., d-decalactone; g-Dodec., g-dodecalactone. EAG responses were normalized to 1 ml of 1% (v/v) cis- 3-hexen-1-ol. Different letters are significantly different at the 5% level by the Tukey test. 268 R. TIAN et al.

Fig. 2. Number of O. excavata moths captured by individ- ual funnel traps baited with the lactone mixture with or with- Fig. 3. Number of O. excavata moths captured by individ- out two green leaf volatile compounds (cis-3-hexen-1-ol and ual funnel traps baited with ripe peach fruit and a mixture of trans-2-hexenal) and paraffin oil from 1 to 22 August, 2005. five lactones from 5 August to 5 September, 2006. No signifi- Significances among test samples (a mixture of five lactones cant difference in the numbers of moths captured by traps on with and without two green leaf volatiles and paraffin oil as a each collecting day was observed, and significant differences control) were analyzed by the Bonferroni test and different let- between test samples were observed by ANOVA. The captured ters show significant difference at the 5% level. Lactone mix- number of moths is shown as the means of data obtained 11 ture; mixture of five lactones (see Materials and Methods). times with 2 traps. Significances among test samples (peach Glv, cis-3-hexen-1-ol and trans-2-hexenal. fruit, a mixture of five lactones, six individual lactones, two green leaf volatiles and paraffin oil as a control) were analyzed by the Bonferroni test and different letters show significant gust 2005, although two green leaf volatile com- difference at the 5% level, although data of five individual lac- pounds (cis-3-hexen-1-ol and trans-2-hexenal tones and trans-2-hexenal are omitted from Fig. 3, because they were zero. g-Hex., g-hexalactone; cis-3-Hex., cis-3- 14 : 29) were added to a mixture of five lactones, hexen-1-ol. there was no significant difference in the numbers of O. excavata moths captured by traps baited with the lactone mixture between with (0.190.05/day/ male antennae responded to plant volatile com- trap) and without (0.210.06/day/trap) green leaf pounds with similar patterns (Van Der Pers, 1981; volatiles (Bonferroni test, p0.05) (Fig. 2). Fur- Hansson et al., 1989; Topazzini et al., 1990; Elke thermore, similar results were obtained in 2006 and Heinz, 1996). (data not shown). Traps baited with ripe peach fruit Among the individual lactones that were specific and a mixture of five lactones captured O. excavata components produced in ripe peach fruit (Jia et al., moths from 5 August to 5 September 2006, but no 1999), EAG responses decreased with increasing moths were captured by traps baited with individ- carbon chain length. However, in the same carbon ual lactones or green leaf volatile compounds chain lactones, there was no significant difference (some data omitted) (Fig. 3). The numbers of in EAG responses between g and d isomers, sug- moths captured by traps baited with ripe peach gesting that both sexes do not discriminate the ring fruit were significantly different from those cap- structures of these lactones on the antenna. tured in traps with the lactone mixture (Bonferroni Plant odors can be classified into general and test, p0.05). specific volatile compounds (Visser, 1986). Al- though insects respond to a wide variety of plant- derived volatile compounds, they often use specific DISCUSSION compounds as cues to find or avoid certain plants. In the present study, no significant difference in In the present experiment, O. excavata moths were EAG responses to the six lactones was observed captured by a trap baited with a mixture of five lac- between females and males. Furthermore, EAG re- tones; however, the green leaf volatile compounds sponses to these lactones showed almost the same did not affect the number of moths captured. These dose-dependent patterns. These results suggest that results show that the moth may use lactones as spe- both females and males of O. excavata can recog- cific cues to find ripe peach fruit as food. However, nize these compounds emitted from ripe peach the number of moths captured by a trap baited with fruits. In several lepidopteran species, female and a mixture of five lactones was almost half of that Responses and Attraction to Peach Fruit Odors in O. excavata 269 captured with ripe peach fruit, showing that other summary). volatile compounds which attract moths are present Muniappan, R., I. U. Silva-Krott and T. S. Lali (1995) Distri- in the fruit. In fact, the moths attack various fruits bution of larval host plants of the fruit piercing moth, Othreis fullonia. Chemoecology 5/6: 75–77. with or without trace lactones (Nomura, 1962; Nakajima, S. and K. Shimizu (1956) Ecological studies on Omori and Mori, 1962; Uchida et al., 1978). Fur- the akebia leaf-like moth, Adrias tyrannus (Guénée) in- thermore, the EAG response to g-hexalactone, juring citrus fruits. Ôyô-Kontyû 12: 30–34. which is one of the major lactones in ripe peach Nomura, K. (1962) Ecological survey on fruit-piercing fruit (Jia et al., 1999), was nearly as high as that to moths in Japan, with special references to their distribu- tion, general bionomics and injury. In Research on the a mixture of five lactones, but no moths were cap- Control of Fruit-Piercing Moths. Japan Plant Protection tured by the compound trap. These results also Association, Tokyo, pp. 19–35 (in Japanese with English indicate that the moths may require multiple com- summary). ponents to find ripe peach fruits. We are currently Ohmasa, Y., S. Wakamura, S. Kozai, H. Sugie, M. Horiike, C. planning to analyze the volatile compounds Hirano and S. Mori (1991) Sex pheromone of the fruit- released from the surface of peach fruit during piercing moth, Oraesia excavata (Butler) (Lepidoptera: Noctuidae): isolation and identification. Appl. Entomol. ripening and monitor these compounds using traps. Zool. 26: 55–62. ACKNOWLEDGEMENTS Omori, H. and S. Mori (1962) Research on the control of fruit-piercing moths. In Research on the Control of Fruit- The authors express their thanks to Professor G. Okamoto, Piercing Moths. Japan Plant Protection Association, Faculty of Agriculture, Okayama University for his helpful Tokyo, pp. 65–80 (in Japanese with English summary). suggestions regarding peach fruit odors. This study was sup- Park, C. G., W. K. Shin, I. G. Kim and C. H. Kim (1988) ported in part by a Grant-in-Aid for Research Project for Uti- Fruit piercing moths collected at an orchard surrounded lizing Advanced Technologies in Agriculture, Forestry and by forest in Gyeongnam province South Korea. Kor. J. 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