Introduction

Many phytophagous insects use odors as cues for orientation to their food resources. Attraction of the insects to their host plants is guided mainly by volatile phytochemicals, which are perceived by chemoreceptor neurons on the antenna (Van Loon 1996). However, knowledge of the mechanisms that guide insects to their host in the field remains limited.

Fruit-piercing moths are distributed worldwide (Muniappan et al. 1995). In Japan, Oraesia excavata (Butler), O. emarginata (Fabricius), and Adris tyrannus amurensis (Staudinger) are the main species that attack ripe fruits, including peaches, oranges, grapes, and pears (Nomura 1962; Omori and Mori 1962; Uchida et al. 1978). Fruit-piercing moths are known to be attracted to various fruit odors (Kohno 1962; Miyazaki et al. 1972; Saito and Munakata 1970; Uchida et al. 1978), in particular, among fruits of various kinds, the moths are strongly attracted to peach fruit (Uchida et al. 1978) and are most often captured in peach orchards (Park et al. 1988). In an earlier study, we clarified the olfactory responses of adult O. excavata males and females to lactones as specific components of ripe peach fruit aromas using electroantennographic (EAG) techniques on moths trapped in the field (Tian et al. 2008). The EAG response to 4-hexanolide, which was present at the highest concentration among the six lactones tested, was not as strong as the response to a mixture of five lactones when 10 % concentrations (v/v) of the lactones were used. In the field-trap test, a mixture of five lactones attracted the moths, but each lactone alone was insufficient to attract the moth. Nevertheless, the number of moths captured using the mixture was almost half that captured using ripe peach fruits. These results suggest that, although they are important as attractants in ripe peaches, other compounds are necessary to attract O. excavata. Actually, peach fruit volatiles include many compounds other than lactones (Derail et al. 1999; Horvat et al. 1990; Jennings and Sevenants 1964; Spencer et al. 1978). The compounds with increased expression in peach fruit during maturation are clearly important for attracting fruit-piercing moths because the moths attack only ripe fruit.

We studied the responses of O. excavata to volatile compounds released by ripe peach fruit. Specifically, we identified peach fruit headspace volatiles during maturation and examined the antennal responses to individual compounds and mixtures using EAG techniques and trap-capturing in the field.

Materials and methods

Insects

The O. excavata larvae were given an artificial diet composed mainly of dried host plant (Cocculus trilobus DC) leaf powder, bean powder, wheat germ, and dried brewer’s yeast at 25 °C under a photoperiod of 16 h light and 8 h day (Ohmasa et al. 1991). Pupae were sexed and placed in screen cages (30 × 30 × 30 cm) until adult emergence. Adult moths were kept in the same screen cages and provided with a whole apple fruit as food until use. The antennae of adult females and males 2–4 days after eclosion were used for the EAG experiments.

Plants and collection of headspace volatile compounds

The ‘Hakutou’ peach fruits (Prunus persica L.) were divided into three stages depending on the degree of maturation. Fruits containing less than 13 % sugar content were classified as immature; those with more than 13 % were classified as ripe. Moreover, ripe fruits kept at room temperature for 3 days were classified as overmature.

A solid-phase microextraction (SPME) triple-phase (divinylbenzene/carboxen/polydimethylsiloxane, 50/30 μm in thickness, 2 cm in length, Sigma-Aldrich Co., St. Louis, Mo, USA) StableFlex fiber was employed for the extraction of volatiles from peach fruit. Each whole fruit (approx. 250 g) was placed in a 500-mL Pyrex® glass jar, covered with aluminum foil, and equilibrated for 30 min at room temperature. The SPME fiber was inserted through the foil and exposed for 30 min. The collected volatiles were then analyzed by gas chromatography and mass spectrometry (GC/MS). The fiber was placed into the injection port of the GC/MS equipment and thermally desorbed for 10 min at 250 °C.

Volatile compound analyses

GC/MS analyses were performed on an Agilent 6890GC/5975MSD (Agilent Technologies, Palo Alto, CA, USA) with a fused silica DB-WAX capillary column (60 m × 0.25 mm, 0.25-μm film thickness, Agilent Technologies). The flow of the helium carrier gas was 1.6 mL/min. The oven temperature was programmed as 50 °C for the first 2 min, followed by an increase of 3 °C/min to 220 °C, then held at 220 °C for 20 min. The injection port, equipped with a 0.75-mm-internal-diameter liner (Sigma-Aldrich Co.), was maintained at 250 °C. The inlet was operated in the splitless mode, and the injection purge on the GC system was turned off for the first 1 min to allow for manual SPME injection. Volatile compounds were identified by comparison of their mass spectra and Kováts retention indices (RIs) with that of an authentic reference standard. When standards were not available, identifications were assigned on the basis of mass spectra and retention indices reported in the Wiley 7th/NIST05 (John Wiley, NY, USA), the NIST Chemistry WebBook (NIST 2011), and VCF Volatile Compounds in Food online database (Nijssen 1963–2014). Each analysis was performed with three repetitions.

Authentic chemicals

All standard compounds were purchased from Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan) and Sigma-Aldrich Co. The following chemical purities were (Z)-3-hexenol, 98 %; ethyl acetate, 99.5 %; propyl acetate, 98 %; ethyl butanoate, 98 %; ethyl hexanoate, 99 %; 3-hydroxy-2-butanone, 95 %; ethyl octanoate, 98 %; 4-hexanolide, 98 %; 4-octanolide, 97 %; 4-decanolide, 96 %; 5-decanolide, 97 %; and 4-dodecanolide, 95 %. Paraffin oil and ethanol were obtained from Nacalai Tesque, Inc. (Kyoto, Japan).

EAG recordings

EAG was measured using the methods of Tian et al. (2008). An antenna was excised at its base and mounted between two metal electrodes using electrically conductive gel (Spectra® 360; Parker Laboratories, Inc., Fairfield, New Jersey 07004, USA). The EAG analyses were performed using a 16-bit analog to digital converter (IDAC-2; Syntech®, Germany) and a PC running EAG ver. 2.7 software (Syntech®). A green leaf volatile compound, (Z)-3-hexenol (1 % v/v), was used as the reference compound for normalization of all responses relative to the responses to the reference compound (100 %). For all experimental procedures, an interstimulus interval of 60–120 s was maintained between successive stimulations. Each antenna was used only once for each compound. Each chemical and the respective mixtures were dissolved in paraffin oil at concentrations of 1 % (v/v). One microliter of the solution was applied to a piece of filter paper (Toyo Roshi Kaisha, Ltd., Japan), which was then inserted into a glass Pasteur pipette. The same amount of paraffin oil was used as a blank control. The tip of the pipette was inserted into the side hole of the mixing tube, where continuous charcoal-filtered airflow (1.2 l/min) was blown through onto the antenna. Using a stimulus controller (Type CS-55, Syntech®), a 0.5-s puff of air (0.6 l/min) was injected through the pipette to simulate an antenna positioned 20 mm from the tube outlet.

Field-trap test

The field-trap test was conducted in areas surrounding peach orchards on a hill and in the peach orchards in Tamashima in Kurashiki city, Japan (34.3°N, 133.4°E). Nine plastic funnel traps (UNITRAP; Sankei Chemicals Co. Ltd., Japan), which had been baited with ripe peach fruit and 0.5 ml of 59.6 % (v/v) ethyl acetate, 0.2 % (v/v) ethyl butanoate, a five-lactone mixture, two-combination mixtures, a mixture of all compounds (the mixture ratio is shown in Table 1), and paraffin oil as a control contained in glass vials (0.8 mm dia. × 40 mm ht.), were hung on branches 1–1.5 m above the ground and at a distance of about 15 m (Tian et al. 2007). Three sets of these nine funnel traps were prepared for this experiment. The glass vial was placed in the bottom of the funnel trap. The ripe peach fruit was replaced three times per week. All chemical compounds were changed weekly. The hanging locations of the traps were randomly changed weekly. Traps were checked three times per week. After the number of captured moths was recorded, moths were removed from the traps. The field test was conducted during 1–31 August 2007 and during 1–31 July 2010.

Table 1 Mixture ratios of peach fruit volatile compounds used for the field-trap test and seasonal survey
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Seasonal survey

A seasonal survey was conducted in the surroundings of peach and orange orchards in Matsuyama city, Japan (33.8°N, 132.8°E). A glass vial with 1 ml of a mixture of all compounds (the mixture ratio is shown in Table 1) was placed in each plastic funnel trap as bait. The traps were hung from tree branches at about 1–1.5 m above the ground around the peach orchard. Three traps were prepared for this experiment. The traps were set up initially in late May 2008 and were maintained until the end of October 2008. The traps were checked and the bait was changed on a weekly basis.

Data analysis

Normalized EAG responses (%) were analyzed using ANOVA after square root transformation of the data. The transformed values were compared using Tukey’s method when ANOVA indicated significance at the 5 % level. The quantities of moths captured by the traps on each collection day were analyzed using ANOVA with factors of the day and test samples (ripe peach fruit, ethyl acetate, ethyl butanoate, five-lactone mixture, two-combination mixtures, a mixture of all compounds, and paraffin oil as the control), followed by Bonferroni’s test.

Results

Peach fruit volatile compounds

In all, 76 compounds of peach fruits that were tested were identified using GC/MS in this study (Table 2). In ripe peach fruits, volatile compounds were found to be more than 0.1 %; relative levels of seven compounds (ethyl acetate, ethanol, propyl acetate, ethyl butanoate, ethyl hexanoate, 3-hydroxy-2-butanone, ethyl octanoate) increased more than five times compared with those in immature fruits. In overmature fruits, the respective ratios of ethyl acetate and ethanol increased. These comprised 90 % of all detected compounds (Table 2).

Table 2 Headspace volatiles of ‘Hakutou’ peach fruit (Prunus persica L.)
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EAG responses

The electroantennogram responses elicited by paraffin oil were 0.88 ± 0.07 mV in females and 0.71 ± 0.06 mV in males (n = 10). The EAG amplitudes in response to (the standard stimulus) 1 μl of 1 % (Z)-3-hexenol were 2.96 ± 0.21 mV (mean ± SE, n = 10) and 2.70 ± 0.15 mV (mean ± SE, n = 10), respectively, on female and male antennae. The EAG responses to the seven compounds (ethyl acetate, ethanol, propyl acetate, ethyl butyrate, ethyl hexanoate, acetoin, and ethyl octanoate) were investigated; of these, only EAG responses to ethyl acetate and ethyl butanoate were stronger than those to paraffin oil (data not shown). Moreover, the EAG response to ethyl butanoate was stronger than that to ethyl acetate (Fig. 1). No significant difference was found in EAG response to ethyl butanoate and ethyl acetate between males and females.

Fig. 1
figure 1

EAG responses (mean ± SE) of O. excavata (n = 10) to 1 μl of ethyl acetate and ethyl butyrate at concentrations of 1 % (v/v) and paraffin oil. The EAG responses were normalized to 1 μl of 1 % (v/v) (Z)-3-hexenol. Different lowercase letters indicate significant differences (p < 0.05) with Tukey’s test after ANOVA. No significant difference was observed between males and females

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Field-trap test

In the field-trap test conducted in August 2007, ethyl acetate, ethyl butanoate, the five-lactone mixture, and each two-combination mixture and a mixture of all compounds were used for bait. A trap baited with ripe peach fruit was also used. Traps baited with ethyl acetate and ethyl butanoate captured O. excavata moths, but fewer moths were captured by those traps than by those with ripe peach fruit (Fig. 2). All two-combination mixtures caught moths. The trap baited with a mixture of ethyl acetate and lactones captured almost as many moths as the ripe fruit. The trap baited with the mixture of all compounds captured the most moths of all traps tested. In the field-trap test conducted in July 2010, the trap baited with the mixture of all compounds captured many more moths than the trap baited with peach fruit before harvest week in the peach orchard. During the harvest week, moths were rarely captured by traps baited with the mixture of all compounds. On the other hand, moths were captured by the traps baited with peach fruit during this same week (Fig. 3).

Fig. 2
figure 2

Number of O. excavata moths captured by individual funnel traps baited with ripe peach fruit, ethyl acetate (ethyl-a), ethyl butanoate (ethyl-b), five-lactone mixture (lactones), two-combination mixtures, and a mixture of all compounds. Each value represents mean ± SE. Different lowercase letters indicate significant differences (p < 0.05) according to Bonferroni’s test after ANOVA

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Fig. 3
figure 3

Number of O. excavata moths captured by individual funnel traps baited with ripe peach fruit and a mixture of all compounds and the yield of peach fruits. Each value for the number of captured moths represents mean ± SE

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Seasonal survey

A seasonal survey was conducted from late May to the end of October 2008. In this experiment, the mixture of all compounds was used as the peach fruit volatile component for bait. Moths were first captured in mid-June. The first small peak was detected at the end of June (Fig. 4). The number of moths captured by the traps increased during the last 10 days of July and in the first 10 days of August with a large peak. Moths were captured continually until mid-October.

Fig. 4
figure 4

Seasonal survey of O. excavata captured by funnel traps baited with a mixture of all compounds. Each value represents mean ± SE, n = 3 replicates

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Discussion

Many studies of the attraction of adult moths by volatile emissions from a host plant have been related to the ovipositional selection of adult females (Fraser et al. 2003; Hern and Dorn 2001; Hern and Dorn 2004; Landolt and Guedot 2008; Natale et al. 2004; Pivnick et al. 1994). Research related to the food search behavior of adult moths that attack host plants directly, as fruit-piercing moths do, is limited. In the present study, we identified the headspace volatiles in ripe peach fruit, measured EAG responses by O. excavata moths to some of these volatile compounds, and verified our findings by field-trapping with these volatiles and mixtures for bait. Moths showed EAG responses to ethyl acetate and ethyl butanoate. They were captured by the trap baited with ethyl acetate and ethyl butanoate. The moths were also captured by the trap baited with a mixture of five lactones, but not by traps with individual lactones, although an EAG response to the individual lactones was shown (Tian et al. 2008). Moreover, much ethyl acetate and ethyl butanoate are contained in the aroma volatiles of the Asian pear cultivar ‘Kousui’ (Pyrus pyrifolia var. culta ‘Kousui’), which is attacked by O. excavata moths similarly to ripe peach fruits (data not shown). Another species of fruit-piercing moth, Adris tyrannus amurensis, which attacks ripe peach fruit, was captured using traps baited with the mixture of ethyl acetate and ethyl butanoate (data not shown). These results indicate that ethyl acetate and ethyl butanoate are more important than individual lactones for the fruit-piercing moth’s food search.

In a previous study, moths were captured using traps baited with a mixture of five lactones, but significantly fewer moths were captured than with ripe peach fruit (Tian et al. 2008). In the present study, when traps baited with the mixture of ethyl acetate, ethyl butanoate, and five lactones were used, about twice the number of moths were captured than when using ripe peach fruit. The mixture is useful year round as an attractant in place of ripe peach fruit. Actually, seasonal survey results obtained in the present study were comparable to those obtained using a trap baited with apple (Ogihara 1997). These results show that the seasonal forecasting of fruit-piercing moths can be performed using this mixture as an attractant. Consequently, it is useful for control of the fruit-piecing moth (e.g., decision at lamplight time of moth-repellent yellow lamp). However, at the time of the seasonal survey in 2008, few moths had been captured by the traps in mid-July. This was peach harvesting time; many ripe peaches and many moths were observed inside the peach orchard (data not shown). Moreover, at the trap test in 2010, the moths were rarely captured by traps baited with the mixture of all compounds inside the peach orchard during the harvesting season. On the other hand, many moths had been captured by the traps baited with ripe peach fruit during this same period. These results suggest that minor compounds in ripe peach volatiles participate in the final step of food orientation by O. excavata. In addition, in the present study, we did not perform a quantitative analysis of the volatile compounds. Therefore, the component ratio of the mixture of all compounds is slightly different from that in a peach fruit because of the characteristics of SPME fibers. Moreover, some lactones have stereoisomers and the ratio of these isomers differs depending on the types of lactone in the peach (Bernreuther et al. 1989; Werkhoff et al. 1993). In the present study, however, we could not determine the isomeric ratio. Therefore, the attraction effect may be improved by using the isomeric mixture whose ratio is similar to that of the natural product. To address these issues, we are planning to perform a quantitative analysis of peach volatile compounds.

The results of the present and previous studies suggest that lactones play an important role in the search for food by O. excavata. Although these lactones are peculiar to ripe peach fruits, they are not present in Asian pears, which are attacked by O. excavata. Moreover, the moths were captured by traps baited with ethyl acetate and ethyl butanoate only outside the orchard, but not inside the orchard. These results also indicate that the respective roles of ethyl acetate, ethyl butanoate, and lactones in the search for food by O. excavata differ. We are currently planning to investigate differences in the behavioral responses of O. excavata to each compound further using Y-tube olfactometers and wind-tunnel testing.