Introduction

Moths rely on their sense of smell to locate mates. Females release relatively small quantities of highly volatile pheromone molecules detected by very sensitive receptors on the male antennae (Allison and Cardé 2016). The high sensitivity and species-specificity of moth sex pheromones and their strong effect on males have made them a cornerstone tool in moth pest control. Pheromones are used to monitor insect-pest occurrence, time insecticide applications, bring the insect in contact with insecticides, or remove a significant part of the population. Foremost, sex pheromones are used to disrupt mating and so reduce population levels and crop damage (Miller and Gut 2015; Witzgall et al. 2010). One way to determine if mating disruption negatively affects male attraction is to use sex pheromone traps to compare male captures in pheromone-disrupted and non-disrupted crops. However, not catching males with pheromone traps does not necessarily imply a failure of males to find females (Knight et al. 2013). In addition, pheromone traps attract only males; thus, no information is available on the mating status of females (Light et al. 2017). One method to facilitate monitoring of males and females in sex pheromone disrupted crops is to use host-plant volatile lures (Miller and Gut 2015). Despite the potential importance of plant volatiles in pest management, there are relatively few commercial plant volatile attractants for moth control (Szendrei and Rodriguez-Saona 2010). One of them is the pear ester, ethyl (E,Z)-2,4-decadienoate, a volatile from ripe pears that is a relatively selective attractant of male and female Cydia pomonella (L.) and is commercialized as a combo lure together with the pheromone for monitoring and increasing the efficiency of mating disruption (Knight et al. 2014; Light et al. 2017).

The oriental fruit moth, Grapholita molesta (Busck) is a worldwide pest of peach (Prunus persica (L.)), apple (Malus domestica Borkh), and other stone and pome fruit tree species (Rothschild and Vickers 1991). Early in the season the larva bores on green shoots, moving to new ones as they are consumed. This feeding hinders the formation of new branches and causes problems in tree nurseries. Later in the season, when shoots start to harden, feeding shifts to newly available fruit, which can cause major economic loss. G. molesta is primarily a pest of peach, but in recent years there have been increasing reports of damage to apple fruit worldwide (Wei et al. 2015). The selection of oviposition locations is thus vital and is aided, at least in part, by host-plant volatiles (Myers et al. 2007; Piñero and Dorn 2007).

The control of G. molesta relies strongly on repeated insecticide applications throughout the season, with the well-known negative impacts on humans and the environment (Guillette and Iguchi 2012). The release of the synthetic pheromone blends from passive dispensers or puffers results in mating disruption and population control (Kong et al. 2014; Witzgall et al. 2010). Although there are no commercial host-plant blends specific to monitor G. molesta, several field and laboratory studies show that host-plant-released volatiles elicit male and female G. molesta responses that are comparable to the natural host. In Australia, a synthetic volatile blend that mimics those emitted by young peach shoots captured up to 130 males per trap, although it did not capture females (Il’ichev et al. 2009). In China, several synthetic volatile blends mimicking pear [Pyrus bretschnei-deri Rehder and Pyrus pyrifolia (Burm.)], and peach (Prun-us persica (L.)) fruit and shoots have been identified (Lu et al. 2012, 2014, 2015). The volatile blend obtained from pear (P. bretschneideri var. Jimi) fruit captured about 50 males and 20 females per trap. In the case of males, this was only five times less than what commercial sex pheromone traps captured in that study (Lu et al. 2012). In addition, this blend resulted in approximately 80% approach and 10% source contact by males in the flight tunnel; this was equivalent to the response of the natural fruit (Lu et al. 2012). In another study in Switzerland, analysis of peach shoot volatiles resulted in a blend that in dual-choice olfactometer tests was as attractive to mated females as the natural host-plant blend (Piñero and Dorn 2007).

Captures of G. molesta in the Australian and Chinese studies were remarkable and paralleled the attraction of C. pomonella to the pear ester (Light et al. 2001). Thus, these new blends could be an invaluable tool for the management of G. molesta. However, G. molesta is a widely distributed species with significant genetic differentiation among world populations (Kirk et al. 2013), and therefore it is crucial to determine if the Australian and Chinese blends, which have been tested only in these two countries, are also attractants in other areas of the geographical distribution of this species. In addition, it would be useful to determine if the two blends are equally attractive. The Swiss blend, which attracts females under laboratory conditions, remains to be tested in the field. The first objective of our study was to compare the attractiveness of the Australian, Chilean, and Swiss blends and to test them at a new location. The three plant blends, plus a new “total” blend made with all the different components from the other three test blends, were tested together in a peach orchard in Chile, a country where G. molesta was first recorded in 1970 (González 2003). The second objective of this study was to explore the potential of the host-plant blends to increase captures of males in sex pheromone traps, as this could improve the monitoring of males under mating disruption (Yu et al. 2014). To this end, we compared captures in traps baited with the pheromone alone and traps baited with the pheromone combined with the host-plant volatiles. Previous flight tunnel studies show that the Chinese blend attracts males and females (Lu et al. 2012), and the Swiss blend does not attract males but synergizes their response to the sex pheromone (Varela et al. 2011). Our third objective was to compare responses between field and laboratory settings. To do so, the plant blends were tested in the flight tunnel alone and in combination with the sex pheromone.

Materials and methods

Chemical stimuli

Host-plant volatiles were purchased from Sigma-Aldrich (Santiago, Chile; chemical purity, product and lot numbers in Table 1). The composition of the host-plant blends followed those reported by the Australian (Il’ichev et al. 2009), Chinese (Lu et al. 2012), and Swiss (Piñero and Dorn 2007) studies (Table 2). The Australian study used (E)-β-farnesene and (E)-β-ocimene, but pure isomers were not available to us at the time of the study, so a mixture of farnesene isomers and a mixture of β-ocimene isomers were used instead (Table 1). A fourth host-plant blend made by combining the components from all three blends was included in the tests (“total” blend, Table 2). To duplicate Australian and Chinese studies, all chemicals were dissolved in n-hexane and loaded in rubber septa (red, i.d × o.d., 3.4 mm × 6.6 mm; Sigma-Aldrich, Santiago, Chile, Product Number Z565709), at approximately similar concentrations as in the original studies (100 mg total and 100 mg major compound, respectively, Table 3). The Swiss blend was prepared the same way as the Chinese blend (100 mg major compound), and the total blend was made with 10 mg of each compound. Plant odor loads for all four blends ranged between 100 and 189 mg (Table 3). With the aim of providing a wider range of release rates, in experiment 1 (see below) the host-plant blends were prepared at an additional tenfold lower concentration, and both high and low concentrations were further dissolved in mineral oil (Sigma-Aldrich product number M8410, Lot Number MKBG7544V, CAS Number 8020-83-5) and loaded in 1.5-mL microcentrifuge tubes fitted with a 15-mm-long × 7-mm-diameter section of dental cotton wick to absorb the chemicals. Mineral or paraffin oil is a mixture of n-alkanes and provides slower and more linear release rates than individual shorter alkanes, like n-hexane, and both are used routinely to deliver plant volatiles in olfactory tests (Andersson et al. 2012). The lids of the microcentrifuge tubes were perforated with a 1.5-mm-diameter hole and were kept closed. Rubber septa were rinsed in n-hexane and then in acetone, and allowed to dry, before use. Microcentrifuge tubes were rinsed in acetone. Host-plant volatiles were loaded in 500-µL volumes in rubber septa and microcentrifuge tubes at the concentrations indicated below. It took about 1 h for the host-plant blends to be absorbed by the rubber septa. For 2–4 h the dispensers were maintained in a well-ventilated area and then placed inside plastic bottles and stored at − 20 °C until taken to the field on the following day.

Table 1 Synthetic host-plant odorants used in the experiments
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Table 2 Odorant ratios in the four tested host-plant blends
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Table 3 Field and flight tunnel experimental details
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In the Chinese study (Lu et al. 2012) commercial sex pheromone rubber septa were used. In our study the three sex pheromone components, (Z)-8-dodecenyl acetate (Z8-12:Ac), E8-12:Ac, and (Z)-8-dodecenol (Z8-12:OH) (Pherobank, Wageningen, the Netherlands, > 99% pure) were diluted in n-hexane at a 100:5.4:10 ratio, respectively (Knight et al. 2015), and loaded in red rubber septa. In the first experiment we used 80 µg, which is an optimal quantity (Knight et al. 2015). Captures were relatively high so in experiments 2 and 3 we reduced the quantity of pheromone to 16 and 8 µg, respectively. This reduction in pheromone load permitted us to test potential synergistic effects of host-plant volatiles on sex pheromone attraction.

Field tests

Experiments were carried out in a peach (Prunus persica var. persica cv. Doctor Davis and Carson) orchard in Chile (Duao, Maule, 35°33′29″S, 71°33′44″W) between December 21, 2012 and February 25, 2013 (Table 3). The Chinese and Australian studies used standard delta traps and funnel-type Efekto fly traps, respectively (Il’ichev et al. 2009; Lu et al. 2012). We used white delta traps (215 mm long × 200 mm wide × 100 mm tall, 340 cm2 adhesive base area, Plastic Delta Trap; Alphascent, West Linn, OR, USA), except for the pheromone treatment in experiment 1, where the traps were red because of a temporary shortage of white traps. This should not have influenced trap catches because trap color does not affect G. molesta captures (Zhao et al. 2013). Traps were placed at 1.7 m high, hanging from 4-cm-diameter blue PVC pipes fitted in the tree branches. Traps within a plot were placed in a transect 15–20 m apart, and plots were at least 15 m apart from each other. Trap floors were lined with removable sticky cards.

In experiment 1 the dispensers, either rubber septa or microcentrifuge tubes, were hung from the ceiling of the trap with a wire, almost touching the trap floor. In the other two experiments the dispensers were placed directly on the sticky floor. Septa and microcentrifuge tubes were labeled with the treatment name using permanent markers. Traps lured with sex pheromone and host-plant odors (experiments 2 and 3) had two septa, one for each stimulus type, which were placed within a few centimeters of each other at the center of the trap. Sticky bottoms were replaced if there were captures. Sex of captured individuals was determined in the laboratory using a stereomicroscope.

Experiment 1 was carried out between December 21, 2012 and January 29, 2013 and tested the four host-plant blends at two doses with two solvents and dispensers (Table 3). Sex pheromone (80 µg) and solvent (n-hexane or mineral oil) were the positive and negative controls, respectively, and were loaded in the corresponding dispensers (septum or microcentrifuge tubes). The 20 plant-volatile treatments [(4 plant treatments × 2 doses + solvent + pheromone) × 2 dispenser types (septum or microcentrifuge tube)] were placed in each of four rows, or plots, at random. There were six weekly trap checks. Sex pheromone and host-plant lures were replaced on the first and second checks. For the remainder of the experiment the pheromone lure was unchanged, while new plant lures were replaced one last time on the fourth check (January 11, 2013).

Experiment 2 was ran between January 29 and February 12, 2013 to determine if the host-plant blends had any effect on male attraction to the sex pheromone. Traps were baited with a sex pheromone septum (16 µg) and a host-plant blend septum at a similar dose as in the original Australian and Chinese studies, which was the same as the “high” dose of experiment 1. This dose resulted in pheromone to host-plant volatile ratios ranging between 1:6250 (Australian blend) and 1:11,812 (Chinese blend) (Table 3). Sex pheromone-only traps served as positive controls and n-hexane-only traps served as negative controls. The 10 treatments (4 host-plant blends; 4 sex pheromone + host-plant blends; solvent; sex pheromone) were replicated in eight plots at random. There were five trap checks every 3–4 days. Sex pheromone septa were not replaced, and new plant lures were replaced on February 5, 2013.

Experiment 3, which was carried out between February 12 and 25, 2013, tested if the inhibitory effect of the host-plant blends observed in experiment 2 (see “Results”) was dose-dependent. It included the two plant blends that caused the strongest inhibition in experiment 2 (Chinese and total, see “Results”), and sex pheromone at 8 µg. The pheromone to plant ratio ranged from the lowest 1:175 (total) to the highest 1:23,625 (Chinese) (Table 3). Two host-plant volatiles, terpinyl acetate and β-ocimene isomer mix, which have shown behavioral activity in previous studies (Cichón et al. 2013; Il’ichev et al. 2009; Knight et al. 2014), were tested alone at 3 mg, as in Knight et al. (2014), and in combination with sex pheromone at a sex pheromone to host-plant volatile ratio of 1:375 (Table 3). In addition, we tested the effect of having one or two septa in the trap, by adding a blank septum to a trap with a pheromone septum. n-Hexane septa were tested alone to control for possible sex pheromone contamination. The 13 treatments (2 host-plant blends with sex pheromone × 3 doses; sex pheromone alone; sex pheromone and n-hexane septum; n-hexane; 2 host-plant volatiles with and without sex pheromone) were placed in four plots at random. There were six checks every 1 or 2 days because there was a population peak during this period. Pheromone-loaded septa were not replaced, but new host-plant lures were replaced on February 18 and 22, 2013.

Flight tunnel test

The colony of G. molesta used in the flight tunnel was established with insects collected at Piacenza, Italy. The population has been maintained at the University of Lleida, Spain, since 2005 without reintroduction of wild individuals. Larvae were reared on a semi-synthetic diet modified from Ivaldi-Sender (1974) under a L16:D8 photoperiod at 24 ± 1 °C. Pupae were separated by sex and placed in 4-L polypropylene containers and provided a cotton ball soaked in 10% sugar water solution. Adults were collected daily and used when 2–4 days old.

The flight tunnel and its methodology have been previously described (Ammagarahalli et al. 2017). The 150 × 45 × 45 cm (length × height × width) tunnel had a 0.35 m s−1 wind flow and the temperature was maintained at 23 ± 1 °C. It was illuminated from above with 36-W fluorescent lamps producing 150-lx white light. Tests were carried out during the last 3 h of the photophase and occasionally into the first hour of the scotophase, in which case the daylight illumination was left on. Males were placed individually in glass tubes and were transferred to the flight tunnel room 30–120 min before the beginning of the test. Test odors were applied in 10-µL loads to 10 × 15 mm filter paper pieces that were allowed to dry for 5–10 min until tested in the flight tunnel 5–180 min later. The male was placed in the flight tunnel after the odor stimulus, on top of a metal-wire platform similar to the one used for the odor source and 1.3 m downwind from it. We recorded for 2 min if the male took flight, started upwind oriented flight (zigzagging upwind flight) or landed on the filter paper containing the stimulus source, and the time it took the males to engage in these behaviors. The stimulus was placed in the tunnel on top of a 25-cm-tall metal-wire platform. Three to five males flew to each filter paper treatment before changing to another treatment paper. At the end of a test day a filter paper had been used with 8–15 males; therefore, the filter papers were outside their individual glass vial and exposed to the wind flow between for 32–60 min before being discarded. On any given day, only one filter paper was used for each treatment. Because of the high number (see below), the treatment order was randomized in two groups and tested on alternate days.

The following treatments were tested in the flight tunnel: Australian, Chinese, and Swiss host-plant blends at 10 µg each, a suboptimal sex pheromone dose of 1 ng [a response curve to doses of 1, 10, 100, and 1000 ng resulted in 34%, 82%, 89%, and 63% source contact respectively, N = 44, similar to Ammagarahalli et al. (2017)], and sex pheromone/host-plant blends (Australian, Chinese, or Swiss) at 1:10, 1:100, 1:1000, and 1:10,000 ratios with sex pheromone at 1 ng (Table 3). We used a suboptimal dose of sex pheromone because the optimal dose results in a high percentage of response. The 10-µg dose of host-plant odors was 50 times lower than that used in the Chinese flight tunnel test with rubber septa (Lu et al. 2012), yet similar to what we have used previously (Varela et al. 2011). To control contamination, 20 insects were tested with n-hexane on random days. The sex pheromone/host-plant blends were prepared using a stock sex pheromone solution so all had identical sex pheromone concentrations. The 1:0, 1:100, 1:1000, and 1:10,000 blends were prepared on January 18, 2013 and the 1:10 blend on February 4, 2013. Flight tunnel tests were carried out between February 8 and 27, 2013 with N = 64.

Statistical analyses

Generalized linear models (GLM) with Poisson family function in the package lme4 of R (R Development Core Team 2015) were used to analyze trap count data (Bolker et al. 2009). Because of the high temporal variation in trap captures, sampling date was included as a random effect in the generalized linear mixed model (GLMM), along with the variation among plots if they contributed significantly to the model after comparing among models with ANOVA. To treatments with zero captures, a random capture was added so the GLMM could converge. The percentage of males which responded in the flight tunnel was analyzed with GLM models using a binomial family function. Behavioral categories (take flight, oriented flight, and contact) were analyzed separately. One response was added to treatments with no responses in a randomly chosen replicate so the GLM model could converge. The time elapsed before insect response inside the flight tunnel was analyzed with a linear model, lm(), in transformed [log(x + 1)] data. Comparisons among treatment pairs in both field and flight tunnel studies were performed with the glht() or lsmeans() functions of R using Tukey’s alpha correction method. Whenever the term “significant” is used in the text regarding treatment comparisons it indicates that p ≤ 0.05. Raw data and R scripts are available at https://repositori.udl.cat/handle/10459.1/59534.

Results

Field tests

In experiment 1 only one female was captured in a trap baited with a high dose of the Swiss host-plant blend diluted in mineral oil. The four traps baited with sex pheromone septa captured a total of 1632 males, whereas the four traps baited with sex pheromone microcentrifuge tubes captured a total of 215 males. All of the host-plant volatile traps combined, which summed 64, captured a total of 64 males in the entire experiment (Table 4). Of these 64 males, 61 were captured in the fourth weekly check, and these captures clustered mainly in three particular traps: 35 males in a trap baited with the low-dose Swiss host-plant blend in n-hexane, 15 males in a trap baited with the high-dose Chinese host-plant blend in mineral oil, and 9 males in a trap baited with a high-dose total host-plant blend in n-hexane. This level of captures in host-plant baited traps was not observed before or after week 4 (only three more males were captured by host-plant-baited traps in the other five weekly checks) or in experiment 2 (see below). Table 4 summarizes total trap captures and shows how the Swiss host-plant blend at the low dose in n-hexane was the only host-plant blend that captured significantly more males (1.46 ± 1.46 males/trap/check, mean ± SEM) than the n-hexane traps, which captured none.

Table 4 Captures of G. molesta in Chile between December 21, 2012 and January 29, 2013 in traps baited with one of four host-plant blends in two doses and either dissolved in n-hexane and loaded in red rubber septa, or dissolved in mineral oil and loaded in microcentrifuge tubes (experiment 1)
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In experiment 2, traps baited with host-plant volatiles or with n-hexane captured no males, whereas sex pheromone-baited traps captured many males (2800 in total, Table 5). Only nine females were captured in this experiment, all of them in traps baited with the Australian host-plant blend combined with the sex pheromone, but these captures were not significantly different than those from the n-hexane traps. The addition of a septum baited with any host-plant blend to a trap baited with a sex pheromone septum significantly decreased the number of males captured with respect to sex pheromone traps. This negative effect was significantly stronger for the Chinese and total host-plant blends than for the Australian and Swiss host-plant blends.

Table 5 Captures of G. molesta in Chile between January 29 and February 12, 2013 in traps baited with either one septum of one of four host-plant blends, a sex pheromone septum, or sex pheromone with a host-plant blend septum (experiment 2)
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In experiment 3, a total of 13,650 males and 17 females were captured. The addition of a septum baited with any of the three doses of the Chinese or total host-plant blends to a trap baited with a sex pheromone septum significantly decreased the number of males captured relative to traps baited with a sex pheromone septum and an n-hexane septum. This effect was more pronounced as the host-plant dose increased (Table 6). Traps baited with a sex pheromone septum and an n-hexane septum captured more males than traps baited with only the sex pheromone septum. n-Hexane traps captured six males in total.

Table 6 Captures of G. molesta in Chile between February 12 and February 25, 2013 in traps baited with one sex pheromone septum and a host-plant blend septum (Chinese or total) at low, medium, or high doses (experiment 3)
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β-Ocimene (mixture of isomers) captured significantly more males than n-hexane traps. Terpinyl acetate added to sex pheromone significantly increased captures relative to traps baited with a sex pheromone septum, but not relative to traps baited with a sex pheromone and a n-hexane septum together (Table 6).

The 17 females caught in experiment 3 were captured by eight different treatments, five of which captured only one female, while three captured more than one female. The treatments that captured more than one female were always a combination of the host-plant blend with the sex pheromone. Of these, the highest captures were in β-ocimene (mix of isomers) plus sex pheromone which captured three females in one plot on three different dates, three females in another plot on two different dates, and one female in another plot. None of the female captures in these treatments were significantly higher than n-hexane traps.

Flight tunnel test

In the flight tunnel experiment several doses of the Australian, Chinese, and Swiss host-plant blends were added to sex pheromone. None of the host-plant blends alone, nor n-hexane, attracted any males, so they were not included in the means comparison test. Pairwise comparisons between each sex pheromone host-plant combination treatment and the isolated sex pheromone treatment showed that the three host-plant blends significantly increased the percentages of flight, oriented flight, and contact, and did so in a dose-dependent manner (Fig. 1a). The Chinese and Swiss host-plant blends significantly increased responses at the 1:100–1:1000 sex pheromone to host-plant ratios, whereas the Australian host-plant blend did so at the 1:1 and 1:10 ratios. The three host-plant blends significantly reduced the time of response to the sex pheromone, and, as with the percentages of response, the effect was stronger at the higher (plant-wise) sex pheromone to host-plant odor ratios (1:1000 and 1:10,000) (Fig. 1b).

Fig. 1
figure 1

Effects of adding various host-plant volatile blends (Australian, Chinese, and Swiss) of different doses on the behavioral responses of G. molesta males to a suboptimal dose of sex pheromone in a flight tunnel (experiment 4). a Percentage of males engaged in each behavioral category (take flight, oriented upwind flight, and source contact). b Time it took males to engage in each behavioral category. Asterisks indicate significant differences between the sex pheromone host-plant combination treatments and the isolated sex pheromone treatment (1:0) by means of planned pairwise comparisons using Tukey’s test (p ≤ 0.05)

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Discussion

Captures by the Australian and Chinese host-plant blends in our study were substantially lower than in the original studies [from here onwards “Australian and Chinese studies” will refer to Il’ichev et al. (2009) and Lu et al. (2012), respectively]. Although the experimental conditions of the Australian and Chinese studies were closely reproduced in Chile, there were some differences that perhaps account for the low response of these host-plant blends in our experiments. The most evident difference is the chemical purity of farnesene and ocimene, which in the Australian study consisted of (E)-β-farnesene and (E)-β-ocimene. The isolated isomers were not commercially available at the time of the tests; therefore, mixtures of isomers of each compound were used in our study. Insects can distinguish odorant isomers both at the sensory and behavioral levels (De Bruyne and Baker 2008), so it is possible that isomeric purity affected reproducibility of the Australian blend in Chile. However, the β-ocimene mix of isomers in our study was one of the few plant stimuli that was more attractive than n-hexane, whereas the Australian blend influenced male response to pheromone, as did the other plant blends. Although our Australian blend did not have the same isomer purity as the original Australian blend, our results show that it was sensed by and affected the behavior of G. molesta. Furthermore, the Chinese blend used a mixture of farnesene isomers both in the original Chinese study and in our study. Even though it was very attractive in China, it performed very poorly in Chile. Therefore, isomer purity should not have contributed to the different performance of the Chinese blend in China and Chile. Little is known about the detection of plant volatiles in G. molesta, but olfactory receptor neurons relatively specific to the same farnesene isomer mix that we used here have been described on the antenna of males (Ammagarahalli and Gemeno 2015).

Although we used the same solvents, concentrations, and dispensers as in the original Australian and Chinese studies, the quantity and proportion of odorants released by the dispensers were not analyzed in any of these studies. Therefore we do not know if the volatile composition and emission rate would have varied among them. By using an additional lower concentration than in the original Australian and Chinese studies, and mineral oil as an extra solvent, we attempted to diversify the stimulus quantity released by the dispensers. Mineral oil probably decreased release rates because pheromone in mineral oil attracted about seven times fewer males than pheromone in n-hexane. A similar solvent effect was probably true for the host-plant volatiles; however, neither mineral oil nor the lower stimulus concentration improved captures relative to the original solvent and concentration. In our tests, dispensers with plant volatiles were loaded a day before use and replaced every 5.6 ± 3.9 days (mean ± SEM) in order to minimize stimulus degradation and depletion during the assay. A final methodological difference among studies was the use of a funnel-type fly trap in the Australian study, whereas in the Chinese and Chilean studies standard delta traps were used. Although trap type could affect moth captures, our sex pheromone traps captured many males; thus, it is unlikely that trap type alone could explain the dissimilar performance of the Australian blend in Australia and Chile. With regard to the flight tunnel test, we used a lower host-plant odor concentration than the Chinese study, and loaded it on filter paper instead of rubber septa, so the difference between the lack of response in our flight tunnel test and the good response in the Chinese flight tunnel test could be related to the use of different stimulus delivery conditions.

Biological variables such as population genetics could also be involved in the differences between the Australian and Chinese studies and our Chilean test. When populations evolve host-specialization, differences in the detection of host-plant volatile signals may arise (Smadja and Butlin 2009). Significant genetic differences among populations of G. molesta could provide the necessary genetic diversity for the evolution of host varieties (Zheng et al. 2015; Wei et al. 2015). Nonetheless, host-plant specialization may be negligible in G. molesta given its recent human-aided expansion and reduced dispersal power (Wei et al. 2015). This is consistent with the lack of worldwide variation in sex pheromone production and response that we have reported in this species (Knight et al. 2015). However, whether this is also true regarding responses to host-plant volatiles remains to be tested.

Background odors in the environment could influence the response of insects to odor stimuli (Cai et al. 2017). Pear ester is more effective in attracting C. pomonella when deployed in walnut orchards than in pear orchards (Light et al. 2001). In China, pear- and peach-odor blends perform relatively better in orchards of the opposite host (Lu et al. 2014, 2015). Captures with a pear blend in a peach orchard were greater than 40 males per trap (Fig. 2A in Lu et al. 2015). With this in mind we expected substantial captures of G. molesta in our peach fields with the Chinese pear blend, but captures were never higher than in control traps. The Australian peach blend was very attractive in a pear background (Il’ichev et al. 2009), so its poor performance in our peach orchard might be related to the differences in host-plant backgrounds. Yet, in China the peach blends attract G. molesta in peach orchards (more than 10 males/trap, Fig. 2A in Lu et al. 2015), and the pear blend is equally effective in peach and pear orchards (Fig. 2B in Lu et al. 2012), so the relative importance of background odors needs to be examined. Background odors, however, could be relatively important to explain differences between laboratory and field studies. Knudsen et al. (2008), for example, show the host-plant volatile compound which attracts the apple fruit moth, Argyresthia conjugella Zeller, in the flight tunnel is different than the one that attracts it in the field, albeit these compounds are released by the host. The authors conclude that the interaction of the plant volatiles with the background odor contributes to different results between field and laboratory tests. The flower volatile phenylacetaldehyde is a generalist noctuid moth attractant (Tóth et al. 2010) that increases the response of male Spodoptera frugiperda Walker to sex pheromone in the flight tunnel (Meagher and Mitchell 1998). However, this flower volatile decreases captures in sex pheromone traps in the field (Meagher 2001). Perhaps the lack of background plant odors in our flight tunnel test could explain why males were more attracted to the pheromone plus host-plant lures than to the pheromone lure alone. However, it seems unlikely that the presence of background plant odors in the field is responsible for the inhibitory effect of the host-plant blends on sex pheromone traps.

An important advantage of host-plant volatiles over sex pheromones is the potential attraction of females, which provides information on the mating status of the population. The Australian study reports that only males were attracted to the plant lures (Il’ichev et al. 2009), and the Chinese study reports male to female ratios in the range of 10:1–3:1 (Lu et al. 2012). In our study the plant odor blends also caught substantially more males than females (122 vs 2 in total, respectively). Interestingly, 24 additional females (92% of all the females caught in the study) were collected in traps baited with a combination of sex-pheromone and host-plant septa. Given recent reports of pheromone autodetection by female moths, and its possible implications in mating disruption (Holdcraft et al. 2016), it may be worth exploring if the sex pheromone plays any role in female moth attraction to host-plant lures.

A field study with G. molesta shows that two plant volatiles [(Z)-3-hexenyl acetate and undecanol] added to the sex pheromone synergize captures at a 1:0.5 pheromone to host-plant ratio, but at the 1:1 and 1:2 ratios the synergism disappears, with a clear trend to become inhibitory at even higher host-plant ratios (Yu et al. 2014). In other moth species where synergism of host-pant volatiles with sex pheromone has been reported, the pheromone to host-plant ratio tested is in the range of 1:1 (e.g., Dai et al. 2008; Dickens et al. 1993; Light et al. 1993), although sometimes it can be as high as 1:40 (Deng et al. 2004). We used much higher sex pheromone to host-plant ratios, around 1: 10,000, in order to maintain the same host-plant concentrations as in the original Australian and Chinese studies. But when we later tested a lower (1:200) ratio in the dose–response test (experiment 3), the effect was also inhibitory relative to traps baited with a sex pheromone and an n-hexane septum. Thus, it remains to be determined if still lower sex pheromone to host-plant ratios of the Australian and Chinese plant-blends could have a non-inhibitory or even a synergistic effect on sex pheromone captures. There are at least two other cases where host-plant odors reduce moth captures in sex pheromone traps. (E)-2-Penten-1-ol reduced captures of the arctiid moth Hyphantria cunea (Drury) when added to the sex pheromone at a 1:1 ratio (Tang et al. 2012), and phenylacetaldehyde reduced pheromone captures of Spodoptera frugiperda (Smith) in traps baited with a commercial sex pheromone septum and different amounts of the host-plant odorant (Meagher 2001).

Increased captures by the presence of a blank septum in sex pheromone traps was unexpected, and could be a visual response because G. molesta flies during the last light hours of the day using visual information (Kuenen and Gilbert 2014). It could also be a response to increased air turbulence, a factor that facilitates male upwind flight (De Bruyne and Baker 2008). Barros-Parada et al. (2016) report a large effect of the location of pheromone and pear ester septa and acetic acid dispensers in the trap on captures of C. pomonella. Our observations highlight the importance of taking into account apparently negligible experimental variables, like the addition of a second septum, which could impact trap captures and hamper the correct evaluation of host-plant attractants.