A Multiple-Choice Bioassay Approach for Rapid Screening of Key Attractant Volatiles

A Multiple-Choice Bioassay Approach for Rapid Screening of Key Attractant Volatiles Abstract Fermentation volatiles attract a wide variety of insects and are used for integrated pest management. However, identification of the key behavior modifying chemicals has often been challenging due to the time consuming nature of thorough behavioral tests and unexpected discrepancies between laboratory and field results. Thus we report on a multiple-choice bioassay approach that may expedite the process of identifying field-worthy attractants in the laboratory. We revisited the four-component key chemical blend (acetic acid, ethanol, acetoin, and methionol) identified from 12 antennally active wine and vinegar chemicals for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). The identification of this blend took 2 yr of continuous laboratory two-choice assays and then similarly designed field trials. This delay was mainly due to a discrepancy between laboratory and field results that laboratory two-choice assay failed to identify methionol as an attractant component. Using a multiple-choice approach, we compared the co-attractiveness of the 12 potential attractants to an acetic acid plus ethanol mixture, known as the basal attractant for D. suzukii, and found similar results as the previous field trials. Only two compounds, acetoin and, importantly, methionol, increased attraction to a mixture of acetic acid and ethanol, suggesting the identification of the four-component blend could have been expedited. Interestingly, the co-attractiveness of some of the 12 individual compounds, including a key attractant, methionol, appears to change when they were tested under different background odor environments, suggesting that background odor can influence detection of potential attractants. Our findings provide a potentially useful approach to efficiently identify behaviorally bioactive fermentation chemicals. fermentation volatile, bioassay, background odor, attractant, spotted wing drosophila Fermentation volatiles produced by yeasts and acetic acid bacteria can attract a wide variety of insects and have been extensively used to manipulate insect pest behavior for integrated pest management, including various species of beetles (Nout and Bartelt 1998), flies (Lee et al. 2012; Landolt et al 2015; Cha et al. 2016b, 2018), moths (Yamazaki 1998, Sussenbach and Fiedler 1999, Laaksonen et al. 2006, Pettersson and Franzén 2008) and vespid wasps (Dvorak and Landolt 2006, Noll and Gomes 2009, De Souza et al. 2011, Landolt et al. 2014a,b). The implementation of management program often favors using synthetic chemical attractants over bait materials, as a well-characterized chemical lure composed of essential attractant components may be easier to deploy, longer lasting, and more selective than fermentation baits (e.g., Cha et al. 2015). For example, chemical lures for Vespula spp. (Landolt 1998) and Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) (Cha et al. 2014) have been developed based on fermentation bait volatiles and are currently being widely used. Despite the large potential benefits, however, the tedious and time-consuming nature of behavioral tests (Turlings et al. 2004), the lack of clarity in choosing different behavioral testing protocols (e.g., subtraction vs. addition approaches; Linn et al. 1984), and the unexpected discrepancies in behavioral responses from laboratory and field experiments (Knudsen et al. 2008) have often slowed the process of identifying key attractant components and the development of a synthetic lure. Therefore, a straightforward behavioral testing method that can be conducted in a relatively simple laboratory setting and at the same time generate field-worthy results would facilitate the development of effective attractant lures. Recently a chemical lure composed of acetic acid, ethanol, acetoin and methionol was isolated, identified, optimized and commercialized from a fermentation bait for D. suzukii (Cha et al. 2012, 2014, 2017, 2018), which is more efficient and selective than the original material of a wine plus vinegar mixture (Cha et al. 2013, 2015). However, the lure development was slow, taking more than 2 yr of continuous laboratory two-choice assays and field-trapping experiments in multiple locations, even after 12 candidate attractant chemicals were determined using gas chromatography-electroantennographic detection (GC-EAD). One of the major reasons behind the delay was discrepancies in behavioral testing results between laboratory choice assays (Cha et al. 2012) and field trapping experiments (Cha et al. 2014; summarized in Table 1). The co-attractiveness (Meagher and Landolt 2008, Landolt et al. 2014a,b) of some of the 12 EAD-active compounds to a mixture of acetic acid and ethanol were different in laboratory and field, resulting in different blend compositions that resulted in different attractiveness in the field. The discrepancy could have been due to the differences in background odor available in two-choice laboratory assays versus field trapping experiments (Cha et al. 2014). As the 12 EAD-active compounds were ubiquitous fermentation volatiles, field sites may have had a mixture of these 12 compounds and even other volatile compounds in the background, released from various materials including fermenting SWD host fruits, while in laboratory only the chemicals tested in a specific two-choice assay were present as the background. Table 1. Comparison of co-attractiveness of D. suzukii candidate attractants with acetic acid (AA) and ethanol (EtOH) in 1) a laboratory two-choice experiment (AA + EtOH vs. AA + EtOH + one of the candidate attractant; Cha et al. 2012), 2) a field ‘add-on’ test (AA + EtOH vs. AA + EtOH + one of the candidate attractants; Cha et al. 2014), and 3) multiple-choice assay conducted in this study Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Positive indicates that the compound added attractiveness to AA + EtOH (P < 0.05), negative decreased the attractiveness (P < 0.05), and neutral had no effect (P > 0.05) based on Tukey-Kramer tests (Cha et al. 2012, 2014 and Fig 1). *(bold) indicates two key chemicals in the four-component essential blend (acetic acid, ethanol, acetoin and methionol). View Large Table 1. Comparison of co-attractiveness of D. suzukii candidate attractants with acetic acid (AA) and ethanol (EtOH) in 1) a laboratory two-choice experiment (AA + EtOH vs. AA + EtOH + one of the candidate attractant; Cha et al. 2012), 2) a field ‘add-on’ test (AA + EtOH vs. AA + EtOH + one of the candidate attractants; Cha et al. 2014), and 3) multiple-choice assay conducted in this study Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Positive indicates that the compound added attractiveness to AA + EtOH (P < 0.05), negative decreased the attractiveness (P < 0.05), and neutral had no effect (P > 0.05) based on Tukey-Kramer tests (Cha et al. 2012, 2014 and Fig 1). *(bold) indicates two key chemicals in the four-component essential blend (acetic acid, ethanol, acetoin and methionol). View Large Fig. 1. View largeDownload slide Mean (±SEM) numbers of Drosophila suzukii flies captured in traps baited with a control (a mixture of 1.6% acetic acid and 7.2% ethanol; AA + EtOH) and 12 different ‘add-on’ blends (AA + EtOH + one of the EAD-active compounds listed in Table 1). AT: Acetoin, EL: Ethyl lactate, IAA: Isoamyl acetate, MBA: 2-methylbutyl acetate, GB: Grape butyrate, PE: 2-phenylethanol, EB: Ethyl butyrate, HX: 1-hexanol; MT: Methionol, IAL: Isoamyl lactate, ES: Ethyl sorbate, DS: Diethyl succinate. Different letters on bars indicate significant differences by Tukey-Kramer tests at P < 0.05. Statistical tests were based on square-root-transformed data. Means from untransformed data are shown. Fig. 1. View largeDownload slide Mean (±SEM) numbers of Drosophila suzukii flies captured in traps baited with a control (a mixture of 1.6% acetic acid and 7.2% ethanol; AA + EtOH) and 12 different ‘add-on’ blends (AA + EtOH + one of the EAD-active compounds listed in Table 1). AT: Acetoin, EL: Ethyl lactate, IAA: Isoamyl acetate, MBA: 2-methylbutyl acetate, GB: Grape butyrate, PE: 2-phenylethanol, EB: Ethyl butyrate, HX: 1-hexanol; MT: Methionol, IAL: Isoamyl lactate, ES: Ethyl sorbate, DS: Diethyl succinate. Different letters on bars indicate significant differences by Tukey-Kramer tests at P < 0.05. Statistical tests were based on square-root-transformed data. Means from untransformed data are shown. The two major fermentation volatiles, acetic acid and ethanol, have been shown to be involved in insect attraction to fermentation sources as individual compounds or as a mixture. In some cases, a mixture of acetic acid and ethanol was sufficiently attractive to explain such attraction, while in other cases additional fermentation volatiles in addition to acetic acid and ethanol were necessary. For example, a mixture of acetic acid and ethanol was as attractive as the original material of yeast-sugar fermenting solution for an avian parasitic fly, Philornis downsii (Cha et al. 2016) or as a wine plus vinegar bait for false stable flies (Landolt et al. 2015). In contrast, in other insects such as D. suzukii, although ethanol and acetic acid were essential for the attraction, additional fermentation volatiles were necessary to achieve the similar level of attraction to the original fermentation material (Landolt et al. 2012b). In particular, acetic acid + ethanol was more attractive than either chemical alone (Landolt et al. 2012a) but less attractive than a mixture of wine and vinegar that contained the same amount of acetic acid and ethanol, suggesting that SWD are responding to some other wine and vinegar chemicals (Landolt et al. 2012b). Here, we report on a simple laboratory multiple-choice assay approach that uses a mixture of acetic acid and ethanol as a basal attractant and tests co-attractiveness of all of the individual EAD-active compounds separately but concurrently. The new assay was conducted by revisiting the 12 EAD-active wine and vinegar chemicals involved in the development of the four-component chemical lure for D. suzukii. In the multiple-choice assay, all of the 12 EAD-active compounds were available to SWD to respond to in the headspace inside the cage, but released from separate individual traps with acetic acid and ethanol as a basal attractant. This laboratory assay allowed us to quickly predict which compounds would be co-attractive with acetic acid plus ethanol for SWD in the field, which was essential to achieve the same level of attraction from D. suzukii to the original benchmarking mixture of wine and vinegar. Materials and Methods Insects For multiple-choice bioassays, a colony of SWD was maintained at the New York State Agricultural Experiment Station, Geneva, New York. Flies were reared at 21.5 ± 0.9°C, 23.7 ± 0.1 % RH, 16:8 (L:D) h on standard cornmeal diet (1 liter distilled water, 40 g sucrose, 25 g cornmeal [Quaker Oats Co., Chicago, IL], 9 g agar [No. 7060, Bioserve, Flemington, NJ], 14 g torula yeast [No. 1720, Bioserve], 3 ml glacial acetic acid [Amresco, Solon, OH], 0.6 g methyl paraben [No 7685, Bioserve], and 6.7 ml ethanol). Chemicals For this study, we used the 12 EAD-active chemicals (Table 1) that were previously identified from a Merlot wine and a rice vinegar mixture (Cha et al. 2012). Ethyl butyrate (99%, CAS No. 105-54-4), 3-hydroxybutan-2-one (acetoin) (≥96%, CAS No. 513-86-0), 3-methylbutyl acetate (isoamyl acetate) (98%, CAS No. 123-92-2), 2-methylbutyl acetate (99%, CAS No. 624-41-9), 3-methylsulfanylpropan-1-ol (methionol) (≥98%, CAS No. 505-10-2), ethyl (2E,4E)-hexa-2,4-dienoate (ethyl sorbate) (≥97%, CAS No. 2396-84-1), and 2-phenylethanol (≥99%, CAS No. 60-12-8) were purchased from Sigma-Aldrich (St. Louis, MO). Ethyl 2-hydroxypropionate (ethyl lactate) (>97%, CAS No. 97-64-3), 3-methylbutyl 2-hydroxypropionate (isoamyl lactate) (>98%, CAS No. 19329-89-6), and diethyl butanedioate (diethyl succinate) (>99%, CAS No. 123-25-1) were purchased from TCI America (Morris, Portland, OR). Ethyl 3-hydroxybutyrate (grape butyrate) (99%, CAS No. 5405-41-4) was purchased from Arcos Organics (New Jersey). Ethanol (200 proof, CAS No. 64-47-5), acetic acid (99.8%, CAS No. 64-19-7), and 1-hexanol (>95%, CAS No. 111-27-3) were purchased from Pharmco (Brookfield, CT), Fisher Scientific (Pittsburgh, PA, USA), and J. T. Baker (Philipsburg, NJ), respectively. Multiple-Choice Assay The multiple choice assay (N = 5) was conducted with an arena design using a dome cage (60 cm W × 60 cm L × 60 cm H; BugDorm-2120 Insect tent; shop.bugdorm.com). Within each arena, 13 traps were positioned in a circle (20 cm diameter) at equal distance with each trap 9.7 cm apart along the circumference. We used an aluminum foil-covered 100-ml glass beaker with a cut centrifuge vial (0.7 cm diameter) inserted in the foil for SWD entry, as a trap (Cha et al. 2012). All 13 traps had 20 ml 1.6% acetic acid + 7.2% ethanol with soap as a basal attractant and drowning solution. One of the traps served as a control and the remaining 12 traps had one of the 12 EAD-active compounds added to the control blend, loaded as described in Cha et al. (2012) and released from 1.5-ml vial with 3-mm hole with a piece of cotton. A cotton ball (3 cm diameter) soaked with distilled water was placed in the center of arena to provide water to SWD. Adult flies (160–235, roughly 1:1 male: female ratio, 7–10-d-old) were released in each arena. Choice assays started at 1:00 p.m. each day (21.5 ± 0.9°C, 23.7 ± 0.1% RH, 16:8 [L:D] h). The number of flies inside treatment and control traps was counted after 20 h. Statistical Analysis A randomized complete block design was used. Trap catches were analyzed with block as a random factor and different odor sources as a fixed factor using Proc Mixed (SAS Institute 2009). Fly catch data were square-root transformed to improve normality and homoscedasticity (Zar 1984). The means were compared using the Tukey-Kramer test (SAS Institute 2009). Results The results from the laboratory multiple-choice test showed that two compounds, acetoin and methionol, were co-attractive with a mixture of acetic acid and ethanol (basal attractant), significantly increasing the number of D. suzukii trapped by 4.5- and 2.6-fold, respectively, compared to the control traps baited with the mixture of acetic acid and ethanol (F12,48 = 37.81, P < 0.0001; Fig. 1). In each experiment listed in Table 1, an EAD-active compound was indicated ‘positive’ (i.e., co-attractive) when adding the compound to the mixture of 1.6% acetic acid and 7.2% ethanol resulted in a significant increase in D. suzukii trap catches (P < 0.05; Cha et al. 2012, 2014 and Fig. 1) compared to the acetic acid and ethanol mixture, ‘neutral’ when adding the compound did not influence trap catches (P > 0.05; Cha et al. 2012, 2014 and Fig. 1), and ‘negative’ (i.e., antagonistic) when the compound significantly decreased D. suzukii trap catches (P < 0.05; Cha et al. 2012, 2014 and Fig. 1). Co-attractiveness of seven EAD-active compounds (acetoin, grape butyrate, 2-phenylethanol, diethyl succinate, ethyl butyrate, 1-hexanol, and isoamyl acetate) did not change over three different tests (Table 1). However, co-attractiveness of the other five compounds changed among different tests. For example, the co-attractiveness of methionol and ethyl sorbate was changed from positive and neutral, respectively, when they were tested in field experiments or in multiple-choice assays, to neutral and negative, respectively, when they were tested in a two-choice assay, suggesting a potential role of background odor on the behavioral implication of a chemical compound. Discussion Acetoin and methionol, along with acetic acid and ethanol, are key attractant components for D. suzukii. Previously these chemicals were discovered after 2 yr of extensive laboratory and field trapping experiments. However, based on the results from this study, the same outcome could have been achieved with 1 wk of laboratory experiments. The main problem was that the laboratory two-choice assay used in Cha et al. (2012) failed to recognize methionol as an attractive blend component for D. suzukii. This resulted in the formulation of a sub-optimally attractive blend that was not statistically different but numerically inferior to the target mixture of wine and vinegar in the field (Cha et al. 2012). Methionol was identified as an attractant component and added to the blend later based on a field repetition of a similar experiment (Cha et al. 2014). Although the exact olfactory mechanisms underlying our finding are still not clear, it appears evident that the background odor available to D. suzukii can modulate the attractiveness of some potential attractant compounds, an important aspect to consider during the process of identifying behavior modifying chemicals. The key to the success of our multiple-choice approach is to first determine a basal attractant that other potential attractants are co-attractive to. For a fermentation bait, acetic acid, ethanol or the mixture are likely candidates for the basal attractant (Landolt et al. 2015, Cha et al. 2016). Comparing the co-attractiveness of multiple compounds simultaneously but released individually from multiple point sources is a novel approach to directly identify key components of behavior modifying chemicals. In this study, acetoin and methionol were co-attractive to acetic acid plus ethanol mixture and this comprised the same four-component blend that was developed previously from extensive trapping experiments (Cha et al. 2012, 2014) and further improved by optimizing proportionality of the blend components (Cha et al. 2017). While there have been several studies employing multiple-choice assays to facilitate screening of attractants, these studies generally compared attractiveness of different mixtures or extracts and involved elaborate special multi-arm glass olfactometer, such as four- (Vet et al. 1983), six- (Turlings et al. 2004), and eight-arm olfactometers (Liu and Şengonca 1994, Abraham et al. 2015). Our design may be more flexible, as it does not require a specialized olfactometer and thus is not as limited in terms of the number of co-attractants that can be tested together. The role of background odor in insect olfactory behavior has received greater attention recently (Webster and Cardé 2017). Although our results did not test the effect of background odor per se (i.e., effect of odor of habitat that includes host plant, non-host plant, soil, microbes, etc), our results show that the responses of D. suzukii to individual potential attractants can be changed by the differential presence of odor (e.g., potential attractants, odor from the habitat, etc.) in the background, and suggest that evaluation of attractiveness of different odorants needs to be done in the context of a background odor. In particular, methionol is a key component of the SWD lure, as removing methionol from the attractant blend significantly reduced attraction in the field (Cha et al. 2014). The fact that this compound was co-attractive to the acetic acid and ethanol mixture when there was additional odor (i.e., in field tests and the multi-choice test) but was not co-attractive when there was only methionol, acetic acid and ethanol as the background in the two-choice assay, highlights the need to consider background odor in the process of attractant development. The background odor environment in a laboratory bioassay is inherently less noisy than in the field, and the difference in the background odor may contribute to discrepancies in the perception of a volatile compound between the laboratory and field. For example, it has been shown that olfactory coding of some behaviorally important individual odorants in the glomeruli of Spodoptera littoralis could change when the compound was presented to the moth with additional odorants as a chemical blend (Saveer et al. 2012). Our approach of testing the co-attractiveness of all or several individual EAD-active compounds might create a background odor that aided in the discrimination of different sources, similar to what would happen in natural habitats, compared to when only one compound is available for the insect to respond to (i.e., two-choice assays). We recognize this approach may not apply to all insects, especially when determining the basal attractant is difficult (i.e., in many cases, including pheromone and host plant attraction, insects typically do not respond to single compounds). However, we expect it will be relatively successful for insects that respond to fermentation baits as well as other baits like decomposing plant or animal material where the same principal would apply. For example, this approach may be useful to identify attractants for insects responding to protein baits with basal attractants such as chemicals containing ammonia (e.g., ammonium acetate, ammonium carbonate, ammonium bicarbonate, etc; Epsky et al. 2004, Yee et al. 2005). This type of interference from background odors may be more common when ubiquitous volatiles from plants, microbes, or food are involved in the attraction, compared to pheromone attraction, which may be chemically more distinctive and less influenced by a background odor. Acknowledgments We thank Gabrielle Brind’Amour and Shinyoung Park for maintaining the insect colony and technical assistance and two anonymous reviewers for insightful comments. 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A Multiple-Choice Bioassay Approach for Rapid Screening of Key Attractant Volatiles

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Entomological Society of America
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Published by Oxford University Press on behalf of Entomological Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US.
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0046-225X
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1938-2936
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10.1093/ee/nvy054
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Abstract

Abstract Fermentation volatiles attract a wide variety of insects and are used for integrated pest management. However, identification of the key behavior modifying chemicals has often been challenging due to the time consuming nature of thorough behavioral tests and unexpected discrepancies between laboratory and field results. Thus we report on a multiple-choice bioassay approach that may expedite the process of identifying field-worthy attractants in the laboratory. We revisited the four-component key chemical blend (acetic acid, ethanol, acetoin, and methionol) identified from 12 antennally active wine and vinegar chemicals for Drosophila suzukii (Matsumura) (Diptera: Drosophilidae). The identification of this blend took 2 yr of continuous laboratory two-choice assays and then similarly designed field trials. This delay was mainly due to a discrepancy between laboratory and field results that laboratory two-choice assay failed to identify methionol as an attractant component. Using a multiple-choice approach, we compared the co-attractiveness of the 12 potential attractants to an acetic acid plus ethanol mixture, known as the basal attractant for D. suzukii, and found similar results as the previous field trials. Only two compounds, acetoin and, importantly, methionol, increased attraction to a mixture of acetic acid and ethanol, suggesting the identification of the four-component blend could have been expedited. Interestingly, the co-attractiveness of some of the 12 individual compounds, including a key attractant, methionol, appears to change when they were tested under different background odor environments, suggesting that background odor can influence detection of potential attractants. Our findings provide a potentially useful approach to efficiently identify behaviorally bioactive fermentation chemicals. fermentation volatile, bioassay, background odor, attractant, spotted wing drosophila Fermentation volatiles produced by yeasts and acetic acid bacteria can attract a wide variety of insects and have been extensively used to manipulate insect pest behavior for integrated pest management, including various species of beetles (Nout and Bartelt 1998), flies (Lee et al. 2012; Landolt et al 2015; Cha et al. 2016b, 2018), moths (Yamazaki 1998, Sussenbach and Fiedler 1999, Laaksonen et al. 2006, Pettersson and Franzén 2008) and vespid wasps (Dvorak and Landolt 2006, Noll and Gomes 2009, De Souza et al. 2011, Landolt et al. 2014a,b). The implementation of management program often favors using synthetic chemical attractants over bait materials, as a well-characterized chemical lure composed of essential attractant components may be easier to deploy, longer lasting, and more selective than fermentation baits (e.g., Cha et al. 2015). For example, chemical lures for Vespula spp. (Landolt 1998) and Drosophila suzukii (Matsumura) (Diptera: Drosophilidae) (Cha et al. 2014) have been developed based on fermentation bait volatiles and are currently being widely used. Despite the large potential benefits, however, the tedious and time-consuming nature of behavioral tests (Turlings et al. 2004), the lack of clarity in choosing different behavioral testing protocols (e.g., subtraction vs. addition approaches; Linn et al. 1984), and the unexpected discrepancies in behavioral responses from laboratory and field experiments (Knudsen et al. 2008) have often slowed the process of identifying key attractant components and the development of a synthetic lure. Therefore, a straightforward behavioral testing method that can be conducted in a relatively simple laboratory setting and at the same time generate field-worthy results would facilitate the development of effective attractant lures. Recently a chemical lure composed of acetic acid, ethanol, acetoin and methionol was isolated, identified, optimized and commercialized from a fermentation bait for D. suzukii (Cha et al. 2012, 2014, 2017, 2018), which is more efficient and selective than the original material of a wine plus vinegar mixture (Cha et al. 2013, 2015). However, the lure development was slow, taking more than 2 yr of continuous laboratory two-choice assays and field-trapping experiments in multiple locations, even after 12 candidate attractant chemicals were determined using gas chromatography-electroantennographic detection (GC-EAD). One of the major reasons behind the delay was discrepancies in behavioral testing results between laboratory choice assays (Cha et al. 2012) and field trapping experiments (Cha et al. 2014; summarized in Table 1). The co-attractiveness (Meagher and Landolt 2008, Landolt et al. 2014a,b) of some of the 12 EAD-active compounds to a mixture of acetic acid and ethanol were different in laboratory and field, resulting in different blend compositions that resulted in different attractiveness in the field. The discrepancy could have been due to the differences in background odor available in two-choice laboratory assays versus field trapping experiments (Cha et al. 2014). As the 12 EAD-active compounds were ubiquitous fermentation volatiles, field sites may have had a mixture of these 12 compounds and even other volatile compounds in the background, released from various materials including fermenting SWD host fruits, while in laboratory only the chemicals tested in a specific two-choice assay were present as the background. Table 1. Comparison of co-attractiveness of D. suzukii candidate attractants with acetic acid (AA) and ethanol (EtOH) in 1) a laboratory two-choice experiment (AA + EtOH vs. AA + EtOH + one of the candidate attractant; Cha et al. 2012), 2) a field ‘add-on’ test (AA + EtOH vs. AA + EtOH + one of the candidate attractants; Cha et al. 2014), and 3) multiple-choice assay conducted in this study Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Positive indicates that the compound added attractiveness to AA + EtOH (P < 0.05), negative decreased the attractiveness (P < 0.05), and neutral had no effect (P > 0.05) based on Tukey-Kramer tests (Cha et al. 2012, 2014 and Fig 1). *(bold) indicates two key chemicals in the four-component essential blend (acetic acid, ethanol, acetoin and methionol). View Large Table 1. Comparison of co-attractiveness of D. suzukii candidate attractants with acetic acid (AA) and ethanol (EtOH) in 1) a laboratory two-choice experiment (AA + EtOH vs. AA + EtOH + one of the candidate attractant; Cha et al. 2012), 2) a field ‘add-on’ test (AA + EtOH vs. AA + EtOH + one of the candidate attractants; Cha et al. 2014), and 3) multiple-choice assay conducted in this study Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Candidate co-attractant (SWD antennally active yeast chemicals) Laboratory two-choice test Field add-on test Multi-choice test 3-hydroxy butan-2-one (acetoin)* Positive Positive Positive 3-methylsulfanylpropan-1-ol (methionol)* Neutral Positive Positive Ethyl 2-hydroxypropionate (Ethyl lactate) Negative Positive Neutral 2-methylbutyl acetate Negative Neutral Negative Ethyl (2E,4E)-hexa-2,4-dienoate (Ethyl sorbate) Negative Neutral Neutral 3-methylbutyl 2-hydroxypropionate (Isoamyl lactate) Neutral Negative Neutral Ethyl 3-hydroxybutyrate (Grape butyrate) Neutral Neutral Neutral 2-phenylethanol Neutral Neutral Neutral Diethyl butanedioate (Diethyl succinate) Neutral Neutral Neutral Ethyl butyrate Negative Negative Negative 1-hexanol Negative Negative Negative 3-methylbutyl acetate (Isoamyl acetate) Negative Negative Negative Positive indicates that the compound added attractiveness to AA + EtOH (P < 0.05), negative decreased the attractiveness (P < 0.05), and neutral had no effect (P > 0.05) based on Tukey-Kramer tests (Cha et al. 2012, 2014 and Fig 1). *(bold) indicates two key chemicals in the four-component essential blend (acetic acid, ethanol, acetoin and methionol). View Large Fig. 1. View largeDownload slide Mean (±SEM) numbers of Drosophila suzukii flies captured in traps baited with a control (a mixture of 1.6% acetic acid and 7.2% ethanol; AA + EtOH) and 12 different ‘add-on’ blends (AA + EtOH + one of the EAD-active compounds listed in Table 1). AT: Acetoin, EL: Ethyl lactate, IAA: Isoamyl acetate, MBA: 2-methylbutyl acetate, GB: Grape butyrate, PE: 2-phenylethanol, EB: Ethyl butyrate, HX: 1-hexanol; MT: Methionol, IAL: Isoamyl lactate, ES: Ethyl sorbate, DS: Diethyl succinate. Different letters on bars indicate significant differences by Tukey-Kramer tests at P < 0.05. Statistical tests were based on square-root-transformed data. Means from untransformed data are shown. Fig. 1. View largeDownload slide Mean (±SEM) numbers of Drosophila suzukii flies captured in traps baited with a control (a mixture of 1.6% acetic acid and 7.2% ethanol; AA + EtOH) and 12 different ‘add-on’ blends (AA + EtOH + one of the EAD-active compounds listed in Table 1). AT: Acetoin, EL: Ethyl lactate, IAA: Isoamyl acetate, MBA: 2-methylbutyl acetate, GB: Grape butyrate, PE: 2-phenylethanol, EB: Ethyl butyrate, HX: 1-hexanol; MT: Methionol, IAL: Isoamyl lactate, ES: Ethyl sorbate, DS: Diethyl succinate. Different letters on bars indicate significant differences by Tukey-Kramer tests at P < 0.05. Statistical tests were based on square-root-transformed data. Means from untransformed data are shown. The two major fermentation volatiles, acetic acid and ethanol, have been shown to be involved in insect attraction to fermentation sources as individual compounds or as a mixture. In some cases, a mixture of acetic acid and ethanol was sufficiently attractive to explain such attraction, while in other cases additional fermentation volatiles in addition to acetic acid and ethanol were necessary. For example, a mixture of acetic acid and ethanol was as attractive as the original material of yeast-sugar fermenting solution for an avian parasitic fly, Philornis downsii (Cha et al. 2016) or as a wine plus vinegar bait for false stable flies (Landolt et al. 2015). In contrast, in other insects such as D. suzukii, although ethanol and acetic acid were essential for the attraction, additional fermentation volatiles were necessary to achieve the similar level of attraction to the original fermentation material (Landolt et al. 2012b). In particular, acetic acid + ethanol was more attractive than either chemical alone (Landolt et al. 2012a) but less attractive than a mixture of wine and vinegar that contained the same amount of acetic acid and ethanol, suggesting that SWD are responding to some other wine and vinegar chemicals (Landolt et al. 2012b). Here, we report on a simple laboratory multiple-choice assay approach that uses a mixture of acetic acid and ethanol as a basal attractant and tests co-attractiveness of all of the individual EAD-active compounds separately but concurrently. The new assay was conducted by revisiting the 12 EAD-active wine and vinegar chemicals involved in the development of the four-component chemical lure for D. suzukii. In the multiple-choice assay, all of the 12 EAD-active compounds were available to SWD to respond to in the headspace inside the cage, but released from separate individual traps with acetic acid and ethanol as a basal attractant. This laboratory assay allowed us to quickly predict which compounds would be co-attractive with acetic acid plus ethanol for SWD in the field, which was essential to achieve the same level of attraction from D. suzukii to the original benchmarking mixture of wine and vinegar. Materials and Methods Insects For multiple-choice bioassays, a colony of SWD was maintained at the New York State Agricultural Experiment Station, Geneva, New York. Flies were reared at 21.5 ± 0.9°C, 23.7 ± 0.1 % RH, 16:8 (L:D) h on standard cornmeal diet (1 liter distilled water, 40 g sucrose, 25 g cornmeal [Quaker Oats Co., Chicago, IL], 9 g agar [No. 7060, Bioserve, Flemington, NJ], 14 g torula yeast [No. 1720, Bioserve], 3 ml glacial acetic acid [Amresco, Solon, OH], 0.6 g methyl paraben [No 7685, Bioserve], and 6.7 ml ethanol). Chemicals For this study, we used the 12 EAD-active chemicals (Table 1) that were previously identified from a Merlot wine and a rice vinegar mixture (Cha et al. 2012). Ethyl butyrate (99%, CAS No. 105-54-4), 3-hydroxybutan-2-one (acetoin) (≥96%, CAS No. 513-86-0), 3-methylbutyl acetate (isoamyl acetate) (98%, CAS No. 123-92-2), 2-methylbutyl acetate (99%, CAS No. 624-41-9), 3-methylsulfanylpropan-1-ol (methionol) (≥98%, CAS No. 505-10-2), ethyl (2E,4E)-hexa-2,4-dienoate (ethyl sorbate) (≥97%, CAS No. 2396-84-1), and 2-phenylethanol (≥99%, CAS No. 60-12-8) were purchased from Sigma-Aldrich (St. Louis, MO). Ethyl 2-hydroxypropionate (ethyl lactate) (>97%, CAS No. 97-64-3), 3-methylbutyl 2-hydroxypropionate (isoamyl lactate) (>98%, CAS No. 19329-89-6), and diethyl butanedioate (diethyl succinate) (>99%, CAS No. 123-25-1) were purchased from TCI America (Morris, Portland, OR). Ethyl 3-hydroxybutyrate (grape butyrate) (99%, CAS No. 5405-41-4) was purchased from Arcos Organics (New Jersey). Ethanol (200 proof, CAS No. 64-47-5), acetic acid (99.8%, CAS No. 64-19-7), and 1-hexanol (>95%, CAS No. 111-27-3) were purchased from Pharmco (Brookfield, CT), Fisher Scientific (Pittsburgh, PA, USA), and J. T. Baker (Philipsburg, NJ), respectively. Multiple-Choice Assay The multiple choice assay (N = 5) was conducted with an arena design using a dome cage (60 cm W × 60 cm L × 60 cm H; BugDorm-2120 Insect tent; shop.bugdorm.com). Within each arena, 13 traps were positioned in a circle (20 cm diameter) at equal distance with each trap 9.7 cm apart along the circumference. We used an aluminum foil-covered 100-ml glass beaker with a cut centrifuge vial (0.7 cm diameter) inserted in the foil for SWD entry, as a trap (Cha et al. 2012). All 13 traps had 20 ml 1.6% acetic acid + 7.2% ethanol with soap as a basal attractant and drowning solution. One of the traps served as a control and the remaining 12 traps had one of the 12 EAD-active compounds added to the control blend, loaded as described in Cha et al. (2012) and released from 1.5-ml vial with 3-mm hole with a piece of cotton. A cotton ball (3 cm diameter) soaked with distilled water was placed in the center of arena to provide water to SWD. Adult flies (160–235, roughly 1:1 male: female ratio, 7–10-d-old) were released in each arena. Choice assays started at 1:00 p.m. each day (21.5 ± 0.9°C, 23.7 ± 0.1% RH, 16:8 [L:D] h). The number of flies inside treatment and control traps was counted after 20 h. Statistical Analysis A randomized complete block design was used. Trap catches were analyzed with block as a random factor and different odor sources as a fixed factor using Proc Mixed (SAS Institute 2009). Fly catch data were square-root transformed to improve normality and homoscedasticity (Zar 1984). The means were compared using the Tukey-Kramer test (SAS Institute 2009). Results The results from the laboratory multiple-choice test showed that two compounds, acetoin and methionol, were co-attractive with a mixture of acetic acid and ethanol (basal attractant), significantly increasing the number of D. suzukii trapped by 4.5- and 2.6-fold, respectively, compared to the control traps baited with the mixture of acetic acid and ethanol (F12,48 = 37.81, P < 0.0001; Fig. 1). In each experiment listed in Table 1, an EAD-active compound was indicated ‘positive’ (i.e., co-attractive) when adding the compound to the mixture of 1.6% acetic acid and 7.2% ethanol resulted in a significant increase in D. suzukii trap catches (P < 0.05; Cha et al. 2012, 2014 and Fig. 1) compared to the acetic acid and ethanol mixture, ‘neutral’ when adding the compound did not influence trap catches (P > 0.05; Cha et al. 2012, 2014 and Fig. 1), and ‘negative’ (i.e., antagonistic) when the compound significantly decreased D. suzukii trap catches (P < 0.05; Cha et al. 2012, 2014 and Fig. 1). Co-attractiveness of seven EAD-active compounds (acetoin, grape butyrate, 2-phenylethanol, diethyl succinate, ethyl butyrate, 1-hexanol, and isoamyl acetate) did not change over three different tests (Table 1). However, co-attractiveness of the other five compounds changed among different tests. For example, the co-attractiveness of methionol and ethyl sorbate was changed from positive and neutral, respectively, when they were tested in field experiments or in multiple-choice assays, to neutral and negative, respectively, when they were tested in a two-choice assay, suggesting a potential role of background odor on the behavioral implication of a chemical compound. Discussion Acetoin and methionol, along with acetic acid and ethanol, are key attractant components for D. suzukii. Previously these chemicals were discovered after 2 yr of extensive laboratory and field trapping experiments. However, based on the results from this study, the same outcome could have been achieved with 1 wk of laboratory experiments. The main problem was that the laboratory two-choice assay used in Cha et al. (2012) failed to recognize methionol as an attractive blend component for D. suzukii. This resulted in the formulation of a sub-optimally attractive blend that was not statistically different but numerically inferior to the target mixture of wine and vinegar in the field (Cha et al. 2012). Methionol was identified as an attractant component and added to the blend later based on a field repetition of a similar experiment (Cha et al. 2014). Although the exact olfactory mechanisms underlying our finding are still not clear, it appears evident that the background odor available to D. suzukii can modulate the attractiveness of some potential attractant compounds, an important aspect to consider during the process of identifying behavior modifying chemicals. The key to the success of our multiple-choice approach is to first determine a basal attractant that other potential attractants are co-attractive to. For a fermentation bait, acetic acid, ethanol or the mixture are likely candidates for the basal attractant (Landolt et al. 2015, Cha et al. 2016). Comparing the co-attractiveness of multiple compounds simultaneously but released individually from multiple point sources is a novel approach to directly identify key components of behavior modifying chemicals. In this study, acetoin and methionol were co-attractive to acetic acid plus ethanol mixture and this comprised the same four-component blend that was developed previously from extensive trapping experiments (Cha et al. 2012, 2014) and further improved by optimizing proportionality of the blend components (Cha et al. 2017). While there have been several studies employing multiple-choice assays to facilitate screening of attractants, these studies generally compared attractiveness of different mixtures or extracts and involved elaborate special multi-arm glass olfactometer, such as four- (Vet et al. 1983), six- (Turlings et al. 2004), and eight-arm olfactometers (Liu and Şengonca 1994, Abraham et al. 2015). Our design may be more flexible, as it does not require a specialized olfactometer and thus is not as limited in terms of the number of co-attractants that can be tested together. The role of background odor in insect olfactory behavior has received greater attention recently (Webster and Cardé 2017). Although our results did not test the effect of background odor per se (i.e., effect of odor of habitat that includes host plant, non-host plant, soil, microbes, etc), our results show that the responses of D. suzukii to individual potential attractants can be changed by the differential presence of odor (e.g., potential attractants, odor from the habitat, etc.) in the background, and suggest that evaluation of attractiveness of different odorants needs to be done in the context of a background odor. In particular, methionol is a key component of the SWD lure, as removing methionol from the attractant blend significantly reduced attraction in the field (Cha et al. 2014). The fact that this compound was co-attractive to the acetic acid and ethanol mixture when there was additional odor (i.e., in field tests and the multi-choice test) but was not co-attractive when there was only methionol, acetic acid and ethanol as the background in the two-choice assay, highlights the need to consider background odor in the process of attractant development. The background odor environment in a laboratory bioassay is inherently less noisy than in the field, and the difference in the background odor may contribute to discrepancies in the perception of a volatile compound between the laboratory and field. For example, it has been shown that olfactory coding of some behaviorally important individual odorants in the glomeruli of Spodoptera littoralis could change when the compound was presented to the moth with additional odorants as a chemical blend (Saveer et al. 2012). Our approach of testing the co-attractiveness of all or several individual EAD-active compounds might create a background odor that aided in the discrimination of different sources, similar to what would happen in natural habitats, compared to when only one compound is available for the insect to respond to (i.e., two-choice assays). We recognize this approach may not apply to all insects, especially when determining the basal attractant is difficult (i.e., in many cases, including pheromone and host plant attraction, insects typically do not respond to single compounds). However, we expect it will be relatively successful for insects that respond to fermentation baits as well as other baits like decomposing plant or animal material where the same principal would apply. For example, this approach may be useful to identify attractants for insects responding to protein baits with basal attractants such as chemicals containing ammonia (e.g., ammonium acetate, ammonium carbonate, ammonium bicarbonate, etc; Epsky et al. 2004, Yee et al. 2005). This type of interference from background odors may be more common when ubiquitous volatiles from plants, microbes, or food are involved in the attraction, compared to pheromone attraction, which may be chemically more distinctive and less influenced by a background odor. Acknowledgments We thank Gabrielle Brind’Amour and Shinyoung Park for maintaining the insect colony and technical assistance and two anonymous reviewers for insightful comments. 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Environmental EntomologyOxford University Press

Published: Apr 13, 2018

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