Advances in the Chemical Ecology of the Spotted Wing Drosophila (Drosophila suzukii) and its Applications

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1 Journal of Chemical Ecology Advances in the Chemical Ecology of the Spotted Wing Drosophila (Drosophila suzukii) and its Applications Kevin R. Cloonan 1 & John Abraham 2 & Sergio Angeli 3 & Zainulabeuddin Syed 4 & Cesar Rodriguez-Saona 1 Received: 15 May 2018 /Revised: 10 July 2018 /Accepted: 18 July 2018 # Springer Science+Business Media, LLC, part of Springer Nature 2018 Abstract Significant progress has been made in understanding the cues involved in the host and mate seeking behaviors of spotted wing drosophila, Drosophila suzukii (Matsumura). This insect pest has been discovered in many fruit growing regions around the world since Unlike closely related Drosophila species, D. suzukii attacks fresh fruit and has become a severe pest of soft fruits including strawberry, cherry, blackberry, blueberry, raspberry, and may pose a threat to grapes. Prior to 2008, little was known about the courtship and host-seeking behaviors or chemical ecology of this pest. Since then, researchers have gained a better understanding of D. suzukii attraction to specific odors from fermentation, yeast, fruit, and leaf sources, and the visual cues that elicit long-range attraction. Several compounds have also been identified that elicit aversive behaviors in adult D. suzukii flies. Progress has been made in identifying the constituent compounds from these odor sources that elicit D. suzukii antennal responses in electrophysiological assays. Commercial lures based on food volatiles have been developed to attract D. suzukii using these components and efforts have been made to improve trap designs for monitoring this pest under field conditions. However, current food-based lures and trap technologies are not expected to be specific to D. suzukii and thus capture large numbers of non-target drosophilids. Attractive and aversive compounds are being evaluated for monitoring, mass trapping, and for the development of attract-and-kill and push-pull techniques to manage D. suzukii populations. This review outlines presently available research on the chemical ecology of D. suzukii and discusses areas for future research. Keywords Spotted wing drosophila. Invasive pest. Attractants. Repellents. Behavior-based technologies. Pheromones. Chemical communication Introduction Since 2008, spotted wing drosophila, Drosophila suzukii (Matsumura), has emerged as a worldwide invasive pest species Kevin R. Cloonan and John Abraham contributed equally to this work. * Kevin R. Cloonan raynecloonan@gmail.com Department of Entomology, Rutgers University P.E. Marucci Center, 125A Lake Oswego Rd, Chatsworth, NJ, USA Department of Conservation Biology and Entomology, School of Biological Sciences, College of Agriculture and Natural Sciences, University of Cape Coast, Cape Coast, Ghana Faculty of Science and Technology, Free University of Bozen-Bolzano, Piazza Università 5, Bozen-Bolzano, Italy Department of Entomology, University of Kentucky, Lexington, KY 40546, USA around fruit growing regions (Walsh et al. 2011; Asplen et al. 2015; Klick et al. 2016). These flies are native to parts of Southeast Asia (Benito et al. 2016; Evans et al. 2017). In addition to infesting many wild host-plants, D. suzukii also infests highvalue crops including stone fruits, blueberry, strawberry, raspberry, blackberry, plums, and apricots (Burrack et al. 2013; Abraham et al. 2015; Klick et al. 2016;Riceetal.2016; Mazzi et al. 2017) and may become a pest of grape (Ioriatti et al. 2015). Female flies have large, saw-like ovipositors on the tip of their abdomens allowing them to pierce the soft flesh of ripening fruit (Atallah et al. 2014; Harris et al.2014). Unlike other drosophilid flies that lay eggs primarily on decaying and rotten fruit, D. suzukii prefers to oviposit in ripening fruit (Karageorgi et al. 2017). Once a female has pierced through the flesh, it lays her eggs and the developing larvae proceeds to feed on the inside of the infested fruits making the fruit unmarketable (Goodhue et al. 2011; Walsh et al. 2011). The wounds caused by females saw-like ovipositors also leave the infested fruits susceptible to secondary microbial infection (Walsh et al. 2011; Rombaut et al. 2017).

2 In the USA, estimates of economic losses due to D. suzukii attack amounted to over million dollars (Bolda et al. 2010; Walsh et al. 2011). Moreover, in Europe additional costs incurred in fruit production due to D. suzukii infestation could reach 64,138/ha (Mazzi et al. 2017). Presently, the only viable tool for reducing economic injury caused by D. suzukii is frequent application of chemical insecticides (Farnsworth et al. 2017). Growers also lack monitoring lures that are 1) specific for D. suzukii and that 2) accurately predict fruit infestation by the fly. This is especially true in the warmer southern growing regions of the USA where fly populations persist during the winter months (Diepenbrock et al. 2016) and are unlikely to enter an overwintering phase. In colder northern latitudes where low temperatures cause populations to overwinter and die-off, monitoring tools may provide growers with information on D. suzukii activity. In the laboratory, only about 2% of adults and pupae exposed to temperatures of 10 C for 84 days survived (Dalton et al. 2011). It was recently proposed by Kirkpatrick et al. (2018a) that once a single female D. suzukii is detected in a commercially-baited trap, it is highly likely that fruit infestation has already occurred. Reliable monitoring tools that accurately predict D. suzukii activity before fruit are susceptible would allow growers to time insecticide applications more effectively (Cha et al. 2018a, b). For various reasons, including high parasitism by native biological control agents (Daane et al. 2016), D. suzukii has not been reported as a severe insect pest of fruit in its native region. Because of this, no effective monitoring tools were available prior to its invasion of North America and Europe in the late 2000s. Thus, we are at the beginning of understanding the chemical ecology of D. suzukii. Much of our knowledge about the attractive behaviors of D. suzukii to various cues comes from an immediate demand to develop monitoring lures for management decisions. Up to now, research has focused on compounds eliciting broad-range attraction and repellency behaviors in adult flies. For instance, several studies suggest that the odor, color, ripeness, firmness, and microbial communities of host plant species contribute to the selection process of host-seeking D. suzukii flies (e.g. Abraham et al. 2015; Kirkpatrick et al. 2016; Mazzetto et al. 2016;Riceetal.2016;Lasaetal.2017). In this review we survey current knowledge on the chemical ecology of D. suzukii.we reportstudies ond. suzukii with the aim of providing current information on food and host choice selection and factors influencing them, chemical cues used in host selection, current knowledge of D. suzukii pheromones, D. suzukii odorant receptor repertoire, visual cues used in host selection, fly response to aversive chemicals, and interactions with other cues such as color. We discuss how this information has been used to develop behaviorbased management strategies for D. suzukii. Lastly, we attempt to build on knowledge gaps in the chemical ecology of D. suzukii for future work with the ultimate goal of reducing economic damage caused by the fly. Host Choice Selection With a broad host range, D. suzukii exploits plants from thirteen reported plant families (Table 1). Adult flies are not attracted to and cannot develop on other crops like cranberry (Steffan et al. 2013). It may be that cranberry fruit are too firm for D. suzukii to penetrate. Using an artificial substrate, Burrack et al. (2013) showed that increasing the mediums firmness decreased female oviposition. The fact that D. suzukii has a wide variety of host plants makes investigating the odor, visual, and mechanical cues used by adults in host choice selection difficult. Normally, gravid females oviposit in ripening fruit. It has been demonstrated that CO 2 plays a significant role in the ability of D. suzukii to differentiate between ripening and ripe fruit because ripening fruit emit more CO 2 than ripe fruit (Krause Pham and Ray 2015). In addition to CO 2 and other olfactory cues, egg-laying flies also use mechanical cues to discriminate between suitable and unsuitable host material such as fruit firmness (Karageorgi et al. 2017). Female flies prefer to oviposit into softer fruits (Kinjo et al. 2013; Lasa et al. 2017), making soft fruit more susceptible to fly infestation. However, it is logical to suggest that gravid females would oviposit in less ripe (relatively hard) fruit in the absence of softer fruit. Lee et al. (2016) found in laboratory studies that oviposition on blueberry increased with decreasing penetration force. Calcium silicate sprayed on fruit increased the firmness and penetration force of the fruit and hence reduced D. suzukii oviposition (Lee et al. 2016). Fruit ripeness also influences host-choice selection. It is possible that not all fruits at the same ripening stage will have the same firmness. For instance, Ehlenfeldt and Martin Jr (2002) found differences in the fruit firmness of different cultivars of blueberries. In a recent study, it was found that female D. suzukii preferentially oviposit into fresh strawberry compared to overripe strawberry (Karageorgi et al. 2017). However, Keesey et al. (2015) showed that adult female flies are more attracted to overripe strawberry versus green, white, blush, and red strawberries. This discrepancy could be due to the physiological status of the flies each research group tested. For example, ovipositing female flies would be more attracted to fresh fruit volatiles, but food- or mate-seeking females would be more attracted to overripe fruit volatiles. Keesey et al. (2015) suggests that D. suzukii are initially attracted to leaf material and subsequently exploit fresh fruit volatiles for oviposition, and that flies attracted to fermenting and rotting fruit do so for feeding and mating purposes. Tochen et al. (2016) showedthat emerged adult D. suzukii had low reserves of glycogen and sugars. Adults exposed to cherry and blueberry blossoms had significantly greater glycogen, sugar, and fructose levels compared to adults exposed to water controls (Tochen et al. 2016), suggesting that adult flies may seek these sugar sources upon emergence. Cultivar type also strongly influences oviposition

3 Table 1 List of hosts used by Drosophila suzukii Common name Genus species Plant family Reference Raspberry Rubus idaeus Rosaceae Abraham et al Cherry Prumus cerasus Rosaceae Revadi et al Strawberry Fragaria x ananassa Rosaceae Revadi et al Blackberry Rubus allegheniensis Rosaceae Elsensohn and Loeb 2018 Black raspberry Rubus occidentalis Rosaceae Elsensohn and Loeb 2018 Milkflower cotoneaster Cotoneaster lacteus Rosaceae Lee et al Wild cherry Prunus avium Rosaceae Lee et al Cherry laurel Prunus laurocerasus Rosaceae Lee et al Portuguese laurel Prunus lusitanica Rosaceae Lee et al Himalaya blackberry Rubus armeniacus Rosaceae Lee et al Salmonberry Rubus spectabilis Rosaceae Lee et al Dewberry Rubus aboriginum Rosaceae Ballman and Drummond 2017 Dogwood Cormus spp. Cornaceae Lee et al Bunchberry Cormus canadensis Cornaceae Ballman and Drummond 2017 Silky dogwood Cormus amomum Cornaceae Elsensohn and Loeb 2018 Blue honeysuckle Lonicera caerulea Caprifoliaceae Lee et al Snowberry Symphoricarpos albus Caprifoliaceae Lee et al Japanese honeysuckle Lonicera japonica Caprifoliaceae Ballman and Drummond 2017 Morrow s honeysuckle Lonicera morrowii Caprifoliaceae Elsensohn and Loeb 2018 Mullberry Morus spp. Moraceae Lee et al Mulberry Ficus carica Moraceae Yu et al Cascara buckthorn Frangula purshiana Rhamnaceae Lee et al Common buckthorn Rhammus cathartica Rhamnaceae Elsensohn and Loeb 2018 Oregon grape Mahonia aquifolium Berberidaceae Lee et al Autumn olive Elaeagmus umbellate Elaeagnaceae Lee et al Spicebush Lindera benzoin Lauraceae Lee et al Black elderberry Sambucus nigra Adoxaceae Lee et al Sweet box Sarcococca confusa Buxaceae Lee et al Bittersweet nightshade Solanum dulcamara Solanaceae Lee et al American pokeweed Phytolacca americana Phytolaccaceae Elsensohn and Loeb 2018 Blueberry Vaccinium corymbosum Ericaceae Abraham et al choice in blackberries, blueberries, raspberries, and wine grapes (Lee et al. 2011a, b). Early ripening cultivars can escape infestation. For example, in northern latitudes in the USA some early ripening highbush blueberry cultivars are harvested before local D. suzukii populations are active and able to lay eggs (Hampton et al. 2014). Other studies have demonstrated that ph and Brix (total soluble sugars) of fruit may influence D. suzukii oviposition behavior. Lee et al. (2016) showed that oviposition increases with increasing ph and Brix. This finding is in line with earlier studies that showed that females lay more eggs on fruits with higher sugar content and less acidity, and larvae develop faster on high-sugar fruit varieties (Lee et al. 2011a, b; Leeet al. 2015). However, a more recent study investigating a different host fruit showed that female D. suzukii preferred to oviposit on fruits with a lower ph and Brix (Little et al. 2017), suggesting that females rely on a combination of cues to discriminate between hosts. Both long- and short- range olfactory cues are important factors in D. suzukii host selection process (see below). The interactions of these mechanical and olfactory cues on D. suzukii host choice selection, however, have not been investigated. Odor Cues While foraging, D. suzukii flies seek habitats suitable for feeding, mating, and oviposition, and volatiles play key roles during this process (Fig. 1). We expect volatiles to be involved in two steps during fly foraging: 1) location of habitats associated with fruit for feeding and mating; and, 2) location of fruit for oviposition. First, flies locate the general habitat where potential host fruit are likely found. For this, similar to other vinegar flies, they may use ubiquitous odors from

4 Fig. 1 A graphical representation of the different foraging choices and odor cues used by virgin and mated female D. suzukii flies. a Newly emerged female flies seek rotten fruit b for sources of sugar and protein. c She likely uses fermentation volatiles (i: ethanol, ii: acetic acid, iii: acetoin, and iv: methionol) to locate areas containing rotten fruit. It may also be that female flies use leaf odors (v: β-cyclocitral) to locate areas where male flies might be. d Mated, gravid female flies then seek intact fresh fruit e for egg-laying purposes. f They likely utilizing a suite of fruit and yeast odors (vi: trans-2-hexanal, vii: hexanol, viii: 3- methyl-2-butenyl-acetate, ix: 3-methyl-2-butanone, x: 2-heptanone, xi: butyl acetate, xii: isoamyl acetate, and xiii: isobutyl acetate) to locate such intact fresh fruits fermentation products (Fig. 1). Volatiles from plant sources (e.g. leaves) and/or yeast might also be used, if available, but likely to a lesser extent. These fermentation products attract flies to places likely for feeding and mating. Next, mated females seek oviposition sites, and fruit volatiles are likely important in this process (Fig. 1). Fruit and yeast volatiles may interact in female attraction to oviposition sites. The volatiles identified from these different sources (i.e. fermentation, fruit, leaves, and yeast) and evidence of their involvement during D. suzukii foraging are discussed next. Several recent studies have identified chemical cues that attract adult D. suzukii to host plants, as well as compounds that its antennae can detect. Specific references of antennally active compounds to D. suzukii are reported in Table 2.Adult flies utilize these different odor cues for distinct behaviors (Table 3). For example, in laboratory assays, Karageorgi et al. (2017) showed that adult flies utilize fruit odors for oviposition behaviors and yeast odors for feeding behaviors. Leaf odors may be involved in host and mate finding and courtship behaviors. Below we have separated various volatile (attractive and aversive) compounds based on a primary source of their emission (e.g. fermentation, fruit, leaf, microbial); however, we recognize that some of these compounds can be produced and emitted from multiple sources. Similarly, these compounds are potentially used parsimoniously by flies as dictated by their physiological state. Also, it is important to note that insect attraction to host material is the result of the central processing of specific ratios of ubiquitous compounds (i.e. odor cues), not the recognition of species-specific compounds solely at the peripheral level (i.e. odorants) (Bruce et al. 2005). Practically, this means that a single compound could elicit an antennal or behavioral response in the laboratory, but this compound may have little ecological significance to a host-, food-, or mate-seeking insect in its natural environment when combined with additional stimuli. Fermentation Odors In an effort to explore D. suzukii attraction to fermentation odors, Landolt et al. (2012a) placed different combinations of wine, apple cider vinegar, ethanol, and acetic acid (the primary components of wine and vinegar, respectively) in blackberry fields. They hypothesized that much or all of the flies response to vinegar and wine were due to acetic acid and ethanol, respectively. They found that mixtures of Merlot wine and apple cider vinegar were more attractive to female D. suzukii than mixtures of ethanol and acetic acid alone (Landolt et al. 2012a), suggesting that compounds other than acetic acid and ethanol contribute to D. suzukii attraction. Soon after these experiments, Landolt et al. (2012b) examined mixtures of wine, vinegar, ethanol, and acetic acid for field attraction. They hypothesized that these mixtures would result in synergistic fly attraction. They found that (wine + vinegar) and (ethanol + acetic acid)

5 Table 2 List of antennally (EAD)-active compounds on Drosophila suzukii found in fruits (including strawberry, raspberry, cherry, blueberry, and blackberry), fruit extracts, foliage, and fermentation products (e.g. Merlot wine and rice vinegar) in different studies Compound Source Reference Acids Acetic acid 1, 2 Revadi et al. 2015, Cha et al. 2012, Mazzetto et al Hexanoic acid 1 Revadi et al Methylpropanoic acid 2 Mazzetto et al Methylbutanoic acid 2 Mazzetto et al Methylbutanoic acid 2 Mazzetto et al Alcohols Ethanol 1, 2 Revadi et al. 2015, Cha et al. 2012, Mazzetto et al Hexanol 1, 3, 2 Revadi et al. 2015, Abraham et al. 2015, Cha et al Z-3-Hexen-1-ol 1, 3, 4 Revadi et al. 2015, Abraham et al. 2015, Keeseyetal.2015 E-2-Hexenol 4 Keesey et al Octanol 1 Revadi et al Octen-3-ol 1, 4 Revadi et al. 2015, Keeseyetal.2015 β-phenylethanol 1 Revadi et al Methyl-1-butanol 3 Abraham et al Heptanol 3 Abraham et al Methyl-5-hepten-2-ol 3, 4 Abraham et al. 2015, Keeseyetal.2015 E-2-nonenol 4 Keesey et al Phenethyl alcohol 4 Keesey et al Nitrophenol 4 Keesey et al Eugenol 4 Keesey et al Methionol 2 Cha et al Phenylethanol 2 Cha et al Propanol 2 Mazzetto et al Aldehydes Hexanal 3 Abraham et al E-2-Hexenal 1, 3 Revadi et al. 2015, Abraham et al Nonanal 1 Revadi et al Benzaldehyde 2 Mazzetto et al Ketone 2-Heptanone 1, 3 Revadi et al. 2015, Abraham et al Acetoin 2 Cha et al Propanone 2 Mazzetto et al Esters Butyl acetate 3 Abraham et al Ethyl acetate 1, 2 Revadi et al. 2015, Cha et al Hexyl acetate 1 Revadi et al. 2015, Keeseyetal.2015 Isoamylacetate 1,2 Revadietal.2015, Cha et al Ethyl butanoate 1 Revadi et al Ethyl hexanoate 1 Revadi et al Ethyl octanoate 1 Revadi et al Methyl hexanoate 1 Revadi et al Methyl octanoate 1 Revadi et al Z-3-Hexenyl acetate 1, 4 Revadi et al. 2015, Keeseyetal Methyl-2-butenyl acetate 3 Abraham et al Ethyl butyrate 2 Cha et al Ethyl lactate 2 Cha et al Methylbutyl acetate 2 Cha et al Grape butyrate 2 Cha et al Isoamyl lactate 2 Cha et al Ethyl sorbate 2 Cha et al Diethyl succinate 2 Cha et al Methyl butyrate 1 Keesey et al Methyl isovalerate 1 Keesey et al Isopentyl acetate 1 Keesey et al Aromatics Methyl salicylate 1, 4 Revadi et al. 2015; Keeseyetal.2015 Norisoprenoids α-ionone 1 Revadi et al β-ionone 1, 4 Revadi et al. 2015; Keeseyetal.2015 Isoprenoids β-cyclocitral 4 Keesey et al Monoterpenes α-phellandrene 1 Revadi et al. 2015

6 Table 2 (continued) Compound Source Reference β-phellandrene 1 Revadi et al Limonene 1 Revadi et al p-cymene 1 Revadi et al Linalool 1, 3 Revadi et al. 2015, Abraham et al Sesquiterpenes E-Caryophyllene 1 Revadi et al , Fruit;2, Fermentation product; 3, Fruit extract; 4, Foliage of fruit crop e.g. strawberry mixtures were more attractive to D. suzukii than either component alone (Landolt et al. 2012b), suggesting that there may be some synergistic attraction with these odors. Building upon these findings, Cha et al. (2012) investigated the headspace of two fermentation by-products (rice vinegar and Merlot wine that are attractive to D. suzukii) to isolate antennally (electro-antennographic detection, EAD)-active compounds to D. suzukii. Rice vinegar contained seven EAD-active compounds, namely ethyl acetate, 3-hydroxybutanone (acetoin), ethyl lactate, isoamyl acetate, 2-methylbutyl acetate, ethyl-3- hydroxybutyrate (grape butyrate), and 2-phenylethanol at a release rateof 1.6 mg/day/100 ml in a mixture. Merlot wine Table 3 List of attractive and aversive cues, both chemical and visual, for Drosophila suzukii from several different sources Attractive cues Merlot wine Apple cider vinegar Cha et al Rice vinegar Cha et al. 2012; Akasaka et al Bakers yeast and sugar mixture Cha et al Acetic acid Revadi et al Ethanol Cha et al Acetoin Cha et al Ethyl lactate Cha et al Methionol Cha et al Mixture of:butyl acetate, hexanol, 2-heptanone, 3-methyl-1-butanone, trans-2-hexanal, Abraham et al methyl-2-butenyl acetate, 2-heptanol, hexanol, cis-3-hexanol, 6-methyl-5-hepten-2-ol, linalool Isoamyl acetate Dekker et al Yeast species Hanseniaspora uvarum in culture Mori et al Acetic acid bacteria Gluconobacter oxydans in culture Mazzetto et al Acetic acid bacteria Gluconobacter kanchanaburiensis in culture Mazzetto et al Acetic acid bacteria Gluconobacter saccharivorans in culture Mazzetto et al β-cyclocitral Keesey et al Red color Kirkpatrick et al. 2016; Riceetal.2016 Black color Kirkpatrick et al Purple color Kirkpatrick et al Aversive cues Geranium essential oil Renkema et al Peppermint essential oil Renkema et al Citronella essential oil Renkema et al Lavender essential oil Renkema et al Thyme essential oil Renkema et al Eastern white pine essential oil Renkema et al White pine essential oil Renkema et al White spruce essential oil Renkema et al Rosemary essential oil Renkema et al Ginger essential oil Renkema et al Eucalyptus essential oil Renkema et al Thymol Renkema et al Citronellol Renkema et al octen-3-ol Wallingford et al. 2016a Geosmin Wallingford et al. 2016a ( )-iridomyrmecin (from Leptopilina boulardi) Ebrahim et al (R)-actinidine (from Leptopilina boulardi) Ebrahim et al Butyl anthranilate Krause Pham and Ray 2015 Methyl N,N-dimethylanthranilate Krause Pham and Ray 2015 Ethyl anthranilate Krause Pham and Ray 2015

7 J Chem Ecol contained thirteen EAG-active compounds; the following six compounds in addition to the previous seven: ethyl butyrate, 1-hexanol, methionol, isoamyl lactate, ethyl sorbate, and diethyl succinate at a release rate of 2.2 mg/day/100 ml in a mixture (Cha et al. 2012). In field assays, a combination of wine and vinegar captured significantly more D. suzukii than mixtures of acetic acid and ethanol, the seven component EAD-active vinegar blend (including acetic acid and ethanol), or the thirteen component EAD-active wine blend (including acetic acid and ethanol) (Cha et al. 2012). These results suggest that either wine and/ or vinegar contain more behaviorally relevant components that were not detected via GC-EAD, or that the relative concentrations of the compounds in wine and vinegar are different than in the synthetic blends. Next, Cha et al. (2012) ran a series of two-choice olfactometer assays in an effort to finetune the seven- and thirteen-component blend to include only attractive compounds. In particular the addition of individual antennally-active compounds to a mixture of acetic acid and ethanol did not always increase D. suzukii attraction. Ethyl acetate, ethyl butyrate, 1-hexanol, isoamyl acetate, 2methylbutyl acetate, and ethyl sorbate decreased D. suzukii attraction to the mixture; while the addition of acetoin significantly increased fly attraction to the mixture. Grape butyrate, methionol, isoamyl lactate, 2-phenelethanol, and diethyl succinate did not alter the number of D. suzukii attracted to the mixture. Thus, Cha et al. (2012) concluded that of the thirteen EAD-active compounds from the headspace of vinegar and wine, only six compounds (acetoin, grape butyrate, 2- phenylethanol, methionol, isoamyl lactate, and diethyl succinate), in addition to acetic acid and ethanol, are responsible for D. suzukii attraction. Also, the fermentation source of different vinegars influences their attraction to D. suzukii. For example, brown rice vinegar is more attractive than apple cider vinegar (Akasaka et al. 2017). It is unclear whether this increased attractiveness is due to differences in the relative quantity of different attractive compounds or due to the differing attractive compounds. In wild blackberry and domesticated blueberry field trials, Cha et al. (2014) experimented with several bait formulations and found that mixtures of acetic acid, ethanol, acetoin, ethyl lactate and methionol (3methylsulfanylpropan-1-ol) caught similar numbers of flies to mixtures of wine plus vinegar. Drop-out studies revealed that if acetic acid, ethanol, acetoin, and methionol were not included in the blend, fewer flies were captured. However, when ethyl lactate was excluded, the four-component blend (acetic acid, ethanol, acetoin, and methionol) still captured similar numbers of flies to the wine and vinegar mixture (Cha et al. 2014). This four-component blend is also as attractive as the most commonly used D. suzukii standard attractant, apple cider vinegar (Cha et al. 2013). The fourcomponent blend is now used in commercially-available lures, such as Pherocon SWD Dual-Lure (proprietary blend, Trécé Inc., Adair, OK, USA) and Scentry (proprietary blend, Scentry Biologicals Inc., Billings, MT, USA) (Fig. 2). Fig. 2 Selected commercially available baits and lures used to trap adult Drosophila suzukii including (a) apple cider vinegar, (b) yeast typically added to sugar and water, (c) torula yeast tablets (ISCATechnologies), (d) Suzukii Trap bait (BioIberica), (e) DrosaLure (Andermatt Biocontrol, Grossdietwil, Switzerland), (f) SPLAT (ISCA Technologies), (g) Pherocon SWD lure (Trécé Inc.), and (h) Scentry lure (Scentry Inc.)

8 In an effort to increase the efficiency of this fourcomponent blend, Cha et al. (2017) found that an increased release rate of acetoin (from 0.5 mg/d to 43 mg/d), acetic acid (from 0.25 to 4%), and ethanol (from 0.08 to 2%) increased D. suzukii trap captures by 104% - 147%. However, an increase in the release rate of methionol (between 0.4 mg/d to 4.9 mg/ d) did not improve trap captures (Cha et al. 2014; Adams et al. 2017). Cha et al. (2017) also found that a synergistic relationship existed between acetic acid and acetoin. In trials where the concentration was increased in only acetic acid or acetoin, D. suzukii captures remained the same (Cha et al. 2017). Similar results were seen with the closely related D. melanogaster (Becher et al. 2017), suggesting that the synergistic relationship between acetic acid and acetoin may be conserved in the melanogaster group. This information may be important for the development of a lure that is selectively attractive for only D. suzukii. In an attempt to develop a more attractive lure, Kleiber et al. (2014) investigated the attraction of structurally related analogs to the previously identified four-component blend in two-choice behavioral assays. For these experiments they tested several different alcohols in neat form (methanol [7.2 ml], ethanol [7.2 ml], propanol [7.2 ml], butanol [7.2 ml], and pentanol [7.2 ml]), carboxylic acids (formic acid [2 ml], acetic acid [2 ml], propionic acid [2 ml], butyric acid [2], and valeric acid [2 ml]), four acetates (ethyl acetate [5.5 μl], propyl acetate [6.4 μl], butyl acetate [7.3 μl], pentyl acetate [8.3 μl]), and three esters of 2-pheynylethanol (phenethyl acetate [880 μl], phenethyl propionate [980 μl], phenethyl butyrate [1070 μl]) compared to water controls. Individual compounds were then dispensed onto cotton rolls and suspended over clear plastic traps containing either apple cider vinegar or neutralized apple cider vinegar. Traps were placed in cherry, blueberry, blackberry, and raspberry fields across Oregon. None of these compounds, singly or in several different combinations, increased D. suzukii attraction to apple cider vinegar in the field (Kleiber et al. 2014). However, the addition of propanol, phenethyl acetate, formic acid, acetic acid, and valeric acid all reduced D. suzukii captures when mixed with apple cider vinegar (Kleiber et al. 2014). Compounds that are deterrent to other Drosophila species (similar to propanol, phenethyl acetate, etc. deterring D. suzukii) may increase the selectivity of commercial D. suzukii lures by excluding other Drosophila bycatches. The above work shows that of the 13 antennally-active compounds in the headspace of fermenting food odors, all four attractant components are from microbial metabolism. Methionol is a byproduct of yeast metabolism (Seow et al. 2010), acetoin is a byproduct of yeast (Romano and Suzzi 1996) and bacterial metabolism (Le Bars and Yvon 2008), acetic acid is a byproduct of lactic acid metabolism (Mas et al. 2014), and ethanol is a byproduct of yeast metabolism (Goold et al. 2017). Microbial Odors Since vinegar (one of the most attractive substances identified thus far) is the byproduct of acetic acid bacterial metabolism, Mazzetto et al. (2016) investigated the attraction of adult D. suzukii to pure cultures of several acetic acid bacterial species under laboratory conditions. In twochoice attraction assays adult flies were more attracted to pure cultures of Gluconobacter oxydans, Gluconobacter kanchanaburiensis, andgluconobacter saccharivorans versus blank media controls. Volatile collections and GC-MS analysis showed that the primary headspace components of these attractive bacterial species were ethanol, acetic acid, 2- propanol, and 2-propanone. Acetobacter cibinongensis and A. persici, the least attractive bacterial cultures, all lacked ethanol in their headspace. Ethanol may have been primarily responsible for D. suzukii attraction to G. oxydans, G. kanchanaburiensis, and G. saccharivorans. Two-choice attraction experiments should investigate the attraction of the other two compounds, 2-propanol and 2-propanone, as single components. Hamby and Becher (2016) provide an extensive review on D. suzukii microbial interactions. They suggest that investigating the associations between D. suzukii and microbial species may help researchers improve the specificity of synthetic lures in the field. Investigating yeast species across the landscape that are in association with D. suzukii may offer a newly, unexplored source of attractive volatiles for exploitation. Yeasts are important for the survival and development of Drosophila species and even increase adult fecundity (Simmons and Bradley 1997). Baker s yeast,saccharomyces cerevisiae, is incorporated into most drosophilid diets (Guo et al. 1996; Skorupa et al. 2008; Daltonetal. 2011), including D. suzukii, because it is necessary for larval growth (Starmer 1981; Starmer and Aberdeen 1990; Becheretal.2017). Thus, it is reasonable to assume that, like other closely related Drosophila species, D. suzukii would be attracted to yeast species present in their environment that may increase larval survival and female fecundity. Hamby et al. (2012) sought to first establish if wild populations of D. suzukii harbored yeast species in their digestive tracks. They identified several yeast species from D. suzukii alimentary canals and larval frass in cherry and raspberry fields including Hanseniaspora uvarum, Pichia kluyveri, and Pichia terricola. Hanseniaspora uvarum was the most abundant yeast species present in fly alimentary canals and larval frass, and this yeast was the most attractive of the 6 yeasts tested both under binary or multi-choice assays for D. suzukii. Subsequent GC-EAD analysis from H. uvarum headspace volatiles revealed isobutyl acetate and isoamyl acetate as the strongest chemostimuli eliciting the greatest antennal responses in D. suzukii (Scheidler et al. 2015). In laboratory two-choice tests, adult flies were attracted to these two yeast volatiles (250 μl, neat) compared to water controls (KRC, unpublished data). However, in the laboratory, isoamyl acetate

9 decreased the attraction of D. suzukii in traps when combined with acetic acid and ethanol compared to acetic acid and ethanol alone (Cha et al. 2012; Cha et al. 2018a, b). Further, in the field, isoamyl acetate did not attract D. suzukii alone or when mixed with acetic acid and ethanol (Cha et al. 2014). Female D. suzukii arereportedlyattractedtoyeaststoincrease egg development and maturation (Mori et al. 2017). Supporting this hypothesis, Mori et al. (2017) found that mated female flies are more attracted to both blueberries and H. uvarum than unmated females in wind tunnel bioassays, and mated females consume more H. uvarum yeast than unmated females. However, blueberries treated with H. uvarum did not elicit greater oviposition versus blueberries without the yeast (Mori et al. 2017). Conversely, female D. suzukii laid more eggs on cherry fruits infested with Candida sp. and S. cerevisiae than un-infested cherries (Bellutti et al. 2017). There is an apparent trade-off between these yeast-feeding and oviposition behaviors in D. suzukii. Under laboratory conditions, mated females laid fewer eggs on blueberries when given access to H. uvarum for feeding versus females that were not given H. uvarum for feeding (Mori et al. 2017). Fruit Odors Onceafemaleflyhaslocatedasourceof fermenting food for egg maturation and mate finding, she cues in on fruit odors to find a suitable oviposition site (Fig. 1). Nutritional geometry experiments showed that gravid D. suzukii preferred to oviposit into artificial media with low protein to carbohydrate (P:C) ratios (Young et al. 2018), suggesting that females prefer to oviposit their eggs in carbohydrate rich environments. Therefore, egg-laying D. suzukii should discriminate between suitable host fruits during this oviposition phase of their lifecycle. Two-, three-, and fivechoice olfactometer assays found that adult flies were attracted to fruits in the following order: raspberry strawberry > blueberry cherry (Abraham et al. 2015). Larval performance assays in the laboratory showed that larvae survive best on fruits in the following order: raspberry > strawberry > blackberry > cherry > blueberry (Bellamy et al. 2013). Taken together, these data suggest that mated D. suzukii females are cueing in on fruit volatiles that signal differentially suitable host material for her developing larvae. In an attempt to characterize these attractive fruit volatiles, Abraham et al. (2015) further collected volatiles from raspberry, the most attractive fruit in the olfactometer assays, and performed GC-EAD assays. D. suzukii antennae could detect 11 compounds from the raspberry extract headspace including: 1) butyl acetate, 2) hexanol, 3) 2-heptanone, 4) 3-methyl- 1-butanone, 5) trans-2-hexanal, 6) 3-methyl-2-butenyl acetate, 7) 2-heptanol, 8) hexanol, 9) cis-3-hexanol, 10) 6-methyl-5-hepten-2-ol, and 11) linalool (Abraham et al. 2015). Several two-choice assays then examined the attraction of male and female D. suzukii to: 1) raspberry extract compared to a blank control; 2) the 11-component synthetic blend compared to a blank control; and 3) raspberry extract versus the 11-component synthetic blend. The synthetic blend was a mixture of the EAD-active compounds (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 in a ratio [by volume] of 1:36:3:5:10:2:7:8:9:2:8, respectively) in mineral oil. In the first experiment, female flies were more attracted to the 11-component synthetic blend compared to the blank control, whereas males were equally attracted to both. However, male and female flies were more attracted to the raspberry extract compared to the 11- component synthetic blend in the second experiment. These results suggest that attractive components in the raspberry extract might be missing in the 11-component synthetic blend. Revadi et al. (2015) performed similar GC-EAD assays to Abraham et al. (2015) except with intact fruit. Volatile collections performed by Abraham et al. (2015) relied on pureed, centrifuged, and frozen fruit extracts, which may have a different volatile profile than whole intact fruit. Revadi et al. (2015) sampled the headspace of intact raspberry, strawberry, blueberry, and cherry fruits. GC-EAD recordings found 20 antennally active compounds in the headspace of raspberry fruit including: 1) acetic acid, 2) hexanoic acid, 3) ethanol, 4) (Z)-3-hexen-1-ol, 5) 1-octanol, 6) 1-octen-3-ol, 7) β- phenylethanol, 8) nonanol, 9) ethyl acetate, 10) isoamyl acetate, 11) ethyl butanoate, 12) ethyl hexanoate, 13) (Z)-3- hexenyl acetate, 14) methyl salicylate, 15) α-phellandrene, 16) β-phellandrene,17) limonene, 18) p-cymene, 19) (±)-linalool, and 20) (E)-caryophyllene. Strawberry, the second most attractive fruit (Abraham et al. 2015) with the second best larval performance index (Bellamy et al. 2013), contained 14 antennally active compounds including six of the EADactive compounds from raspberry (β-phenylethanol, ethyl acetate, isoamyl acetate, ethyl hexanoate, (Z)-3-hexenyl acetate, (±)-linalool, and (E)-caryophyllene) with the addition of six compounds not found in the headspace of raspberry: hexanol, (E)-2-hexenal, 2-heptanone, ethyl octanoate, methyl hexanoate, and methyl octanoate. Similar trends were seen for blueberry and cherry (Revadi et al. 2015). Raspberry, the most attractive fruit, and the fruit with the best larval performance, is the only fruit whose headspace contained several antennally-active monoterpenes (α-phellandrene, β- phellandrene, limonene, and p-cymene). Although these antennally-active monoterpenes were in relatively low quantities in the headspace of raspberry fruits (Revadi et al. 2015), they may provide gravid D. suzukii with information on host quality. Leaf Odors As indicated above, previous studies suggest that host seeking D. suzukii cue in on fermenting fruit and yeast odors to find suitable hosts for egg-laying and feeding purposes. However, less is known about their attraction to cues from other tissues of host plants like leaves, stems, and roots. Keesey et al. (2015) found that D. suzukii are attracted to the strawberry leaf odor constituent β-cyclocitral in laboratory

10 two-choice trap assays. The authors hypothesized that this chemical is used as a possible long-range cue in selectively attracting D. suzukii to the vicinity of a fruiting plant (Fig. 1). Pheromone Odors Sex pheromones mediate the mate-seeking and courtship behaviors in many insect species (Witzgall et al. 2010). In the closely related species D. melanogaster and D. sechellia, females are known to produce the male attractants 7,11-heptacosadiene and 7,11-nonacosadiene (Antony et al. 1985), while monoenes, such as 7-tricosene, are mostly found on males (Everaerts et al. 2010). In D. suzukii, no clear indication of the function of male- or female-associated cuticular hydrocarbons has been proven so far. In D. melanogaster, the male-produced pheromone, 11-cisvaccenyl acetate (cva) influences many behaviors including aggregation of both females and males (Kurtovic et al. 2007; Weng et al. 2013), increasing unmated females acceptance of males (Weng et al. 2013), reducing courtship among mated males (Zawistowski and Richmond 1986; Ejima et al. 2007; Kurtovic et al. 2007), and reducing the courtship behaviors towards mated females (Scott 1986; Ejima et al. 2007; Ziegler et al. 2013). A recent study has reported that D. suzukii does not produce cva and its production site, the ejaculatory bulb, is much smaller in D. suzukii relative to that of D. melanogaster. When cva was applied to the cuticle of male D. suzukii their mating rate decreased (Dekker et al. 2015). This implies that, the drosophila sex pheromone, cva, disrupts mating in D. suzukii. Drosophila suzukii can detect cva because, although its production site is reduced, the cva sensitive odorant receptor, Or67d is functional, and its sensillum (T1) are functional on D. suzukii antennae (Dekker et al. 2015). Aversive Odors Repellent compounds can be used as part of an integrated pest management program to control important insect pests in agriculture (Cook et al. 2007). Several aversive compounds have been investigated for use in controlling D. suzukii under laboratory and field settings (Table 3). In the laboratory, Renkema et al. (2016) found that the essential oils from geranium, peppermint, citronella, lavender, thyme, eastern white cedar, white pine, white spruce, rosemary, ginger and eucalyptus were repellent to female D. suzukii adults. They then investigated the most abundant components of these oils for repellency by placing odor-impregnated laminate polymer flakes (10% w/v) around fresh raspberries in the laboratory. Thymol, the most abundant component in thyme oil, was the only individual compound tested that showed a significant level of repellency to male and female D. suzukii for 24 h. Citronellol, the most abundant compound in geranium oil, and geraniol were repellent to female flies only up to 6 h after application (Renkema et al. 2017); whether the chemicals dissipated or the flies became acclimated is unknown. The microbial odors geosmin and 1-octen-3-ol repelled D. suzukii females at relatively high concentrations (10 1 and 10 2 in mineral oil [v/v]) under laboratory and field conditions (Wallingford et al. 2016a). When incorporated at a 20% concentration into a controlled-release clay substrate called SPLAT (specialized pheromone and lure application technology; ISCA Technologies, Riverside, CA), 1-octen-3-ol reduced D. suzukii infestation in raspberries for 4 days after application in two separate field trials (Wallingford et al. 2016b). These two treated field plots had roughly 28.8 and 49.5% fewer larvae reared per gram of fruit versus the control, untreated plots (Wallingford et al. 2016b). No difference between control and treated plots was observed after seven days of 1-octen-3-ol application. Video recording assays in the laboratory suggested that 1-octen-3-ol acts as a long-range repellent, while geosmin acts as a short-range contact deterrent (Wallingford et al. 2017). Drosophila suzukii larvae and adults are also repelled and deterred by other natural products including extracts from its parasitoid Leptopilina boulardi (Barbotin) (Hymenoptera: Figitidae) (Ebrahim et al. 2015) (Table3). Odorant Receptors Odorant molecules largely determine the odor space of an insect (Grabe et al. 2016). To investigate the composition of D. suzukii odorant receptors (ORs), Hickner et al. (2016) compared the OR repertoire of D. suzukii with the closely related species D. biarmipes and D. takahashii. They found that D. suzukii lost three ORs (Or74a, Or85, and 98b) and have had two OR expansions (Or23a and Or67a) with positive selection suggested for Or67a. In a similar study, Ramasamy et al. (2016) found positive selection for Or85a and Or22a in D. suzukii. Or67a and Or22a are also expressed on the D. melanogaster antennae. Or67a is expressed in the ab10 sensilla in D. melanogaster (Couto et al. 2005). The broadly tuned Or67a in D. melanogaster shows strong excitatory responses in single sensillum recordings (SSR) to phenethyl alcohol, benzaldehyde, and pentyl acetate (Hallem and Carlson 2006). Hickner et al. (2016) suggests that this expansion of Or67a may be responsible for D. suzukii s increased antennal sensitivity to isoamyl acetate compared to D. biarmipes and D. takahashii. Or23a is expressed in the at2b sensilla in D. melanogaster, and its best ligand reported thus far is 1-pentanol (Münch and Galizia 2015), a byproduct of yeast metabolism. The expanded Or22a has an affinity for fermentation products (Ramasamy et al. 2016) and may be responsible for D. suzukii attraction to several fermentation volatiles. Since D. suzukii is attracted to ripening fruits, unlike the saprophytic D. melanogaster, the differences between the olfactory receptor repertoire of the two species and their functional expression/response profiles may provide crucial

11 information about differential attraction and avoidance in these two species. Interaction between Multiple Sensory Cues Several studies have demonstrated that olfactory and visual cues synergize to increase insect attraction to their host plants (Campbell and Borden 2006; Wenninger et al. 2009; Burger et al. 2010; Tasin et al. 2011). As a polyphagous species, D. suzukii likely employs multiple sensory modalities to detect and discriminate suitable host material. Basoalto et al. (2013) showed that red-baited traps caught more D. suzukii compared to baited traps that were clear, and that the use of alternating bands of red, black, and red (called Zorro traps) near the trap entrance significantly increased D. suzukii catches in the field. To investigate the ability of D. suzukii to discriminate between different colors, Kirkpatrick et al. (2016) exposed adult flies to several different colors of odorless paper disks and monitored their landing behavior. They found that adult flies will only land on disks that are red, black, and purple. In a similar study, Rice et al. (2016) found that adult D. suzukii are attracted by black and red spheres. In an effort to develop a visual trap, Rice et al. (2016) investigated the use of an insecticidal red spherical trap under laboratory, semi-field, and field conditions. These wax-based red traps that were attractive to D. suzukii flies contained an insecticide plus sugar mixture that could be ingested by flies. Larger spheres caught more flies than smaller spheres, and the shape of these visual traps had no significant effect on trap capture (Rice et al. 2016). The combination of visual and olfactory stimuli may increase D. suzukii attraction in the field. In fact, Iglesias et al. (2014) found that the addition of a yellow stimulus to sugar and yeast baited traps increases D. suzukii attraction versus clear baited traps in blueberry fields. Similarly, red sphere traps baited with the commercial Scentry Lure captured more D. suzukii adults than clear traps baited with the lure in cherry fields (Kirkpatrick et al. 2017). Management Strategies Effective insect traps should exploit the unique host-seeking behavior of the target insect pest. For a D. suzukii trap to be useful, it needs to: a) specifically attract only the target insect pest, b) be effective at capturing and retaining the majority of pest insects that come in contact with the trap, c) provide early detection of the pest insect, and d) correlate trap catch with subsequent fruit infestation. Currently the most effective means to prevent economic injury caused by D. suzukii are calendar-based insecticide sprays (Beers et al. 2011; Lee et al. 2011a, b). Due to the potential development of insecticide resistance, non-target effects, pesticide drift, and consumer acceptance issues, behavior-based tools to reduce D. suzukii infestation are desired. Below we describe current efforts evaluating these strategies to monitor and manage D. suzukii populations. Monitoring Significant efforts have been made to improve the physical aspects of D. suzukii traps including color, size, entry-hole placement, and entry-hole size. Field trapping experiments placed across seven US states found that traps baited with vinegar caught more flies if they had more entry points versus traps with fewer entry points (Lee et al. 2012). Lee et al. (2012) evaluated the efficiency of seven traps for monitoring D. suzukii on farms for early detection. Among all the traps, a Rubbermaid container with a mesh lid and rain tent trap (Haviland trap) caught the greatest numbers of D. suzukii flies (Lee et al. 2012). In a bid to improve trap designs for monitoring D. suzukii, Lee et al. (2013) evaluated traps with different colors, two different bait surface areas, and two different entry positions. Yellow traps with a large surface area for baits and side entry points caught more D. suzukii than any other traps (Lee et al. 2013). Presently, the main issues with traps and lure technology for D. suzukii are two-fold: low specificity and low correlation with fruit infestation. First, the commercial D. suzukii lures developed so far are based on fermenting volatiles which are not supposed to be specific to D. suzukii; thus, they attract large numbers of non-target drosophila flies in the field. In fact, commercially available lures capture between % non-d. suzukii drosophilids (Lee et al. 2013), and sorting these non-d. suzukii flies from the target D. suzukii requires a large amount of time and labor. Examples of these lures/baits include: DROSUZ (Alpha Scents Inc., West Linn, OR, USA), Scentry Lure, Suzukii Trap (a mixture of organic acids and hydrolyzed protein; BioIberica, Barcelona, Spain), and the Pherocon SWD (Fig. 2). Second, these commercially available lures do not accurately predict fruit infestation in most USA fruit growing regions. However, in some northern fruit growing latitude blueberries, where winters are able to knock-down D. suzukii populations, the commercially available Scentry Lure can detect adults 1 5 weeks before fruit infestation (Cha et al. 2018a, b). Lure efficiency will vary depending on region and crop, however (Shawer et al. 2018). For example, although the Scentry Lure detects D. suzukii 1 5 weeks before fruit infestation in northern blueberry, the lure detects D. suzukii the same week of fruit infestation in raspberry (Cha et al. 2018a, b). Further, early detection is not meaningful or feasible in southern fruit growing regions of the USA because D. suzukii populations are present year-round in these warmer climates. Trap specificity for commercially available D. suzukii lures and baits are still relatively weak and capture large numbers of non-target Drosopholids. For example, in a large, multi-state comparison of different lures in the USA, the commercial

12 Pherocon SWD lure suspended over apple cider vinegar captured the largest number of flies (Burrack et al. 2015), however, the trap selectivity for D. suzukii was still low for these lures (less than half of all captured flies were D. suzukii) (Burrack et al. 2015). In a separate example, adding apple cider vinegar to the Pherocon SWD trap increased D. suzukii attraction in the field (Frewin et al. 2017), though it did not increase trap specificity. In Italian sweet cherry field trials, the most effective traps were baited with the commercial Suzukii Trap mixed with 75% apple cider vinegar and 25% wine, though the selectivity of these traps was still low and only 24% of the total flies caught were D. suzukii (Tonina et al. 2018). One chemical ecology-approach to improve specificity of a lure is to define key essential chemical components for an attraction or deterrence behavior. By eliminating nonessential (e.g. redundant) attractant components from a blend, one could reduce noisy attraction from non-target insects that use some of the non-essential chemical components as their attractant. This type of improved specificity has been shown in D. suzukii (Cha et al. 2015), although the refined lure is still not highly specific due to the nature of the attractant chemical all drosophilids are attracted to microbial volatiles. Mass Trapping Mass trapping is a behavior-based management tool that uses an attractive chemical lure or pheromone to trap large numbers of insect pests (Faccoli and Stergulc 2008). In an effort to investigate the viability of mass trapping as a tool to control D. suzukii populations, Hampton et al. (2014) deployed large numbers of red cup traps baited with vinegar, yeast, and wheat flour in blueberry fields. Sampled berries surrounding deployed traps were infested with higher numbers of D. suzukii larvae than those plots containing nonbaited traps (Hampton et al. 2014). This increased infestation was likely due to low trap retention and spill-over effects. Only 10 30% of the attracted flies actually ended up in the drowning solution and thus about 70% of the attracted flies remained in the surrounding of the berries and oviposited upon the fruit (Hampton et al. 2014). A similar trend was seen using red cups baited with yeast plus sugar in wild blueberries across the US state of Maine. Alnajjar et al. (2017) found that incidence of infested fruit increased as the density (one trap per every 0.9 m - one trap per every 2.7 m) of baited traps increased. This increase in fruit infestation was also likely due to a spill-over effect (Alnajjar et al. 2017). These data suggest that currently available trapping technologies, with low pest retention, are ill suited for mass trapping D. suzukii. Attract-And-Kill Attract-and-kill is a control method similar to mass trapping except that it does not rely on pest retention inside a trap (Gregg et al. 2018). Here, a lure attracts an insect pest to a point source which also contains a killing agent that the attracted insect acquires through touch or feeding. Unlike mass-trapping, attract-and-kill technology can reduce the risk of a spill-over effect (El-Sayed et al. 2009). An attract-and-kill regime for D. suzukii could incorporate insecticidal compounds (Rice et al. 2017), entomopathogenic fungi (Becher et al. 2017; Gao et al. 2017), or double stranded RNA into attractive food material like yeast (Murphy et al. 2016). This type of attract-and-kill method has been used to control the codling moth, a pest of pome fruit, where a pathogenic virus was mixed with an attractive yeast and killed the pest (Knight and Witzgall 2013). One promising control strategy being developed is attractive red spheres impregnated with high-doses of chemical insecticides. In the laboratory, Rice et al. (2017) showed that these attractive red spheres impregnated with several chemical insecticides including dinotefuran, spinetoram, spinosad, permethrin, lambda-cyhalothrin, and lambda-cyhalothrin at 1.0% active ingredient (a.i.) killed 100% of D. suzukii that came into contact with them. In raspberry fields in West Virginia, spheres impregnated with 1.0% a.i. dinotefuran decreased D. suzukii fruit infestation in treated plots (Rice et al. 2017). Another attract-and-kill strategy under development is HOOK SWD (ISCA Technologies, Riverside, CA, USA), a SPLAT-based formulation (Fig. 2f). One exciting attract-and-kill approach for D. suzukii management may lie in using very specific RNA silencing technology. Murphy et al. (2016) showed that adult D. suzukii flies fed double-stranded RNA (dsrna) molecules targeting y- Tubulin died faster than control flies. They then genetically engineered yeast that expresses the y-tubulin targeting dsrna molecules and fed them to adult D. suzukii flies. Treated flies laid fewer eggs and exhibited less movement than control flies. Ingestible insecticides could also be incorporated into attractive yeast species for attract and kill. For example, adult D. suzukii fed H. uvarum yeast combined with the bio-insecticide spinosad experienced 26% greater mortality than adults fed un-treated H. uvarum yeast (Mori et al. 2017). In another example, the addition of the insect growth regulator, Lufenuron, to D. suzukii diet at a rate of ppm reduced larval pupation success between 90 and 99% (Sampson et al. 2017). Furthermore, the addition of a phagostimulant, a compound that increases feeding, to these killing agents could increase their efficacy. Cowles et al. (2015) showed that flies fed on mixtures of sucrose and spinosad died 120 min faster compared to those flies fed on spinosad alone. Lastly, mixtures of the sugar alcohol erythritol and sucrose increased adult fly mortality in blueberry fields (Choi et al. 2017). An attract-and-kill regime could also attract flies only to the borders of crop fields designated for pesticide applications. Iglesias et al. (2014) found that spraying a combination of the chemical insecticides pyrethrin and azadirachtin only on the border of a blackberry field decreased fruit infestation in

13 the entire field. Thus, a successful attract-and-kill strategy may deploy lure-baited traps only on the borders and treat only the borders with chemical insecticides. The combination of a point-source attractant, killing agent, and phagostimulants could reduce the number of broad insecticide applications. Push-Pull Another potential control tool could incorporate attractive lures and aversive odors in a push-pull system. This system includes a lure, or plant, that attracts the pest insect away from a crop (pull). At the same time the insect pest is repelled away from the target crop (push) and into a trap-crop or lure-baited trap (Cook et al. 2007). Push-pull systems have been used to control several insect pest species including the crucifer flea beetle, Phyllotreta cruciferae, a pest of broccoli (Parker et al. 2016). Wallingford et al. (2017) recently investigated a push-pull system in the laboratory and field as a potential control tool for D. suzukii. Under laboratory conditions the push treatments alone (25% 1-octen-3-ol in mineral oil) had a 66.2% reduction in egg deposition on raspberry fruit, pull treatments alone (baited with a fermenting wheat and apple cider vinegar mixture) saw a reduction of 69.6% oviposition on raspberry fruit, and the push-pull treatments together had an additive reduction of 87.6% oviposition on raspberry fruit (Wallingford et al. 2018). Field assays were also carried out with potted raspberry plants using the commercially available Scentry Lure as the attractive pull component. Plots receiving the push treatment alone (25% 1-octen-3-ol in mineral oil) had a 56.7% reduction in egg deposition compared to control plots receiving the push-pull treatments together (25% 1-octen-3-ol in mineral oil plus Scentry Lure baited traps) which had a 57.4% reduction in egg deposition compared to control plots, and those receiving the pull treatments alone (Scentry Lure baited traps) had an increase of 44.1% more eggs than control plots (Wallingford et al. 2018). Like in the previous mass trapping examples where treated plots saw greater fruit infestation (Hampton et al. 2014; Iglesias et al. 2014), Wallingford et al. (2018) also saw a significant increase in D. suzukii oviposition in those plots with attractive lures. This, again, was likely due to a spill-over effect. Future Directions Although we have made significant advances in the chemical ecology of D. suzukii in the last decade, from physiological and behavioral assays to the development of behavior-based strategies to manage this pest, knowledge gaps still exist in our understanding of the host, food, and mate-seeking behaviors of D. suzukii. These gaps leave fruit growers with few alternatives for viable D. suzukii control besides calendar-based applications of synthetic and organic insecticides. Filling these gaps will help improve the efficacy and reliability of existing lures and the implementation and adoption of behavior-based strategies at field and farmwide scales. Current lure and trapping technologies (Fig. 3) are not particularly selective for D. suzukii and do not always correlate with fruit infestation. Despite this, progress has been made in providing growers with tools for D. suzukii monitoring. For example, the commercially available Pherocon SWD and Scentry lures, utilizing a four-component blend developed by Cha et al. (2014), can provide growers (particularly those in the northern latitudes) with information on D. suzukii activity before fruit infestation. However, in sweet cherry, Kirkpatrick et al. (2018a) found that one fly in a Scentry-baited trap correlated with 192 D. suzukii per trapping area of 2.7 ha (26 per acre). They suggest that by the time D. suzukii are detectable in these traps, the fly population already exceeds the tolerable damage threshold (Kirkpatrick et al. 2018a). Future work should attempt to correlate commercial lure catches with fruit infestation for different crops in different latitudes. Current trapping systems are not recommended for mass trapping, attract-and-kill, or push-pull regimes because of their low pest retention. Future work should investigate host odor-pheromone synergisms in D. suzukii, as has been done in the closely related D. melanogaster (Das et al. 2017), as one mechanism to increase lure specificity and retention. Including compounds that are repellent to other insect species, but not D. suzukii, to lure formulations may also help reduce non-target insect captures. As mentioned in the BFruit odors^ section of this review, the headspace of raspberry (the most attractive fruit for egg-laying D. suzukii) contains several monoterpene compounds including limonene and p-cymene. Essential oils containing large amounts of limonene have been used as natural sources of insect repellents (Maia and Moore 2011). In another example, the Peruvian peppertree, Schinus molle, extracts repel the stored grain pests Trogoderma granarium and Tribolium castaneum (Abdel-Sattar et al. 2010). The primary component in the headspace of the Peruvian peppertree is p-cymene. As long as they don t act as an attractant for different non-targets, these compounds may offer some potential as additives to lure formulations that will repel non-target insect species but will still attract the target D. suzukii. The addition of anti-aggregation pheromone components from closely related drosophilids in currently available lures may also increase their specificity for D. suzukii. To produce more specific lures, future research should also focus on how the physiological status of adult D. suzukii influences their attraction to different types of odor cues. Progress is being made in this area. For example, Swoboda- Bhattarai et al. (2017) showed that adult female flies collected on the surface of ripe raspberry and blackberry carried more mature eggs than female flies collected on or in red cup traps baited with yeast and sugar. Further, recent work by Wong et al. (2018) showed that female flies with fewer egg loads were more attracted to fermentation odors. As females matured and

14 J Chem Ecol Fig. 3 Selected homemade and commercially available traps used to monitor and control adult Drosophila suzukii. a Clear, plastic, 20 oz. deli-cup trap baited with apple cider vinegar with red mesh coverings on the two side openings, b a similar clear, plastic, deli-cup trap containing apple cider vinegar and a sticky card to capture flies. c Another homemade trap except that this trap utilizes a red 12 oz. Solo cup trap with numerous holes punctures around the lid as entry points, d Pherocon trap (Trécé Inc.), e Drosal Pro trap (Andermatt Biocontrol), f ISCA trap (ISCA Technologies), g Contech trap (Contech Enterprises Inc.. British Columbia, Canada), and h the Droso-Trap (Biobest Inc., Westerlo, Belgium) gained higher egg loads, they were almost exclusively attracted to fruit odors (Wong et al. 2018). In another recent example, Kirkpatrick et al. (2018b) showed that Bwinter morphs,^ or adults reared under winter conditions (10 C, 45% R.H., 12:12 [L:D]), had a decreased antennal response to several microbial odors including acetic acid, isoamyl acetate, and geosmin. Winter morphs were also not repelled by geosmin in two-choice laboratory assays, whereas Bsummer morphs,^ or adults reared under summer conditions (24 C, 45% R.H., 16:8 [L:D]), were repelled by geosmin. Future work should build on these studies and examine the influence of D. suzukii age, mating status, and feeding status on their response to different stages in the phenology of fruit crops. This work may help explain the differential D. suzukii attraction to the same lure in different crops and in different growing regions. In order for behavior-based control tools (e.g. mass trapping, attract-and-kill, and push-pull) to provide growers with viable alternatives to the use of insecticides or to reduce their applications, future research must focus on the conditions under which these technologies are most effective. Potential areas of future work could evaluate the influence of D. suzukii densities, fruit density, trap density, landscape conditions, and fruit phenology on the efficacy of these behavior-based tools. Future research should also focus on the methods of deployment and formulations for these technologies. Concluding Remarks The ability to infest fresh fruit makes D. suzukii one of the most economically-important invasive insect pest worldwide. In the last decade, we have learned important aspects about the odor space of D. suzukii. Adult flies target high value crops such as strawberry, blueberry, raspberry, blackberry and cherry, and several non-crop plants act as suitable hosts and refuge sites, and may also serve as overwintering sites. Like most polyphagous insect species, D. suzukii relies on a combination of odor cues to find suitable oviposition, feeding, and mating sites including fermentation, fruit, yeast, and leaf odors. Odorants eliciting antennal and behavioral responses have been isolated from these odor sources in attempts to produce an attractive D. suzukii lure for monitoring purposes. Color also influences adult attraction and may synergize with odors to increase D. suzukii attraction to host plants. Compared to other closely related drosopholid flies, D. suzukii has experienced an expansion of ORs having affinities for isoamyl acetate, short-chain esters, and fermentation products. Solutions for current trapping systems need to address issues of lack of selectivity, spillover effects/poor retention, and poor correlation with fruit infestation. Challenges also exist on the implementation of behaviorbased technologies such as mass-trapping, attract-and-kill, and push-pull in cropping systems that have a zerotolerance for fruit infestation.