Identification of Active Components from Volatiles of Chinese Bayberry, Myrica Rubra Attractive to Drosophila Suzukii

Arthropod-Plant Interactions (2018) 12:435–442 https://doi.org/10.1007/s11829-018-9595-z

ORIGINAL PAPER

Identification of active components from volatiles of Chinese bayberry, Myrica rubra attractive to Drosophila suzukii

Yan Liu1,2 · Wenxia Dong1 · Feng Zhang3 · Marc Kenis4 · Frans Griepink5 · Jinping Zhang3 · Li Chen2 · Chun Xiao1

Received: 11 September 2017 / Accepted: 29 January 2018 / Published online: 21 February 2018 © Springer Science+Business Media B.V., part of Springer Nature 2018

Abstract Drosophila suzukii (Diptera: Drosophilidae) is native to Southeast Asia and now has become a severe pest of several soft fruits in Europe and the Americas. It causes considerable damage to Chinese bayberry, Myrica rubra, in China. In the present study, we employed gas chromatograph–electroantennographic detection (GC–EAD) together with behavioural bioassays and trapping experiments to identify volatile semiochemicals emitted by Chinese bayberry attracting D. suzukii. Electro- physiological experiments revealed the presence of six EAD-active compounds from ripe bayberry fruits, including methyl (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate, α-ylangene, α-humulene and an unidentified compound that elicited consistent antennal response. In two-choice bioassays, bayberry fruits attracted all responding flies, and significantly more flies responded to the volatile extract of bayberry fruits. Four EAD-active compounds were attractive to mated female D. suzukii at lower doses (0.01 and 0.1 µg), but showed repellency at higher doses (10 and 100 µg). Mixtures of these four compounds at different ratios attracted D. suzukii flies at all test doses (0.1, 1 and 10 µg). Both male and female flies were trapped by a mixture of synthetic methyl (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate and α-humulene in a ratio of 1:1.3:1:6.4 in the field trapping experiment. Significantly more males than females were captured in the trap baited with the synthetic blend, and the percentages of D. suzukii captured out of all flies by the traps baited with lure were higher than that baited with blank control. Our findings may provide insights into the olfactory responses of D. suzukii to specific host plant volatiles, and contribute to further development of an effective lure for monitoring D. suzukii in the field.

Keywords Fruit fly · Host plant volatile · Olfactory response · Attraction · Host location · GC–EAD

Introduction Handling Editor: Jarmo Holopainen. For most herbivore insects, olfactory cues are of great impor- Yan Liu and Wenxia Dong contributed equally to this work. tance for host plant finding (Bruce and Pickett 2011). Vola- tiles released from host plant are important in host location * Li Chen [email protected] by herbivores (Bruce and Pickett 2011; Bruce et al. 2005). Numerous studies have shown that insects use volatile cues * Chun Xiao [email protected] to locate their host plants from a long distance for feeding, mating or oviposition. For instance, volatiles from Chinese 1 Plant Protection College, Yunnan Agricultural University, liquorice root are used by newly emerged larvae of Porphy- Kunming 650201, China rophora sophorae as chemical cues for host plant location 2 State Key Laboratory of Integrated Management of Pest (Liu et al. 2016). The aroma of fruits or leaves of various Insects and Rodents, Institute of Zoology, Chinese Academy Citrus species has been shown to enhance the mating com- of Sciences, Beijing 100101, China petitiveness of males of the Mediterranean fruit fly, Ceratitis 3 MoA‑CABI Joint Laboratory for Bio‑safety, Institute of Plant capitata (Wiedemann) (Diptera: Tephritidae) (Kouloussis Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China et al. 2013). Mated female moths of tea looper caterpillar, Ectropis obliqua Prout are more attracted to and preferen- 4 CABI, 2800 Delémont, Switzerland tially oviposit on infested tea plants (Sun et al. 2014). 5 Pherobank BV, 3960 BA Wijk bij Duurstede, The Netherlands

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Drosophila suzukii (Matsumura) (Diptera: Drosophili- bayberry fruits and to test their attractiveness to D. suzukii dae), a fruit fly of Asian origin, has been a serious threat both in the laboratory and in the field. for cultivated and wild soft-skinned fruits including blueberry, blackberry, cherry, mulberry, raspberry, strawberry and other wild, ornamental and crop plants (Cini et al. Materials and methods 2012; Kenis et al. 2016; Lee et al. 2011; Walsh et al. 2011) in Europe and the Americas (Asplen et al. 2015; Calabria Insects et al. 2012; Cini et al. 2012; Goodhue et al. 2011). Unlike most Drosophila flies ovipositing on overripe and rot- The culture of D. suzukii used in the laboratory experiments ten fruits, D. suzukii is one of the few Drosophilids with was established with field-collected populations from an serrated ovipositors that enable females to oviposit on orchard of Chinese bayberry M. rubra, in Yunnan Province, unwounded fresh fruit (Mitsui et al. 2006). The adult flies China. The flies were reared on peeled bananas in the labo- of D. suzukii reach maturity 1 or 2 days after emergence ratory under controlled conditions (24 ± 1 °C, 65 ± 5% RH, and then start to lay eggs 1 day after mating. The larvae 14:10 h L:D). hatch inside the fresh fruit (Cini et al. 2012), causing dam- Newly eclosed flies were kept in a cage (10 × 10 × 10 cm) age to the fruit and rendering it unmarketable. with access to food and water, and allowed to mate for Chinese bayberry (Myrica rubra), a widely cultivated 3 days. Then the adult flies were starved for approximately subtropical fruit crop in the region of the Yangtze River 24 h. Only 5-day-old females were used in behavioural and (Cheng et al. 2008; Kang et al. 2012), is one of the main electrophysiological experiments. The mating status of hosts to D. suzukii (Liu et al. 2017; Wu et al. 2007; Zheng the test fly was confirmed by dissecting the spermathecae et al. 2015). The temperature and humidity are high during out of the abdomen to check the presence of toroidal mass fruit-ripening and harvest period of Chinese bayberry in (Avanesyan et al. 2017). May and June, providing suitable climatic conditions for D. suzukii development. D. suzukii can cause serious dam- Extraction, identification and quantification age to Chinese bayberry fruits when the fly populations of volatiles from Chinese bayberry fruit reach their peak. In field, 30–40% of the fruits are infested by D. suzukii at the beginning of the harvest season and up Fresh bayberry fruits (Dongkui cultivar) at the ripe stage to 100% in the late fruit-ripening period (Zhao et al. 2017). (2.5 kg) were enclosed in a glass vessel (24 cm diam., 21 cm Male D. suzukii can mate multiple times, and a single high). Air entering the vessel was drawn through a filter female can lay up to 140 eggs (Liu et al. 2017). There- filled with activated charcoal and allochroic silica gel, and fore, male trapping with sex pheromone is not an effec- was pumped out from the vessel for 4 h at 500 mL/min tive tactic to control D. suzukii. It is practical to monitor through an adsorbent tube (5 mm internal diam.; 500 mg D. suzukii by traps baited with various mixtures of vin- Porapak Q, Waters Corporation, Ireland). All connections egar, wine, sugar water, and baker’s yeast (Asplen et al. were made with Teflon tubing. Volatiles were eluted from 2015), or apple cider vinegar with 5% acidity (Lee et al. the Porapak Q trap with 3 mL hexane (98%, Merck, Ger- 2012). However, insects other than Drosophilids can be many). Volatile sample was concentrated to 300 µL under a trapped by these traps (Lee et al. 2012). Previous studies mild stream of nitrogen at room temperature, and was stored have demonstrated that D. suzukii uses volatiles from rasp- in a freezer (− 20 °C) until used for chemical analysis and berry, blackberry, cherry, blueberry, strawberry, or from electrophysiological experiments. Eight replications of the fermented baits to select oviposition sites (Abraham et al. volatile collections from bayberry fruits at the same ripe 2015; Keesey et al. 2015; Revadi et al. 2015) and use vola- stage were conducted as described above. Four volatiles col- tiles from wine and vinegar to search for food (Cha et al. lections were combined for GC–MS, GC–EAD analyses and 2014). Female flies were attracted to synthetic blends of behavioural experiments, and the other four collections with active volatile compounds identified from raspberry fruits addition of internal standard were subjected to quantitative with coupled gas chromatography–electroantennographic GC–FID analysis. detection (GC–EAD) (Abraham et al. 2015). The identifi- The combined volatile sample (2 µL) was analysed with cation of specific volatiles emitted by host plants attractive an Agilent 7890A GC coupled to a 5975C mass selective to D. suzukii may aid current efforts in the development of detector (70 eV) equipped with a non-polar HP-5ms col- more selective and efficient traps (Cha et al. 2013; Keesey umn (30 m × 0.25 mm × 0.25 µm film thickness; Agilent). et al. 2015; Revadi et al. 2015). The oven temperature was held at 40 °C for 1 min and pro- In this study, we aimed to identify electrophysiologi- grammed at 5 °C/min to 180 °C, then 10 °C/min to 240 °C cally active compounds from the headspace of Chinese for additional 10 min holding time. The injector and transfer line temperature was set at 230 and 250 °C, respectively,

1 3 Identification of active components from volatiles of Chinese bayberry, Myrica rubra… 437 with helium (1.0 mL/min) as the carrier gas. Scan range was placed into two separated glass bulbs covered with dark from 50 to 500 m/z. Mass spectra were compared to those cloth. For bioassays of volatile samples, 10 µL solution was reported in the NIST08 database, and structure assignments impregnated onto a piece of round filter paper (φ = 1.5 cm), were confirmed by comparing retention times and mass and the solvent was allowed to evaporate before the trail. spectra with those of authentic standards. Filter paper treated with hexane only was used as control. Gas chromatography (GC) analyses were conducted using Ten mated females were introduced to the olfactometer at an Agilent 7890A GC equipped with a HP-5ms capillary the entrance of the main stem at one time, and were observed column in splitless mode. The column configurations and until the fly made a ‘choice’ (entering into side arms) or until GC conditions were the same as described above. Nonyl ace- 15 min elapsed. Flies that remained in the main stem were tate (99.0%, Sigma-Aldrich) was used as an internal standard recorded as ‘no choice’. The arms together with bulbs were for chemical quantification by comparing peak areas (Revadi switched after a trial to minimize any spatial effects. The et al. 2015). olfactometer was cleaned with ethyl alcohol and baked at 200 °C after every two trials (Revadi et al. 2015). Ten rep- Coupled gas chromatography– licates (ten female flies each replicate) were tested for each electroantennographic detection (GC–EAD) treatment, and the bioassay experiments were conducted in a laboratory under 24 ± 1 °C and 60–70% RH. GC–EAD measurements were performed as described by Based on the GC–MS and GC–EAD results of Chinese Chen et al. (2009). The above combined volatile sample of bayberry fruit extracts, the authentic samples of identi- the Chinese bayberry fruits (2 µL) was injected in a Shi- fied compounds were purchased from commercial sources, madzu GC-2010 plus equipped with an HP-5ms column including methyl (E)-3-hexenoate (97%, Sigma-Aldrich), programmed from 40 °C (held for 2 min) to 120 °C at 5 °C/ methyl (E)-2-hexenoate (98%, Sigma-Aldrich), ethyl (E)- min and then to 240 °C (held for 4 min) at 15 °C/min and 2-hexenoate (99%, Pherobank) and α-humulene (96%, interfaced with the electroantennogram (EAG) apparatus. Sigma-Aldrich). Due to its non-availability from commercial Helium was used as the carrier gas. The column effluent was source, α-ylangene was not tested. split at 1:1 ratio, with one part to flame ionization detector (FID) of GC, and the other to a heated line into a humidified Bioassay I: Fresh Chinese bayberry fruits versus blank, airstream (400 mL/min) which was directed at the antenna and head space volatile extract from fresh Chinese bay- preparation. EAG recordings were made using Ag–AgCl berry fruits versus hexane. glass microelectrodes filled with Beadle–Ephrussi Ringer Bioassay II: Different doses of the EAD-active compound solution (750 mg NaCl, 35 mg KCl, 29 mg CaCl 2·2H2O, versus hexane. All the compounds were prepared in hex- 100 mL demineralized H2O). The antenna of the isolated ane and diluted to 10, 1, 0.1, 0.01 and 0.001 µg/µL. head was positioned between two glass electrodes. The ref- Bioassay III: Mixture of four EAD-active compounds at erence electrode was connected to the neck of the isolated different ratios versus hexane. head, while the recording electrode was connected to the tip of antenna. The signals generated by the EAD and FID were A mixture of these four compounds was prepared at natu- passed through a Syntech IDAC-2 high-impedance amplifier ral ratios of 1:1.3:1:63.7. Two more mixtures with decreased and analysed with Syntech GC–EAD software. FID peaks proportion of α-humulene were also prepared (1:1.3:1:31.8 that elicited EAD responses for at least five runs were con- and 1:1.3:1:6.4). These mixtures were diluted with hexane sidered electrophysiologically active and marked for identi- to 1, 0.1 and 0.01 µg/µL for behavioural bioassays. fication by GC–MS. Field trapping experiments Y‑tube olfactometer bioassays Because the proportion of α-humulene did not affect the Evaluation of the olfactory responses of mated D. suzukii attractiveness of the mixtures in the laboratory tests, we used females to the fruit volatiles extract and the EAD-active the volatile mixture with the lowest ratio of α-humulene, i.e. compounds were performed in a Y-tube olfactometer (stem the mixture of methyl (E)-3-hexenoate, methyl (E)-2-hex- 15 cm; arm length 10 cm; arm angle 30°; internal diam. enoate, ethyl (E)-2-hexenoate and α-humulene in a ratio of 2 cm) housed in a large plastic box (43 × 30 × 18 cm) whose 1:1.3:1:6.4 (0.1 µg/µL hexane solution), as a trap lure for top was left open (Liu et al. 2017). Each arm of the Y-tube field trials. Two millilitres of the mixture were loaded into an was connected to a glass bulb (100 mL) with an open end open-ended plastic tube dispenser (φ = 1 cm, length = 3 cm) (φ = 2 cm). Activated charcoal-filtered and humidified clean filled with cotton. The prepared dispenser was kept open for air was pumped uniformly through the arms at 200 mL/min. 10 min to allow evaporation of the solvent and was subse- Intact fresh bayberry fruits (25 g) versus blank control were quently hung inside cylinder-shaped traps made from blue

1 3 438 Y. Liu et al. sticky traps (25 × 15 cm, Jida Environmental Protection Results Technology Co. Ltd, Cixi City, Zhejiang Province, China). Dispenser with hexane only was used as control. Bioactive volatile compounds identified Field trials were conducted in September 9–19, 2016 in a from Chinese bayberry sandal wood (Osyris wightiana Wall. ex Wight) orchard in Kunming City, Yunnan Province (102.73N, 25.04E, altitude Six compounds in the Chinese bayberry fruit volatile col- 1895 m). A randomized design was used with five repli- lections elicited consistent antennal responses in mated cates per treatment and the control. Traps were hung on tree D. suzukii females (Fig. 1). Five of the EAD-active com- branches 1–1.5 m above the ground. The distance between pounds were identified as methyl (E)-3-hexenoate, methyl the traps was approximately 20 m. The number of male and (E)-2-hexenoate, ethyl (E)-2-hexenoate, α-ylangene and female D. suzukii as well as other Drosophila flies captured α-humulene (Table 1). In Chinese bayberry volatile extracts, in traps was counted every 2 days, and at the same time β-caryophyllene (peak I in Fig. 1) was a predominant con- dispensers were replenished. stituent, but did not elicit any antennal response (Fig. 1).

Statistical analyses Behavioural responses of D. suzukii in the Y‑tube olfactometer The percentage of mated female D. suzukii flies that have made a choice in the olfactometer bioassays was analysed In the Y-tube olfactometer bioassays, approximately with Mann–Whitney U test at 0.01 level using SPSS 20.0 80% of tested females made choices. The responding (SPSS Inc., Chicago, IL, USA). Field trapping data were D. suzukii females all chose the arm with fresh Chinese transformed by square root before being analysed with one- bayberry fruits (P < 0.0001, Fig. 2A), and were signifi- way ANOVA followed by Tukey’s test (P = 0.05). Student’s cantly attracted to volatiles extract of bayberry fruits t test was used to compare the percentages of D. suzukii out (P < 0.0001, Fig. 2A). Mated females showed a similar of all flies captured between treatment and control traps. response to individual volatile compounds. In general,

Fig. 1 GC–EAD responses of mated D. suzukii females to bayberry fruit volatiles. The chemical identity of GC peaks was listed in Table 1. The largest peak “I” was β-caryophyllene

Table 1 GC–EAD-active Peak no. Compounds RIa Concentration (ng/µL) Ratio (%) compounds to mated female D. suzukii emitted from ripe fruit 1 Methyl (E)-3-hexenoate 735 3.16 ± 1.01 0.03 of bayberry, M. rubra (Lour.) 2 Methyl (E)-2-hexenoate 763 4.15 ± 1.08 0.04 3 Ethyl (E)-2-hexenoate 836 3.17 ± 0.98 0.03 4 Unidentified 1286 0.98 ± 0.28 0.01 5 α-Ylangene 1382 2.01 ± 0.67 0.02 6 α-Humulene 1544 201.06 ± 20.93 1.91

a Kovats Retention Indexes (RI) were obtained from analysis on a HP-5MS column. Compounds 1, 2, 3, 6 were confirmed with synthetic standards. α-Ylangene was tentatively identified

1 3 Identification of active components from volatiles of Chinese bayberry, Myrica rubra… 439

Fig. 3 Field evaluation of female and male D. suzukii captured in traps baited with a mixture of methyl (E)-3-hexenoate, methyl (E)- 2-hexenoate, ethyl (E)-2-hexenoate and α-humulene at ratio of 1:1.3:1:6.4 (0.1 µg/µL) (treatment), or with hexane (control) from 9 to 19 September 2016 in Yunnan Province, China. Different letters above bars indicate significant difference by Turkey’s test (P = 0.05). Statistical tests were based on square-root transformed data, and means from untransformed data are shown. Single asterisk means significant difference by Students’ t test at 0.05 level

Field trials

The total number of male and female D. suzukii captured in traps baited with the mixture of methyl (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate, α-humulene in the ratio of 1:1.3:1:6.4 was significantly more than that Fig. 2 Olfactory responses of mated female D. suzukii (n = 100) in control traps. Furthermore, significantly more males to bayberry fruits and their head space volatiles extract ( ), sin- A than females were captured by the traps baited with volatile gle compound (B), and mixtures of methyl (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate, α-humulene at the ratios of mixture (P < 0.0001) (Fig. 3). Except D. suzukii, other flies 1:1.3:1:63.7 (mixture A), 1:1.3:1:31.8 (mixture B), and 1:1.3:1:6.4 (mainly D. melanogaster) were attracted by these traps as (mixture C) versus solvent control (hexane) (C). The double asterisk well. The percentages of D. suzukii captured by the traps indicates significant difference between the test sample and control at the level of 0.01 (Data were analysed by Mann–Whitney U test) baited with the volatile mixture were higher than that by the blank control traps (Fig. 3). the volatile compounds were attractive to mated females Discussion at lower doses, but showed repellency at higher doses (Fig. 2B). Methyl (E)-3-hexenoate, ethyl (E)-2-hexenoate In recent years, volatile compounds from Chinese bayberry and α-humulene attracted mated female D. suzukii at fruits have been studied extensively (Cheng et al. 2008, doses of 0.01, and 0.1 µg (P < 0.0001), while all com- 2015a, b, 2016). Esters and sesquiterpenes, such as methyl pounds significantly repelled flies at doses of 10, and 3-hexenoate, α-humulene and β-caryophyllene were pre- 100 µg (P < 0.0001, Fig. 2B). dominant compounds released by mature Chinese bayberry Mixtures of methyl (E)-3-hexenoate, methyl (E)- (Cheng et al. 2015a; Kang et al. 2012; Soares et al. 2007). 2-hexenoate, ethyl (E)-2-hexenoate and α-humulene Our GC–EAD results showed that three esters (methyl at three different ratios (1:1.3:1:63.7, 1:1.3:1:31.8 and (E)-3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hex- 1:1.3:1:6.4) significantly attracted mated female D. enoate), two terpenes (α-ylangene and α-humulene) and suzukii at all tested doses of 0.1, 1 and 10 µg (P < 0.0001) one unidentified compound from Chinese bayberry vola- (Fig. 2C). tiles were detected by mated females of D. suzukii. Among

1 3 440 Y. Liu et al. the six EAD-active compounds, α-humulene is a common locate and select their host plants by the volatile compounds component from plant leaves (da Silva et al. 2016; Lemos released from plants through the air and detected at some et al. 2015; Mariscal-Lucero et al. 2015) and fruits (Cheng distance from the source (Knolhoff and Heckel 2014). These et al. 2015a; Kang et al. 2012), and methyl 3-hexenoate is host plant volatiles usually regulate insects’ behaviour, for a specific component in bayberry fruits compared to other instance, attracting them towards the host plants, promoting berry fruits (Cheng et al. 2015a; Kang et al. 2012). The other their feeding or oviposition behaviour and enabling insects three EAD-compounds, methyl (E)-2-hexenoate, ethyl (E)- to distinguish host plants from non-host plants (Clavijo 2-hexenoate and α-ylangene, which have not been detected Mccormick et al. 2014). The percentage of responding from berries previously, are present as a major component flies to the mixtures is similar to or greater than that to the from soursop (Carlos et al. 2011; de Santana et al. 2017), volatiles from Chinese bayberry, suggesting that the four fresh-cut peki fruit (Damiani et al. 2009) and fresh schizan- compounds are the main components from the head space dra fruit (Kim et al. 2008), respectively. Therefore, methyl volatiles extract in attracting D. suzukii. 3-hexenoate, methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate Four EAD-active compounds (methyl (E)-3-hexenoate, and α-ylangene can be considered as unique bayberry vola- methyl (E)-2-hexenoate, ethyl (E)-2-hexenoate and tiles eliciting a response from D. suzukii. Although these α-humulene) from Chinese bayberry volatiles attracted compounds are present in other fruits, their specific ratio in mated female flies at lower doses, but showed repellency the bayberry volatile blend could be more important, and at higher doses. This dose-dependent olfactory response any change in the specific ratio may lead to the loss, at least pattern in D. suzukii (Wallingford et al. 2016) is similar to partly, of the bioactivity as suggested by previous studies reported olfactory responses in Drosophila spp. (Kleiber (Bruce and Pickett 2011; Li et al. 2016). et al. 2014; Semmelhack and Wang 2009; Stensmyr et al. These EAD-active compounds, triggering significant 2003). Semmelhack and Wang (2009) found an odour con- GC–EAD in D. suzukii, were minor components from centration-dependent neuronal mechanism underlying the Chinese bayberry volatiles compared to β-caryophyllene, olfactory behavioural switch in Drosophila. Apple cider vin- indicating that D. suzukii might rely on these compounds egar at a low concentration triggers robust attraction by acti- to locate their host plants. They are apparently different vating several glomeruli that mediate attraction. At high con- from the EAD-active compounds from fresh fruits of straw- centrations, vinegar activates an additional glomerulus that berry (Keesey et al. 2015), raspberry, blackberry, cherry, mediates the behavioural switch and becomes less attractive blueberry (Keesey et al. 2015), fermented food (Cha et al. to adult flies. Further research is required to understand the 2014), and headspace volatile extracts of wine and vinegar neuronal basis of the switch of behavioural response of D. (Cha et al. 2012, 2013). β-Caryophyllene present in volatile suzukii to host plant volatiles at different doses. extracts of raspberry, blueberry, blackberry and strawberry The blend of plant volatiles at natural ratios instead of has been demonstrated to elicit significant EAD responses individual compounds helps insects locate their hosts (Wijk in mated D. suzukii females (Revadi et al. 2015). In the pre- et al. 2011). The mixture of four EAD-active compounds at sent study, however, no antennal response was recorded for different ratios and different doses attracted more D. suzukii β-caryophyllene, despite much higher amounts of this vola- females in olfactometer bioassays compared to individual tile released from Chinese bayberry fruits. Although bay- compound alone. α-Humulene is a widely distributed vola- berry fruits may emit some of the same volatiles found in tile compound from the plants of more than 40 families and other berry fruits like β-caryophyllene, it is likely that they is known as an attractant or a synergist for more than one do not emit them in the right quantity to be attractive to D. insect family (Niogret et al. 2011). In our study, the reduc- suzukii. These differences might be due to divergence of tion of α-humulene concentration in volatile mixtures did Chinese and European populations of D. suzukii that have a not have any influence on the attractiveness of the mixture, high degree of olfactory plasticity adapting to different habi- indicating a general function as an attractant rather than a tats in invaded regions (Allada and Chung 2010; Dubruille synergist to female D. suzukii. and Emery 2008). Our field trials confirmed the attraction of volatile mix- Attraction to host fruit volatiles is very common for fruit tures. Field traps baited with a combination of the four EAD- flies such as Bactrocera invadens (Siderhurst and Jang 2006) active compounds captured both male and female flies. The and D. suzukii (Revadi et al. 2015). Our laboratory behav- fact that a lower number of D. suzukii females were cap- ioural bioassays demonstrated that almost all mated female tured in the control traps and our annual monitoring traps flies responded to Chinese bayberry fruits and fruit head suggests a significant male bias of D. suzukii population in space volatiles, providing further evidence that olfaction September. Although apparently more males were caught in plays an important role in D. suzukii host selection (Cha the traps baited with synthetic Chinese bayberry fruit vola- et al. 2012; Keesey et al. 2015; Revadi et al. 2015). This phe- tile compounds, it seems unlikely that male flies are more nomenon is common for herbivorous insects as they usually responsive to synthetic volatiles than female flies. A similar

1 3 Identification of active components from volatiles of Chinese bayberry, Myrica rubra… 441 phenomenon was observed by Rossi-Stacconi et al. (2016) Carlos JMC, José RCV, Osorio C, Jimenez AM (2011) Volatile com- Annona muri- that male D. suzukii were more abundant in spring and pounds during the ripening of colombian soursop ( cata L. cv. Elita). Vitae 18:245–250 autumn in an Italian mountain region. Our annual monitor- Cha DH, Adams T, Rogg H, Landolt PJ (2012) Identification and field ing study with a lure of vinegar, sugar and wine in the same evaluation of fermentation volatiles from wine and vinegar that region demonstrated that autumn population of D. suzukii mediate attraction of spotted wing drosophila, Drosophila suzukii. was apparently male-biassed (Unpublished data). This dif- J Chem Ecol 38:1419–1431 D. suzukii Cha DH, Hesler SP, Cowles RS, Vogt H, Loeb GM, Landolt PJ (2013) ference in numbers of male and female could be Comparison of a synthetic chemical lure and standard fermented explained by crop phenology (Hammack 2003) and the phys- baits for trapping Drosophila suzukii (Diptera: Drosophilidae). iological status of both sexes, during which more males are Environ Entomol 42:1052–1060 searching for mating sites and proportionally more females Cha DH, Adams T, Werle CT, Sampson BJ, Adamczyk JJ Jr, Rogg H, Landolt PJ (2014) A four-component synthetic attractant for Dros- are entering into ovarian diapause states. Our annual moni- ophila suzukii (Diptera: Drosophilidae) isolated from fermented toring data indicated that the number of other flies trapped bait headspace. 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A slow-release formulation of our Cheng H, Chen J, Chen S, Wu D, Liu D, Ye X (2015a) Characteriza- tion of aroma-active volatiles in three Chinese bayberry (Myrica volatile blend could be developed as an effective lure for rubra) cultivars using GC-MS-olfactometry and an electronic monitoring D. suzukii. Closely related fly species can have nose combined with principal component analysis. Food Res Int different odour preferences. For instance, D. suzukii and D. 72:8–15 biarmipes were found more responsive to leaf odours than Cheng H, Chen J, Li X, Pan J, Xue SJ, Liu D, Ye X (2015b) Differ- D. melanogaster entiation of the volatile profiles of Chinese bayberry cultivars (Keesey et al. 2015). Further work is also during storage by HS-SPME-GC/MS combined with principal required to investigate chemical cues emitted from same host component analysis. 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