Identification and field verification of an aggregation pheromone from the white-spotted flower chafer, Protaetia brevitarsis Lewis (Coleoptera: Scarabaeidae)

The white-spotted flower chafer (WSFC), Protaetia brevitarsis Lewis, is native to East Asia. Although their larvae are considered a potential resource insect for degrading plant residues, producing protein fodder, and processing to traditional medicine, adult WSFCs inflict damage to dozens of fruit and economic crops. The control of the WSFC still relies heavily on pesticides and the inefficient manual extraction of adults. Here, we report the identification and evaluation of the aggregation pheromone of WSFCs. From the headspace volatiles emitted from WSFC adults, anisole, 4-methylanisole, 2-heptanone and 2-nonanone were identified as WSFC-specific components. However, only anisole and 4-methylanisole elicited positive dose–response relationship in electroantennography tests, and only 4-methylanisole significantly attracted WSFCs of both sexes in olfactometer bioassays and field experiments. These results concluded that 4-methylanisole is the aggregation pheromone of WSFCs. Furthermore, we developed polyethylene vials as long-term dispensers of 4-methylanisole to attract and kill WSFCs. The polyethylene vial lures could effectively attracted WSFCs for more than four weeks. Pheromone-based lures can be developed as an environmentally friendly protocol for monitoring and controlling WSFC adults.

The white-spotted flower chafer (WSFC), Protaetia brevitarsis Lewis (Coleoptera: Scarabaeidae: Cetoniinae) (NCBI: txid348688) is native to East-Asia and distributed throughout Japan, Korea, Mongolia and China 1 . Since its larvae usually feed on humus and decomposed plant tissues in topsoil 2 , WSFCs are cultivated as a potential resource insect to produce high-quality organic fertilizer by converting herbaceous and ligneous plant residues 3,4 . In China and Korea, WSFC larvae are also utilized to produce protein fodder and traditional medicine [5][6][7] . However, WSFC adults, which cause up to 5-30% yield loss to corn, grape and sunflower in the absence of control measures, are considered important agricultural and horticultural pests 8,9 . Although there have been outbreaks of the WSFCs that have caused damage to economic crops in China during the last three decades, there are currently no reliable control strategies for use against WSFCs. Commonly, WSFC larvae spend most of their lives underground often distant from orchards 3 , while adults are excellent flyers that have the ability to colonize feeding and breeding sites rapidly 10 . Another issue that weakens the control efficacy is that the adults preferentially attack ripe or nearly ripe fruits that cannot safely be sprayed with insecticides 10 . To date, control of WSFCs still relies heavily on environmentally damaging pesticides and inefficient manual extraction of adults, which is impossible for large-scale production. Therefore, novel and effective strategies for controlling and monitoring WSFCs are urgently needed.
Trapping and killing of insect pests based on chemical communication cues, such as insect pheromones and food attractants, has been demonstrated as an environmentally friendly method to control agricultural pests 11 . A

Results
Traps baited with live beetles. Both male and female WSFCs were attracted to the bucket traps baited with live beetles, and the captures of WSFC males and females were approximately equal in all treatments ( Fig. 1), suggesting that aggregation pheromones rather than sex pheromones, existed in the WSFC population. Neither female nor male WSFCs were caught in the blank traps during any of the field experiments. In the field experiment of verifying the existence of long-distance pheromones, no deaths or escapes of the marked beetles were recorded over a one-day interval, indicating that the captured WSFCs had no opportunities to escape from the bucket traps. Among five traps for two weeks, only two females and two males were captured in the traps baited with food alone. The captures among traps baited with virgin males, virgin females, mated males and mated females were not significantly different (male captures: F 3,16 = 0.129, P = 0.941; female captures: F 3,16 = 0.265, P = 0.850) (Fig. 1), indicating that both male and female WSFCs produce and release the aggregation pheromone. It was also concluded that both virgin and mated beetles attracted similar numbers of WSFCs (Fig. 1).
Volatile identification. Considering that WSFCs are diurnal animals, we collected the volatiles during the photoperiod. The GC-MS analysis revealed that the volatiles from both female and male WSFCs were not large in amounts, their total ion chromatogram (TIC) peaks were easily confused by noisy signals (including column bleeding caused by moisture from collection surroundings, and impurity compounds from environment and solvent). To distinguish the pheromones from beetles, we compared the volatiles of WSFCs with those of blank control (Fig. 2). The comparison results showed that four compounds were identified from the volatile extraction from both male and female WSFCs, including one major component (4-methylanisole) and three minor components (anisole, n-heptanone and n-nonanone). All compounds were verified with authentic samples through retention time and mass spectrometry.

Electroantennography (EAG) analysis.
The EAG tests showed that the antennae of both male and female WSFCs responded strongly to 4-methylanisole, and responded mildly to anisole (Fig. 3). The EAG

Y-tube behavior tests.
In the Y-tube bioassay, 4-methylanisole was the most attractive candidate at a con-
Field trials. To screen the aggregation pheromone of WSFCs, we evaluated the attractiveness of the EAGactive candidates as well as their blends with EAG-inactive components in the field. As the most EAG-active compound, 4-methylanisole, both separately and contained in binary blends, captured > 4 female and > 4 male WSFCs, while blank control, sole n-heptanone and n-nonanone displayed no attractiveness to WSFCs (Table S1). The ratio of captured male to female beetles was near 1:1 (Table S1). The addition of anisole, n-heptanone and n-nonanone to 4-methylanisole failed to improve its attractiveness (male captures: F 3,16 = 0.307, P = 0.820; female  We concluded that 4-methylanisole was the sole candidate aggregation pheromone of WSFC. In the field experiment of dose optimization, loading higher amounts of 4-methylanisole, from 0.5 to 20 mg, significantly increased the WSFC captures (male capture: F 4,20 = 17.716, P < 0.001; female capture: F 4,20 = 17.125, P < 0.001. Table S2). The maximum WSFC captures were 11.25 beetles per week at a 4-methylanisole dose of 100 mg, and more 4-methylanisole (> 20 mg) did not contribute significantly to increasing the catch numbers (male captures: F 2,12 = 0.995, P = 0.398; female captures: F 2,12 = 0.588, P = 0.571).
Lure duration. We further evaluated the long-term performance of 4-methylanisole lure from 15 July to 2 September, 2018. There were large differences in the weekly captures of both the fresh lures and test lures, presumably due to the seasonal variation in the WSFC population (Fig. 6). Compared with the fresh lures, however, the test lures trapped almost the same numbers of WSFCs (sum of the captured females and males) in the first four weeks (Fig. 6, 4th week, F 1,8 = 1.043, P = 0.337). Starting from the 5th week, the test lures attracted significantly lower numbers of WSFCs than the fresh lures (Fig. 6, 5th week, F 1,8 = 7.511, P = 0.025). Nevertheless, the test lures still trapped approximately three beetles even after aging for 6 weeks, suggesting that the vial lures baited with 4-methylanisole could be an efficient method for long-term monitoring WSFCs.

Discussion
In the present study, we identified and verified 4-methylanisole as an aggregation pheromone emitted from both sexes of WSFCs. Although producing and emitting aggregation pheromones by both sexes is unusual in beetle species, this phenomenon is reported in Cydia pomonella and Locusta migratoria and Colletes bees [21][22][23] . Synthetic 4-methylanisole elicited a strong response from adult antennae in the electrophysiology tests, and attracted female and male beetles both in the Y-tube olfactometers and in the field experiments. To our knowledge, 4-methylanisole has not been reported as an aggregation pheromone of any insect.
Although 4-methylanisole is uncommon as an insect-originated volatile, it has been reported in bouquets of many plant species, such as Melodorum fruticosum 24 , Mimusops elengi 25 , and Gelsemium sempervirens 26 . In some species, 4-methylanisole plays crucial roles in the plant-pollinator relationship. As the dominant aromatic volatile in the flowers of Ficus semicordata and Hydrocleys martii, 4-methylanisole has been proven to contribute to the attraction of pollinators 27,28 . 4-Methylanisole has also been reported in the flowers of phytelephantoid palms (Arecaceae) 29 and Cymbopetalum brasiliense 30 , and was inferred to be well perceived by various insects, especially pollinators 28 . In the inflorescences of the macauba palm, the dominant floral fragrance was identified as 4-methylanisole (> 97% relative amount), which showed exclusive attractiveness to a florivorous chafer, Cyclocephala forsteri 31 . In this study, we found that 4-methylanisole showed significant attractiveness to the WSFCs, a Cetoniinae species. It is well known that Cetoniinae beetles, the most frequent flower visitors, not only play an important role in plant pollination [32][33][34] but also are serious pests of flowers and fruits because they usually attack and feed on blooming flowers and ripe fruits 35 . Polyphagous WSFC adults feed on the flowers and fruits of dozens of plant species 1 , resulting in the possibility that 4-methylanisole identified from WSFC volatiles originated in their foods sources. To control for this, we fed the WSFCs in volatile collection with only water. Therefore, the 4-methylanisole detected in the WSFC adults was produced and released autonomously. To investigate where 4-methylanisole secretes, we separated the midguts, hindguts and accessory (colleterial) glands, which were the potential pheromone-producing organs of Scarabeidae beetles 36,37 . Majority of 4-methylanisole and anisole www.nature.com/scientificreports/ were detected in the accessory glands of both sexes, and they were also detected in hindguts, while only trace of anisole was found in the midguts (Fig. S2). Our Y-tube bioassays and field experiments suggest that synthetic 4-methylanisole displayed significant attractiveness to both male and female WSFCs. These results could not exclude the possibility that 4-methylanisole is an aggregation-sex pheromone, which also induces attraction of both sexes [38][39][40] . WSFCs displayed no obvious courtship behavior (e.g. mounting) towards 4-methylanisole in both Y-tube and arena bioassay, indicating that 4-methylanisol was an aggregation pheromone of WSFCs rather than an aggregation-sex pheromone. WSFCs are active in daytime, flying or crawling around the arena unless they are feeding or resting. To settle WSFCs in the behavioral arena, we used odorless agar-gel as food and resident place. The results of aggregation index (Fig. 5) were fully according with the Y-tube tests (Fig. 4) and field trapping (Table S1-S2), collectively confirming that 4-methylanisole was significantly attractive to both sexes of WSFCs. Combined with food, aggregation pheromones also show higher attractiveness in other chafers 16 . It makes sense that the beetles release chemical signals to summon conspecifics during eating, which could provide conspecific individuals with more opportunities to find food sources and mate partners. It provided future possibility to develop more efficient WSFC attractants by the combination of pheromones and host volatiles.
Few reports have documented 4-methylanisole as an insect-derived sex pheromone or aggregation pheromone, but anisole, the homologue of 4-methylanisole, has been identified as the main component of the sex pheromone of the flower chafers Holotrichia reynaudi 3,41 and H. consanguinea 42 . In particular, anisole was used to capture a large number of female and male H. consanguinea, leading to aggregation and copulation behavior 6,42 . Anisole was also detected in the volatiles emitted from WSFC adults, but it elicited only a relatively small antennal response (Fig. 3A) and did not result in the enhancement of the attractiveness of 4-methylanisole (Table S1). Whether anisole has biological function will be further assayed in our future study.
It is well documented that flower-derived 4-methylanisole attracts flower-visiting insects, including various kinds of bees and beetles 28,29,31 . This means that traps baited with 4-methylanisole could capture pollinators in the field. During our experiment in a vineyard, a variety of nontarget insects, such as wasps, chafers, ladybirds, spiders, flies, lacewings, moths, hoverflies, leafhoppers and plant bugs, were caught. Compared with the traps baited with sunflower oil only, the traps with 4-methylanisole trapped significantly more halictid bees, Xylocopa appendiculata and Oxycetonia jucunda Faldermann (unpublished data), which are well-known pollen consumers [43][44][45] . The capture numbers of other nontarget species were not different between the traps baited with 4-methylanisole and the control lure (unpublished data), suggesting that the capture of these bycatches was not associated with WSFC pheromone.
The capture of beneficial insects would potentially decrease the application of WSFC lures. Novel traps might be a way to reduce the negative effects of control measures on nontarget species. To monitor and control Scarabaeidae pests, many traps, such as cone trap cross-vane panel trap 46 , pitfall trap 47 , funnel trap 48,49 and Trece trap 50,51 , have been designed and applied. Few studies have reported the use of sticky traps for flower chafers, presumably because chafers are strong enough to escape from sticky cards, a behavior that has been observed in WSFCs. Additionally, color of traps is a significant factor on bycatch rate of nontarget beneficial insects, including honey bees, bumblebees, solitary bees and ladybirds 52,53 . In our further work, we will design and develop more efficient traps with non-attraction for beneficial insects.
Our results indicate that 4-methylanisole, the exclusive field-active component among the volatiles released from the WSFCs, is the sole aggregation pheromone. The vial lures loaded with 4-methylanisole significantly trapped both sexes of WSFCs, suggesting they could be developed as potential method for monitoring this pest. Our future studies will focus on modifying the structure of 4-methylanisole to increase its selectivity in favor of target over non-target insects.

Methods and materials
Insects and sample collection. The WSFC population was reared at Cangzhou Technical College (Hebei Province, China). The pupae were separated and individually kept in glass vials until adult emergence. The newly-emerged adults were sorted by sex based upon differences in morphology of their abdomens and reared individually in small cages. Both the male and female adults were given sucrose water (5%) as food after their emergence. WSFCs used for collecting volatiles were fed with only water for three days before volatile collection, to ensure no food odor in the tested beetles. The beetles used in the EAG test and behavioral experiment were fed fresh apple slices, which were replaced every day. The male and female WSFCs were reared in different rooms to avoid an effect of interaction on pheromone collection. The rearing room was maintained at 25 ± 2 °C and 50-70% RH with a 12:12 h L:D.
The head space method 54 was used to collect the volatiles emitted from virgin WSFCs. Briefly, groups of 50 males and females (~ 10 days old, at the onset of showing aggregation habit 23 ) were placed separately in glass aeration chambers (30 cm height × 5.5 cm diam) after rinse in distilled water and natural air-dry for 1 h. The volatiles from the caged beetles were drawn through the chamber using a diaphragm pump at a flow rate of 200 mL·min −1 , and adsorbed on a purified absorbent (Porapak Q, Sigma-Aldrich, USA). Subsequently, the adsorbed volatiles were eluted with distilled hexane. Air through an empty chamber was also collected as a blank control. The samples were stored at − 20 °C until GC-MS analysis. The collection of volatiles was conducted during the photo period (from 4 to 8 h), considering that the WSFCs naturally feed and mate in the daytime 55 . The volatiles from each sex were collected in triplicate. EAG bioassays. The electrophysiological responses of WSFC antennae to the four volatiles from the beetles were monitored using an EAG apparatus (Syntech Ltd., Kirchzarten, Germany). The EAG preparation followed that used in Zhang et al. 56  The odor compounds were dissolved in paraffin oil. Paraffin oil was used as a negative control. In the Y-tube olfactometer, compound solution and paraffin oil on strips of filter paper (3 cm × 0.5 cm) were placed into the arms, and a WSFC individual (10-20 days old) was released into the stem during the photophase (WSFCs are diurnal insects 23 ). The orientation of the beetle was recorded after 15 min at 25-27 °C. The beetles over midpoint of control or odor arms were defined as making choice, and others were recorded as no choice. To eliminate positional bias, the odor arms were rotated after five trials. The Y-tube was washed with ethanol after each test. In each treatment, 60 beetles were introduced into Y-tube individually. The attractive compounds were screened by loading 10 μL of the beetle-produced compounds at a concentration of 1.0 μg·μL −1 as odor sources. Then, the dose-dependent response to the candidate aggregation pheromone was evaluated by loading 0.5, 1.0, 5.0, 10 and 100 μg of pure compound in 10 μL of paraffin oil.

Aggregation index.
To determine the aggregation index of WSFCs to the aggregation pheromones, we conducted bioassays in an arena of two-choice olfactometer (180 cm × 90 cm × 10 cm) with a gauze to prevent escape (Fig. 7). In the center of treated compartment, a 9-cm petri dish with agar-gel (1.5%) and 2 μg or 20 μg of pure 4-MA was added, while a petri dish with agar-gel was set in the control compartment. The WSFCs in a group of ten were introduced in the center of the arena, and their numbers in each section were counted after 15 min. Uncommitted beetles in the middle arena were recorded as non-responders. Each treatment was replicated 10 times (a total of 100 WSFCs). The aggregation index was calculated as (T − C) × 100/N, where T and C are the numbers of beetles stayed in the treated and control compartments respectively, and N represents the total number of tested WSFCs 57 .
Lures. Pheromone dispensers were prepared using polyethylene (PE) vials (25 mm × 6 mm × 0.25 mm thick; Pherobio Technology Co. Ltd., Beijing) following previous procedure with slight modifications 58 . Briefly, the PE vials were loaded with appropriate amounts of the candidate aggregation pheromone components in 200 μL of sunflower oil and heat-sealed. The volatiles slowly permeated the vial walls and were released. Setup for behavioural preference of Protaetia brevitarsis in a dual-choice arena system. The yellow and blue part represented treated and control compartment, respectively. The beetles were initially positioned in the center of the arena. Two short cylinders represent the 9-cm petri dish which is filled with 1.5% agar-gel. 115.49°E). Traps were hung ~ 1.5 m above the ground between the rows of vines with a minimum of 20 m apart. In each experiment, traps were randomly arranged in the vineyard. Traps were examined weekly, and the numbers of captured beetles were counted and recorded by sex. After removing the trapped WSFCs and renewing the lures, the positions of the traps were rerandomized. Five replicates were tested for each treatment.
Verification of WSFC pheromones. Live beetles were used to verify the existence of long-distance pheromones in WSFCs from 26 July to 9 August 2016. Five live WSFCs (~ 10 days old) were marked with red markers colors and caged directly in every trap. Fresh apple slices were also added as food for the WSFCs. Six treatments were conducted: blank, food only, virgin females, virgin females, mated females and mated males. Every morning, the live beetles and their food were renewed, and the traps washed. Long-term assessment of lures. The longevity of the vial lures were estimated by continuously testing the attractiveness of the lures from 15 July to 2 September 2018. All the vial lures were prepared by loading 100 mg of 4-methylanisole. Due to the lack of efficient measures to monitor the WSFC population, a treatment baited with fresh lures (FLs) that were renewed after each check was set to avoid the deviation of WSFC captures originating from the population variation.
Statistical analysis. SPSS 19.0 was used for all the statistical analyses. The Y-tube and binary-choice arena data were analyzed using χ 2 goodness-of-fit tests after excluding the unresponsive individuals. The differences in captures between pairs of treatment were analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test. P < 0.05 was considered statistically significant. The data used for ANOVA did not include the allzero values from controls and failed treatments, and were validated with Levene' test.