Screening and Field Evaluation of Synthetic Volatile Blends Attractive To

Chemoecology DOI 10.1007/s00049-012-0110-1 CHEMOECOLOGY

RESEARCH PAPER

Screening and ﬁeld evaluation of synthetic volatile blends attractive to adults of the tea weevil, Myllocerinus aurolineatus

Xiao-Ling Sun • Guo-Chang Wang • Yu Gao • Zong-Mao Chen

Received: 1 June 2012 / Accepted: 29 June 2012 Ó Springer Basel AG 2012

Abstract Plant volatiles are known to play a role in host host, and is a strong candidate for an attractant that could location in many herbivorous insects. Although a few be used to control the weevil. studies have determined the role of specific chemicals from herbivore-induced plant volatiles (HIPVs) in mediating Keywords Attractant Á Synthetic volatile blends Á interactions between conspecifics in insects belonging to Myllocerinus aurolineatus Á Tea plant Curculionidae, little is known about how this process works when different components are used. By measuring the behavioral responses of the tea weevil, Myllocerinus Introduction aurolineatus (Voss) (Coleoptera: Curculionidae), to a ser- ies of chemicals in a Y-tube olfactometer, we found that a Herbivore-induced plant volatiles (HIPVs) have both blend containing (E/Z)-b-ocimene and (Z)-3-hexenyl ace- repellent and attractive roles in host location by conspecific tate was attractive to male M. aurolineatus adults, and a and nonspecific herbivorous insects. For example, the two- blend containing 2-phenylethanol and (Z)-3-hexenyl ace- spotted spider mite Tetranychus urticae (Dicke 1986), the tate was attractive to female M. aurolineatus adults; both tobacco budworm Heliothis virescens (De Moraes et al. blends were as attractive to the insects as the volatiles 2001) and the western flower thrips Frankliniella occi- emitted by the tea plants infested with adult weevils. A net dentalis (Delphia et al. 2007) are reportedly repelled by cage experiment in the laboratory showed that traps baited plant volatiles induced by conspecifics. Ka¨nnaste et al. with (E/Z)-b-ocimene plus (Z)-3-hexenyl acetate attracted (2009) found that methyl salicylate and (-)-linalool, the male herbivores, whereas the traps baited with released from mite-infested spruce trees, significantly 2-phenylethanol and (Z)-3-hexenyl acetate did not. Field decreased the attractiveness of the tree to pine weevils experiments verified that tea plants exposed to a blend of Hylobius abietis (L.). The attractiveness of HIPVs to her- (E/Z)-b-ocimene plus (Z)-3-hexenyl acetate attracted sig- bivorous insects has been observed mainly for beetles nificantly more weevils than did the control plants or sticky (Coleoptera) with aggregative behavior, which has been traps baited with the above lure. These results suggest that discussed in detail by Sun et al. (2010). Surprisingly, most the blend of (E/Z)-b-ocimene and (Z)-3-hexenyl acetate is of these studies have measured only behavioral responses an important signal which the tea weevil uses to locate its in Curculionidae beetles. To our knowledge, only a few have determined the role of specific chemicals from HIPVs in mediating interactions between conspecifics. For instance, Diaprepes abbreviatus (Linnaeus) and Myllocer- X.-L. Sun (&) Á Y. Gao Á Z.-M. Chen Tea Research Institute, Chinese Academy of Agricultural inus aurolineatus Voss have been verified to be attracted Sciences, Hangzhou 310008, China by the specific volatiles emanating from host plants infes- e-mail: [email protected] ted by conspecific weevils in the laboratory (Ota´lora-Luna et al. 2009; Sun et al. 2010), while Sitophilus granarius G.-C. Wang Henan Institute of Science and Technology, (L.) adults were verified to have the ability to respond Xinxiang 453000, China behaviorally to a wide range of cereal volatiles. Moreover, 123 X.-L. Sun et al. that response may change as a function of concentration Experimental Plantation (Tea Research Institute, Chinese (Germinara et al. 2008). But whether these chemicals are Academy of Agricultural Sciences, Hangzhou Xihu still attractive to herbivores in the field remains unclear, Longjing First-degree Reserve, China). The insects were except that (E)-2-hexen-1-ol and (Z)-3-hexen-1-ol were separated according to their morphological characteristics found to be attractive to the gray corn weevil, Tanymecus into groups of males and females and maintained in sepa- (Episomecus) dilaticollis Gyllenhal, when presented indi- rate net cages (75 9 75 9 75 cm) with fresh tea shoots. vidually in the field (Toshova et al. 2010). Net cages were kept in a controlled climate room pro- The tea weevil, M. aurolineatus (Voss) (Coleoptera: grammed at 25 ± 2 °C, 70 ± 5 % r.h., and a photoperiod Curculionidae), is native to China. This species has one of 13:11 h (L:D). Two weeks later, weevils were chosen generation per year, and overwinters as larvae in the soil. It for experiments. Before testing, the weevils were starved is a leaf-feeding pest of the tea plant Camellia sinensis O. for 2 h. Ktze., and has aggregative behavior that can seriously reduce tea yield and quality (Zhu et al. 1988). In recent Olfactometer test years, outbreaks of this weevil have occurred in eastern and western regions of China. Though chemical controls are Responses of M. aurolineatus adults to different odors effective against this weevil, they pose health and envi- released from different sources or combinations of syn- ronmental risks. Alternative strategies for controlling this thetic compounds were measured in a Y-tube olfactometer, weevil are urgently needed. Our previous study showed which was described in detail by Sun et al. (2010). Briefly, that the infestation of M. aurolineatus adults increased the the olfactometer consisted of a Y-shaped glass tube of emission of more than 40 volatiles. Among these, c-ter- 2.5 cm i.d. The base tube and two arms of the olfactometer pinene, benzyl alcohol, (Z)-3-hexenyl acetate, myrcene, were each 12 cm long. Each arm was connected to an odor benzaldehyde and (Z)-3-hexenal were found to be attrac- source container. An air stream was generated by a vacuum tive to both males and females, (E/Z)-b-ocimene and pump and divided, and each secondary air stream was sent (E,E)-a-farnesene were attractive only to male adults, and through a flowmeter, a tube with an active charcoal filter, a (E)-4,8-dimethyl-1,3,7-nonatriene, 2-phenylethanol, linal- humidifier bottle, and one of the odor containers. Subse- ool, and (Z)-3-hexenol were attractive only to females (Sun quently, the two airstreams passed through the two arms of et al. 2010). However, nothing is known about the attrac- the Y-tube olfactometer at 800 ml min-1. The Y-tube tive activity of these chemicals in the field. In order to olfactometer was placed in a green box (1.5 m long 9 1m explore the tea weevil’s attractants, we first determined the wide 9 1.5 m high). Each edge of the box top was illu- attractiveness of mixtures of some of these chemicals to the minated by a single 25 W lamp. All bioassays were herbivore and compared this attractiveness with those of conducted between 15:00 and 20:00. During experiments, the herbivore-induced tea volatiles in the lab. Then, we the temperature in the room was maintained at 25–27 °C. tested the attractiveness of the candidate mixtures to this Weevils were allowed to walk upwind towards the odor weevil in the field. sources. Each weevil was individually introduced into the base tube. An odor source was considered to have been chosen when a weevil crossed a line 8 cm after the division Methods and materials of the base tube and remained there for at least 1 min. A weevil that did not make a choice within 5 min was con- Tea plants sidered a non-responding individual and recorded as ‘‘no response’’. After 2 weevils were tested, the olfactometer Longjing tea plants were individually planted in plastic tube was washed with 98 % acetone and then heated at pots (14 cm i.d. 9 15 cm high), irrigated once every other 100 °C for 5 min. The position of odor source containers in day, and fertilized with rapeseed cake once a month. Plants relation to each arm was reversed after each replication to were kept in a controlled climate room, which was main- eliminate directional bias. To avoid contamination, the tained at 26 ± 2 °C, 70-80 % r.h., and 12-h photophase. connections of the odor chamber to the flow meter were The light intensity for plants was about 450 lmol pho- rinsed with 98 % acetone and then heated at 100 °C for tons m-2 s-1) during the photophase. One-year-old plants 5 min after each replication. were used for experiments. Response of M. aurolineatus adults to synthetic blends Insects of volatile compounds

Adult weevils of mixed age and sex and of unmated status Our previous study showed that (E/Z)-b-ocimene and were collected from damaged plants on the Tea 2-phenylethanol were abundant components in the 123 Synthetic volatile blends attractive to adults of the tea weevil herbivore-induced tea volatiles. In search of potent attrac- 2-phenylethanol (10 mg); (6) PMC-F versus 2-phenyletha- tants, we chose (E/Z)-b-ocimene or 2-phenylethanol as a nol (10 mg); (7) PMC-M versus (E/Z)-b-ocimene (10 mg): control chemical, added one or two chemicals out of the 6 (Z)-3-hexenyl acetate (0.6 mg); (8) PMC-F versus (E/Z)- active chemicals in the control and then measured the b-ocimene (10 mg):(Z)-3-hexenyl acetate (0.6 mg); (9) difference in attractiveness of the mixtures compared to the PMC-M versus 2-phenylethanol (10 mg):(Z)-3-hexenyl control chemical. The behavioral responses of the adult acetate (0.7 mg); (10) PMC-F versus 2-phenylethanol weevils to the above pairs of odors, belonging to two (10 mg):(Z)-3-hexenyl acetate (0.7 mg). The synthetic groups (shown in Figs. 3 and 4), were tested in the Y-tube odor sources were prepared as above. olfactometer. In this experiment, the synthetic volatile odor sources Net cage experiment were prepared according to the method described by Sun et al. (2010). After individual silicon rubber septa (used as The experiment was conducted in the controlled climate odor dispensers) were filled with 10 llof(E/Z)-b-ocimene room programmed at 25 ± 2 °C, 70 ± 5 % r.h. and a or 2-phenylethanol, different quantities of one or two other photoperiod of 13:11 h (L:D), with two traps in one cage. chemicals were added. Septa filled only with 10 ll(E/Z)-b- A trap, so-called bucket-type, was composed of a plastic ocimene or 2-phenylethanol were used as controls. After bucket, a plastic top and a plastic cap (Fig. 1). Two traps, airing for 5 h, the septa were stored at -20 °C until being one baited with septa containing a blend and the other placed in odor containers where they were used as odor baited with empty septa, were put into opposite corners of a sources for bioassays. Each septum was used for experi- net cage (75 9 75 9 75 cm). Thirty male or female adult ments for less than 3 h. During this period, the release weevils were released into the center of the cage. One day ratio was relatively stable (Sun et al. 2010). Purity and later, the number of weevils in each trap was recorded. The commercial sources of chemical standards are as follows: experiment was replicated six times. In this experiment, the (E/Z)-b-ocimene ([90 %, E:Z = 1:1), b-myrcene (90 %), following pairs of odors were tested: (1) the blend con- 2-phenylethanol ([99 %), c-terpinene (98 %), benzyl taining (E/Z)-b-ocimene and (Z)-3-hexenyl acetate versus alcohol ([99 %), benzaldehyde (98 %), (Z)-3-hexenyl the control; (2) the blend containing 2-phenylethanol and acetate (97 %) and (Z)-3-hexenal (98 %) were obtained (Z)-3-hexenyl acetate versus the control. from Fluka (Buchs, Switzerland), Acros (Eccles, UK), TCI America (Portland, USA), Sigma-Aldrich Inc. (St. Louis, Attractiveness of synthetic volatile blends in the field MO, USA) or Carl Roth (USA). Field trials were conducted at the Tea Experimental Plan- Comparison of the attractiveness of plant–M. tation, which is located in Hangzhou Xihu Longjing First- aurolineatus complex and synthetic volatile blends degree Reserve, China. In this experiment, the same blends tested in the net cage experiment were used. The trials were In this experiment, the following odor sources were set up in a paired design for treatment and control, with two prepared: septa 2 m apart. For the treatment, septa filled with one of the two blends were individually placed in the center of the 1. Intact plants: Potted plants were put in cylindrical glass tubes (18 cm i.d. 9 25 cm high), one pot one plant, as described in detail by Sun et al. (2010). Four days later, the plants were used as an odor source for bioassays. 2. Plant–M. aurolineatus adult complex (PMC): Plants were prepared according to the above treatment (1) and 3 days later were individually infested with 40 female (PMC-F) or male (PMC-M) M. aurolineatus adults. One day later, the plants carrying 40 female or male M. aurolineatus adults were used as odor sources for bioassays. The behavioral responses of the weevils to the following pairs of odors were tested in the Y-tube olfactometer: (1) PMC-M versus intact plant; (2) PMC-F versus intact plant; (3) PMC-M versus (E/Z)-b-ocimene (10 mg); (4) PMC-F Fig. 1 Bucket-type trap. 1 plastic bucket, 2 plastic top, 3 silicon versus (E/Z)-b-ocimene (10 mg); (5) PMC-M versus rubber septa (empty or containing volatile compound), 4 plastic cap 123 X.-L. Sun et al. tops of tea plants. Septa without chemicals were used as weevils in each trap was recorded once a day at 8:00 am, controls. The experiment was replicated eight times totally for 3 days. The number of weevils caught in each (Fig. 2). The distance between replications within a site trap was summed over the course of the experiment and was 15 m, and the distance between the experimental sites then analyzed. of the two blends was 30 m. The number of weevils in 1m2 of tea plants, in which the septa were placed in the Statistical analysis center, was counted at 5:00 a.m. on the morning before the septa were installed and then 2 days later. The increased The differences between the number of M. aurolineatus number between days were calculated, and then analyzed. males and females entering each arm of the olfactometer for each paired treatment were analyzed by the Kruskal– Attracting capacity of traps baited with synthetic Wallis test (v2 approximation). Differences in the behav- volatile blends ioral responses of M. aurolineatus adults to pairs of odors were determined by student’s t test. Data were analyzed In this experiment, yellow sticky trap was used. Two traps, with Statistica (Statistica, SAS, Institute Inc., Cary, NC, one baited with septa containing the blend of (E/Z)-b-o- USA). cimene plus (Z)-3-hexenyl acetate, and the other baited with empty septa, were randomly deployed in a tea experimental plantation. The distance between traps was Results approximately 15 m. The bottom of the traps was approximately 1 cm higher than the top of the tea plants. Response of M. aurolineatus adults to synthetic blends The experiment was replicated ten times. The number of of volatile compounds

Twenty-two synthetic volatile blends, which were divided into two groups, were tested in the Y-tube olfactometer (Figs. 3, 4). M. aurolineatus male adults were signiﬁ- cantly more attracted to the blend containing (E/Z)- b-ocimene plus (Z)-3-hexenyl acetate than to neat (E/Z)-b-ocimene; however, no increased attractiveness was found when (Z)-3-hexenal, benzaldehyde, b-myrcene, benzyl alcohol, or c-terpinene was added. Female adults were signiﬁcantly more attracted by the blend containing 2-phenylethanol and (Z)-3-hexenyl acetate than to the blend containing only 2-phenylethanol. Interestingly, the blend of 2-phenylethanol plus (Z)-3-hexenyl acetate and c-terpinene was repellent for male adults. The blends containing 2-phenylethanol plus (Z)-3-hexenal, benzaldehyde, b-myrcene, benzyl alcohol, or c-terpinene had the same attractiveness to the herbivore as did 2-phenylethanol alone.

Comparison of the attractiveness of plant–M. aurolineatus complex and synthetic volatile blends

The volatiles emitted from PMC-M or PMC-F were more attractive to the adult weevils (both male and female) than were the volatiles emitted from either intact tea plants, (E/Z)-b-ocimene or 2-phenylethanol (Table 1). However, there was no signiﬁcant difference in attractiveness to the Fig. 2 Design of ﬁeld plot to test the attractiveness of synthetic adult weevils (both male and female) between the volatiles volatile blends. The trials were set up in a paired design for treatment from PMC-M or PMC-F and the volatiles from the blend and control, with two septa 2 m apart. The experiment was replicated containing (E/Z)-b-ocimene plus (Z)-3-hexenyl acetate or eight times. The distance between replications within site was approximately 15 m, and the distance between the experimental sites the blend containing 2-phenylethanol and (Z)-3-hexenyl of the two blends was at least 30 m acetate (Table 1). 123 Synthetic volatile blends attractive to adults of the tea weevil

Fig. 3 Behavioral responses of a M. aurolineatus (a) male and (b) female adults to eleven blends (each compared to (E/Z)-b-ocimene) in a Y-tube olfactometer. Numbers the number of M. aurolineatus adults choosing an odor source. N [32 per treatment. v2 test: * P \ 0.05; ** P \ 0.005; n.s. P [ 0.05

Synthetic compounds μl 1 2 3 4 5 6 7 8 9 10 11 (Z )-3-hexenal 2.4 benzaldehyde 0.5 β-myrcene 0.2 (Z)-3-hexenyl acetate 0.6 benzyl alcohol 0.4 terpinene 0.1 (Z/E)-ocimene 10

Net cage experiment controls (Fig. 6a); however, there was no signiﬁcant difference between the number of adult weevils on the tea Compared to traps baited with empty septa, traps baited plants baited with the septa containing the blend of with septa containing (E/Z)-b-ocimene and (Z)-3-hexenyl 2-phenylethanol and (Z)-3-hexenyl acetate and the number acetate caught signiﬁcantly more male adults but not on control plants (Fig. 6a). females; traps baited with a blend of 2-phenylethanol and (Z)-3-hexenyl acetate did not catch more adult weevils Attracting capacity of traps baited with synthetic (either males or females) (Fig. 5). volatile blends

Attractiveness of synthetic volatile blends in the field According to the field results from tea plants that were baited with blends, the blend of (E/Z)-b-ocimene and In the field, significantly more adult weevils were found on (Z)-3-hexenyl acetate was chosen to test with the traps. In the tea plants that were baited with the septa containing the the field, more adult weevils were found on the traps blend of (E/Z)-b-ocimene and (Z)-3-hexenyl acetate than that were baited with the septa containing the blend of on the control plants; the number of herbivores on the (E/Z)-b-ocimene and (Z)-3-hexenyl acetate than on the blend was 17.76-fold higher than the number on the control traps; the number of the weevil on the traps with

123 X.-L. Sun et al.

Fig. 4 Behavioral responses of a M. aurolineatus (a) male and (b) female adults to eleven blends (each compared to 2-phenylethanol [phenyl-]) in a Y-tube olfactometer. Numbers = the number of M. aurolineatus adults choosing an odor source. N [32 per treatment. v2 test: * P \ 0.05; ** P \ 0.005; n.s. P [ 0.05

Synthetic compounds µl 1 2 3 4 5 6 7 8 9 10 11 (Z )-3-hexenal 2.8 benzaldehyde 0.6 β-myrcene 0.2 (Z )-3-hexenyl acetate 0.7 benzyl alcohol 0.3 γ-terpinene 0.1 2-phenylethanol 10 the blend was 5.39-fold higher than the number on the apple maggot fly Rhagoletis pomonella (Walsh) (Linn et al. controls (Fig. 6b). 2005) and the grapevine moth Lobesia botrana Denis & Schiffermu¨ller (Tasin et al. 2007). This phenomenon was also substantiated by field bioassay results, e.g., grape berry Discussion moth Paralobesia viteana (Cha et al. 2008). Carrying out experiments in the lab and field, we also found that the Herbivorous insects locate their host plants through the mixture of (E/Z)-b-ocimene plus (Z)-3-hexenyl acetate perception of species-specific compounds or of specific attracted M. aurolineatus adults (Figs. 3, 5, 6), suggesting ratios of ubiquitous compounds (Bruce et al. 2005). The that both chemicals play an important role in the weevils’ absolute release rate of volatiles may also play a role in ability to locate a host. The emission of (Z)-3-hexenyl locating appropriate hosts (Byers 1992; Byers et al. 1985, acetate, an important component of green-leaf volatiles, 2000). Specific blends mixed with several compounds have may be elicited by physical damage (Shimoda 2010), been found to be used as host location cues by some her- herbivore infestation (McCall et al. 1994; Shimoda 2010; bivorous insects in the lab, such as the fruit fly Drosophila Sun et al. 2010) and exogenous application of (Z)-3-hex- melanogaster (Zhu et al. 2003), the orange wheat blossom enol (Farag et al. 2005) or methyl jasmonate (Rodriguez- midge Sitodiplosis mosellana (Birkett et al. 2004), the Saona et al. 2001; Ibrahim et al. 2005). It has been reported

123 Synthetic volatile blends attractive to adults of the tea weevil

Table 1 Comparison of behavioral responses of M. aurolineatus 16 adults to synthetic volatile blends versus the plant M. aurolineatus adult complex by Y-tube olfactometer 14 Comparison Sex No. of no No. of P responses responses 12 AB AB 10 PMC- Intact plant Male 9 28 12 * * M Female 4 23 9 * 8 PMC- Intact plant Male 3 23 9 * 6 F Female 7 27 15 * Mean catch per trap PMC- (E/Z)-b-Ocimene Male 2 23 9 * 4 M Female 4 24 8 ** PMC- (E/Z)-b-Ocimene Male 6 25 9 * 2 F Female 3 26 11 * PMC- 2-Phenylethanol Male 4 24 9 * 0 M Female 2 24 9 * B 1 B 2 PMC- 2-Phenylethanol Male 7 26 11 * F Female 1 28 12 * Fig. 5 Means (±SE) of M. aurolineatus adult males and females PMC- (E/Z)-b- Male 6 17 15 n.s. caught in traps baited with Blend 1 or Blend 2 (white) versus control (gray). This experiment was conducted in net cages located in a M Ocimene ? (Z)-3- Female 6 13 19 n.s. hexenyl acetate controlled climate room programmed at 25 ± 2 °C, 70 ± 5 % r.h., and a photoperiod of 13:11 h (L:D). Asterisks indicate significant PMC- (E/Z)-b- Male 5 19 13 n.s. differences between members of a pair (blend vs. control, P \ 0.05, F Ocimene ? (Z)-3- Female 11 17 15 n.s. student’s t test). Blend 1 (B1) contained a mixture of (E/Z)-b-ocimene hexenyl acetate (10 mg) and (Z)-3-hexenyl acetate (0.6 mg), and Blend 2 (B2) PMC- 2-Phenylethanol ? (Z)- Male 1 19 13 n.s. contained a mixture of 2-phenylethanol (10 mg) and (Z)-3-hexenyl M 3-hexenyl acetate Female 7 19 13 n.s. acetate (0.7 mg) PMC- 2-Phenylethanol ? Male 7 19 14 n.s. F (Z)-3-hexenyl Female 9 14 18 n.s. acetate confirm a common phenomenon, namely, that the blend N[32 per treatment. v2 test: * P \ 0.05; ** P \ 0.005; n.s. P [ 0.05 attractive to an insect in the laboratory may be not attractive to the insect in the field. For example, significantly more Rhynchophorus cruentatus (F.) were attracted to that (Z)-3-hexenyl acetate may act as a signal between females plus palm tissue than to the palm tissue or females plants (Engelberth et al. 2004; Kost and Heil 2006; Heil in the laboratory bioassay, but females plus palm tissue did et al. 2008), an attractant to herbivores (Zhang et al. 2011), not attract more weevils than palm or females in the field a synomone attracting natural enemies of the herbivores experiment (Weissling et al. 1993). Many factors, such as (James 2003; 2005), or as an agent with synergistic activ- the concentration of the blend, temperature, humidity, ities on sex or aggregation pheromones (Dickens et al. surrounding plants and so on, will result in the difference in 1993; Reddy and Guerrero 2000; Deng et al. 2004). Our attractiveness of the blend to the insect in the laboratory results demonstrated that the addition of (Z)-3-hexenyl and in the field. Thus, that the blend of 2-phenylethanol and acetate may enhance the attractiveness of (E/Z)-b-ocimene (Z)-3-hexenyl acetate did not attract the herbivore in the to the herbivore (Fig. 3), suggesting a synergistic effect. field might be due to the reasons stated above. For the In lab experiments, we found that the blend containing blend containing (E/Z)-b-ocimene plus (Z)-3-hexenyl ace- (E/Z)-b-ocimene plus (Z)-3-hexenyl acetate attracted only tate, given its attractiveness to both male and female adults male M. aurolineatus adults, whereas the blend of in the field, we speculate that male adults attracted by the 2-phenylethanol and (Z)-3-hexenyl acetate attracted only blend may produce an aggregation pheromone that subse- female adults (Figs. 3, 4). However, when the two blends quently, together with the blend, attracts both male and were tested in the net cage and in the field, the result was female adults. As has been reported previously, almost all interesting: the former attracted only male M. aurolineatus male insects of Curculionidae, such as Sitona lineatus (L.) adults in the net cage experiment (Fig. 5), but in the field, (Blight et al. 1984), Rhynchophorus cruentatus (Fabricus) both male and female adults were attracted (Fig. 6); the (Weissling et al. 1993), Rhabdoscelus obscur (Giblin- latter blend did not attract either male or female weevils in Davis et al. 2000), Anthonomus rubi Herbst (Innocenzi either the cage or field experiments (Figs. 5, 6a). This may et al. 2001), Scyphophorus acupunctatus Gyllenhal 123 X.-L. Sun et al.

a The net cage bioassay in the laboratory showed that 60 traps baited with the blend containing (E/Z)-b-ocimene plus (Z)-3-hexenyl acetate were more attractive than traps 50 * containing empty septa (Fig. 5). Moreover, field experiments verified that this blend attracted significantly more 40 weevils than did the control (Figs. 6). This suggests that the blend consisting of (Z)-3-hexenyl acetate and (E/Z)-b- 30 ocimene can be used to develop attractants, thus helping eradicate this pest. 20

Acknowledgments We thank Emily Wheeler for editorial assistance. 10 We thank Liqun Chen and Biao Gao, who came from Jilin Agricultural University and Yangtze University as summer students to our group, for No. of weevils attracted to tea plants 0 collecting and rearing weevils. We thank Zongxiu Luo, who drew the B1 Con B2 Con draft of trap. The study was sponsored by National Program on Key Basic Research Project (973 Program) (2012CB114104), National Natural Science Foundation of China (31171862), the Division of b 2.5 * Science and Technology of Zhejiang Province (2011C22043), and the Planning Subject of ‘the Twelfth Five-Year-Plan’ in National Science and Technology (2011BAD01B02-5). 2.0

1.5 References

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Mean catch per trap 7:1319–1328 .5 Blight MM, Pickett JA, Smith MC, Wadhams LJ (1984) An aggregation pheromone of Sitona lineatus—identification and initial field studies. Naturwissenschaften 71:480 0.0 B 1 Con Bruce TJA, Wadhams LJ, Woodcock CM (2005) Insect host location: a volatile situation. Trends Plant Sci 10:269–274 Fig. 6 Means (±SE) of M. aurolineatus adults (a) attracted to tea Byers JA (1992) Attraction of bark beetles, Tomicus piniperda, plants baited with Blend 1 (B1) or Blend 2 (B2) versus control (Con), Hylurgops palliatus, and Trypodendron domesticum and other and (b) caught in traps baited with B 1 versus control (Con). B1 a insects to short-chain alcohols and monoterpenes. J Chem Ecol mixture of (E/Z)-b-ocimene (10 mg) and (Z)-3-hexenyl acetate 18:2385–2402 (0.6 mg); B2 a mixture of 2-phenylethanol (10 mg) and (Z)-3-hexenyl Byers JA, Lanne BS, Lo¨fqvist J, Schlyter F, Bergstro¨m G (1985) acetate (0.7 mg). This experiment was conducted on the Tea Olfactory recognition of host-tree susceptibility by pine shoot Experimental Plantation. Asterisks indicate significant differences beetles. Naturwissenschaften 72:324–326 between members of a pair (blend versus control, P \ 0.05, student’s Byers JA, Zhang QH, Birgersson G (2000) Strategies of a bark beetle, t test) Pityogenes bidentatus, in an olfactory landscape. Naturwissens- chaften 87:503–507 Cha DH, Nojima S, Hesler SP, Zhang AJ, Linn CE, Roelofs WL, (Ruiz-Montiel et al. 2003), and Anthonomus eugenii Cano Loeb GM (2008) Identification and field evaluation of grape shoot volatiles attractive to female grape berry moth (Paralo- (Szendrei et al. 2011) produce aggregation pheromones, besia viteana). J Chem Ecol 34:1180–1189 and these attract both males and females to traps in the De Moraes CM, Mescher MC, Tumlinson JH (2001) Caterpillar- field. Moreover, compared to the host volatiles alone, induced nocturnal plant volatiles repel conspecific females. aggregation pheromone plus host plant volatiles have been Nature 410:577–580 Delphia CM, Mescher MC, De Moraes CM (2007) Induction of plant verified to significantly enhance trap captures of the boll volatiles by herbivores with different feeding habits and the weevil Anthonomus grandis Boheman (Dickens 1989), the effects of induced defenses on host-plant selection by thrips. diamondback moth Plutella xyllostella (L.) (Reddy and J Chem Ecol 33:997–1012 Guerrero 2000) and the beet armyworm Spodoptera exigua Deng JY, Wei HY, Huang YP, Du JW (2004) Enhancement of Spodoptera exigua ¨ attraction to sex pheromones of by volatile (Hubner) (Deng et al. 2004). Our previous experiments also compounds produced by host plants. J Chem Ecol 10:2037–2045 found that both male and female M. aurolineatus weevils Dicke M (1986) Volatile spider-mite pheromone and host-plant were attracted by the odor emitted from 40 male weevils kairomone, involved in spaced-out gregariousness in the spider (unpublished data). This suggests that male M. aurolinea- mite Tetranychus urticae. Physiol Entomol 11:251–262 tus Dickens JC (1989) Green leaf volatiles enhance aggregation phero- adults may also produce an aggregation pheromone, a mone of boll weevil, Anthonomus grandis. Entomol Exp Appl phenomenon that needs to be further studied in the future. 52:191–203

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