Enhanced Attraction of Plutella Xylostella (Lepidoptera: Plutellidae) to Pheromone-Baited Traps with the Addition of Green Leaf Volatiles

BIOLOGICAL AND MICROBIAL CONTROL Enhanced Attraction of Plutella xylostella (Lepidoptera: Plutellidae) to Pheromone-Baited Traps With the Addition of Green Leaf Volatiles

1 2 1,3 PENGYAN LI, JUNWEI ZHU, AND YUCHUAN QIN

J. Econ. Entomol. 105(4): 1149Ð1156 (2012); DOI: http://dx.doi.org/10.1603/EC11109 ABSTRACT Diamondback moth, Plutella xylostella (L.) (Lepidoptera: Plutellidae), is one of the most serious pests of Brassicaceae crops worldwide. Electrophysiological and behavioral responses of P. xylostella to green leaf volatiles (GLVs) alone or together with its female sex pheromone were investigated in laboratory and Þeld. GLVs 1-hexanol and (Z)-3-hexen-1-ol elicited strong electroan- tennographic responses from unmated male and female P. xylostella, whereas (Z)-3-hexenyl acetate only produced a relatively weak response. The behavioral responses of unmated moths to GLVs were further tested in Y-tube olfactometer experiments. (E)-2-Hexenal, (Z)-3-hexen-1-ol, and (Z)-3- hexenyl acetate induced attraction of males, reaching up to 50%, signiÞcantly higher than the response to the unbaited control arm. In Þeld experiments conducted in 2008 and 2009, signiÞcantly more moths were captured in traps baited with synthetic sex pheromone with either (Z)-3-hexenyl acetate alone or a blend of (Z)-3-hexenyl acetate, (Z)-3-hexen-1-ol, and (E)-2-hexenal compared with sex pheromone alone and other blend mixtures. These results demonstrated that GLVs could be used to enhance the attraction of P. xylostella males to sex pheromone-baited traps.

KEY WORDS electroantennogram, trap catch, green leaf volatile, Plutella xylostella, sex pheromone

In recent years, several case studies have demon- was used previously as a three-component blend of strated that green leaf volatiles (GLVs) not only stim- (Z)-11-hexadecenal, (Z)-11-hexadecenyl acetate, and ulate insect pheromone production and release but (Z)-11-hecadecen-1-ol at a ratio of 1:1:0.01 (Reddy also can act to synergize behavioral responses to sex and Guerrero 2000). Recently, several studies have pheromones (Landolt and Phillips 1997, Reddy and shown that the use of sex pheromones with host plant Guerrero 2004). There has been considerable focus on volatiles can increase pheromone trap catches signif- understanding the interactions between host plant icantly in several lepidopteran species (Dickens et al. GLVs and sex pheromones of phytophagous insects 1993a,b; Light et al. 1993; Ochieng et al. 2002; Deng et for the use in enhancing the pheromone trap catches al. 2004a,b; Yang et al. 2004), including diamondback by mixing the two together in lure systems (Dickens moth (Reddy and Guerrero 2000, Dai et al. 2008). A et al. 1993a,b; Light et al. 1993; Deng et al. 2004a,b). series of GLVs with a straight six carbon-chain length Therefore, the combination of insect sex pheromones are emitted from the host plant (Brassica oleracea ssp. with GLVs in trapping systems can be used to increase capitata)ofP. xylostella, and these compounds elicit the efÞcacy of detection, monitoring, and mass trap- signiÞcant behavioral responses in laboratory bioas- ping technologies in integrated pest management says (Reddy and Guerrero 2000, Reddy et al. 2002). (IPM) strategies. Here, we report on 1) electroantennogram (EAG) Diamondback moth, Plutella xylostella (L.) (Lepi- response of P. xylostella to selected host plant GLVs, doptera: Plutellidae), is a destructive pest that has 2) Y-tube olfactometer behavioral tests, and 3) results caused signiÞcant economical losses in Brassicaceae of Þeld trapping experiments with combined phero- crops worldwide (Talekar and Shelton 1993). This mone and GLVs. The effectiveness of using two dis- pest is hard to control due to its high reproductive rate penser materials for GLVs to enhance the pheromone and the level of resistance it has developed to many trap catches also is discussed. insecticides. Among all IPM programs currently used in China, mass trapping using synthetic sex phero- Materials and Methods moneÐbaited traps seems to be the most practical method for controlling this pest (Wang et al. 2003, Li Insects Rearing and Maintenance. P. xylostella lar- et al. 2011). The female sex pheromone of P. xylostella vae were collected from cabbage (Brassica oleracea L.) plants in Beijing (39.5ЊN, 116.2ЊE), China, from 1 Department of Entomology, China Agricultural University, Bei- July to August in 2007, and they were reared in an jing 100193, China. environmental chamber at 26 Ϯ 2ЊC, 60Ð70% RH, and 2 Agroecosystem Management Research Unit, USDAÐARS, Lin- coln, NE 68583. a photoperiod of 14:10 (L:D) h until pupation. Two- 3 Corresponding author, e-mail: [email protected]. week-old radish (Raphanus sativus L.) plants with two

0022-0493/12/1149Ð1156$04.00/0 ᭧ 2012 Entomological Society of America 1150 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 4

fully expanded true leaves were used as the larval food. Y-Tube Olfactometer Assays. A Y-tube olfactometer The pupae were separated by sex before adult eclo- was used to investigate behavioral responses of moths sion. After emergence, adult moths were transferred to different GLVs with the absence of any visual cues. individually to glass tubes (20 by 3 cm) and fed with All experiments were conducted under red light at 10% sucrose solution under the same conditions de- 25 Ϯ 2ЊC and 60% RH. The Y-tube olfactometer was scribed above until experiments. Two-day-old virgin modiÞed according to those described in Ibrahim et al. moths were used for EAG tests and laboratory behav- (2005) and Wei et al. (2007). They system consisted ioral bioassays. of a stem (10 cm in length) and arms (23 cm in length), Chemicals. (Z)-11-Hexadecenal (Z11Ð16:Ald) and with a 60Њ angle at a diameter of 2.3 cm, connected to (Z)-11-hexadecenyl acetate (Z11Ð16:Ac) were syn- the observation chamber. Charcoal-Þltered and mois- thesized and puriÞed according to methods described turized air was pumped from an air pump (Ya Rong in Dai et al. (2008) in the College of Science, China Biochemical Instrument Factory, Shanghai, China) Agricultural University, Beijing, China. (Z)-11-Hexa- through jars containing GLVs and then entered to the decen-1-ol (Z11Ð16:OH) was purchased from Shin- Y-tube olfactometer. Airßow through each of the ol- Etsu Chemical Industry (Tokyo, Japan). The purity of factometer arms was maintained at 250 ml/min by a these compounds was Ͼ95% measured by gas chro- ßow regulator. A piece of Whatman Þlter paper (1 by matographic analyses. The three pheromone com- 2 cm; Fuyuan Papermill Company, Hangzhou, Zheji- pounds were mixed as the pheromone lure at a ratio ang, China) was impregnated with 10 ng of test ␮ of 1:1:0.01. GLVs (Z)-3-hexenyl acetate, 1-hexanol, GLV(s) stimulus (in 10 l of hexane), and then the (Z)-3-hexen-1-ol, hexanal, (E)-2-hexenal, hexyl ace- Þlter paper was air-dried for 3 min and the introduced tate, and 1-hexen-3-ol were purchased from Sigma- into the jars. The control jars were provided with the Aldrich (St. Louis, MO), with purity Ͼ95%. Hexane same volume of hexane only. Each stimuli and control (purity Ն99.5%) and parafÞn oil were purchased from Þlter paper was used only once and then replaced with Chinese Medical Company (Shanghai, China). Dia- new papers for another individual. Moths were indi- mondback moth pheromone lure was prepared by vidually introduced into the Y-tube at the entrance of impregnating 200 ␮gofZ11Ð16:Ald, Z11Ð16:Ac, and the stem with a choice of the test odor and the control. Z11Ð16:OH hexane solution (at a ratio of 1:1:0.01) in The position of the arms containing treated odors and controls was reversed to minimize directional bias a green-colored rubber septum (1 by ⌽0.5 cm) man- after every Þve individuals had been tested. After 10 ufactured by Zhongjiesifang Company (Beijing, moths had been tested, the olfactometer was discon- China). nected and thoroughly washed with soap and water EAG Responses. EAG responses of P. xylostella to Þrst, rinsed with 70% ethanol, and Þnally dried in the GLVs were measured from 2-d-old unmated male and oven at 120ЊC overnight. Each individual was allowed female moths, according to the methods described in 10 min to respond in the olfactometer. Moths were Fang and Zhang (2002) and Dai et al. (2008). Each tested between 1900 and 2400 hours, and each indi- antenna was excised from the head. The base of the vidual was used only once in the experiments. A “Þrst isolated antenna was connected with an electrode choice” was declared whenever the moth moved Ͼ5 (ground) Þlled with RingerÕs physiological saline, and Ϸ cm into either arm. A “no choice” was recorded when 0.5 mm of terminal segment was cut off with a pair an individual remained inactive for 10 min. Prelimi- of microscissors; the cut end was then connected to nary tests had shown that P. xylostella did not respond another recording electrode. EAG signals were re- to the solvent control (hexane) in the Y-tube olfac- corded with a head-stage preampliÞer and processed tometer bioassay. Each GLV stimulus was tested with with a PC-based signal processing system (Syntech, at least 15 moths and repeated four times. Hilversum, The Netherlands). Field Trapping. To investigate whether catch efÞ- Pheromone and GLVs were dissolved in parafÞn oil. cacy was enhanced from the GLV-added sex phero- ␮ We applied 10 l of the test solution at a concentration mone-baited traps, four attraction-choice Þeld exper- Ϫ3 of 2.5 ϫ 10 ␮g/␮l to a piece of Þlter paper (5 by 0.5 iments (Table 1) were conducted at the Organic cm), allowed the solution to dry, and then inserted the Vegetable Science Research Farm in Yanqing District, Þlter paper into a glass Pasteur pipette (10 cm in Beijing (40.4ЊN, 115.9ЊE). The traps used in all Þeld length). The tested stimuli were then blown onto the experiments were yellow water pans (20 cm in diam- prepared antenna via a charcoal-Þltered, humidiÞed eter by 10 cm in depth) Þlled with 0.1% diluted de- airstream delivered at a ßow rate of 200 ml/min by an tergent solution (8 cm in depth). Traps were placed 10 electronically controlled stimulus ßow controller cm above either cabbage or caulißower (Brassica ol- (CS05, Syntech). Intervals between stimulations were eracea L. variety botrytis) plants. Sex pheromone was set to at least 1 min. released from rubber septa lures contained a three- The responses to test stimuli were recorded from component blend (Z11Ð16:Ald:Z11Ð16:Ac:Z11Ð16: antennae of 10 virgin male and 10 female moths in a OH) at a ratio of 1:1:0.01 (in hexane), and GLVs were randomized order and were repeated three times. No released from 500-␮l plastic centrifuge tubes at doses response was observed from antennae to the control, of 2 or 20 mg (dissolved in 200 ␮l of parafÞn oil). Both parafÞn oil. Absolute EAG responses were measured lures were hung side by side at 1 cm above the water at millivolts by using Syntech EAG-program surface. A 2-mm-diameter hole was predrilled in the (Synthec). cap of the centrifuge tube used for the GLV lure to August 2012 LIETAL.: SEX PHEROMONE WITH GLVs FOR TRAPPING P. xylostella 1151

Table 1. Experimental periods, trapping ﬁelds, lures, and dispensers used in ﬁeld trapping experiments

Experimental Trapping Expt. Blend used for Þeld trapping tests Dispenser period Þeld I 16Ð30 Sept. 2008 Cabbage Ph (3-component blend at 200 ␮g) Pheromone in rubber septa (Ph) and ϩ ␮ Ph Z3-C6:Ac (20 mg) GLVs in 500- l ϩ Ph Z3-C6:Ac (2 mg) centrifuge tube ϩ Ph C6:OH (20 mg) ϩ Ph C6:OH (2 mg) ϩ Ph Z3-C6:OH (20 mg) ϩ Ph Z3-C6:OH (2 mg) ϩ Ph E2-C6:Ald (20 mg) ϩ Ph E2-C6:Ald (2 mg) II 1Ð10 Oct. 2008 Cabbage Ph ϩ Ph Z3-C6:Ac (20 mg) ϩ Ph Z3-C6:OH (20 mg) ϩ Ph E2-C6:Ald (20 mg) ϩ ϩ ϩ Ph Z3-C6:Ac Z3-C6:OH E2-C6:Ald ϩ ϩ ϩ ϩ Ph Z3-C6:Ac C6:OH Z3-C6:OH ϩ E2-C6:Ald 1-C6-3-ol ϩ ϩ ϩ ϩ ϩ Ph Z3-C6:Ac C6:OH Z3-C6:OH C6:Ald ϩ ϩ E2-C6:Ald C6:Ac 1-C6-3-ol III 1Ð16 June 2009 Caulißower Ph ϩ Ph Z3-C6:Ac ϩ ϩ ϩ Ph Z3-C6:Ac Z3-C6:OH E2-C6:Ald ϩ ϩ ϩ ϩ Ph Z3-C6:Ac C6:OH Z3-C6:OH ϩ E2-C6:Ald 1-C6-3-ol ϩ ϩ ϩ ϩ ϩ Ph Z3-C6:Ac C6:OH Z3-C6:OH C6:Ald ϩ ϩ E2-C6:Ald C6:Ac 1-C6-3-ol IV 17 JuneϪ3 July 2009 Caulißower Ph M1: pheromone in rubber septa (Ph) ϩ ϩ ϩ ␮ Ph Z3-C6:Ac Z3-C6:OH E2-C6:Ald and GLVs in 500- l centrifuge tube M2: both pheromone (Ph1) and GLVs in rubber septa M3: both pheromone (Ph2) and GLVs in 500-␮l centrifuge tube

Abbreviations: Z3-C6:Ac, (Z)-3-hexenyl acetate; C6:OH, 1-hexanol; Z3-C6:OH, (Z)-3-hexen-1-ol; C6:Ald, hexanal; E2-C6:Ald, (E)-2-hexenal; C6:Ac, hexyl acetate; 1-C6-3-ol, 1-hexen-3-ol; Ph, sex pheromone lures purchased from a company; Ph1, self-made sex pheromone lures in rubber septa; and Ph2, self-made sex pheromone lures in centrifuge tubes. release the volatiles. Traps were placed 10 m apart in the pheromone and a three-component GLV blend as a random order and exchanged to a same new lure it might affect moth trap capture (see descriptions for every 3 d when traps were checked. The captured M.1, M.2, and M.3 below). moths were identiÞed and counted. Three replicates For M.1, the sex pheromone dispenser (Ph) used were placed at 50 m apart. The average emission rates was a rubber septa purchased from Zhongjesifang of the GLVs were measured by weighing plastic cen- Company. The GLV lure (500-␮l plastic centrifuge trifuge tubes at 7 p.m. every night during a 5-d exper- tube) was made of adding a 20-mg blend of GLV imental period and then compared the differences in compounds dissolved in 200 ␮l of parafÞn oil. The two weight of each tube. The average loss of GLVs in the dispensers were hung close to each other and set at 1 tubes was 0.7Ð24 ␮g/h. Due to the observed high losses cm above the water surface of the yellow pan trap. of GLVs, the GLV lures were replaced with a new one For M.2, rubber septa were used for both sex pher- in the pheromone traps after 3 d. omone and GLV lures. The sex pheromone dispenser The Þrst Þeld experiment conducted in cabbage (Ph1) was prepared at a dose of 200 ␮g of sex pher- Þeld in 2008 was aimed to see whether adding different omone in a green rubber septum. A 20-mg GLV lure doses of EAG-active GLVs to the pheromone-baited was made by impregnating 20 ␮l of GLV (pure) in to traps could enhance the trap catches, compared with a rubber septum (left at room temperature overnight those baited with pheromone alone. until all GLVs absorbed inside the rubber septum). In the second and third experiment, traps were The two lures were placed in the trap the same as baited with standard pheromone lures with either described for M.1. individual or GLV blends at 20-mg doses dissolved in For M.3, 500-␮l plastic centrifuge tubes were used 200 ␮l of parafÞn oil and compared with traps baited as dispensers for the pheromone (Ph2) and GLV. Ph2 with pheromone alone (same dose as the commer- was prepared by adding 200 ␮g of sex pheromone cially available pheromone lure, 1 ␮g/␮l in hexane). dissolved in 200 ␮l of hexane in the 500-␮l plastic We conducted the second Þeld experiment in the centrifuge tube. Pheromone was released through a same research farm in a cabbage Þeld in 2008 but in the predrilled (⌽2-mm) hole in the cap. GLVs were di- third Þeld experiment in a caulißower Þeld. rectly dissolved in 200 ␮l of parafÞn oil and Þlled in the In the fourth experiment, we compared the efÞcacy plastic tube. The two tubes were suspended within the of using three different dispenser systems containing trap as described above. 1152 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 4

Table 2. EAG responses of antennae of unmated male and to host plant GLVs (30 ؍ female P. xylostella moths (n

EAG response (mV) Volatilea Male antennae Female antennae (Z)-3-Hexenyl acetate 0.22 Ϯ 0.02b 0.23 Ϯ 0.01b 1-Hexanol 1.13 Ϯ 0.09f 1.09 Ϯ 0.08 g (Z)-3-Hexen-1-ol 1.02 Ϯ 0.04e 1.00 Ϯ 0.07f hexanal 0.82 Ϯ 0.04d 0.76 Ϯ 0.05de (E)-2-Hexenal 0.77 Ϯ 0.03d 0.78 Ϯ 0.07e Hexyl acetate 0.34 Ϯ 0.01c 0.35 Ϯ 0.01c 1-Hexen-3-ol 0.81 Ϯ 0.10d 0.73 Ϯ 0.08de Control 0.00 Ϯ 0.00a 0.00 Ϯ 0.00a

Values are mean Ϯ SEM. Means followed by different letters are signiÞcantly different at P Ͻ 0.05 based on one-way ANOVA followed by DuncanÕs tests. a All chemicals were tested in a dose of 25 ng (10 antennae, three replicates).

Statistical Analyses. Statistical analyses were per- formed using the SPSS version 11.5 for Windows (SPSS, Inc., Chicago, IL). The distribution of insect responses from the Y-tube bioassays was analyzed with a chi-square test. One-was analyses of variance Fig. 1. Y-tube percentage of behavioral responses of un- (ANOVAs) followed by Duncan multiple range tests mated male (A) and female (B) P. xylostella moths to a 10-ng Ͻ dose of host plant GLVs. Data followed by asterisks show (P 0.05) were used to analyze the EAG and Þeld signiÞcant difference from control (hexane, chi-square test) trapping results, except that the fourth experiment in each arm (*, P Ͻ 0.05; **, P Ͻ 0.01). that was analyzed using an independent-sample t-test (P Ͻ 0.01). hexenal] was used at a ratio of 1:100, not 1:10 or other ratios (P Ͻ 0.05; Table 3). Results Seven treatments were then compared in the sec- EAG Responses. All host plant volatiles tested elic- ond Þeld experiment to test whether adults of P. xy- ited signiÞcant EAG responses from both unmated lostella were attracted more to traps baited with pher- male and female P. xylostella antennae, relative to the omone combined with individual GLVs or blend ϭ Ͻ control parafÞn oil) (males: F7,72 65.67, P 0.05 and mixtures of GLVs. Pheromone traps baited with either ϭ Ͻ females: F7,72 74.97, P 0.05) (Table 2). Among all added with (Z)-3-hexenyl acetate alone or a mixture the tested GLVs, 1-hexanol elicited the strongest EAG blend containing (Z)-3-hexenyl acetate, 1-hexen-ol, response, whereas (Z)-3-hexenyl acetate gave the and (E)-2-hexenal were found signiÞcantly more at- lowest. In general, the orders of the responses were tractive to males P. xylostella, with increases of moth Ͼ Ͼ ϭ alcohols aldehydes acetates. No signiÞcant dif- captures by 54 and 37%, respectively (F6,77 11.37; ferences in EAG responses to the GLVs were found P Ͻ 0.05) (Fig. 2), compared with numbers of males between male and female antennae (Table 2). trapped with pheromone alone. Y-Tube Behavioral Assays. In Y-tube bioassays, male and female moths showed different behavioral responses to host plant GLVs (Fig. 1). (Z)-3-Hexenyl Table 3. Numbers of male moths caught in P. xylostella sex acetate and (E)-2-hexenal were more attractive to pheromone traps synergized by baited with different doses of host both sexes, relative to the rest of treatments (chi- plant GLVs in 2008 Ͻ square test, P 0.05). But 67.8% of male moths re- Lure Ratioa Mean Ϯ SE sponded to (Z)-3-hexen-1-ol, compared with 48.44% ϩ Ϯ of females (chi-square test, P Ͻ 0.01). Neither sex Ph (Z)-3-hexenyl acetate 1:100 63.25 6.84d Ph ϩ (Z)-3-hexenyl acetate 1:10 51.25 Ϯ 2.20bc showed a response to 1-hexanol, hexanal, hexyl ace- Ph ϩ 1-hexanol 1:100 45.67 Ϯ 5.44ab tate, and 1-hexen-3-ol (chi-square test, P Ͼ 0.05). Ph ϩ 1-hexanol 1:10 39.83 Ϯ 3.85a Field Trapping. Although relatively lower EAG re- Ph ϩ (Z)-3-hexen-1-ol 1:100 39.00 Ϯ 4.42a ϩ Ϯ sponses were elicited from moth antennae from (Z)- Ph (Z)-3-hexen-1-ol 1:10 39.25 4.87a Ph ϩ (E)-2-hexenal 1:100 56.50 Ϯ 6.50cd 3-hexenyl acetate and (E)-2-hexenal, 50% more male Ph ϩ (E)-2-hexenal 1:10 45.17 Ϯ 7.42ab moths were caught in traps baited with pheromones Ph 45.00 Ϯ 3.42ab with these two GLVs compared with those with the sex pheromone only in the Þrst Þeld test (F ϭ 2.52; Values are mean Ϯ SE every 3 d. Means followed by different letters 8,99 Ͻ P Ͻ 0.05) (Table 3). Furthermore, the signiÞcant in- are signiÞcantly different at P 0.05 based on one-way ANOVA followed by DuncanÕs test. creases were only observed for a mixture of sex pher- a Relative ratios of synthetic sex pheromone at a 200-␮g dose versus omone and GLVs [(Z)-3-hexenyl acetate or (E)-2- GLVs. August 2012 LIETAL.: SEX PHEROMONE WITH GLVs FOR TRAPPING P. xylostella 1153

Fig. 2. Effect of individual or blended host plant GLVs on the numbers of male moths caught in P. xylostella sex pheromone traps. Data are means Ϯ SE every 3 d. Bars with different letters indicate signiÞcant difference at P Ͻ 0.05 based on one-way ANOVA followed by DuncanÕs test.

In 2009, the highest captures of P. xylostella from traps baited with sex pheromone combined with a blend of three-component GLVs was further con- Þrmed, with a 22% increase relative to traps with ϭ Ͻ pheromone alone (F4,70 5.03; P 0.05) (Fig. 3). Although 17% more P. xylostella males were captured from traps baited with pheromone and (Z)- 3-hexenyl acetate, they were not signiÞcantly different from the control with pheromone alone (P Ͼ 0.05; Fig. 3). In the fourth Þeld experiment, the differences in trap catches were compared by using three different dispenser systems for sex pheromone and the highest attractant blend containing pheromone and three-component GLVs revealed from the previous Þeld experiments. SigniÞcantly higher captures were found from the best lure system using rubber septa impregnated with sex pheromone and plastic centrifuge tubes added with GLVs (P Ͻ 0.01; Fig. 4). Rubber septum was not a suitable carrier for loading GLVs for enhancing moth attraction. Although more male moths were caught in traps baited with lures of centrifuge tube-added pheromone and Fig. 3. Effect of host plant GLVs on the capture of male GLVs, but the captures were signiÞcantly lower moths in P. xylostella sex pheromone traps in 2009. Data are than traps with the combined lures using rubber means Ϯ SE every 3 d. Bars with different letters show septum for pheromone and plastic tubes for GLVs signiÞcant difference at P Ͻ 0.05 based on one-way ANOVA (P Ͻ 0.01; Fig. 4). followed by DuncanÕs test. 1154 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 105, no. 4

Fig. 4. Comparisons of P. xylostella pheromone-baited trap captures combined with a three-component blend of GLVs with different disperser systems. Ph, sex pheromone lures purchased from company; Ph1, self-made sex pheromone lures in rubber septa; and Ph2, self-made sex pheromone lures in centrifuge tubes. Pheromone lures contained a three-component blend of the synthetic sex pheromone of P. xylostella in a 1:1:0.01 ratio of Z11Ð16:Ald:Z11Ð16:Ac:Z11Ð16:OH. Data are means Ϯ SE every 3 d. Bars with an asterisk indicate signiÞcantly more moths captured than control at P Ͻ 0.05 based on independent- sample t-test.

Discussion more work needs to be carried out for a better understanding, such as using single-cell recording tech- Our study demonstrates that (Z)-3-hexenyl acetate niques to Þnd enhanced olfactory responses, or to is the most active host plant GLV compound that can clone and study the function speciÞc binding proteins increase behavioral responses of P. xylostella in both laboratory bioassays and Þeld trapping experiments. located on the antennae that are speciÞc to the sex This result supports Þndings reported previously in pheromone and GLV blend compounds. Raina et al. Reddy and Guerrero (2000) and Dai et al. (2008). A (1992) proposed that host plant odors inßuences sex relatively lower antennal response to (Z)-3-hexenyl pheromone biology of phytophagous insects. They acetate observed from the current study is contrary to have found that GLVs may stimulate pheromone pro- that described in Dai et al. (2008). This may be due to duction in Helicoverpa zea (Boddie) and Helicoverpa the different release rate of these compounds, the age phloxiphaga (Grote & Robinson). Similar results have of moths, or dosages of the stimuli used between the been reported in some coleopteran species (Tumlin- two studies. (Z)-3-Hexenyl acetate is a ubiquitous son et al. 1969, Dickens 1986, Jaffe et al. 1993, Lextrait GLV compound released from plants and elicits EAG et al. 1995). Pittendrigh and Pivnick (1993) have fur- responses from various moth antennae (Light et al. ther conÞrmed that the production and release of sex 1993, Rojas 1999, Deng et al. 2004b). Deng et al. pheromone in P. xylostella requires the stimulation of (2004a) have shown that (Z)-3-hexenyl acetate elicits host plant volatiles, especially GLV. a low EAG response from male Helicoverpa armigera Several GLVs, e.g., 1-hexanol, 1-hexan-3-ol, and (Hu¨ bner) antennae but increases moth orientation hexanal, also elicit strong EAG responses but fail to rates in laboratory behavioral tests. The increases in increase trap catches in Þeld experiments. A signiÞ- adults captures of P. xylostella found from pheromone cant response of males to (Z)-3-hexen-1-ol was ob- traps with GLVs suggest that females use additional served in the laboratory bioassay but not from the Þeld chemical cues (such as GLVs) for searching suitable trapping tests. However, Dai et al. (2008) reported host plants. Similar cases have been reported in He- that the addition of (Z)-3-hexen-1-ol to the phero- liothis virescens (F.) and Spodoptera exigua (Hu¨ bner) mone-baited trap signiÞcantly increased trap catches. by Dickens et al. (1993). The discrepancy between these two studies may be (Z)-3-Hexenyl acetate may not act as a behavioral contributed by different dosages, ratio of pheromones, stimulant alone. But its synergistic effect is evident and bait carriers used (200 mg in parafÞn oil versus 43 from traps of combined pheromone and GLVs, with mg in peanut oil; the ratio of synthetic sex pheromone enhanced behavioral responses of both sexes of P. at 1:1:0.01 on green rubber septum versus 7:3:1 on red xylostella observed, relative to those with pheromone rubber septum). In addition, the intertrap distance alone (Reddy and Guerrero 2000, Dai et al. 2008). The used in the study (10 m) may lead to some trap in- underlining mechanisms of the GLV synergism to sex terference between treatments with high release rates pheromone-mediated orientation have not been well of volatile GLVs. (E)-2-Hexenal elicits a moderately studied. Ochieng et al. (2002) have suggested that strong EAG response from antennae of both sexes, but such an increase could result from the development of it only enhances behavioral responses from laboratory pheromone-speciÞc detection pathways. However, bioassays and Þeld trapping tests by using as one of the August 2012 LIETAL.: SEX PHEROMONE WITH GLVs FOR TRAPPING P. xylostella 1155 blended compounds. No increase in captures is found Dickens, J. C., J. H. Visser, and J.N.C. Vanderpers. 1993b. from traps baited with pheromone with (E)-2-hexenal Detection and deactivation of pheromone and plant odor added individually in Þeld trapping tests. Only a blend components by the beet armyworm, Spodoptera exigua of (Z)-3-hexenyl acetate, (E)-2-hexenal, and (Z)-3- (Hu¨ bner) (Lepidoptera: Noctuidae). J. Insect Physiol. hexen-1-ol added to pheromone-baited traps enhance 39: 503Ð506. adult behavioral responses. Fang, Y. L., and Z. N. Zhang. 2002. Inßuence of host-plant Different types of dispenser systems have been in- volatile components on oviposition behavior and sex pheromone attractiveness to Helicoverpa armigera. Acta vestigated for using host plant GLVs as synergistic Entomol. Sin. 45: 63Ð67. compounds to increase pheromone trap catches. Our Ibrahim, M. A., A. Nissinen, and J. K. Holopainen. 2005. study has shown that the most effective dispenser Response of Plutella xylostella and its parasitoid Cotesia system for increasing trap catches in P. xylostella with plutellae to volatile compounds. J. Chem. Ecol. 31: 1969Ð added GLVs is plastic centrifuge tubes with GLVs. The 1984. use of different dispenser systems, particularly with Jaffe, K., P. Sanchez, H. Cerda, J. V. Hernandez, R. Jaffe, N. added GLVs in pheromone traps, may play important Urdaneta, G. Guerra, R. Martinez, and B. Miras. 1993. roles in future insect pest management involving tools Chemical ecology of the palm weevil Rhynchophorus pal- such as mass trapping in the Þeld. marum L. (Coleoptera: Curculionidae): attraction to host In conclusion, we have demonstrated that adult P. plants and to a male produced aggregation pheromone. xylostella are attracted to volatiles released by their J. Chem. Ecol. 19: 1703Ð1720. host plants, including (Z)-3-hexenyl acetate and a Landolt, P. J., and T. W. Phillips. 1997. Host plant inßuences on sex pheromone behavior of phytophagous insects. blend of GLVs. Addition of GLVs [either (Z)-3-hex- Annu. Rev. Entomol. 42: 371Ð391. enyl acetate or a blend of (Z)-3-hexenyl acetate, (Z)- Lextrait, P., J. C. Biemont, and J. Pouzat. 1995. Pheromone 3-hexen-1-ol, and (E)-2-hexenal] to the pheromone- release by the two forms of Callosobruchus maculates bated traps can enhance trap catches in the Þeld. This females: effects of age, temperature, and host plant. study provides useful information for future develop- Physiol. Entomol. 20: 309Ð317. ing economically sound monitoring and control strat- Li, Z. Y., A. D. Chen, J. M. Zhang , Q. S. Lin, H. Y. Chen, D. Y. egies in IPM. Zhang, Z. D. Hu, F. Yi, and X. Feng. 2011. Study on trapping effects of different kinds of sex pheromone to diamondback moth. Chin. J. Appl. Entomol. 48: 324Ð327. Acknowledgments Light, D. M., R. A. Flath, R. G. Buttery, F. G. Zalom, R. E. Rice, J. C. Dickens, and E. B. Jang. 1993. Host-plant We thank Prof. Chenzhu Wang (Institute of Zoology, green-leaf volatiles synergize the synthetic sex phero- Chinese Academy of Science, Beijing, China) for technical mones of the corn earworm and codling moth (Lepidop- help with EAG recordings. 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