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Keywords:

  • Anoplophora glabripennis;
  • Asian longhorned beetle;
  • cuticular hydrocarbons;
  • kairomone;
  • oxidation;
  • pheromone

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Abstract  Volatiles from female Asian longhorned beetle (ALB), Anoplophora glabripennis, were evaluated as candidate sex pheromone components. Previous studies on ALB have revealed several antennally active compounds from virgin females; however the origins and activity of these compounds were not apparent and require further investigation. We tested the hypothesis that one or more of the ALB contact sex pheromones is a precursor that undergoes abiotic oxidation to yield volatile pheromone components, and evaluated the activity of these compounds using laboratory and field bioassays. Gas chromatography coupled electroantennography detection (GC-EAD) analysis indicated the presence of three antennally active aldehydes (heptanal, nonanal, and hexadecanal) in female cuticular extracts exposed to ozone or UV and visible light. In laboratory bioassays using a Y-tube olfactometer, males were preferentially attracted to ozonized female body washes over crude body washes. Similarly, synthetic formulations of these compounds were preferred over controls in the olfactometer. Field trapping experiments conducted from 2006 to 2008 in Ningxia, China showed that synthetic lures of the three aldehydes formulated in a ratio simulating that of virgin females attracted more beetles compared to controls, and that combinations of these aldehydes, linalool oxide, and host kairomones captured more beetles than controls, and captured significantly more males.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The Asian longhorned beetle (ALB), Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae: Lamiinae), is a serious pest of hardwood trees in China, North America and Europe. It is native to China and the Korean Peninsula and was introduced to North America and Europe in the nineteen-nineties and the first decade of the twenty-first century, respectively. In North America, it was first detected in New York City (1996), followed by Chicago (1998), Toronto (2003), New Jersey (2003), Massachusetts (2008), and most recently, Bethel (Clermont County), Ohio (2011) (Cavey et al., 1998; Poland et al., 1998; Haack et al., 1997, 2006, 2010; Balser, 2011). In North America, ALB has a broad host range, attacking and killing at least 24 species of trees in 13 genera, with the most highly favored tree species being six maple (Acer) species (Morewood et al., 2004; Sawyer, 2008; Zhang et al., 2008). In all the ALB infestations in North America and Europe, Acer is the most commonly attacked genus (Haack et al., 2010). In northern China, ALB is a serious pest of shelterbelts, plantations and urban forests (Yin & Lu, 2005), while in South Korea, ALB exists in low population densities in natural hardwood stands in riparian forests (Williams et al., 2004).

As of October 2011, in the United States, where ALB is under eradication management, approximately 70 000 infested and high-risk trees have been removed as sanitation is the cornerstone of ALB control. Furthermore, over 866 000 trees have been prophylactically treated with the systemic insecticide imidacloprid (Haack et al., 2010). In Toronto, Canada, successful eradication in 2003 involved the removal of 25 000 high-risk and infested trees followed by annual surveys (Haack et al., 2010). In Europe, established populations of ALB were first reported in Austria in 2001 and later in France, Germany and Italy. The number of ALB-infested trees destroyed in Europe has been a modest 475 as of 2008 (Haack et al., 2010).

In contrast, the damage caused by ALB in China has been staggering. ALB outbreaks in China began in the early 1980s and have spread throughout much of the country. The problems have been most severe in plantations and shelterbelts in the arid northern provinces, where Asian species have been planted outside their natural range. For example, 80 million trees were lost in Ningxia and another 11 million in Inner Mongolia (Yang, 2005). Urban forests have also been severely impacted; for example, in a single year (2004) the northern city of Harbin lost 70 000 Acer negundo L. (Li et al., 2008).

Management of existing ALB infestations is based on detection, sanitation and insecticidal control. Prevention is based on the selection of resistant varieties and species of trees for planting and avoidance of site and stand conditions that favor outbreaks (e.g., monocultures). Improvements in the management of ALB outbreaks are needed in the areas of chemical attractants for improved efficiency of detection and biological control in the native range of ALB where eradication is unrealistic (Hu et al., 2008; Haack et al., 2010). The combination of chemical attractants with biological control agents, such as fungal pathogens specific to ALB (Dubois et al., 2004a,b; Hajek et al., 2006), or with insecticides could generate environmentally benign control options by minimizing non-target effects.

Long-range mate location mediated by pheromones has been documented in five subfamilies of the Cerambycidae (Cerambycinae, Lamiinae, Lepturinae, Spondylidinae, and Prioninae). In the Cerambycinae, male-produced sex pheromones have been reported in Anaglyptus subfasciatus Pic (Nakamuta et al., 1997), Curius dentatus (F.) (Lacey et al., 2004), Hylotrupes bajulus (L.) (Reddy et al., 2005a), and Xylotrechus quadripes Chevrolat (Hall et al., 2006). The primitive Prionus californicus Motschulsky (Prioninae) and Ortholeptura valida (LeConte) (Lepturinae) use female-produced sex pheromones (Rodstein et al., 2011; Ray et al., 2011). Among other Cerambycoidea, species in two genera of Vesperidae have been shown to use female-produced sex pheromones (Leal et al., 1994; Boyer et al., 1997). Both male- and female-produced long range attractants for ALB have been investigated. Male ALB produce two functionalized dialkyl ethers, 4-(n-heptyloxy)butanal and 4-(n-heptyloxy)butan-1-ol, that elicit GC-EAD responses in females (Zhang et al., 2002) and are attractive to females in laboratory assays (Zhang et al., 2001, 2002; Nehme et al., 2009). Captures of ALB in traps baited with these compounds were modest but significantly greater than to unbaited control traps in field assays in China (Nehme et al., 2010). Francese (2004) reported the production of volatile, virgin female compounds that elicited GC-EAD responses in ALB; however, behavioral responses to those volatiles were not apparent in laboratory or field behavioral assays.

Female contact sex pheromones have been reported in several species of longhorned beetles (Kim et al., 1992; Ginzel & Hanks, 2003; Ginzel et al., 2003a,b, 2006) including ALB. Zhang et al. (2003) reported that female ALB cuticular hydrocarbon extracts contained five olefins, (Z)-9-tricosene, (Z)-9-pentacosene, (Z)-7-pentacosene, (Z)-9-heptacosene, and (Z)-7-heptacosene, that elicited copulatory behavior in males. Three of these compounds, Z-9-tricosene, Z-9-pentacosene, and Z-9-heptacosene are also cuticular hydrocarbon components of the sawfly Cephus cinctus Norton (Hymenoptera: Cephidae) (Bartelt et al., 2002; Cosséet al., 2002). Although they do not elicit antennal activity, these unsaturated hydrocarbons and other unsaturated cuticular lipids are precursors to antennally active volatiles, including a pheromone component (Bartelt et al., 2002; Cosséet al., 2002). In C. cinctus as well as three other species of Hymenoptera, the yellow-headed spruce sawfly, Pikonema alaskensis Rohwer, (Tenthredinidae) (Bartelt & Jones, 1983), Macrocentrus grandii Goidanich (Braconidae) (Swedenborg & Jones, 1992), and the pine false webworm, Acantholyda erythrocephala (L.) (Pamphiliidae) (Staples et al., 2009), unbranched alkenes or alkenediol acetates undergo abiotic oxidation to form functionalized, volatile pheromone products. The conversion of relatively non-volatile cuticular hydrocarbon or lipid components to volatile pheromones likely occurs widely in insects (Bartelt et al., 2002). Francese (2004) reported the presence of volatile antennally-active aldehydes and linalool oxide in aeration extracts of females, however the origins of the compounds were unclear. ALB adults are diurnal and are active in the crowns of trees. Thus, female exposure to sunlight and air may facilitate auto- or photo-oxidation of specific, oxidation-sensitive cuticular hydrocarbon components to form volatile oxidation products. In this study, we test the hypothesis that one or more of the ALB contact sex pheromones identified by Zhang et al. (2003) serve as a precursor to candidate volatile pheromone components, and evaluate the activity of these compounds using laboratory and field bioassays.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Source of insects

Adult ALB males and females were obtained from the Sarkaria Arthropod Research Laboratory (SARL), Cornell University, Ithaca, NY, USA. Body wash extracts of cuticular hydrocarbons from individual females were prepared at SARL; males were shipped in sturdy metal containers, in accordance with USDA-APHIS-PPQ permit and quarantine regulations, to SUNY-ESF, Syracuse, NY, USA for GC-EAD and behavioral assays. For the field experiments, ALB adults were obtained by felling infested trees near the field site and placing the infested logs in indoor emergence cages.

Oxidation of female cuticular hydrocarbon extracts

Female extracts (n = 5 for virgin beetles, n = 5 for mated beetles) were prepared by immersing a live female beetle in 2 mL dichloromethane for 1 min and then concentrating the extracts to 0.5 mL by evaporation under nitrogen. Extracts were oxidized by bubbling ozone from an ozone generator (Model GL-1, PCI Ozone and Control Systems, Inc., West Caldwell, NJ, USA; flow rate 50–100 mL/min) through a Pasteur pipette in the solution for 5 sec at −77°C, until the solution turned blue, and then reducing the ozonides with dimethyl sulfide (DMS) (Attygalle et al., 1989). Photo-oxidations (n = 5 for virgin beetles, n = 5 for mated beetles) of one ALB female equivalent (FE) were performed by spreading the extract on a glass microscope slides over a 4 cm2 area and allowing the solvent to evaporate. Following 30 min exposure to direct sunlight in Syracuse, New York, on June 2006 from 12:00–12:30 in full sun, the extract was reconstituted by washing the slide with 2 mL dichloromethane. The expected predominant aldehyde products were heptanal, nonanal, tetradecanal, hexadecanal, octadecanal, and eicosanal (7AL, 9AL, 14AL, 16AL, 18AL, 20AL, respectively).

GC-EAD

Samples were analyzed by GC-EAD on an HP 5890 Series II GC (Hewlett-Packard, Sunnyvale, CA, USA) with an injection port temperature at 280°C, equipped with a DB-1 capillary column (30 m × 0.25 mm × 0.25 μm film; J & W Scientific, Folsom, CA, USA), in splitless mode, and the GC oven was programmed from 40°C (held for 1 min), to 300°C at 10°C/min (held for 10 min). Column effluent was split 1 : 1 to the flame ionization detector (FID) and electroantennographic detector (EAD) with two deactivated capillary columns of equal length as transfer lines and a universal capillary Y connector (Sigma-Aldrich, St. Louis, MO). The column to the EAD preparation passed through the wall of the GC via a heated transfer line fitted with a glass-lined insert (SGE Analytical Science, Austin, TX), heated to 250°C. A male ALB antenna, severed just above the scape, was mounted in the well of a custom-built acrylic holder. The well was filled with Beadle-Ephrussi Ringer's solution (Bjostad, 1998), and equipped with a pure gold wire indifferent electrode. The apex of the antenna was pierced with an electrolytically sharpened tungsten wire and mounted on a glass electrode set inside an acrylic holder. The glass electrode was made from a glass capillary (World Precision Instruments Inc., Sarasota, FL) and formed on a Narishige electrode puller (Narishige Inc., East Meadow, NY), filled with Beadle-Ephrussi Ringer's solution with a pure gold wire recording electrode. Output signals from antennae were amplified by a custom-built single-step high-input impedance DC amplifier described by Nojima et al. (2003).

FID and EAD signals were digitized and recorded using a PowerLab data acquisition system with Chart 4.0 software signal rendering (AD Instruments Inc., Colorado Springs, CO) connected to a PC. An additional software-controlled digital low-pass filter was set at a cut-off frequency of 10 Hz. A minimum of three consecutive antennal responses (n = 3 males, 9 recordings total) were recorded before a compound was scored as antennally active.

Identification and synthesis of female volatiles

To tentatively identify the antennally active peaks, GC-MS was performed on subsamples using the same column type, carrier gas and temperature program. The volatile compounds derived from the ozonized or photooxidized female extracts, corresponding to the antennally active peaks, were identified using coupled gas chromatography-mass spectrometry (GC-MS) on an HP 5890 Series II GC interfaced to an HP 5971 Mass Selective Detector (MSD) (Hewlett-Packard, Sunnyvale, CA, USA), with electron ionization (70 eV), source temp. 180°C, using the same column and GC conditions described above.

To verify identification of the antennally active compounds, the samples were coinjected with compounds acquired from a commercial source or synthesized in a single step synthesis from commercially available precursors. 14AL, 16AL, 18AL, and 20AL were synthesized from 1–2 mmol/L of the corresponding primary alcohol by oxidization at room temperature (25°C) in pyridinium dichromate (PDC) in dichloromethane (Corey & Schmidt, 1979). GC-EAD revealed three antennally active compounds: 7AL, 9AL, and 16AL (see results).

Bioassay of activated extracts and synthetic compounds

To assess the activity of the oxidized female extracts and synthetic formulations of the products, the following odor stimuli were assayed for behavioral responses in adult male ALB: (i) oxidized female ALB extract vs. crude female ALB extract (n = 20 males), (ii) synthetic blend of all six predominant aldehyde products (7AL, 9AL, 14AL, 16AL, 18AL, 20AL) vs. control (n = 17 males), (iii) synthetic blend of all six predominant aldehyde products vs. the three antennally active aldehydes (7AL, 9AL, and 16AL) (n = 20 males), and (iv) the three antennally active aldehydes vs. control (n = 20 males). The quantity of compounds used approximated one FE per hour. FEs were estimated by calculating the theoretical quantities of the six predominant volatile products from five parent ALB olefins (Z)-9-tricosene, (Z)-9-pentacosene, (Z)-7-pentacosene, (Z)- 9-heptacosene, and (Z)-7-heptacosene, using the oxidation rates of Z-9-tricosane reported from C. cinctus by Bartelt et al. (2002). In a pilot study (n = 10 mated females), we confirmed that the ratios of five olefins produced by our insects matched those of Zhang et al. (2003). An equal volume of dichloromethane applied to filter paper was used as control.

All experiments were done in the laboratory in a glass Y-tube at a 15° incline under a 500 W halogen lamp. The olfactometer arms (30 cm × 6 cm dia.) formed a 70° angle; the central tube was of the same dimensions. Charcoal filtered house air was introduced at 200 mL/min. Test stimuli were placed on filter paper and were introduced into the airstream of the respective arms of the olfactometer. The olfactometer was cleaned with dichloromethane after each trial resulting in a positive response, and at the end of each day. The positions of the control and treatment were randomized with every trial. ALB males were placed in the central tube of the olfactometer and allowed to walk upward toward the fork in the “Y”. A positive or negative response was recorded after the beetle stayed in the distal half of the treatment or control chamber (respectively) for ten consecutive minutes. Individual male beetles were assayed a maximum of once per day. Although bioassays had no maximum time limit, beetles choosing the treatment or control did so before 90 min; otherwise a no response was scored if the beetle spent 10 consecutive minutes in the central tube of the olfactometer.

Field responses of ALB to female and host volatiles

We evaluated the attractiveness of suspected pheromones and host kairomones in five randomized complete block field experiments using flight-intercept panel traps (IPM Technologies, Portland, OR, USA) in July and August of 2006–2008 (summarized in Table 1). The traps were washed and coated with Rain-X® (Sopus Products, Houston, TX) prior to use. Compounds were released from red rubber septa and release rates were measured gravimetrically for each compound (n = 5) (Table 2). In 2007 and 2008, volatiles were released from rubber septa placed inside a 4 mL amber vial with a septum cap (National Scientific, Rockwood, TN, USA). A piece of 0.076 3 mm thick amber polyethylene (part #90–812C7, National Bag Co. Inc., Hudson, OH) was placed under the cap in place of the septum. Release rates were determined by collecting volatiles on Super-Q adsorbent (Alltech Assoc., Deerfield, IL, USA) in the laboratory at 22°C and were quantified using GC-MS and with decanal as internal standard (conditions described above). Each compound was released from a separate device.

Table 1.  Summary of compounds used in field trapping experiments for Asian longhorned beetle (Anoplophora glabripennis) from 2006–2008, in Ningxia, China. Each column represents the volatiles used in one treatment; volatiles were released in separate devices. Controls in all experiments were unbaited traps.
Test attractantsExperiments
12345
Treatments
123123456123456123451234567
  1. †Compounds 7AL, 9AL and 16AL, in proportions (1 : 7 : 1) approximating those of virgin female ALB. ‡Compounds 7AL, 9AL and 16AL, in proportions (2 : 11 : 1) approximating those of mated female ALB. §cis-3-Hexen-1-ol, camphene, delta-3-carene, linalool, and trans-caryophyllene; release rates as in Table 2. ††Virgin female extract. ‡‡Mated female extract.

7AL,9AL,16AL (V)†x  xx    x    xx  x x   xxx
7AL,9AL,16AL (M)‡ x                         
16AL  x                        
Host kairomones§    x xx   xx x x xxx   xxx
7AL,9AL,14AL, 16A,18AL, 20AL     xxxx xx               
Linalool oxide       xx  xxxx xxx x   xxx
Ozonized VFE††                     x  x  
Photo-oxidized                      x    
 VFE††                         x 
Photo-oxidized MFE‡‡                       x  x
Table 2.  Release devices and release rates of compounds used in field experiments as potential attractants of Anoplophora glabripennis.
CompoundPurityNatural sourceSourceRelease deviceRelease rate
  1. ‡In 2006 rubber septa were charged every day for heptanal and every three days for nonanal; in 2007 and 2008 rubber septa were loaded with compound and placed inside 4 mL amber vials with amber polyethylene sheeting sealed under an open screw cap (area of polyethylene exposed 0.385 cm2). ‡Compounds were loaded on cotton and placed inside a 20 mL vial that was sealed with amber polyethylene sheeting under an open screw cap (area of polyethylene exposed 1.767 mm2). §Compounds were loaded on cotton and placed inside an amber 4 mL vial that was sealed with amber polyethylene sheeting under an open screw cap (area of polyethylene exposed 0.385 cm2). ††The compound cis-3-hexen-1-ol was loaded on blotter paper and placed in amber polyethylene bag, other compounds were loaded on cotton batting and placed in amber polyethylene bag. ‡‡The abbreviation FE stands for female equivalent. §§The quantitiy released is estimated to be ∼15 FE/day.

Heptanal98%Female/hostAldrichRubber septa† 4.3 μg/day§§
Nonanal98%Female/hostAldrichRubber septa†15.7 μg/day§§
Nonanal (virgin formulation)98%Female/hostAldrichRubber septa†30.4 μg/day§§
Tetradecanal89.93%FemaleSynthesizedRubber septa 1.4 μg/day§§
Hexadecanal92.40%FemaleSynthesizedRubber septa 2.9 μg/day§§
Hexadecanal (virgin formulation)92.40%FemaleSynthesizedRubber septa 4.4 μg/day§§
Octadecanal94.25%FemaleSynthesizedRubber septa14.3 μg/day§§
Eicosanal50.22%FemaleSynthesizedRubber septa 1.4 μg/day§§
2008 virgin female compounds100%FemaleFemale extractQuartz cuvette∼1 FE‡‡/day
2008 virgin female compounds – ozonized100%FemaleFemale extractRubber septa∼1 FE‡‡/day
2008 mated female compounds100%FemaleFemale extractQuartz cuvette∼1 FE‡‡/day
cis-3-hexen-1-ol98%HostAldrichAmber bag‡‡500 mg/day
trans-caryophyllene80%HostAldrichAmber bag‡‡500 mg/day
Linalool97%HostAldrichAmber bag‡‡500 mg/day
Camphene95%HostAldrichLarge vial‡500 mg/day
delta-3-carene99%HostAldrichLarge vial‡500 mg/day
Linalool oxide95%Female/hostTCI (Japan)Small vial§ 15 μg/day

The field site was located at Guang Xia, Ningxia Autonomous Region, China at 38°06′83.2″N and 105°92′49.1″E; it was a vineyard with interspersed rows of poplar trees. Poplars were even aged (∼20 years), 5–7 m tall, moderately to heavily-infested with ALB, with the same site characteristics as other moderately infested ALB trapping sites (see Nehme et al., 2010). Rows were approximately 800 m long and traps were hung in living, infested trees about 2 m above the ground and placed 15–20 m apart. Collection cups contained a 50 : 50 (v/v) solution of ethylene glycol and water to kill trapped beetles. Every 3–6 days the lures were changed, their positions were re-randomized and beetles were collected.

Field experiment 1 The field responses of ALB to blends of three antennally active oxidation products (7 AL, 9 AL and 16 AL) in ratios corresponding to those of mated (2 : 11 : 1) and virgin (1 : 7 : 1) females were assessed. Three synthetic formulations were compared in this experiment: (1) 7 AL, 9 AL, and 16 AL in a ratio that approximated that of virgin females (1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively), (2) 7 AL, 9 AL, and 16 AL in a ratio that approximated that of mated females (2 : 11 : 1, released at 4.3 μg, 15.7 μg, and 2.9 μg/day, respectively), (3) 16AL alone (released at 4.4 μg/day), and (4) trap with no lure. 16 AL was tested alone because it is the only antennally active aldehyde produced by ALB that is not also produced by hosts (Francese, 2004; Wickham, 2009). Due to high volatility of 7 AL, rubber septa were recharged daily with 4.3 μg 7 AL. For each temporal replication, red rubber septa were loaded with 47.1 μg and 91.2 μg 9 AL for mated and virgin formulations. The traps were baited with lures on July 22, 2006, and lures were replaced and re-randomized on July 24. Captured beetles were collected on July 24 and 27. There were six spatial and two temporal replicates.

Field experiment 2 Oxidation products were tested alone and with a host kairomone blend (cis-3-hexen-1-ol, camphene, delta-3-carene, linalool, and trans-caryophyllene) (see Wickham, 2009), and linalool oxide. Each compound used a separate release device. Cis-3-hexen-1-ol was loaded onto blotter paper and linalool and trans-caryophyllene were loaded onto cotton batting and placed inside amber polyethylene bags. Camphene and delta-3-carene were loaded onto cotton batting and placed inside 20 mL amber vials that were sealed with polyethylene sheeting under an open screw cap (area of polyethylene exposed 1.767 mm2). Linalool oxide was prepared the same and placed inside 4 mL amber vials that were sealed with polyethylene sheeting under an open screw cap (area of polyethylene exposed 0.385 mm2) (see Table 2). The treatments were: (1) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively), (2) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend, (3) six compound blend alone (with 7AL, 9AL, and 16AL in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively), (4) six compound blend (with 7AL, 9AL, and 16AL in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomones blend, (5) six compound blend (with 7AL, 9AL, and 16AL in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, (6) six compound blend (with 7AL, 9AL, and 16AL in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + linalool oxide, and (7) trap with no lure. Due to high volatility of 7AL, rubber septa were recharged daily with 4.3 μg 7AL. For each temporal replication, red rubber septa were loaded with 47.1 μg and 91.2 μg 9AL for mated and virgin formulations. The traps were baited with lures on July 22, 2006 and lures were replaced and re-randomized an on July 24. Captured beetles were collected on July 24 and 27. There were six spatial and two temporal replicates.

Field experiment 3 The goal of this experiment was similar to experiment 2 (to investigate the possible synergistic effects of adding host kairomones and linalool oxide to the oxidation products in a ratio typical of virgin females); however, we refined the release rates for 7AL and 9AL. The release devices for 7AL and 9AL, which have high volatility, were formulated with more constant release rates by enclosing the loaded rubber septa in 4 mL amber vials sealed with amber polyethylene sheeting, sealed under an open screw top, so lures did not require frequent recharging in the field. Release rates were confirmed by GC-MS using decanal as an internal standard. External calibration curves were calculated using standards of 7AL, 9AL, and decanal at 0.01. 0.1, 1, 10, 100 μg/μL (R2 = 0.99, 0.98, and 0.99 for each compound respectively). The treatments were: (1) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively), (2) six compound blend (with 7AL, 9AL, and 16AL in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively), (3) six compound blend (with 7AL, 9AL, and 16AL in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, (4) host kairomone blend + linalool oxide, (5) linalool oxide, (6) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, and (7) blank traps. Traps and lures were placed in the field on August 4, 2007, and lures were replaced and re-randomized on August 8. Beetles were collected from the traps on August 8 and August 12 and sorted to sex. There were eleven spatial and two temporal replicates.

Field experiment 4 The treatments were: (1) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively), (2) host kairomone blend + linalool oxide, (3) linalool oxide, (4) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, (5) host kairomone blend, and (6) unbaited traps. The release devices for 7AL and 9AL were the same as described in field experiment 3. The experiment was set up on 15 July 2007, and re-randomized on July 21. Beetles were collected from the traps on July 20 and July 26 and sorted to sex. There were seven spatial and two temporal replicates.

Field experiment 5 The objective of this experiment was to determine if there were additional pheromone components that were not identified and tested in the previous experiments. Two formulations of female compounds were prepared and tested by: (i) allowing natural photo-oxidation of one FE of maturation fed 11–17 d virgin beetles (taken from emergence cages in Ningxia) or mated (field-collected beetles; mating status verified by spermathecal dissection and microscopic inspection after extraction) female extract, or (ii) ozonized extracts of fed 11-day old virgin female beetles (extracted at SARL) and applying ca. 1 FE to rubber septa. The treatments were: (1) 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, (2) ozonized virgin female extracts (1 FE), (3) photo-oxidized virgin female extracts in quarts cuvettes (1 FE), (4) Photo-oxidized mated female extracts in quartz cuvettes (1 FE), (5) ozonized virgin female extracts (1 FE) + 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, (6) photo-oxidized virgin female extracts in quartz cuvettes (1 FE) + 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, (7) photo-oxidized mated female extracts in quartz cuvettes (1 FE) + 7AL, 9AL, and 16AL (in virgin female ratio 1 : 7 : 1, released at 4.3 μg, 30.4 μg, and 4.4 μg/day, respectively) + host kairomone blend + linalool oxide, and (8) unbaited traps. The release devices for 7AL and 9AL were the same as described in field experiments 3 and 4. The experiment was set up on July 15, 2008 with six spatial replicates and lures were changed and re-randomized on July 21. Captured beetles were collected on July 20 and 26.

Statistical analyses

Because all raw and log transformed trap captures were heteroscedastic, a 2-way non-parametric Kruskall-Wallis ANOVA, blocked by spatial and temporal replication, was used for all analyses. To test for differences between treatments and controls, Kruskall-Wallis ANOVA comparisons with experimentwise error control (Bonferroni) were completed. To compare male and female trap catches within treatments, a χ2 goodness-of-fit test was used. Statistical analyses were done using STATISTICA Version 6, (Statsoft, Inc. 2003). To test male responses in Y-tube olfactometer bioassays, a χ2 goodness-of-fit test was used.

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Oxidation of female extracts and GC-EAD activity

Ozone treatment of the mated female extract yielded six functionalized products (7AL, 9AL, 14AL, 16AL, 18AL, 20AL) in the approximate ratio of 3 : 11 : 1 : 2 : 10 : 1, with the average relative quantities of individual compounds within females ≤0.2 SE (data not shown). 7AL, 9AL, and 16AL elicited antennal responses from male antennae (Table 3). Ozone treatments of the virgin female extracts yielded the same products in the approximate ratio of 3 : 22 : 1.5 : 3 : 10 : 1, with the average relative quantities of individual compounds within females ≤0.2 SE (data not shown). Photo-oxidations of mated and virgin female extract done by exposure to sunlight on glass microscope slides also yielded antennally active quantities of aldehydes in the same approximate ratios and variation as described above.

Table 3.  Male Anoplophora glabripennis EAD responses (n= 6) to Aldehyde products of oxidized female cuticular hydrocarbons.
CompoundRetention timeMale antennal response (mV ± SE)
  1. ‡Not detectable by GC-EAD. ‡Not detectable by GC-FID and barely detectable by GC-MS.

Heptanal 7.380.31 ± 0.05
Nonanal11.100.61 ± 0.12
Tetradecanalnd‡nd‡
Hexadecanal21.250.15 ± 0.02
Octadecanal26.44nd‡
Eicosanalnd‡nd‡

Bioassay of oxidized cuticular extracts and synthetic aldehydes

In the Y-tube olfactometer, males typically walked upside down through the central tube to the junction of the side arms and paused there with an antenna in each side arm. They typically entered each side arm and then doubled back to again rest at the Y juncture with an antenna in each arm. They frequently repeated this behavior several times until finally making a choice as described above. In this olfactometer assay, (1) ozonized female cuticular extracts were significantly more attractive to males than were crude extracts, (2) the six aldehyde component blend was more attractive than solvent controls, and (3) 7AL, 9AL, and 16AL had the same activity as the six component blend (Table 4). There were no differences in male attraction to the six component synthetic formulation compared to the 7AL, 9AL, and 16AL formulation (Table 4).

Table 4.  Male Anoplophora glabripennis responses to female volatiles in a Y-tube olfactometer.
No. beetles testedTreatment stimulusNo. beetles respondingControl stimulusNo. beetles respondingχ2P-value
  1. ‡7AL, 9AL, 14AL, 16AL, 18AL, and 20AL correspond to heptanal, nonanal, tetradecanal, hexadecanal, octadecanal, and eicosanal.

20Ozonized female extract15female extract550.05
177AL, 9AL, 14AL, 16AL, 18AL, 20AL‡16solvent113.240.001
207AL, 9AL, 14AL, 16AL, 18AL, 20AL‡97AL, 9AL, 16AL110.10.75
207AL, 9AL, 16AL‡18solvent212.80.001

Field responses of ALB to female volatiles

Field experiment 1 Only traps baited with 7AL, 9AL, plus 16AL released in a ratio approximating that of a virgin female captured significantly more beetles than the control (Fig. 1, Kruskal-Wallis ANOVA test: H3,48 = 8.398, P = 0.038 5; Kruskal-Wallis ANOVA pairwise test: H1,48 = 7.700, P = 0.005 5 [P < 0.05/4 = 0.012 5 Bonferroni experimentwise error control]).

image

Figure 1. Mean Asian longhorned beetle trap catches in panel traps baited with female aldehydes in rations approximating those of virgin and mated females in Ningxia, China, July 2006 (Experiment 1). Bars with the same letter are not significantly different (Kruskal-Wallis ANOVA test: H3,48= 8.398, P= 0.0385; Kruskal-Wallis ANOVA pairwise test: H1,48= 7.700, P= 0.005 5; P < 0.05/4 = 0.0125 Bonferroni experimentwise error control).

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Field experiment 2 The treatment with the host kairomone blend added to the six aldehyde blend formulated to approximate a virgin female captured more ALB than controls (Fig. 2, Kruskal-Wallis ANOVA test: H6,84 = 10.218, P = 0.115 8; Kruskal-Wallis ANOVA pairwise test: H1,24 = 6.785, P = 0.006 2 [P < 0.05/7 = 0.007 1 Bonferroni experimentwise error control]).

image

Figure 2. Mean Asian longhorned beetle trap catches in panel traps baited with combinations of virgin female aldehydes, host kairomones (hk) and linalool oxide (lo) in Ningxia, China, July 2006 (Experiment 2). Bars with the same letter are not significantly different (Kruskal-Wallis ANOVA test: H6,84= 10.218, P= 0.115 8; Kruskal-Wallis ANOVA pairwise test: H1,24= 6.785, P= 0.006 2; P < 0.05/7 = 0.007 1 Bonferroni experimentwise error control).

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Field experiment 3 Traps baited with lures formulated with more stable release rates of synthetic pheromones in the virgin female ratio plus host kairomones and linalool oxide captured more beetles than controls (Fig. 3, Kruskal-Wallis ANOVA test: H6,154 = 8.345 455, P = 0.213 9; Kruskal-Wallis ANOVA pairwise test: H1,44 = 6.769 231, P = 0.003 3 [P < 0.05/7 = 0.007 1 Bonferroni experimentwise error control]), and captured significantly more males (Fig. 3; χ2 = 16, df = 1, P < 0.001).

image

Figure 3. Mean Asian longhorned beetle trap catches in panel traps baited with combinations of female aldehydes (in a ratio simulating a virgin female), host kairomones (hk), and linalool oxide (lo), August 2007, in Ningxia, China (Experiment 3). Bars with the same letter are not significantly different (Kruskal-Wallis ANOVA test: H6,154= 8.345, P= 0.213 9; Kruskal-Wallis ANOVA pairwise test: H1,44= 6.769, P= 0.003 3; P < 0.05/7 = 0.007 1 Bonferroni experimentwise error control). †Treatment caught significantly more males than females, χ2= 4, df = 1, P < 0.05.

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Field experiment 4 Traps baited with lures containing synthetic pheromones in the virgin female ratio plus host kairomones and linalool oxide captured more ALB than controls (Fig. 4; Kruskal-Wallis ANOVA test: H5,84 = 14.136 49, P = 0.014 8; pairwise Kruskal-Wallis ANOVA test: H1,28 = 7.576 981, P = 0.005 9 [P < 0.05/6 = 0.008 3 Bonferroni experimentwise error control]), and captured significantly more males than females (Fig. 4; χ2 = 4, df = 1, P < 0.05). No other lure treatment captured significantly more ALB than the control.

image

Figure 4. Mean Asian longhorned beetle trap catches in panel traps baited with female aldehydes, host kairomones (hk), and linalool oxide (lo), July 2008, in Ningxia, China (Experiment 4). Bars with the same letter are not significantly different (Kruskal-Wallis ANOVA test: H5,84= 14.136, P= 0.014 8; pairwise Kruskal-Wallis ANOVA test: H1,28= 7.577, P= 0.005 9; P < 0.05/6 = 0.008 3 Bonferroni experimentwise error control). †Treatment caught significantly more males than females (χ2= 16, df = 1, P < 0.001).

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Field experiment 5 The lures containing photo-oxidized virgin female extracts plus synthetic 7AL, 9AL, and 16AL in the virgin female ratio, plus the host kairomone blend and linalool oxide captured more male beetles than females (Fig. 5; χ2= 6, df = 1, P < 0.05), but the mean trap catch was not significantly greater than that of the control (Kruskal-Wallis test: H7,96= 10.782 42, P= 0.148 4; Kruskal-Wallis test: H1,24= 4.561 983, p= 0.032 7 [P > 0.05/8 = 0.006 3 Bonferroni experimentwise error control]).

image

Figure 5. Mean Asian longhorned beetle trap catches in panel traps baited with combinations of oxidized virgin female extracts, oxidized mated female extracts, female aldehydes, host kairomones (hk), and linalool oxide (lo), July 2008, in Ningxia, China (Experiment 5). Trap catches by treatment were not significantly different (Kruskal-Wallis test, H7,96= 10.782, P= 0.1484). †Treatment caught significantly more males than females (χ2= 6, df = 1, P < 0.05).

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Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Ozone treatment of mated female ALB cuticular extracts yielded six functionalized products, of which three were antennally active to male ALB (Table 3). Using the ratios of the five olefins reported by Zhang et al. (2003), and the oxidation rates of Z-9-tricosene reported by Bartelt et al. (2002), we calculated the expected ratio of aldehyde products from female ALB. The 1 : 2 : 2 : 8 : 1 ratio of (Z)-9-tricosene, (Z)-9-pentacosene, (Z)-7-pentacosene, (Z)-9-heptacosene, and (Z)-7-heptacosene reported by Zhang et al. (2003) would be expected to produce a 7AL, 9AL, 14AL, 16AL, 18AL, and 20AL in the ratio of 3 : 11 : 1 : 2 : 10 : 1, which was confirmed by ozonolysis and photooxidation of mated female extracts This ratio of the synthetic aldehydes was used for Y-tube olfactometer bioassays and the three EAD-active aldehydes (7AL, 9AL, 16AL) accounted for the activity in ozonized mated female extracts and six aldehyde (7AL, 9AL, 14AL 16AL, 18AL, 20AL) blends.

Just prior to the first field experiments, we determined virgin female extracts had altered ratios of the aldehyde products. Field experiment 1 determined that the blend of 7AL, 9AL, and 16AL released in a ratio approximating that of a virgin female was more attractive than (1) the same compounds released in a ratio approximating that of mated females, (2) 16AL alone, and (3) controls. In experiment 2, the six aldehyde (7AL, 9AL, 14AL 16AL, 18AL, 20AL) blend plus the host kairomone blend (cis-3-hexen-1-ol, camphene, delta-3-carene, linalool, and trans-caryophyllene) was more attractive to ALB than to the control. Experiments 3 and 4 repeated experiment 2 with improved release devices for 7AL and 9AL, and demonstrated a similar level of attraction to aldehydes plus host kairomones plus linalool oxide, with the only major difference being that significantly more males than females were attracted. This provided the first field evidence of a female pheromone Another major difference was that linalool oxide also captured significantly more males than females in experiment 3, suggesting this female-produced compound is of importance in the chemical ecology of ALB. Because analyses of virgin female extracts indicated the presence of several unsaturated compounds (unpublished data), Experiment 5 included potential oxidation products of these compounds, using photo-oxidation and ozonolysis of natural extracts of virgin and mated beetles, to enhance the best performing synthetic lures in Experiments 2, 3, and 4. The treatment of photo-oxidized virgin female cuticular extracts plus synthetic aldehydes (7AL, 9AL, and 16AL in virgin female formulation) plus the host kairomone blend plus linalool oxide captured more males than females, but the treatment means were not significantly different. The lack of enhancement of the lures was likely because too little source material, only one female equivalent of virgin female beetle extract for photo-oxidations (in quartz cuvettes) and ozonolysis (in rubber septa) per treatment, was used. Future field experiments should investigate these compounds as additional pheromone components.

The urgency of the ALB problem in China, North America and Europe, has prompted several studies on the mechanisms of mate attraction. Nehme et al. (2010) suggest ALB follows the model proposed by Saint-Germaine et al. (2007), where males respond to host kairomones to locate suitable hosts, and then virgin females respond to a combination of host kairomones and male-produced pheromones. Indeed, the male-produced pheromone elicits behavioral responses from virgin females in laboratory assays (Zhang et al., 2002; Nehme et al., 2009), and field assays have demonstrated statistically significant but limited attraction (Nehme et al., 2010). Alternatively, Smith et al. (2007, 2008) suggest females select hosts; evident from strong female to male bias of attraction to sentinel trees, followed by increased male attraction to an unknown cue, which our evidence suggests is likely to be a combination of host kairomones and volatiles from females.

Female-produced, long-range sex pheromones have not been reported for other species in the Lamiinae. The only other female cerambycids reported to produce a sex pheromone are P. californicus (Prioninae), which produces 3,5-dimethyldodecanoic acid (Rodstein et al., 2011), and O. valida (Lepturinae), which produces cis-vaccenyl acetate (Ray et al., 2011). In the Lamiinae, male-produced aggregation pheromones are an important component of the mating system (Pajares et al., 2010). Male Monochamus galloprovincialis (Olivier) and M. alternatus Hope (Lamiinae) produce an aggregation pheromone, 2-undecyloxy-1-ethanol, which is structurally similar to the dialkyl ethers 4-heptyloxy-1-butanol and 4-heptyloxy-butyraldehyde produced by male ALB and 2-(4-heptyloxy-1-butyloxy)-1-ethanol produced by male Monochamus leuconotus (Pascoe) (Zhang et al., 2002; Hall et al., 2006; Pajares, 2010; Teale et al., 2011).

At least two strategies of long-range mate attraction that depend largely on the condition of the larval host (Hanks, 1999) have emerged (Allison et al., 2004; Millar et al., 2009). For species utilizing stressed hosts, which tend to be ephemeral resources, both sexes are attracted to host kairomones (Ginzel & Hanks 2005), male-produced pheromones (Hanks et al., 2007; Lacey et al., 2007a,b, 2008b, 2009; Ray et al., 2009a,b), host kairomones plus male produced aggregation pheromones (Silk et al., 2007), or host and bark beetle kairomones plus male produced aggregation pheromones (Pajares et al., 2010). In a few species, females are attracted to male-produced sex pheromones (Lacey et al., 2004; Hall et al., 2006; Hanks et al., 2007), or a combination of host kairomones plus male produced sex pheromones (Fonseca & Garbin, 2009). In contrast, ALB attacks apparently healthy trees, females appear to initially find and select hosts (Smith et al., 2007, 2008), and then virgin females attract males with pheromones (Fig. 6). We suggest that ALB males produce pheromone following attraction to hosts containing virgin females, and that the pheromone acts over a relatively short range, such as within the tree or up to several meters from the male. In both strategies, the chemically mediated reproductive behaviors are completed by contact chemoreception of female cuticular compounds and mating (Allison et al., 2004; Ginzel et al., 2006; Lacey et al., 2008a; Fonseca & Garbin 2009; Ibeas et al., 2008, 2009). Exceptions include the old-house borer, Hylotrupes bajulus, which utilizes dead wood (Reddy et al., 2005a,b, 2007), and P. californicus, which appears to be unique in its use of a pheromone gland on the ovipositor (Barbour et al., 2006; Cervantes et al., 2006).

image

Figure 6. Hypothesized four-step mating sequence of Asian longhored beetle reproductive behaviors and the semiochemicals that mediate them (center), compared to hypothesized three-step mating sequence for cerambycid beetles of Ginzel and Hanks (2005).

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The hypothesis that ALB females select host trees is supported by the initial colonization of Acer mono Maxim by females. Smith et al. (2007, 2008) observed that the initial 3.5 : 1 (F : M) ratio of ALB arriving at uninfested A. mono trap trees shifted to 2 : 1 after females began feeding in the tree. We suggest that after females arrive at the tree and begin feeding, they release volatile cues that attract males, at which point male arrival rates increase. In our 2007–2008 field experiments, intercept panel traps baited with lures containing synthetic virgin female aldehydes and a host kairomone blend captured more beetles than did the controls and captured more males than females. As for the male pheromone components, other studies support our hypothesis that it operates over a closer range. Y-tube olfactometer tests demonstrate virgin females are attracted to the pheromone components (Nehme et al., 2009), and field tests comparing two trap designs baited with the same lure (flight-intercept traps designed to capture flying beetles vs. collar traps designed to capture walking beetles) suggest virgin females are more likely walk to, rather than fly to, the male-produced pheromone (Nehme et al., 2010). Future research should investigate the effective range of both male and female pheromones.

The role of semiochemicals in mate location in ALB is apparently complex and the exact sequence of events remains unclear. Male and female pheromones and host kairomones are all involved as mates call and search for each other. In high density populations, it is plausible that random contact or visual cues might be sufficient to bring the sexes together. Such conditions are largely the result of anthropogenic disturbance (plantations of susceptible hosts in China, or introduction to susceptible urban forests in the U.S.) and are unlikely to resemble conditions under which the ALB mating system evolved. Under more natural conditions such as those that presently occur in the temperate, natural forests of South Korea, ALB populations are relatively sparse (Williams et al., 2004) and likely require an efficient system of mate attraction, which may also hold true in natural hardwood forest stands in North America (Dodds & Orwig, 2011). The presence of female and male pheromones suggests an additional step to the 3-step mating sequence proposed by Ginzel and Hanks (Fig. 6, Wickham, 2009). Given the evidence, we cannot exclude host volatiles at proxies, or as the case with heptanal and nonanal, as kairomonal enhancers of pheromones at this time (Tooker et al., 2002); however, further investigations are needed to elucidate most attractive female volatile and host kairomone combination. Recent analyses suggest that besides altered ratios of contact sex pheromones, many previously unknown unsaturated cuticular hydrocarbons vary with age, sex, and mating status in ALB (unpublished data), and more research is needed to understand the precursors and oxidation and release rates of their associated volatile products under natural conditions. In light of many unique unsaturated virgin female ALB compounds (unpublished data), our data presented here suggest we uncovered the basic mechanism of female volatile pheromone production, however the pheromone blend we tested is incomplete. Additional pheromone components may lead to an operational-ready lure for management of ALB. Finally, we cannot rule out the importance of the two male pheromone components in the sequence of mating behaviors for ALB (Zhang et al., 2001, 2002). Perhaps a multi-component trap, designed to use semiochemicals from multiple steps in the mating sequence, as used for Emerald Ash Borer (Poland & McCullough, 2007), will provide a badly needed tool for pest management, survey and detection, and control for this important forest pest.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

We thank Y. Luo, Beijing Forestry University, V. Mastro, D. Lance, and B. Wang, USDA-APHIS-PPQ, and Li Shunping of the Qingtongxia City Forestry Bureau (postal code 751600), for access to field sites and logistical support in China; the students of Beijing Forestry University, W. Xu, and M. Nehme for technical support during field studies; F. Webster SUNY-ESF for access to the ozone generator in his laboratory; and two anonymous reviewers for their helpful comments on the manuscript. We thank J. Francese, USDA APHIS-PPQ, and A. Hajek, SARL & Cornell University for supplying male ALB. This work was supported by a grant from the Alphawood Foundation to SAT and by the National Science Foundation East Asia and South Pacific Summer Institute (NSF-EAPSI) under Grant No. OISE-0813023 to JDW. All experiments were done in China and USA according to the rules of the ethical boards for animal experiments and abided with the current laws of both countries.

Disclosure

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

The authors disclose no conflicts of interest, financial or otherwise, that bias our work in any way.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information
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Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. Disclosure
  9. References
  10. Supporting Information

Oxidation scheme of female ALB contact sex pheromone (Zhang et al., 2003) to volatile products and candidate long-range sex pheromones &lsqb;illustration by author, based on (Bartelt et al., 2002)&rsqb;.

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INS_1504_sm_suppmat.doc89KSupporting info item

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