TETL-D-09-03443
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Supplementary Material
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Experimental procedures
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Volatile collection (New Zealand). Between 500 and 1000 female mealybugs on
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sprouted seed potatoes were housed in a glass container (c. 1 L volume) per
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collection. A charcoal–filtered air stream was pulled over the females and the
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headspace was collected on an adsorbent trap containing 50 mg of Tenax-GR 35/60
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(Alltech Associates Inc.) in a 15 mm long x 10 mm diameter glass tube. Tenax traps
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were thermally conditioned at 200°C under a stream of nitrogen before use. The
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airflow in the headspace collection system was 1 L/min and each collection session
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lasted for 4-7 days. The charcoal filter used to clean the incoming air was thermally
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activated before use in an oven at 150 °C. Control samples that contained only
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sprouted seed potatoes were collected from the above system to distinguish between
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female compounds and ambient contaminants. Immediately after volatile collection,
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Tenax traps were extracted with 1 mL of hexane (5 x 200 µl aliquots, n-hexane
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Suprasolv, Merck, Darmstadt, Germany). Samples were sealed and stored at -20 °C
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until used. Sample volumes were reduced to 10 µl at ambient temperature under a
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stream of argon before GC-MS analysis.
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Volatile collection (Chile). Cohorts of virgin females on butternut squash were
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placed in a glass aeration chamber of c. 5 L volume. All tubing was of Teflon.
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Charcoal-purified air was passed through the chamber at c. 2.5 L/min. Volatiles were
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trapped on c. 250 mg activated charcoal (50-200 mesh, Fisher Scientific) in a 4 mm
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i.d. glass tube, held in place by glass wool plugs. The trapped volatiles were eluted
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every 3-5 days using 1.5 mL hexane (Suprasolv, Merck). Extracts were stored in
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screw cap vials at -20ºC until use.
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GC-MS. GC-MS analysis for both female headspace volatiles and synthetic
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chemicals were conducted on a Saturn 2200 GC-MS (Varian Walnut Creek, CA,
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USA) using an ionization voltage of 70 eV and a mass range of 40 to 650, equipped
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with two different capillary columns: 1) a 30 m × 0.25 mm i.d. × 0.25 μm film VF-
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5ms non-polar capillary column (Varian); 2) a 30 m × 0.25 mm i.d. × 0.25 um VF-
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23ms polar capillary column (Varian) with splitless injection; 3) a 30 m × 0.25 mm
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i.d. × 0.25 um CycloSil-B chiral capillary column (Agilent J&W Scientific) with
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splitless injection. The oven was programmed from 40°C (held for 2 min) to 220°C at
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4°C /min, held for 5 min. Alternatively, a Shimadzu GC-17A-GCMS-QP5050A
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combination equipped with a 30 m x 0.25 mm HP-5 fused silica capillary column was
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used. High resolution mass spectra analysis was carried out using an Agilent 6890 N
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GC coupled to a Waters GCT time of flight (ToF) mass spectrometer. Samples were
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analyzed by splitless injection (1.0min) into a HP6890 PTV cryogenic injection port
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at – 220°C. The oven temperature was programmed from 40°C (held for 2 min) to
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240°C at 4°C /min, held for 5 min.
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Hydrolysis and acetylation. A collected sample (1000 female day equivalents,
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hereafter called FDE) of the unknown extract in hexane was concentrated under a
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stream of argon, and 5% potassium hydroxide in methanol solution was added. The
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mixture was left to react for two hours, and then distilled water was added. The
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organic phase containing the hydrolysed product was extracted with hexane and
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analysed by GC-MS.
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After GC-MS analysis, the remaining hydrolysed extract was treated with acetic
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anhydride, and left to react overnight at room temperature. The reaction was quenched
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with distilled water and the acetylated product was extracted with hexane. The organic
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layer containing the acetate was concentrated under a stream of argon before GC-MS
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analysis.
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Fractionation and laboratory bioassays. A fractionation procedure based on
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polarity of the compounds was developed in order to aid isolation and identification of
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the pheromone, and bioassays were carried out with the fractions. Collected extracts
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were fractionated by column chromatography. 200 μL of crude sample (containing c.
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60 FDE) were separated using 400 mg of silica gel 60 (0.063-0.200 mm, Merck,
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Darmstadt, Germany), successively eluting with 1.5 mL of hexane (fraction 1), 5%
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ether in hexane (fraction 2), 10% ether in hexane (fraction 3), 50% ether in hexane
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(fraction 4) and ether (fraction 5), respectively. About 20 males were released in a
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Petri dish (9 cm diameter) and after 5 min, two pieces of filter paper (2 cm x 2 cm),
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treated with the extract (or fraction or hexane) and with hexane, respectively, were
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placed in the dish. The number of males attracted to each of the filter paper pieces was
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recorded every minute during 30 min. For the evaluation of the sensitivity of males to
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the pheromone, a dilution series of extracts containing 30, 3, 0.3, and 0.03 FDE was
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prepared and assayed. Fractions were assayed as obtained and compared with an
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unfractionated extract containing c. 60 FDE. For statistical analysis, the proportion of
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males responding (males at treatment /total males) was considered. Differences
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between treatments were determined using the general linear model (GLM) repeated
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measures analysis (SPSS 17.0, Chicago, IL, USA) followed by post-hoc comparisons
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using the Tukey’s Honestly Significant Difference (Tukey’s HSD) test. A P-value less
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than 0.05 was considered significant in all instances. The results showed that the
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males significantly preferred the filter papers treated with aliquots of the moderately
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polar to polar fractions 3, 4, and 5 over the solvent control and the attractiveness did
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not differ significantly from that of the unfractionated extract, indicating the presence
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of the pheromone in these fractions (P<0.05) (Fig. 4). The activity of the non-polar
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fractions 1 and 2 did not differ from that of the control (P<0.05). Compounds eluting
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in the non-polar fractions are generally alkanes, alkenes, and aromatic hydrocarbons.
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Examples for typical compound classes eluting in the more polar fractions are
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aldehydes, ketones, and esters (fractions 3 and 4, depending on chain length), and
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alcohols (fraction 5).
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Synthesis of chrysanthemyl 2-acetoxy-3-methylbutanoate (Scheme 1)
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334 mg (2,82 mmol) 2-hydroxy-3-methylbutanoic acid were dissolved together with a
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catalytic amount of 4-dimethylaminopyridine (DMAP) in 10 mL of dry pyridine.
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Acetic anhydride (2.00 mL; 21.2 mmol) was added and the solution was stirred for 2 h
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at room temperature. After the addition of water, the mixture was acidified to pH 3 by
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addition of 2M HCl and extracted twice with diethyl ether. The combined organic
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phases were washed with water and brine, dried over MgSO4 and concentrated to
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yield 280 mg (62%) of a colourless oil. 250 mg (1.56 mmol) of the crude 2-acetoxy-3-
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methylbutanoic acid were dissolved in 1.5 mL benzene and 0.22 mL (2,5 mmol)
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oxalyl chloride and 1 L DMF were added. After 1.5 h stirring at room temperature,
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the solvent and excess reagent were removed by evaporation. The residue was
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dissolved in 1.5 mL benzene and a solution of 0.3 mL (1.66 mmol) chrysanthemol
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(Aldrich, 65:35 trans/cis mixture of isomers) and 0.2 mL pyridine in 1.5 mL benzene
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was added dropwise. After 1 h of stirring at room temperature, the solvent was
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evaporated and water was added. The mixture was extracted twice with diethyl ether,
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the combined organic layers were washed with water, 1M HCl, and brine, dried over
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MgSO4, and concentrated. The crude product was purified by column
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chromatography on silica (10% ethyl acetate in hexane) to yield 270 mg (58%) of
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pure 3. 1HNMR (400 MHz, CDCl3):  = 4.92-4.86 (m, 0.3H), 4.85-4.79 (m, 1.7H),
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4.45 (dd, 0.3H, J = 6.8, 11.7 Hz), 4.31 (dd, 0.3H, J = 6.6, 11.7 Hz), 4.23 (dd, 0.2H, J =
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7.6, 11.7 Hz), 4.13 (dd, 0.3H, J = 6.6, 7.6 Hz), 4.05 (dd, 0.5H, J = 9.0, 11.8 Hz), 3.94
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(dd, 0.3H, J = 9.0, 11.7 Hz), 2.26-2.16 (m, 0.8H), 2.12 (s, 3H), 1.69 (s, 3H), 1.65 (s,
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3H), 1.40 (t, 0.3H, J = 8.3 Hz), 1.17 (dd, 0.7H, J = 5.2, 7.6 Hz), 1.13-0.94 (m, 12.5H),
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0.91-0.84 (m, 0.8H) ppm. 13C-NMR (CDCl3, 100.6 MHz):  = 170.68, 169.92,
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169.78, 135.86, 133.66, 122.72, 118.46, 118.42, 76.87, 76.84, 66.34, 66.29, 63.90,
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63.85, 30.82, 30.73, 30.02, 29.14, 28.91, 28.48, 26.42, 26.38, 26.36, 25.62, 25.58,
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25.55, 22.27, 21.51, 21.44, 20.58, 18.65, 18.60, 18.47, 18.23, 17.22, 17.16, 15.49,
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15.45 ppm., MS (70 eV) m/z: 43(100), 55(17), 57(12), 67(10), 81(17), 82(18), 93(16),
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95(12), 115(13), 121(39), 123(28), 143(10), 296(<1, M+).
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Field experiment protocol. Five doses of the racemic chrysanthemyl 2-acetoxy-3-
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methylbutanoate (0.1, 1, 10, 100, and 1000 µg) loaded into red rubber septa were
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tested for the attraction of P. calceolariae in vineyards near Hawke’s Bay, New
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Zealand for two weeks in February 2009, using a randomized complete block design.
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Septa were loaded with 0.1, 10, 100, and 1000 µg of chrysanthemyl 2-acetoxy-3-
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methylbutanoate dissolved in 200 µl hexane, and the solvent was allowed to evaporate
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in a fume hood. The septa were stored at -20°C until ready for use. Traps were placed
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in five rows with five replications for each treatment at 15 m spacing between
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trapping stations and treatment rows. Septa were placed in the centre of the white
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sticky base lying on the sticky surface. Traps baited with virgin females were used as
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positive control, while traps baited with a blank lure were used as negative controls.
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One of each of the five treatments was randomly assigned to a trap tree within each
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row of trees. The significance in the quantity of the treatment effect in the field
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experiments was tested using ANOVA (Statview, SAS Institute Inc., Cary, North
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Carolina). Significantly different means were identified using Fisher’s Protected Least
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Significant Difference (PLSD). In Chile, a field test was conducted from 3-7 March
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2009 in a raspberry plantation (0.43 ha; 5714 plants/ha) near Nogales (Valparaíso
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Region, Central Chile) using a randomized complete block design. White rubber septa
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were loaded with 100 µg of chrysanthemyl 2-acetoxy-3-methylbutanoate in hexane
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(treatment) or hexane (control). The solvent was allowed to evaporate in a fume hood
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and septa were stored at 5°C until use. Septa were placed in the centre on the sticky
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surface inside the delta traps. One control and one pheromone trap were placed in the
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same row, at 20 m spacing between traps, in four different rows (replicates) that were
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separated at least by 9 m. Males caught in traps were counted 48 hours after setting
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the experiment.
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Attraction of male P. calceolariae to fractions obtained by chromatographic
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separation of an extract of conspecific virgin females (60 FDE) in a Petri dish assay.
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The graph shows the proportion of males responding after 10 min (no. of males at
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stimulus/total no. of males). The bold line inside the boxes indicates the median,
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while upper and lower limits of the boxes correspond to the first and third quartile of
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responses, n = 5. Different characters above the boxes indicate significant differences
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between the treatments (GLM, Tukey’s HSD, P<0.05)
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Abundance
Female headspace
Racemic chrysanthemyl 2-acetoxy-3-methylbutanoate
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Time (min)
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GC-MS traces on chiral column of extracts of headspace collected from mature female citrophilus mealybug and racemic chrysanthemyl
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2-acetoxy-3-methylbutanoate.
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