Methyl salicylate

Insects for Electrophysiological Experiments Male and female sorghum chafers were collected at Rasa (09°55′N, 40°05′E), located 255 km northeast of Addis Ababa, Ethiopia. Adult beetles were sexed based on the presence of a ventral, abdominal groove in males (Rigout 1989 ), and kept separately. After transport to Alnarp, Sweden, adults were kept in clear plastic boxes (30 × 12 × 22 cm, Cofa Plastics AB, Stockholm, Sweden) with a 1:1:1 mixture of planting soil (Yrkesplantjord, Weibull Trädgård AB, Hammenhög, Sweden), peat (Växa trädgårdstorv, Econova Garden AB, Åse, Sweden) and composted cow dung (Simontorps Bas, Weibull Trädgård AB). Boxes were kept at 25°C, 70% relative humidity, and a L16:D8 h cycle. The beetles were fed with apples and bananas ad libitum.
Headspace Plant Volatile Collection Volatiles were collected from the developmental stage of the plant most attractive to the beetles, during the time of day when the beetles feed intensively, i.e., 10 am to 4 pm. For abutilon, the top 20 cm of a single abutilon plant, including flowers, seed pods, and leaves, was enclosed for each collection. For sorghum, volatiles were collected from a single panicle at the soft dough stage (milky stage). Polyacetate bags (35 × 43 cm; Toppits Scandinavia AB, Sweden) sealed with steel wire around the stem of the plant were used for aerations. An activated charcoal filter was placed next to the stem to filter incoming air. Volatiles were collected on glass tube columns (3.5 mm i.d. × 50 mm) packed with 25 mg SuperQ^®, mesh 80/100 (Alltech, Deerfield, IL, USA) with glass wool and Teflon stoppers at both ends (Birgersson and Bergström, 1989 (link)). The filter was placed in the polyacetate bag and connected by PVC tubing to a small battery operated pump (PAS-500 Personal Air Sampler, Supelco, Bellefonte, PA, USA). The flow of the pump was 200 ml/min, and collections were made in the field for 2 h. Immediately after collection, the columns were rinsed with 200 µl of redistilled hexane into 1.1 ml tapered glass vials (1.1 STVG, Chromacol Ltd., Welwyn Garden City, UK). Vials with extracts were kept in an icebox for transportation to the laboratory, and thereafter kept at —18°C until analysis.
Gas Chromatograph-Coupled Electroantennographic Detection (GC-EAD) The response of P. interrupta antennae to volatiles was studied by GC-EAD using an Agilent Technologies gas chromatograph (GC), model 6890, equipped with a fused silica capillary column (30 m × 0.2 mm) coated with Innowax (0.25 µm film thickness) (Agilent Technologies Inc., Santa Clara, CA, USA). For each run, 2 µl of sample were injected in splitless mode. Hydrogen was used as mobile phase at a linear velocity of 45 cm/sec. The oven temperature was programmed from 40°C (5 min hold) to 230°C at 5°C/min. Compounds eluting from the column were split 1:1 in a four-way splitter, with nitrogen as make up gas (20 ml/min), and delivered to the FID and to the antenna. Compounds were carried to the antenna through a glass tube by a charcoal-filtered and humidified air stream at 0.5 m/sec. Antennae were mounted according to Leal et al. (1992 ) and Wolde-Hawariat et al. (2007 (link)). The antenna was excised with fine forceps and placed in an antennal holder (Hillbur 2001 ; JoAC, Lund, Sweden), and the signal was amplified (JoAC) and analyzed with GC-EAD software (Syntech, Hilversum, The Netherlands). EAD responses to FID peaks were defined as repeatable deflections of the baseline. Each extract was tested on five different antennae per sex, for a total of ten antennae per extract.
Chemical Identification Samples of plant volatile collections were analyzed by combined gas chromatography and mass spectrometry (GC-MS) Hewlett Packard 6890 GC and 5973 MS (Agilent Technologies Inc.). Extracts were injected with an HP 7683 auto injector in splitless mode. The GC was fitted with the same column under the same conditions as for GC-EAD, but usinig helium (35 cm/sec) as carrier. Peaks were matched between GC-EAD and GC-MS by retention index. Identifications of compounds were confirmed by comparison of mass spectra in the NIST 1998 and Wiley 1998 commercial mass spectral databases, and with those of authentic GC standards, except for methyl anthranilate, which was not available at the time when GC-MS analysis was done.
Synthetic Compounds Synthetic standards for all experiments were purchased from Sigma-Aldrich (for purity and CAS number, see Table 1). A total of 82 compounds were used in single sensillum recordings (Table 1). The compounds include volatiles commonly found in flowers (Knudsen et al. 2006 (link)), volatiles from tropical fruit (Macku and Jennings 1987 (link); Ibáñes et al. 1998 (link); Boudhrioua et al. 2003 (link); Carasek and Pawliszyn 2006 (link); Clara et al. 2007 (link); Pandit et al. 2009 (link)), and volatiles related to microbial degradation and fermentation (Chatonnet et al. 1992 (link); Fischer et al. 2000 (link); Xiao and Ping 2007 (link)). Approximately half of the compounds used previously have been found to elicit behavioral or electrophysiological activity in the sorghum chafer or related scarab beetles (Stensmyr et al. 2001 (link); Larsson et al. 2003 (link); Wolde-Hawariat et al. 2007 (link)).
Table 1

Synthetic compounds used for single cell screening

#	Compound	S	CAS	%
*1	4-Ethylphenol	A	123-07-9	99
*1	4-Methylphenol	A	106-44-5	99
*2	(E)-2-Hexenal	H	6728-26-3	98
*2	(E)-2-Hexen-1-ol	H	928-95-0	96
*2	(E)-2-Hexenyl acetate	H	2497-18-9	98
*2	(E)-3-Hexen-1-ol	H	928-97-2	98
*2	(Z)-3-Hexen-1-ol	H	928-96-1	98
*2	(Z)-3-Hexenyl acetate	H	3681-71-8	98
3	Hexanal	H	66-25-1	98
*3	1-Hexanol	H	111-27-3	98
*3	Hexyl acetate	H	142-92-7	98
*3	Nonanal	H	124-19-6	95
3	1-Nonanol	H	143-08-8	99,5
*3	1-Octanol	H	111-87-5	99,5
*3	(±)-3-Octanol	H	589-98-0	99
*3	(±)-1-Octen-3-ol	H	3391-86-4	98
*4	Anethole	H	4180-23-8	99
*4	Benzaldehyde	H	100-52-7	99,5
*4	Benzylalcohol	H	100-51-6	99
*4	Eugenol	H	97-53-0	98
*4	Methyl benzoate	H	93-58-3	99
4	Methyl anthranilate	H	134-20-3	99
*4	2-Phenylethanol	H	60-12-8	98
*4	2-Phenylethyl propionate	H	122-70-3	98
*5	(±)-Acetoin	A	513-86-0	97
*5	racemic 2,3-Butanediol	A	513-85-9	99
5	Carvacrol	A	499-75-2	98
5	Cinnamic aldehyde	A	104-55-2	98
*5	Methyl cinnamate	A	103-26-4	99
*5	Methyl salicylate	A	119-36-8	99
*5	Phenylacetaldehyde	A	122-78-1	90
*5	Phenylacetonitrile	A	140-29-4	99
*5	Thymol	A	89-83-8	99,5
*6	Butyric acid	H	107-92-6	99
*6	N-Caproic acid	H	142-62-1	99,5
*6	Isovaleric acid	H	503-74-2	98
6	Valeric acid	H	109-52-4	99,8
*7	Isoamyl alcohol	H	123-51-3	98
*7	6-Methyl-5-hepten-2-one	H	78-70-6	99
7	Tetradecane	H	629-59-4	99,5
7	Tridecane	H	629-50-5	99,5
*8	(±)-beta-Caryophyllene	H	87-44-5	98,5
*8	(-)-trans-Citronellol	H	106-22-9	95
*8	Geraniol	H	106-24-1	98
*8	Geranyl acetate	H	105-87-3	98
*8	(±)−Linalool	H	78-70-6	97
*8	Linalool oxides	H	n/a	97
8	Methyl jasmonate	H	1211-29-6	95
8	Nerolidol	H	7212-44-4	98
9	(±)−delta-Decalactone	H	705-86-2	98
9	(±)−gamma-Decalactone	H	706-14-9	97
9	(±)−gamma-Hexalactone	H	695-06-7	98
*9	(±)−gamma-Nonanlactone	H	104-61-0	97
9	(±)−gamma-Octalactone	H	104-50-7	97
9	(±)−gamma-Undecalactone	H	104-67-6	99
10	(±)−Ethyl 3-hydroxybutyrate	H	5405-41-4	97
*10	(Z)-3-Hexenyl butyrate	H	16491-36-4	98
*10	(Z)-3-Hexenyl isobutyrate	H	41519-23-7	98
*10	(Z)-3-Hexenyl tiglate	H	67883-79-8	97
*11	Butyl butyrate	H	109-21-7	98
*11	Ethyl butyrate	H	105-54-4	99
*11	Ethyl hexanoate	H	123-66-0	99
11	Ethyl propionate	H	105-37-3	99
*11	Hexyl butyrate	H	2639-63-6	98
11	Methyl butyrate	H	623-42-7	99
*11	Methyl hexanoate	H	106-70-7	99
*11	Methyl octanoate	H	111-11-5	99
11	Methyl propionate	H	554-12-1	99
*11	Propyl butyrate	H	105-66-8	99
12	Butyl isobutyrate	H	97-87-0	97
12	Hexyl hexanoate	H	6378-65-0	97
*12	Isoamyl acetate	H	123-92-2	98
*12	Isoamyl butyrate	H	106-27-4	98
12	Isobutyl acetate	H	110-19-0	99,8
*12	Isobutyl isobutyrate	H	97-85-8	99
12	Isopentyl isobutyrate	H	2050-01-3	98
*12	Isopropyl acetate	H	108-21-4	99,8
13	Acetic acid	P	64-19-7	99
13	Acetone	P	67-64-1	99,9
13	Ethanol	P	64-17-5	99
*13	Ethyl acetate	P	141-78-6	99,5
13	Propionic acid	P	79-09-4	99,5

*, compound active in single sensillum recordings

#, screening blend

S, solvent used (A, acetone, H, hexane, P, paraffin oil)

CAS, Chemical Abstracts Service number

%, minimum purity in percent

Single Sensillum Recordings (SSR) Single synthetic compounds were diluted to 1 μg/μl in acetone or hexane, depending on polarity (Table 1). Highly volatile compounds were diluted to 1 μg/μl in paraffin oil. Blends of 2–10 compounds, with each component at the same concentration as in the single compound dilutions, were also prepared for screening purposes (see below; Table 1). Stimuli were prepared by applying 10 μl of 1 μg/μl solution to a 1.5 × 1 cm piece of Whatman filter paper (No. 3, Whatman, Maidstone, United Kingdom) that was placed in a disposable Pasteur pipette (150 mm soda lime glass, VWR International, Stockholm, Sweden). For compounds diluted in hexane or acetone, solvent was allowed to evaporate before stimuli were used in experiments. After evaporation of solvent, 1 ml pipette tips were put on the wide end of the Pasteur pipettes, to reduce any further evaporation of the test compound(s). Between trials, stimulus pipettes were kept at —18 ºC, to avoid evaporation. For comparison, stimulus pipettes containing only solvent as well as empty pipettes were prepared. To ensure that stimulus pipettes were not exhausted, new ones were prepared once per week (after having been used a maximum of ten times), except for screening pipettes, where new ones were prepared each day.Insects were restrained with Parafilm (PM-992, Pecheney plastic packaging, Menasha, WI, USA) and fixed on microscope slides (ca. 76 × 26 mm, Menzel-Gläser, Braunschweig, Germany) using dental wax (Surgident periphery wax, Heraeus Kulzer GmbH, Hanau, Germany), with the lamellae held open on a wax surface using 2-3 mm long pieces of thin tungsten wire. A silver grounding electrode was inserted into the abdomen. Sensilla were contacted with a tungsten electrode (diam 0.12 mm, Harvard Apparatus Ltd, Edenbridge, United Kingdom) electrolytically sharpened in a saturated KNO₂ solution (Hubel 1957 (link)), using a DC-3K Rechts PM-10 piezo micromanipulator (Märzhäuser Wetzler GmbH, Wetzler, Germany). The signal from the ORNs was registered and amplified 10 times with a probe (INR-02, Syntech), amplified 200 times with a Syntech UN-06 AC/DC amplifier, and transferred to a computer through an IDAC-4-USB (Syntech), where it was visualized and analyzed with the software Autospike v. 2.2 (Syntech).A constant flow of 0.5 m/sec of charcoal-filtered and humidified air was delivered through a glass tube with its outlet approximately 15 mm from the antenna. Stimuli were presented to the insect by inserting the stimulus pipette through a hole in the glass tube, and blowing an air puff of 2.5 ml during 0.5 sec through the pipette into the air stream, using a stimulus controller (Syntech SFC-1/b). Control stimuli were delivered first, followed by screening stimuli containing multiple compounds (screening blends listed in Table 1). For all screening stimuli that elicited a positive response of approximately ≥ 40 Hz, the pipettes loaded with all compounds in the blend(s) were brought from the freezer and tested individually after thawing at room temperature for 5 min.The net response to a stimulus was obtained by counting action potentials (spikes) during 0.5 sec starting from the time after the stimulation period at which the earliest response for the neuron was found, and deducting the number of action potentials during 0.5 sec immediately prior to the response. Each neuron was also subjected to blank stimuli (i.e., only solvent), and the net response to the blank was deducted from the response to the test compounds. The resulting value was doubled to obtain a value corresponding to spikes/sec (Hz). The time between the start of the stimulation period and the onset of a response, i.e., increased number of action potentials, sometimes varied between different recording sessions, due to slight variations in the air flow. For each neuron, counting of action potentials was started from the time at which the earliest response in that neuron occurred.
Field Experiments Related to GC-EAD Field experiments with the sorghum and abutilon compounds were carried out at Rasa, Ethiopia (see above). A complete randomized block design with N = 10 was used. The distance between traps was 10 m, and blocks were separated by at least 50 m. Dispensers were placed in cardboard holders (78 × 37 mm, Silvandersson AB, Knäred, Sweden) fitted into a slot in the vanes of Japanese beetle traps (Trécé, Palo Alto, CA, USA), which were suspended approximately 3 m above ground from wooden poles. The traps were emptied daily, and lures were replaced in the morning before the onset of activity for adult P. interrupta. Unbaited traps were used as a negative control.Experiments were performed during two periods: July 11–16 and October 7–13, 2006. The latter tests were done during the cropping season, when the sorghum had seeds in the milky stage. The July experiments were carried out in a grazing area characterized by scattered Acacia trees. In October, traps were placed along the borders of five sorghum fields located approximately 500 m from the July test sites. Compounds were applied to dental cotton rolls (No. 3, Q-dent, Germany). The individual sorghum compounds, (Z)-3-hexen-1-ol, tridecane, 1-octen-3-ol, and 1-octanol, were applied at a dose of 100 mg each. In addition to the sorghum compounds, eugenol and methyl salicylate also were tested. Two of the sorghum-related blends were tested both in July and in October: a blend of the four sorghum compounds with the same total dose (100 mg) as for the individual compounds and in a ratio mimicking what was found in the sorghum headspace, i.e., 10 mg (Z)-3-hexen-1-ol + 30 mg tridecane + 30 mg 1-octen-3-ol + 30 mg 1-octanol, and the same sorghum blend with the addition of 30 mg methyl salicylate. In addition to these, three more blends were tested in October: the sorghum blend with the addition of 30 mg eugenol, the sorghum blend with the addition of 30 mg eugenol and 30 mg methyl salicylate, and a blend of 50 mg eugenol and 50 mg methyl salicylate.The individual abutilon compounds were also tested at a dose of 100 mg: (Z)-3-hexen-1-ol (the same traps as in the sorghum experiment), tetradecane, methyl anthranilate, and methyl salicylate. Methyl anthranilate was tested only individually in October. An abutilon blend at a total dose of 100 mg and with ratios mimicking the headspace collections was also tested as follows: 20 mg (Z)-3-hexen-1-ol + 20 mg tetradecane + 5 mg methyl anthranilate + 55 mg methyl salicylate. In addition, a blend without methyl salicylate was tested. Furthermore, in October, a blend consisting of the abutilon blend with the addition of 30 mg eugenol was added to the experiment.
Field Experiments Related to SSR The materials and methods used in SSR-related field experiments were the same as those used for field experiments related to GC-EAD, except N = 5 and treatments were moved one step within blocks each day to minimize any impact of possible position effects. Some previously untested compounds also were applied to new dispenser types (see below).Six novel compounds selected by SSR were tested on 4–9 July 2008 on unused farmland with sparse vegetation near the village of Embuay Bad in Ethiopia (09°48′N, 40°00′E), 1206 m above sea level, 265 km northeast of Addis Ababa, Ethiopia. Five of the novel compounds tested (anethole, benzaldehyde, racemic 2,3-butanediol, isoamyl alcohol, and methyl octanoate) were selected on the basis that they elicited strong SSR response in separate ORN classes that did not respond to compounds previously tested in the field. For comparison, we also included eugenol and methyl salicylate. Methyl benzoate was tested for reasons different from the other compounds—it was included since it activated the same ORN type as methyl salicylate, which previously had been shown to be highly attractive. Olfactory receptor neurons responding to eugenol did not respond to other compounds included in the screening process. Practical limitations forced us to forego testing of some compounds as it was not possible to acquire necessary quantities in suitable purity, and other compounds were not included since they are more commonly associated with foliage than fruit or flowers.A dose of 100 mg of pure compound (for purity and CAS number, see Table 1) was loaded onto a dispenser that was matched to the volatility (as indicated by boiling point) of the compound. This rough estimation was used to obtain comparable evaporation rates. Cotton rolls (no. 2 dental cotton roll, Demedis GmbH, Langen, Germany) were used as dispensers for anethole, eugenol, methyl benzoate, methyl octanoate, and methyl salicylate. For dispensing benzaldehyde and 2,3-butanediol, cotton rolls were pushed into 4 ml vials (45 × 14.7 mm, clear, Skandinaviska GeneTec AB, Västra Frölunda, Sweden) until the cotton was level with the rim of the opening of the vial. Compound was applied to the cotton roll after it had been placed in the vial. For isoamyl alcohol, a dispenser was made where a cotton roll was put inside a vial closed with a cap (black, closed top, 13 mm, Skandinaviska GeneTec AB). A hole of approximately 2 mm diam was made in the cap. The cotton roll was placed so that it was in direct contact with the cap when the cap was screwed tight to the vial. The chemical was not applied to the cotton roll directly beneath the hole in the cap, but instead towards the edge of the vial, before screwing on the cap.
Statistical Analysis For field experiments, data for total catch of P. interrupta (cumulative over the field testing period) per trap was square root-transformed (√(× + 1)). Data was analyzed with a General Linear Model (GLM), with treatment (type of lure) as a fixed effect, and block as a random effect (Minitab 14 for Windows). Significant GLMs were followed by Tukey’s b post hoc test. The significance level used in all tests was α = 0.05. Trap catch data is presented in graphs as untransformed means with error bars denoting standard error of the mean.