Acetophenone

The following chemicals were purchased at highest purities available from Sigma Aldrich, Bangalore, India: 2-methyltetrahydro-3-furanone, acetic acid, benzaldehyde, 1-octanol, (R)-1-octen-3-ol, ethyl butyrate, ethyl acetate, geranyl acetate, methyl salicylate, methyl laurate, isopentyl acetate, hexanoic acid, 2-methylphenol, geosmin, butyraldehyde, 1,4-diaminobutane, phenyl acetalydehyde, phenylethylamine, pyridine, ammonia solution and mineral oil. 11-cis-vaccenyl acetate was purchased from Cayman Chemical Company, Michigan, United States. Cis-3-hexenyl acetate, 2,3-butanedione, butyric acid, linalool, and acetophenone were purchased from Fluka, Sigma-Aldrich, Bangalore, India. The compound 1-hexanol was purchased from TCI, nonanal was purchased from Acros Organics. Propionic acid was obtained as a gift from the Max Planck Institute for Chemical Ecology, Jena, Germany. Fluo-4AM was purchased from Life Technologies, Stockholm, Sweden.

Olfactory stimuli were delivered with a custom-built olfactometer comprising eight odor channels and controlled via a custom-made LabVIEW (National Instruments, Austin, TX, USA) interface, which synchronized stimulation protocol with the imaging acquisition [21 ]. For this study, we used six ecologically relevant odorants known to elicit distinct responses in the antennal lobe glomeruli: 1-hexanol (1HEX), 3-hexanol (3HEX), 1-nonanol (1NON), isoamyl acetate (ISOA), acetophenone (ACTP), and benzaldehyde (BZDA) (all from Sigma-Aldrich) [2 (link),9 (link)]. Additionally, they provide different degrees of structural and functional variability: 1-hexanol and 3-hexanol have the same carbon length but vary for the position of the hydroxyl group; 1-hexanol and 1-nonanol are primary alcohols with different chain lengths; acetophenone and benzaldehyde both have a benzene ring, but with a ketone and an aldehyde group, respectively; isoamyl acetate is the major component of the honey bee alarm pheromone [27 (link)], while all other odorants are typical floral scent components. Stimuli were presented in a 1 s ON/9 s OFF protocol and each odorant was delivered for 30 consecutive trials. All odorants were diluted 1:200 in mineral oil (Sigma-Aldrich). These concentrations were chosen to be well above the receptor sensitivity threshold and below the saturation level [5 (link)].

Authentic standards of n-alkanes (C₇–C₃₆), acetophenone, octan-3-one, 5-methyl-heptan-3-one, 6-methyl-5-hepten-2-one, 1,4-naphthoquinone and 2-methoxy-1,4-naphthoquinone for a comparison of gas chromatographic–mass spectrometric data to components found in the Carinostoma-secretion were purchased from Sigma (Vienna, Austria). 6-Methyl-1,4-naphthoquinone was prepared according to Bruce and Thomson (1952 (link)); as oxidizing reagent we used CAN (=cerium IV ammonium nitrate from Sigma, Austria). For two further compounds, namely 2-methoxy-6-methyl-1,4-naphthoquinone and 4-chloro-1,2-naphthoquinone, we used known natural sources for reference, namely the secretions of P. quadripunctatum (Raspotnig et al. 2010 (link)) and Cyphophthalmus (formerly Siro) duricorius (Raspotnig et al. 2005 (link)). O-Methyl oximes of ketones in the Carinostoma-extracts were prepared by adding 200 μl of methoxamine (MOX) reagent (=2 % methoxyamine-hydrogen chloride in pyridine, Thermo Scientific, Vienna, Austria) to 60 μl of ketone-rich extracts in hexane, followed by incubation at 70° for 1 h. The products were washed twice with water, dissolved in 60 μl of hexane, and an aliquot (1.5 μl) was directly used for GC–MS.

Acetophenone, aliphatic ketones, 2-propanol, triethanolamine (TEA), citric acid, dipotassium phosphate, and methyl tert-butyl ether (MTBE) were obtained from Sigma-Aldrich, Seelze, Germany. Magnesium chloride, potassium dihydrogen phosphate, mandelonitrile, glucose, and concentrated hydrochloric acid were purchased from Merck, Darmstadt, Germany. Sodium cyanide was received from Fluka Chemika AG, Buchs, Switzerland. In addition, mandelonitrile was stored at -18°C to minimize decomposition to benzaldehyde and hydrogen cyanide. Sodium hydroxide was purchased from VWR International, Darmstadt, Germany and calcium carbonate was obtained from Fisher Scientific, Loughborough, United Kingdom. NOA81 was obtained from Thorlabs, Dachau, Germany. All chemicals were obtained in highest available purity and used as received. The hydroxynitrile lyase from Manihot esculenta was a gift from Jülich Fine Chemicals (now Codexis). Alcohol dehydrogenase from Lactobacillus kefir (LkADH) and glucose dehydrogenase (GDH) were purchased from evocatal, Monheim, Germany. Deionized water was produced with an Ultra Clear Reinstwassersystem by SG Water (now Evoqua, Guenzburg, Germany) and used throughout this study.

The chemicals used included: 6-methyl-5-hepten-2-one (Aldrich, 99%), acetophenone (Sigma-Aldrich, ≥ 99%); geranylacetone (Aldrich, 65% geranylacetone and 35% nerylacetone), heptanal, octanal, nonanal and decanal (Aldrich, 95%).