The following odors and agonists were used: pentyl acetate (Sigma-Aldrich, Cat. Nr. 109584, 99%), butyl acetate (Fluka, Cat. Nr. 45860, ≥99.5% (GC), ACS Reagent), methyl hexanoate (Fluka, Cat. Nr. 21599, 99.8%, analytical standard grade), propyl acetate (Sigma-Aldrich, Cat. Nr. 133108, 99%; Fluka, Cat. Nr. 40858, ≥99%, analytical standard grade), 3-methylthio-1-propanol (Sigma-Aldrich, Cat. Nr. 318396, 98%; Sigma-Aldrich, Cat. Nr. W341509, ≥98% synthetic, FG grade), hexyl acetate (Sigma-Aldrich, Cat. Nr. 108154, 99%), isobutyl acetate (Fluka, Cat. Nr. 94823, 99.8%, analytical standard grade), 2-heptanone (Sigma-Aldrich, Cat. Nr. W254401, 98% synthetic, FG grade), 3-octanol (Sigma-Aldrich, Cat. Nr. 218405, 99%), (±)-geosmin (Sigma-Adrich, Cat. Nr. UC18, ≥98% [GC]), VUAA1 (CAS Nr. 525582-84-7) was synthesized by the group “Mass Spectrometry/Proteomics” of the Max Planck Institute for Chemical Ecology (Jena, Germany). Stimuli stocks consisted of 100 mM solutions in dimethyl sulfoxide (DMSO, Sigma-Aldrich, Cat. Nr. D8418); working solutions were prepared fresh diluting 100 mM stocks in SES to the desired concentration just before the start of the experiment. Negative controls consisted of the equivalent maximum volume of DMSO used to prepare odor stimuli solutions diluted in SES.
Geosmin
Manufactured by Merck Group
Sourced in United States
Geosmin is a chemical compound used in various laboratory equipment. It functions as a highly sensitive detector for the presence of bacteria, fungi, and other microorganisms in a variety of samples.
Automatically generated - may contain errors
Lab products found in correlation
2 heptanone, by Merck Group (3 mentions)
Pentyl acetate, by Merck Group (2 mentions)
Geranyl acetate, by Merck Group (2 mentions)
Cis 3 hexenyl acetate, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (1 mentions)
Propyl acetate, by Merck Group (1 mentions)
3 octanol, by Merck Group (1 mentions)
3 methylthio 1 propanol, by Merck Group (1 mentions)
Hexyl acetate, by Merck Group (1 mentions)
Bromophenol blue, by Merck Group (1 mentions)
2 propanol, by Merck Group (1 mentions)
Fluo 4 am, by Thermo Fisher Scientific (1 mentions)
Benzaldehyde, by Merck Group (1 mentions)
Butyric acid, by Merck Group (1 mentions)
Ethyl acetate, by Merck Group (1 mentions)
Linalool, by Merck Group (1 mentions)
Acetophenone, by Merck Group (1 mentions)
Hexanoic acid, by Merck Group (1 mentions)
2 3 butanedione, by Merck Group (1 mentions)
1 octanol, by Merck Group (1 mentions)
9 protocols using geosmin
1
Odorants and Agonists for Receptor Studies
2
Visualization and Gradient Formation
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Bromophenol blue (Sigma-Aldrich, St. Louis, MI) was employed to visualize the gradient and to estimate the diffusion of the odor along the length of the dig-and-dive chamber. We injected 1 μL of BPB (1 mM) at the bottom of agarose chamber (11 mm in depth) using a Hamilton syringe and diffusion of BPB molecules in time was monitored. By taking a picture of the assay, we confirmed that a stiff gradient of BPB was formed after 20 min. The gradient remained stable for 30 min (Figure 5—figure supplement 1 ). The same procedure was applied to establish gradients of odors (40 mM ethyl acetate and 0.5 mM geosmin, Sigma-Aldrich) and an insect repellent, DEET (10 mM, Sigma-Aldrich): the chemicals were diluted in distilled water and injected into chambers 20 min before the introduction of larvae.
3
Analysis of Geosmin and 2-MIB in Water Samples
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Geosmin (> 97%) was purchased from Sigma-Aldrich Ltd (Dorset, UK). 2-MIB (97.7%) was purchased from Chemservice, Inc (Merseyside, UK). cis-decahydro-1-napthol (Sigma-Aldrich Ltd, UK) was used as an internal standard. Ethanol, 2-propanol, methanol, and n-hexane organic solvents were all HPLC or GC grade (Sigma-Aldrich Ltd, UK). Samples were prepared using ultra-pure type 1 water. Raw water samples were collected by United Utilities water and wastewater services in the northwest of England (Warrington, UK).
4
Potentiometric Sensor Development
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Isoborneol (IB) was purchased from Sigma-Aldrich (São Paulo, Brazil), (±)-geosmin and 2-methylisoborneol (GE/MIB) (100 μg/mL in methanol) were purchased from Sigma-Aldrich (Rome, Italy). Dimethyl sulfoxide (DMSO), methanol (MeOH), and tetrahydrofuran (THF) solvents were obtained from Carlo Erba Reagents (Rome, Italy). Membrane components, high molecular weight Poly(vinyl chloride) (PVC), bis(2-ethylhexyl) sebacate (DEHS) plasticizer, tridodecylmethyl ammonium chloride (TDMACl), potassium tetrakis-(4-chlorophenyl)borate (TpClPBK) lipophilic additives, and nonactine ionophore were purchased from Sigma-Aldrich (Rome, Italy). THF was distilled prior to use and 5,10,15,20-tetraphenylporphyrin manganese(III) chloride ionophore (Mn(TPP)Cl) was synthesized and fully characterized according to the literature procedure [30 ]. Millipore grade water was used for aqueous solution preparation. All the other chemicals were of analytical grade and used without further purification.
5
Generation of Or7a Knockout Mutants
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Accelerated homologous recombination was performed according to Baena-Lopez LA et al. (Baena-Lopez et al., 2013 (link)). Briefly, P-element insertion lines containing the Or7a knockout construct were crossed to hs-Flp, hs-SceI (BS#25679) and heat-shocked at 48 and 72 hr after egg-laying (1 hr duration each time). Female progeny with mottled eyes were crossed to ubi-Gal4[pax-GFP] (Baena-Lopez et al., 2013 (link)) in order to select against flies containing non-homologous recombination events. Stocks were generated from candidate flies that contained both w+ and GFP markers. Or7a mutants were verified by single sensillum recordings and PCR (Figure 4F, G , Figure 4—figure supplement 3 ). In order to identify the ab4 sensillum, 30 μl of geosmin (Sigma #16423-19-1), an odor that specifically activates only ab4B (Or56a) (Stensmyr et al., 2012 (link)), was used (Figure 4F,G ).
Primers used for verification: G4polyA_FOR: TCG ATA CCG TCG ACT AAA GCC; gOr7a_REV:TCG CCG TTG AGT TTT CAG AG
The Or7a-Gal4 knockin was generated by co-injection of the pRIV-Gal4 donor plasmid (Baena-Lopez et al., 2013 (link)) with PhiC31 integrase to target GAL4 to the attP site within the knockout locus, as described in (Baena-Lopez et al., 2013 (link)).
Primers used for verification: G4polyA_FOR: TCG ATA CCG TCG ACT AAA GCC; gOr7a_REV:TCG CCG TTG AGT TTT CAG AG
The Or7a-Gal4 knockin was generated by co-injection of the pRIV-Gal4 donor plasmid (Baena-Lopez et al., 2013 (link)) with PhiC31 integrase to target GAL4 to the attP site within the knockout locus, as described in (Baena-Lopez et al., 2013 (link)).
6
Comprehensive Chemical Compound Synthesis
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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.
7
Synthesis and Purification of Volatile Compounds
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Auranofin (C20H34AuO9PS), ethyl hexanoate, (±)-Geosmin, 2-heptanone, E2-hexanal, geranyl acetate and pentyl acetate were purchased from Sigma Aldrich (Steinheim, Germany). 11-cis-Vaccenyl acetate (cVA) was purchased from Pherobank (Pherobank B.V., The Netherlands). All chemicals have ≥ 97% purity.
8
Odorant Delivery Protocol for Fly Neurobiology
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The following odorants were used in the study: vinegar (Balsamic vinegar, Alnatura, Germany), yeast (Fermipan, Italy; 1g/5ml water), citronella, peppermint (both from Aura Cacia, USA), 3-octanol, ethanol, 4-methylcyclohexanol (MCH), geosmin (0.01% in paraffin oil), isoamyl acetate, 1-Hexanol, 2-Heptanone (all from Sigma-Aldrich, Germany), 11-cis-Vaccenyl acetate (cVA) (Pherobanks, the Netherlands). Odors were diluted in water or paraffin oil to 1% with the exception of yeast and geosmin according to their solubility. For high vinegar stimulation vinegar was diluted to 80% in water to reach 32 ppm. A custom-made odor delivery system with mass flow controllers (Natec sensors, Garching) controlled by a MATLAB script were used for odor delivery. Throughout the experiments, a charcoal filtered continuous air stream of 1,000 mL/min was delivered through an 8 mm Teflon tube positioned 10 mm away from the fly antenna. Odor was delivered into the main air stream by redirecting 30% of main air flow for 1 s through a head-space glass vial containing 5 mL of diluted odorant. The odor delivery was automatically triggered by counting the time frame output from the scanning microscope by a custom written MATLAB script.
9
Analytical Standards for Volatile Organic Compounds
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Analytical standards (>98% purity) of 2-methylisoborneol, geosmin, 2-isopropyl methoxypyrazine (IPMP), 2-isobutyl methoxypyrazine (IBMP), 2,4,6-trichloroanisole (2,4,6-TCA), 2,4,6-tribromoanisole (2,4,6-TBA), β-cyclocitral, β-ionone, cis-3-hexenyl acetate (c3HA), cis-3-hexen-1-ol (c3HO), dimethyl disulfide (DMDS), dimethyl trisulfide (DMTS), 2,6-nonadienal (2,6-ND), 2,4-heptadienal (2,4-HD), 2-ethyl-2-methyl-1,3-dioxolane (EMD), hexanal, indole, naphthalene-d8 (N-d8) and 2,4,6-trichloroanisole-d5 (2,4,6-TCA-d5) were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2,3,6-trichloroanisole (2,3,6-TCA) was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Stock and working standard solutions were prepared in methanol (≥99.9% purity, Sigma-Aldrich, St. Louis, MO) and were stored at 4°C. Reagent grade sodium chloride (NaCl) was purchased in bulk (12 kg plastic drum) from EMD Millipore (Burlington, MA, USA).
For SPME, 1 cm dual-coated (50/30 µm) DVB/CAR/PDMS fibers were purchased from Supelco (Bellefonte, PA, USA). GC inlet liners that are compatible with SPME fibers were purchased from Agilent Technologies (Santa Clara, CA, USA). 20 mL amber headspace screw-top vials with PTFE/silicone magnetic caps were used for holding 10 mL samples for GC-MS/MS analysis (Agilent Technologies, USA).
For SPME, 1 cm dual-coated (50/30 µm) DVB/CAR/PDMS fibers were purchased from Supelco (Bellefonte, PA, USA). GC inlet liners that are compatible with SPME fibers were purchased from Agilent Technologies (Santa Clara, CA, USA). 20 mL amber headspace screw-top vials with PTFE/silicone magnetic caps were used for holding 10 mL samples for GC-MS/MS analysis (Agilent Technologies, USA).
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