Isoamyl alcohol

Benzaldehyde, ®-citronellol or Isoamyl alcohol (Sigma-Aldrich) were diluted as described in water. Odor-exposures were done by suspending a 4 μl drop of these dilutions on the lids of worm culture dishes at least during 24 h from the egg stage at 20°C, covering the critical plasticity period corresponding to the first 12 h of post-hatch development (Remy and Hobert, 2005 (link)).

Genomic DNA was extracted from overnight yeast cultures according to PowerSoil^® DNA Isolation Kit (MO BIO laboratories Inc., Carlsbad, CA USA). An additional cleaning step with Phenol: Chloroform: Isoamyl Alcohol (25: 24: 1) (Sigma-Aldrich) was performed before DNA isolation. Genomic libraries were generated using the TruSeq DNA PCR-Free Library Prep Kit (Illumina Inc., San Diego CA) and Covaris S2 (Woburn, MA) for a 550-bp average fragment size. Libraries were loaded onto the flow cell provided in the NextSeq500 Reagent kit v2 (150 cycles) (Illumina Inc., San Diego CA) and sequenced on a NextSeq500 (Illumina Inc., San Diego CA) platform with a paired-end protocol and read lengths of 151 bp at the CRIBI Biotechnology Center (Padova, Italy) to determine the copy number of the integrated temA and temG_Opt genes.

Reagents and solvents chloroform, methanol, dimethyl sulfoxide (DMSO), isoamyl alcohol, hexane, anhydrous sodium sulfate (Na₂SO₄), linoleic acid, sodium carbonate (Na₂CO₃), and butylated hydroxytoluene (BHT) were purchased from Sigma‐Aldrich. Tween 40, tween‐80, silica gel‐G 60, 2,2‐diphenyl‐1‐picrylhydrazil (DPPH), and β‐carotene were purchased from Sigma‐Aldrich. Culture media DRBC agar (Dichloran, Rose Bengal, Chloramphenicol), PDA (Potato Dextrose Agar), and SMKY liquid media (Sucrose, Magnesium sulfate, Potassium nitrate, Yeast extract) were obtained from Sigma‐Aldrich.
Hydrodistillation apparatus, gas chromatography–mass spectrometry (GC‐MS; Agilent Technologies; model 6,850 and 5,973), centrifugation apparatus (Jouan E76), UV lamp (CN‐6, VILBER LOURMAY), spectrophotometer (6,705 UV/Vis, JENWAY), and a light microscope (Motic: BA210) were used in the present investigation.

Chemotaxis assays were performed on N2, odr-10 (e1515), and dmd-10 (gk1131) animals similarly to previously described (Bargmann, Hartwieg & Horvitz, 1993 (link)). Assay plates were prepared 2 days prior to the assay 10 ml of CTX agar (2% agar, 5 mM KPO₄ pH = 6, 1 mM CaCl₂, 1 mM MgSO₄) in 10 cm plates. The day of the assay CTX plates were spotted with 1 μl of 1 M NaN₃ on opposite sides of the plate. The same locations were then spotted with 1 μl of either chemoattractant (0.1% diacetyl or 1% isoamyl alcohol (Sigma-Aldrich, St. Louis, MO, USA)) or vehicle (ethanol). Assays were performed using populations of C. elegans that had been laid 3 days prior to the experiment. The C. elegans were washed twice with M9 buffer and once with distilled deionized water. Between 50 and 300 individuals were pipetted on the midline of the plate and excess liquid was removed with a kimwipe. After 1-h incubation at 20 °C the number of individuals within a 2 cm circle centered on each spot (chemoattractant and vehicle) and the total number of individuals on the plate were recorded (individuals that had not moved after being placed on the plate were not counted).

All the chemicals and reagents used in this study were of analytical grade. GPI0363 was obtained from the chemical library of the Drug Discovery Initiative at the University of Tokyo and its purity was confirmed by ultra-performance liquid chromatography-high resolution mass spectrometry (m/z: 343.1968 [M + H]⁺; calculated for C₁₉H₂₄N₄O: 343.1934) (ESI Fig. 7†). Rifampicin (potency: 1029 μg mg⁻¹), ammonium acetate, and HPLC grade acetonitrile were obtained from Fujifilm Wako Pure Chemical Industries, Ltd., Osaka, Japan. Fidaxomicin (purity: ≥95%) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Salmon sperm DNA, actinomycin D (purity: ∼98%), and phenol : chloroform : isoamyl alcohol (125 : 24 : 1, v/v/v) were obtained from Sigma Aldrich, Tokyo, Japan. Tryptone, tryptic soy broth (TSB), yeast extract, and Mueller-Hinton broth (MHB) were obtained from Becton, Dickinson and Company (Franklin Lakes, NJ, USA). RNase inhibitor was obtained from Applied Biosystems (Beverly, MA, USA). ATP, CTP, GTP, UTP, and yeast tRNA were obtained from Ambion (Austin, TX, USA). [α-³²P] UTP was obtained from PerkinElmer, Waltham, MA, USA. The E. coli RNAP holoenzyme and core enzyme were obtained from New England Biolabs (Ipswich, MA, USA), and TALON® magnetic beads were obtained from Takara Bio USA Inc. (Mountain View, CA, USA).