Pentadecanoic acid

After an appropriate amount of time for consuming nitrogen, strain JSC4 cells were harvested from the culture medium by centrifugation (7,000 rpm for 2 min). The cells were washed twice with deionized water, lyophilized, and weighed. The lipid composition was determined as fatty acid methyl esters (FAMEs) following direct transesterification of lipids according to the method described in Ho et al. [7 (link)]. FAMEs were analyzed by gas chromatography/mass spectrometry (GC/MS) on a GCMS-QP2010 Plus instrument (Shimadzu). Samples were injected onto a DB-23 capillary column (60 m, 0.25 mm internal diameter, 0.15-μm film thickness; Agilent Technologies, Palo Alto, CA, USA). Helium was used as the carrier gas at a flow rate of 2.3 mL min⁻¹. The injector, ion source, and interface source temperatures were set at 230°C, 230°C, and 250°C, respectively. The oven temperature was initially set at 50°C for 1 min, increased from 50°C to 175°C at a rate of 25°C/min, increased from 175°C to 230°C at a rate of 4°C/min, and held at 230°C for 5 min. Purified FAMEs were identified based on retention time and the pattern of fragmentation by electron impact analysis. Supelco 37 Component FAME Mix (Sigma-Aldrich Co., St. Louis, MO, USA) was utilized as a quantitative standard, and pentadecanoic acid (Sigma-Aldrich Co.) was used as an internal standard.

Chemical compounds used for electroantennography (EAG) and oviposition bioassays were previously identified from extracts of immature stages of Ae. aegypti (3^rd and 4^th larval instars and pupae) [27 (link)]. The isovaleric acid, myristoleic acid, myristic acid and pentadecanoic acid synthetic compounds (purity ≥ 99%; Sigma Aldrich Inc., St-Louis, MO, USA) were diluted in n-hexane (HPLC grade; Carlo Erba reagents, Milano, Italy).

Fatty acid composition and content were determined as FA methyl esters (FAMEs), following the direct transmethylation of freeze-dried cell pellets and triacylglycerols, isolated from total lipid extracts. Samples placed in glass vials were transmethylated with 2% (v/v) sulfuric acid (H₂SO₄) in anhydrous methanol at 80°C for 1.5 h under an argon atmosphere with continuous stirring. Pentadecanoic acid (C₁₅:0; Sigma-Aldrich, United States; 0.5 mg/ml in stock solution) was added as an internal standard. The transmethylation reaction was terminated by cooling to room temperature and the addition of 1 ml of H₂O. FAMEs were extracted with hexane and quantified on a Trace GC Ultra (Thermo, Italy) equipped with an FID and a programmed temperature vaporizer (PTV) injector. The PTV injector was programmed to increase the temperature from 40°C at the time of injection to 300°C at sample transfer. The separation was achieved on a fused silica capillary column (SUPELCOWAX 10, Sigma-Aldrich, United States, 30 m × 0.32 mm) using the following oven temperature program: 1 min at 130°C, followed by a linear gradient to 220°C, and finally 10 min isocratic at 220°C. Helium was used as the carrier gas at a flow rate of 2.5 ml min⁻¹. The detector temperature was set at 280°C. FAMEs were identified by co-chromatography with commercial standards (Sigma-Aldrich, United States).

Biomass samples were harvested by filtration and washed thrice with distilled water. Collected mycelia were frozen overnight at − 80 °C, freeze-dried and the CDW was determined gravimetrically. The lipid extraction was carried out using the method of [53 (link)] with minor modifications and the analysis was conducted using the procedure reported in our previous work. Approximately 20 mg lyophilized biomass was mixed with chloroform/methanol (2:1, v/v), and pentadecanoic acid (15:0, Sigma) was added as an internal standard. 10 % (v/v) methanolic HCl was added to the extracted lipid for 3 h at 60 °C. The resultant fatty acid methyl esters (FAMEs), extracted with n-hexane, were analyzed by GC equipped with a 30 m ×0.32 mm DB-Waxetr column with 0.25 µm film thickness (Shimadzu Co., Ltd., Kyoto, Japan). The program used for GC machine was as follow: 120 °C for 3 min, then ramp from 120 °C to 200 °C at the speed of 5 °C/min, then ramp to 220 °C at 4 °C/min and hold for 2 min.

The chemical standards of l-2-chlorophenylalanine, pentadecanoic acid, and unsaturated fatty acid methyl ester mixes were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Supelco (Bellefonte, PA, USA). The silylating reagents of methoxylamine hydrochloride (MOX) and N-O-bis-(trimethylsilyl)-trifluoroacetamide + trimethylchlorosilane (BSTFA + 1% TMCS) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and Tokyo Chemical Industry (TCI) Chemicals (Kita-ku, Tokyo, Japan), respectively. Chromatographic pure hexane, methanol, chloroform, and deionized water (DW) were procured from J.T. Baker, Inc. (Phillipsburg, NJ, USA). Toluene (≥99.8%, Sigma-Aldrich, St. Louis, MO, USA) and pentane (≥99%, TCI Chemicals, Kita-ku, Tokyo, Japan) were used as received.

Korean perilla and sesame cultivars were grown at the National Institute of Crop Science, Rural Development Administration, Wanju-gun, Korea, during the 2018 growing season (June to November). Chinese perilla and sesame samples were procured from a local market in Xinzhou and JiangXia district (Wuhan city), China. The Chinese samples including perilla and sesame were from the recent harvests of November 2017 and 2016, respectively. Three biologic replicates were prepared for each sample. 5α-Cholestane, ribitol, pentadecanoic acid, fatty acid methyl ester (FAME) mixture, N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) and pyridine were purchased from Sigma-Aldrich (St. Louis, Mo, USA). All other chemicals used in this study were reagent grade unless stated otherwise.

All solvents used were of LC–MS analytical grade. Acetonitrile was purchased from Carlo Erba (Val De Reuil, France), isopropanol and methanol from Fisher Scientific (Laughborough, UK) and formic acid 98–100% from Chem-Lab (Zedelgem, Belgium). Lauric acid was purchased from Acros Organics (>99%), myristic acid from Sigma Aldrich (>99.5%), myristoleic acid from Sigma Aldrich (>99%), pentadecanoic acid from Sigma Aldrich (>99%), palmitic acid from Fluka (analytical standard), 9-palmitoleic acid from Fluka (analytical standard), margaric acid from Sigma Aldrich (>98%), 10-Z-heptadecenoic acid from Cayman Chemical Company (>98%), stearic acid from Fluka (analytical standard), oleic acid from Fluka (analytical standard), linoleic acid from Sigma Aldrich (>99%), linolenic acid from Sigma Aldrich (>99%), arachidic acid from Cayman Chemical Company (>98%), bihomo-γ-linolenic acid from Cayman Chemical Company (>98%), arachidonic acid from Sigma Aldrich (>99), 5,8,11,14,17-cis-eicosapentanoic acid from Fluka (analytical standard), 7,10,13,16,19-cis-docosapentaenoic acid from Cayman Chemical Company (>98%), 4,7,10,13,16,19-cis-docosahexaenoic acid from Sigma Aldrich (>98%) and lignoceric acid from Cayman Chemical Company (>98%).