Methyl salicylate

A commercial formulation of Methyl salicylate (Methyl salicylate, Sigma-Aldrich) was used for all experiments. The rice varieties ‘IR 20, IR 50, IR 64, ASD 16, ASD 19 and ADT 46’ were used for this study. ‘IR 20, IR 50, IR 64’ are susceptible, conventional, semi-dwarf, long-grain aromatic variety with relatively high seedling vigor, whereas ‘ASD 16, ASD 19 and ADT 46’ are moderate resistance, conventional, semi-dwarf long grain variety with low seedling vigor. Rice were surface disinfected by immersing in 2% sodium hypochlorite for about two minutes and washed several times with sterilized water, then dried in sterilized filter paper.
Salicylate formulations and treatments consisted of increasing levels of 0, 25, 50, 75 and 100, mg/L of MeSA. A stock solution of MeSA 100 mg/L was used for successive dilutions in distilled water. The seeds were treated by applying 2 ml/kg of seed in each respective MeSA concentration according to methodology described by Tavares et al.28 . The solution of MeSA with the different doses was directly placed at the bottom of the zip lock plastic bag. Then 250 g of seeds were added into each bag. They were agitated for 10 minutes. The seeds were spread out on clean paper and left to dry at room temperature for 24 hours. Treated seeds were used for further experiments.

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.

Twenty types of compounds, derived from tea plants and plant-derived semiochemicals attracted to other thrips, were used in our trials [4 (link),24 (link),25 (link),26 (link)]. In particular, 4-acetylpyridine, p-anisaldehyde, decanal, eugenol, farnesene (mixture of isomers, α-farnesene, and (E)-β-farnesene), geraniol, (Z)-3-hexenol, (Z)-3-hexenyl butyrate, limonene, methyl anthranilate, methyl benzoate, 3-methyl butanal, methyl isonicotinate, methyl salicylate, β-myrcene, nonanal, (E)-β-ocimene, (−)-α-pinene, (+)-α-pinene, and γ-terpinene were all purchased from Sigma-Aldrich (St. Louis, MO, United States) (Table S1). Hexane (HPLC grade, CNW Technologies GmbH (Düsseldorf, Germany)) was chosen as solvent, and the abovementioned synthetic volatiles were diluted to a specific concentration.

Brains were removed from the fixative solution and washed 3 times in 0.01 M of PBS (10 min each). Brains were then dehydrated in ascending concentrations of ethanol (50, 70, 90, 95 and 3 × 100% for 10 min each) and clarified in methylsalicylate (Sigma-Aldrich, Steinheim, Germany) for at least 3 days. The samples were then mounted on aluminum slides with a central hole filled with methylsalicylate and covered by thin coverslips on both sides.
Antennal lobes were scanned with a laser-scanning confocal microscope (LSM-700; Carl Zeiss, Jena, Germany). Using a water immersion objective (20 × plan-apochromat 1.0 NA), optical sections were acquired at 1 µm intervals (z), with a resolution ranging from 0.52 to 0.69 µm/pixels (x,y), depending on the size of the AL of each species. Given the large size of the hornet AL, complete scans were obtained by stitching adjacent “tiles” (512 × 512 pixels) of optical sections with the tile function of the ZEN software (Carl Zeiss, Jena, Germany). Fluoro-ruby labeled neurons were visualized using a 555 nm solid-state laser, while Lucifer yellow was excited at 488 nm.

4-Hydroxy-2,5-dimethylfuran-3(2H)-one (HDMF), 2 (or 5)-ethyl-4-hydroxy-5 (or 2)-methylfuran-3(2H)-one (EHMF), 4-hydroxy-5-methylfuran-3(2H)-one (HMF), naringenin, eugenol, vanillic acid, salicylic acid, 1-naphthol, farnesol, sorbic acid, nerolidol, geraniol, fisetin, vanillin, chlorogenic acid, 2-naphthol, caffeic acid, methyl salicylate, ferulic acid, 1-octanol, and myricetin were purchased from Sigma-Aldrich (Shanghai, China). UDP-glucose, UDP-galactose, and UDP-glucuronic acid were purchased from Promega (Madison, USA). UDP-Xylose was purchased from Angfei Biotech Co., Ltd. (Guangzhou, China).

Fifty-five days after the seedling, the median portion of the main stem leaflet was used to perform the comparative microscopy analysis. The solution of formalin-acetic acid-alcohol was utilized to fix the samples Paolillo and Zobel (2002) (link). The preserved stem and leaflet were divided into 5 mm segments cut horizontally into thin cross sections. Johansen’s pigments were used to stain the cross sections Johansen, 1940 . As a clearing solution, ethanol and xylene: methyl salicylate (1:2, v/v) (99% purity, Sigma-Aldrich, St. Louis, MO, USA) were used to clarify the samples for better images. Finally, brief observations were done under a microscope, and delicate images were obtained using the EVOS FL Cell Imaging System (Thermo Fisher Scientific).

Authentic standards of (E)-2-hexenal, p-xylene, α-pinene, β-pinene, trans-isolimonene, β-myrcene, 2-carene, α-phellandrene, 3-carene, α-terpinene, p-cymene, β-phellandrene, (Z)-β-ocimene, (E)-β-ocimene, γ-terpinene, terpinolene, n-nonanal, allo-ocimene, methyl salicylate, β-elemene, (Z)-jasmone, (E)-β-caryophyllene, and α-humulene (>95% purity) were purchased from Sigma-Aldrich (St. Louis, MO, United States). Dichloromethane (99.9% purity) was purchased from Merck (Germany).