For NMR spectroscopy the product was redissolved in CDCl 3 and measured directly afterward. This residual product was partly redissolved in hexane and stored at ‒28 ☌ until GC/MS or HPLC analyses and further experiments. Finally, hexane was removed under a nitrogen atmosphere, resulting in 1.2 g crude, unpurified product (yield of ∼50%, containing hexane and carboxylic acid derivatives as byproducts, calculated based on NMR data Figs. The organic phase was washed three times with saturated sodium bicarbonate solution and dried with sodium sulfate. HCl was added, and the mixture was extracted with hexane. After heating the mixture for 10 min to 50 ☌ under exclusion of moisture (using a drying tube filled with anhydrous CaCl 2), the reaction was terminated with ice water. Two grams (4.2 mL, 15 mmol, 2 eq) hexanoyl chloride were added drop-wise while stirring at 0 ☌. Mandelonitrile hexanoate (MNH IUPAC name is Cyano-(phenyl)-methyl hexanoate) was synthesized from mandelonitrile (2-hydroxy–2-phenylacetonitrile) and hexanoyl chloride by an Einhorn acylation ( 49): 1 g (0.9 mL, 7.5 mmol, 1 eq) mandelontrile was dissolved in 6 mL pyridine. Retention indices ( 46) and literature mass spectra ( 47, 48) for GC, as well as authentic standards for GC and HPLC, were used for compound identification. Chromatograms were recorded using a VWD-UV/VIS detector at λ = 245 nm (0–5 min) and at λ = 490 nm (5–10 min). The separation of 20-µL sample aliquots was performed using an isocratic elution with 40% phosphate-buffer (pH = 3.2) and 60% methanol:water mixture (85:15 vol/vol) for 10 min with a flow rate of 1 mL/min, on a Discovery HS C18 (15 cm × 4 mm, 5 µm) column (Sigma-Aldrich), at 25 ☌. The breakdown of MNH was monitored using an Agilent 1100 HPLC system (Agilent Technologies Fig. Crude aqueous extracts of the mites (in vivo) and 1 µL synthetic MNH (in 50 µL water in vitro) were further hydrolyzed from 30 min up to 1 h. Electron ionization mass spectra were recorded at 70 eV from m/z = 40–400. Crude hexane sample aliquots (2–5 µL) were analyzed with a GC/MS-QP2010 Ultra gas-chromatography mass-spectrometry system (Shimadzu) equipped with a ZB-5MS column (Phenomenex) according to a protocol given elsewhere ( 45). In addition, synthetic MNH (see below) was directly hydrolyzed in water as an in vitro control ( Fig. As a control, mites were directly immersed in hexane without mechanical stimulation. ( ii) To simulate a predator attack with aqueous saliva contact, mites were submerged in water. ( i) To simulate predator attacks, without aqueous saliva contact, mites were mechanically stressed using a fine brush (equals slight disturbance) or briefly shaken in an otherwise empty GC vial using a vortex mixer (equals strong disturbance) before hexane extraction. The two extraction solvents related to tests of two potential defense reactions-in water-free or humid conditions-and their corresponding MNH breakdown pathways.
After 3 min the solvent was separated from the mites. Oil gland exudates were extracted by submersing groups of 15–45 living mites in 20–50 µL solvent (hexane or water), a well-established method to obtain oil gland components ( 21, 28, 29, 45).