Synthetic carvacrol

Antennae of Ma. arion were excised at the base of the flagellum. The tip of the terminal segment was cut using micro-scissors, and the antennae (n = 8) were connected to an electrophysiology apparatus. The excised tip was placed on the antennal holder (two 1.7 mm diameter wells containing 0.1 M KCl); the recording electrode was connected to the base of each antenna with the reference electrode connected to its tip. Baseline responses of antennae were established from exposure to clean GC-grade air, and compared with responses when fluxing ant allomones, plant volatiles and synthetic pure compounds or mixtures. VOCs were fluxed towards the mounted antenna on the electroantennogram (EAG) apparatus with clean air as carrier gas. Synthetic carvacrol, thymol and Z-3-hexen-l-ol were obtained from Sigma-Aldrich (Milan, Italy) (10 µg per desiccator). VOCs from treated and untreated O. vulgare from 4 l glass jars (see above), synthetic mixtures and extracts from solitary ant nests were flowed. Electrical signals were pre-amplified and their intensity measured using a 0.052 V step input; the amplified output was 6.49 V. Responses were expressed as mean differences to the standard (Z-3-hexen-l-ol), Dimethyl sulfoxide (Sigma-Aldrich, USA) and clean air after exposing the antenna alternately, every 30 s, to a test chemical or to clean air.

Oral carvacrol treatment via the drinking water was initiated two days after initial C. jejuni infection. Therefore, synthetic carvacrol (purchased from Sigma-Aldrich, Munich, Germany) was dissolved in Tween 80 (0.2% v/v) and added into autoclaved tap water to a final concentration of 500 mg/L (ad libitum) resulting in a daily dose of 100 mg per kg of body weight considering a body weight of approximately 25 g per mouse and a daily drinking volume of approximately 5 mL. The placebo control animals received a vehicle via the drinking water instead. Treatment solutions were changed every other day until the end of the experimental period (i.e., day 6 p.i.; Figure 1).