In Vivo Feeding Studies – Artificial Diets.

For artificial diets, individual monoterpenes or the monoterpene mixture was added to pelleted rabbit chow at 1% of DW. Camphor and camphene are solids at room temperature and cannot be added homogenously to rabbit chow, whereas α-pinene, β-pinene, and 1,8-cineole are liquid and can be directly added to rabbit chow. Pure camphor (260 mg/mL) and camphene (248 mg/mL) were therefore dissolved together in methylene chloride (≥ 99.8% pure, Sigma Aldrich, St. Louis, MO). The methylene chloride mixture was thoroughly mixed with rabbit chow in a glass jar at a concentration of 25 μg/g DW of chow. The treated rabbit chow was then spread in a single layer in a fume hood for six hours to allow the highly volatile solvent to evaporate. The time needed for evaporation of the solvent relative to individual monoterpenes was determined by analyzing the concentration of methylene chloride and the camphor and camphene dissolved in methylene chloride added to rabbit chow over time until concentrations of methylene chloride were less than 1.0 μg/g DW of chow. The evaporation of camphor and camphene during the six hour period was minimal relative to the solvent, resulting in the desired final concentrations of monoterpenes (Table 1). In a preliminary study, we determined that pygmy rabbits and mountain cottontails did not discriminate between control rabbit chow and chow that was mixed with methylene chloride only (no camphor and camphene) and allowed to evaporate for six hours (Nobler 2016). After the solvent was evaporated, the remaining liquid monoterpenes were thoroughly mixed with the rabbit chow already treated with camphor and camphene in a glass jar. To prevent the volatilization of monoterpenes, all treated chow was stored at −20° C until offered to rabbits. Samples of treated rabbit chow were saved in sealed scintillation vials at −20° C before being analyzed for concentrations of monoterpenes via gas chromatography.