Ligustilide

Menthol and allyl isothiocyanate (AITC) were purchased from Acros Organics (Geel, Belgium), ligustilide from Alomone Labs (Jerusalem, Israel), and ASP 7663 from Tocris (Minneapolis, MN). Rotifers were hatched and isolated into 24 well plates with 15 ppt Instant Ocean and 6 × 10⁵ cells/ml T. suecica, as above. For the Menthol exposure, Menthol was added to cultures at a final concentration of 10 μM, 50 μM, or 100 μM, and plates were maintained at 21 °C. In a second experiment, 50 mM AITC in ethanol was added to cultures at a final concentrations of 1 μM, 5 μM or 10 μM; plates with AITC were tested at both 21 °C and 16 °C. To prevent egg hatching, 20 μM FUDR was used in these experiments. For the third experiment, 50 mM ligustilide in ethanol was added to a final concentration of 50 μM, 100 μM, or 200 μM, and 50 mM in ethanol of ASP 7663 to 0.5 μM, 2 μM, or 5 μM, all without FUDR. The concentrations used were based on the EC 50 in cell or mammal studies. The controls for AITC, ligustilide, and ASP 7663 contained 0.4% ethanol. All agonist and control treatments were maintained in a 12:12 h light:dark cycle. Menthol, AITC, food, and FUDR were renewed every 3 days throughout life. For ligustilide and ASP 7663, transfers to fresh food and agonists were performed daily. Survivorship was scored as described above.