Fig. 1.
Repellents citronellal and citronellol stimulate scorpion avoidance response via depolarizing its VNC ganglion neurons. (A) Schematic representation of scorpion avoidance response assay. Two pieces of gauze were preimmersed with water (control) or a solution with the desired concentration of the test drug and placed at one side of the container (100 cm × 50 cm × 30 cm) with a layer of clean sand. Each gauze was covered by a piece of clay tile. For each trial, 100 scorpions were placed at the center of the container covered with a piece of glass. After about 1 h, the numbers of scorpions under and outside the tiles were separately counted. (B) Summary of the assay shown in A. Note, in the citronellal and citronellol groups, >50% of scorpions were outside of the tiles. Data from five or six independent experiments per group are presented. Error bars indicate SD. ***P = 2.56E−5 for citronellal, 2.70E−8 for citronellol, and 5.23 E−6 for DEET; P = 0.106 for camphor and 0.120 for menthol versus control (ANOVA). (C) Schematic diagram of scorpion VNC showing a total of seven ganglia (Left) and an example anatomical picture (Right). (D) Representative whole-cell recordings of scorpion VNC neurons in response to repeated applications of citronellol (0.1 mM, Top) and citronellal (0.5 mM, Bottom), respectively. Each drug application lasted for ∼15 s. Holding potential (Vh) was −60 mV. A comparison between the responses of cells to the first and the 10th applications is shown in Inset. The dotted line indicates zero current level. (E) Time courses of peak currents elicited by repeated applications of citronellol (n = 7) and citronellal (n = 8). Currents were normalized by the maximum values after sensitization. (F) Concentration-response curves of citronellal and citronellol after full sensitization. The solid lines are fits to the Hill equation with EC50 = 0.27 ± 0.02 mM, nH = 2.0 ± 0.2 for citronellol (n = 6); and EC50 = 0.50 ± 0.02 mM, nH = 3.0 ± 0.4 for citronellal (n = 6). (G) Representative current-clamp responses of isolated scorpion VNC neurons consecutively challenged with citronellal (0.2 and 2 mM) and citronellol (0.2 and 2 mM). Note, the injected current was set to zero. (H) Statistics plot of membrane potential. The addition of different concentrations of citronellal or citronellol caused neurons to undergo different degrees of depolarization. RMP, resting membrane potential. Number of cells is indicated in parentheses. ***P = 3.38E−4 for 0.2 mM citronellal, 2.57E−8 for 2 mM citronellal, 4.53E−7 for 0.2 mM citronellol, 2.57E−8 for 2 mM citronellol versus RMP (ANOVA). (I) Concentration-response curves of inhibitory effects of RR and 2-APB on isolated VNC neurons in the presence of citronellol (2 mM). Solid lines indicate fits with the Hill equation, which yielded IC50 = 0.78 ± 0.02 μM, nH = 1.5 ± 0.1 for RR (n = 7); and IC50 = 182.45 ± 13.15 μM, nH = 1.4 ± 0.1 for 2-APB (n = 8). Error bars represent SEM.