Fig. 3.
Algogenic, membrane-permeabilizing, insecticidal, and anticoagulant effects of asp caterpillar venoms. (A) Activation of SH-SY5Y cells by membrane disruption induced by M. crispata venom, visualized with [Ca2+]i-sensitive dye and propidium iodide (Left). Membrane permeabilization and calcium influx were concentration-dependent, but all activity was lost after treatment by heat, exposure to acetonitrile, or proteases (Right, P < 0.001 in each case, unpaired Student’s t test, each point n = 3). (B) Activation of DRG cells by application of venoms (50 µg/mL) visualized with [Ca2+]i-sensitive dye. Graphs on the right show data for all cells, median (dark blue) and interquartile range (light blue). (C) Application of 1 µg/mL M. crispata venom to DRGs was ameliorated by treating venom with acetonitrile, heat, or protease (P < 0.001 in each case, Kruskal–Wallis test, n = 100 cells per sample at 250 s). (D) Pain behavior, erythema, and edema induced by injection of M. crispata venom (100 ng) into mouse hindpaw were inhibited by prior treatment of venom with acetonitrile. *P < 0.05, unpaired Student’s t test. **P < 0.005, paired (within mouse) or unpaired (between mouse) Student’s t test, n = 3. In the Middle panel, red and blue arrows indicate the injection of untreated and acetonitrile-treated venoms, respectively, and “i” and “ui” indicate injected and uninjected paws, respectively. (E) Fractionation of 50 µg M. crispata venom by size exclusion chromatography (Top) followed by testing of each individual fraction (second panel) located potent membrane-permeabilizing activity in fractions 5 and 6 (datapoints intersected by dotted lines connected to fractions 5 and 6 in the first panel). Numbers indicate fractions as in Top panel; PI, propidium iodide. Lower panels, concentration–response curves obtained by diluting these fractions. (F) Insecticidal activity of asp caterpillar venoms (Top), each point n = 8 flies; and anticoagulant of M. crispata venom on human plasma (Bottom), **P < 0.005 Student’s t test, n = 3.