Effect of Wilforine on the Calcium Signaling Pathway in Mythimna separata Walker Myocytes Using the Calcium Imaging Technique

Although the action site of wilforine is located in the muscle tissue of insects, the insecticidal mechanism of wilforine is not yet clear. This research explored the effects of wilforine on the calcium signaling pathway using the calcium imaging technique to reveal the insecticidal mechanism. It was confirmed that wilforine had strong cytotoxicity to Mythimna separata myocytes with the IC₅₀ values of 25.14 and 19.65 mg/L using CCK-8 and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide methods, respectively. The morphological development of M. separata myocytes was also affected. The calcium imaging technique showed that the intracellular calcium ion concentration ([Ca²⁺]_i) increased by 23.45% of the initial value after being treated with 100 nM wilforine. However, wilforine did not increase [Ca²⁺]_i after the myocytes were preincubated with thapsigargin, and the [Ca²⁺]_i could not be decreased by 50 μM ryanodine after being treated with 100 nM wilforine. These results indicated that the targets of wilforine are located in the sarcoplasmic reticulum, and ryanodine receptor (RyR) is an important action target of wilforine. Furthermore, wilforine can also activate the inositol triphosphate receptor (IP₃R), which was confirmed through the use of 2-aminoethyl diphenylborinate, an inhibitor of IP₃R. Connected with previous research studies, it can be concluded that wilforine affects the calcium signaling pathway by combining with RyR and IP₃R, causing calcium dyshomeostasis, which results in insect paralysis and death.