Use of Sf9 cells identifies V-ATPase as a target of pyridalyl

In modern agriculture, pesticides play a critical role in maintaining steady crop production. Insecticides protect crops from insect pests that cause yield losses. Pyridalyl is a structurally unique insecticide that is particularly effective against Lepidoptera and Thysanoptera. However, the mode of action remains unclear. We sought to identify the molecular target of pyridalyl using Sf9 cells, a cell line derived from Spodoptera frugiperda. Pyridalyl (1 μM) suppressed the growth of cells, but was not lethal. It induced cell cycle arrest at the G2/M phase and inhibited the cellular uptake of small molecules such as [3H]leucine. Vacuoles containing damaged organelles accumulated in cells cultured with pyridalyl, indicating autophagy induction. Pyridalyl-treated cells were intensely stained with monodansylcadaverine (a marker for autophagosomes), but not with acridine orange (a probe for the lysosomal H+ pump), indicating the failure of autophagosome acidification, which is necessary for the degradation of autophagosome contents. Pyridalyl treatment did not enhance the expression of SfAtg8 (an autophagy marker), suggesting blockage or stagnation of autophagy. Exposure of Sf9 cells to pyridalyl led to the inhibition of vacuolar-type H+-adenosine triphosphatase (V-ATPase) activity, with a submicromolar IC50 value. Overall, we demonstrated that pyridalyl inhibits intracellular V-ATPase; this results in autophagosome formation, followed by the blockage of autophagic flux in cells. Given the diverse physiological roles of V-ATPase, the insecticidal activity of pyridalyl may arise from its interference with V-ATPase throughout the insect body rather than by blocking autophagy.