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Plenty of insect cells are sensitive to azadirachtin, an ideal biopesticide. However, the mode of action of azadirachtin on insect cells still remains unexplained. In this study. Trichoplusia ni BTI-Tn-5B1-4 cell line was used as the model system to evaluate induction of programmed death and cytoskeletal damage by azadirachtin. The result of colorimetric [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide] (MTT) assay indicated that the effect of azadirachtin on cells growth was time-dependent with different concentrations (0.75, 1.5 and 3 mu g/mL). As autophagic vacuoles were observed obviously by transmission electron microscopy, azadirachtin could induce autophagy in cells. Furthermore, lysosomal function was affected after treatment with azadirachtin in time-dependent manner. Aberrant morphological figures appeared in treated cells, such as contraction and turning round, especially the appearance of apoptotic bodies and membrane blebbing, which was the evidence of azadirachtin-induced apoptosis, with apoptosis rate of 13.98% at 1.5 mu g/mL for 120 h. As the stress fibers changed after treatment, the cytoskeletal function could be influenced. The effect on cell cycle was also analyzed by flow cytometry, with the result that azadirachtin enhanced cell cycle arrest at the G2/M phase gradually. Crown Copyright (C) 2010 Published by Elsevier Inc. All rights reserved.